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MULTIDRUG RESISTANCE PROTEIN 1 (MDR1)
AND GLYCOSPHINGOLIPIDS BIOSYNTHESIS:
ADVANTAGES FOR THERAPEUTICS
by
María Fabiana De Rosa
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by María Fabiana De Rosa (2009)
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Multidrug Resistance Protein 1 (MDR1)
And Glycosphingolipids Biosynthesis:
Advantages for Therapeutics
Doctor of Philosophy, 2009
María Fabiana De Rosa
Department of Laboratory Medicine and Pathobiology
University of Toronto
ABSTRACT
ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic
molecules from the inner to the outer leaflet of the plasma membrane. We have previously
reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms
responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic,
glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a
whole new spectrum of innovative therapeutic options. We found that cell surface MDR1
partially co- localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition
of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We
speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be
functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating
MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on
MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from
MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also
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able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide
had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its
favourable in vivo features make it a possible choice for inhibition of MDR1 to increase
bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific
GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1
inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this
disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of
accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with
third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of
untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach
not only for Fabry disease given the extraordinary cost of conventional enzyme replacement
therapy, but also for other neutral GSL storage diseases, such as Gaucher disease.
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ACKNOWLEDGEMENTS
I would like to specially thank my supervisor, Dr. Clifford Lingwood, for his firm and
warmth valuable guidance through the fascinating world of cell biology, his constant and
unconditional support, and his patience along this project.
I would like to thank all my committee members, Dr. David Clarke, Dr. Peter Dirks, and
Dr. Reinhart Reithmeier for their support and their brilliant input in this project along these
years.
I would also like to specially thank my external examine, Dr. Inka Brockhausen, for her
valuable appraisal of this thesis.
I would also like to thanks all the collaborators in this project, Dr. Shinya Ito, Dr Jeffrey
Medin, Bernice Wang, Cameron Ackerley, Aina Tulips, Vanessa Rassaiah, and Xin Fan that
bring their invaluable expertise on specific aspects of this project.
And finally, I specially would like to thank all past and present members from Dr.
Lingwood’s laboratory, especially Dr. Mylvaganam, Beth Binnington and Shirley Thompson
that not only bring me their unconditional help but also their constant support in good and not so
good times along this project.
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To the beloved memory of my parents, Emma and Jose Maria, for their loving
encouragement and support in the pursuit of my career’s dream.
To the beloved memory of my parents-in-law, Gloria and specially Ricardo, who always
believed in my scientific capabilities and for his support in my decision to pursue a PhD degree.
To the beloved memory of my aunt, Maria Elvira, who initiates me in the intriguing and
challenging principles of biology.
To my beloved husband, for his unconditional love, his loving support and strength in
weakest times, for his comforting hugs that hold me tightly and do not let me fall, for sharing
side by side the precious and not the so precious moments of this difficult path of pursuing a
PhD, and for being that wonderful dad, always but even more when mommy had those
abominable deadlines.
To my children, Ricardo, Thomas, and my little princess, Gloria that enlighten every
minute of my life with their smiles and laughs, and for their big hugs and loving kisses that kept
me going even in the most difficult times along this project.
To my brothers-in-law, Francisco and Juan Manuel, that make me feel as their special
sister and friend and in whom they believe and encourage in the pursuit of her dreams.
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TABLE OF CONTENTS
ABSTRACT
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF ABBREVIATIONS
LIST OF TABLES
LIST OF FIGURES
LIST OF SCHEMAS
CHAPTER ONE. INTRODUCTION
1.1. Multidrug Resistance
1.1.1. Multidrug Resistance in Cancer
1.1.2. Cellular Mechanisms of Multidrug Resistance
1.2. ATP-Binding Cassette Transporters
1.2.1. Structural Organization and Mechanism of ABC Transporters
1.2.2. ABC Transporters in Normal Cells
1.2.3. ABC Transporters in Cancer Cells
1.2.4. ABC Lipid Transporters
1.3. P-glycoprotein – MDR1
1.3.1 Structure of P-glycoprotein
1.3.2 Mechanism of Action
1.3.3 P-glycoprotein Substrates and Inhibitors
1.3.4 P-glycoprotein Silent Polymorphisms
1.3.5 P-glycoprotein Regulation
1.3.6 P-glycoprotein Knockout Mice
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1.3.7 Other Approaches in Modulation of Multidrug Resistance
1.3.8 P-glycoprotein and Stem Cells
1.4. Glycosphingolipids
1.4.1. Structure of Glycosphingolipids
1.4.2. Classification of Glycosphingolipids
1.4.3. Biosynthesis and Degradation of Glycosphingolipids
1.4.4. Inhibitors of Glycosphingolipids Biosynthesis
1.4.5. Lipid Rafts
1.4.6. Biological Functions of Glycosphingolipids
1.4.7. Globotriaosylceramide
1.4.7.1. Structure of Globotriaosylceramide
1.4.7.2. Biological Functions of Globotriaosylceramide
1.4.7.2.1. Antineoplastic Potential of Verotoxin
1.4.8. Lysosomal Storage Diseases
1.4.8.1. Pathogenesis
1.4.8.2. Fabry and Gaucher Diseases
1.4.8.3. Therapeutic Approaches
1.5. Multidrug Resistance and Glycosphingolipids
1.5.1. MDR1 and Ceramide Metabolism
1.5.2. MDR1 and Lipid Flippase
1.5.2.1.Alternative Mechanisms for GlcCer Transport into the
Golgi
1.5.3. MDR1, Cholesterol and Lipid Rafts
CHAPTER TWO. HYPOTHESIS AND SPECIFIC AIMS
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2.1. Rationale
2.2. Hypothesis
2.2.1. Specific Aims
CHAPTER THREE. INHIBITION OF MULTIDRUG RESISTANCE BY
ADAMANTYLGb3, A GLOBOTRIAOSYLCERAMIDE ANALOG
3.1. Abstract
3.2. Introduction
3.3. Materials and Methods
3.3.1. Materials
3.3.2. Cell Culture
3.3.3. Immunostaining of MDR1
3.3.4. Post-embedding Immunogold Cryolectron Microscopy
3.3.5. Neutral Glycolipids Extraction and Analysis
3.3.6. Verotoxin 1 Thin Layer Chromatography Overlay
3.3.7. Cytotoxicity Assay
3.3.8. MDR1-MDCK Raft Isolation
3.3.9. Western Blot Analysis of MDR1
3.3.10. Rhodamine 123 Efflux Assay
3.3.11. Drug Transport
3.3.12. Disulfide Cross-Linking Analysis
3.4. Results
3.4.1. MDR1 in Part Co-localizes with Gb3
3.4.2. AdaGb3 Prevents Multidrug Resistance in MDR1-MDCK and
SK VLB Cells
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3.4.3. AdaGb3 Inhibits Cell Surface MDR1 Expression in the Long Term
in MDR1-MDCK and SK VLB Cells but Increases Intracellular
MDR1 as PPMP, VT1B, and CsA
3.4.4. Effect of Adamantyl Analogs on GSL Levels
3.4.5. MDR1 Distribution in Lipid Rafts
3.4.6. AdaGb3 Inhibits the Efflux of Rhodamine 123 in MDR1-MDCK
and SK VLB Cells
3.4.7. Verotoxin Treatment Inhibits MDR1-mediated Rhodamine 123
Efflux
3.4.8. AdaGb3 Inhibits MDR1-mediated Vinblastine Efflux in Polarized
Gastrointestinal Epithelial Cells
3.4.9. AdaGb3 Differentially Binds the MDR1 Drug Binding Pocket as
Compared with Verapamil and Cyclosporin A
3.5. Conclusions
CHAPTER FOUR. INHIBITION OF ABC DRUG TRANSPORTER, MDR1, AS
POSSIBLE THERAPY FOR FABRY DISEASE
4.1. Introduction
4.2. Materials and Methods
4.2.1. Materials
4.2.2. Cell Culture
4.2.3. Neutral Glycolipids Extraction and Analysis
4.2.4. Gangliosides Extraction and Analysis
4.2.5. Verotoxin 1 Thin Layer Chromatography Overlay
4.2.6. Metabolic Labeling of MDR1-MDCK Glycosphingolipids
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4.2.7. Cytotoxicity Assay
4.2.8. Treatment of Neonatal Fabry Mice
4.2.9. Tissue Extraction of Neutral GSL
4.2.10. VT1 Tissue Staining
4.2.11. Epitope Unmasking Treatment
4.2.12. Anti-Gb3 Tissue Staining
4.2.13. HPLC Quantitation of Gb3
4.3. Results
4.3.1. MDR1 Inhibition in LSD Cell Lines
4.3.2. Accmulation of Gb3 in Fabry Mice by 10 Weeks
4.3.3. Inhibition of MDR1 Prevents Buildup of Gb3 in Fabry Mice Livers
4.3.4. Inhibition of MDR1 Prevents Buildup of Gb3 in Fabry Mice Hearts
4.3.5. Quantitative Liver Gb3 Analysis by TLC and HPLC in Treated vs.
Untreated Mice
4.3.6. Cholesterol Depletion and Anti-Gb3 Liver Staining
4.4. Conclusions
CHAPTER FIVE. MDR1 DEPENDENT AND INDEPENDENT MECHANISMS FOR
GOLGI GLUCOSYL CERAMIDE TRANSLOCATION IN HeLa CELLS
5.1. Introduction
5.2. Materials and Methods
5.2.1. Materials
5.2.2. Purification and Amplification of Plasmids
5.2.3. Cell Culture and Transfection
5.2.4. Neutral Glycolipids Extraction and Analysis
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5.2.5. Gangliosides Extraction and Analysis
5.2.6. Metabolic Labeling of MDR1-MDCK Glycosphingolipids
5.3. Results
5.3.1. Restriction Analysis for CerS1 (uog1)
5.3.2. C18 Ceramide Synthesis (CerS1- uog 1) is Channeled into Neutral
GSLs but not into Gangliosides in HeLa Cells
5.3.3. C18 Ceramide Synthesis (CerS1- uog1) is Inhibited by CsA in
HeLa Cells
5.4. Conclusions
CHAPTER SIX. DISCUSSION
6.1. Conclusions
6.2. Future Directions
REFERENCES
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LIST OF ABBREVIATIONS
ABC ATP-binding-casette
AdaGalCer adamantyl galactosylceramide
AdaGb3 adamantyl globotriaosyl ceramide
AdaGlcCer adamantyl glucosylceramide
ATP adenosine triphosphate
BCRP breast cancer resistance protein
BMT bone marrow transplantation
CerS ceramide synthase
CFTR Cystic fibrosis transmembrane
conductance regulator
CMT cell-mediated therapy
CsA cyclosporin A
CYP3A cytochrome P450, family 3,
subfamily A
DES dihydroceramide desaturase
DDTB 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide
DNJ deoxynojirimycin
ER endoplasmic reticulum
ERT enzyme replacement therapy
FB1 fumonisin B1
FBS fetal bovine serum
GalCer galactosylceramide
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Gb3 globotriaosylceramide
Gb4 globotetraosylceramide
GluCer glucosylceramide
GM3 monosialoganglioside
GSLs glycosphingolipids
HPLC high-performance liquid
chromatography
IAM immobilized artificial membrane
IEM immunoelectronmicroscopy
KOT knock-out
LacCer lactosylceramide
Lass longevity assurance
LRP lung resistance-related protein
LSDs lysosomal storage disorders
Mabs monoclonal antibodies
MDCK Mabin-Darby canine kidney
MDR multidrug resistance
MRP multidrug resis tance protein
NBD nucleotide binding domain
NBDNJ N-butyldeoxygalactonojirimycin
PAF platelet-activating factor
PAMPA parallel artificial membrane
permeability assay
PC phosphatidylcholine
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PDMP 1-phenyl-2-decanoylamino-3-
morpholino-propanol
PE phosphatidylethanolamine
P-gp P-glycoprotein
PI3K phosphoinositol-3-kinase
PKA protein kinase A
PKC protein kinase C
PMF proton-motive force
PPMP 1-phenyl-2-hexadecanoylamino-3-
morpholino-propanol
Rho123 rhodamine 123
SM sphingomyelin
SNP single-nucleotide polymorphism
SPT serine palmitoyltransferase
SRT substrate reduction therapy
TGN trans-Golgi network
TLC thin layer chromatography
TM transmembrane
TMD transmembrane domain
TRH translocating chain-asssociating
membrane protein homolog
UOG upstream of growth and
differentiation factor
VT1 verotoxin 1
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VT1B verotoxin 1 B subunit
VT2 verotoxin 2
YB-1 Y-box binding protein
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LIST OF TABLES
TABLE 3.1. Summary of Effects of AdaGb3 on MDR1-mediated [3H] Vinblastine
Transport.
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LIST OF FIGURES
FIGURE 3.1. Cell Surface Colocalization of MDR1 and FITC-VT1B in MDR1-MDCK
and SK VLB Cells.
FIGURE 3.2. Intracellular Colocalization of MDR1 and FITC-VT1B in SK VLB Cells.
FIGURE 3.3. Plasma Membrane MDR1 and Glycosphingolipids.
FIGURE 3.4. Double Label Cryo immunoelectronmicroscopy of Intracellular MDR1
and Internalized VT1B.
FIGURE 3.5. Effect of AdamantylGb3 on Cell Resistance to Vinblastine.
FIGURE 3.6. Effect of AdamantylGb3 on MD1 Expression in MDR1-MDCK and
SK VLB Cells.
FIGURE 3.7. Treatment of Vero Cells with Ada-GSLs Analogs.
FIGURE 3.8. Verotoxin Cytotoxicity Assays Before and After Treatment with
AdaGalCer and P4.
FIGURE 3.9.A. MDR1 Distribution in Rafts Fractions.
B. Caveolin Distribution in Rafts Fractions.
FIGURE 3.10. Inhibition of Efflux of Rho123 from Adamantyl Gb3-treated Cells.
FIGURE 3.11. Verotoxin-mediated Internalization of Gb3 Inhibits MDR1-dependent
Rhodamine 123 Efflux.
FIGURE 3.12. Effect of Adamantyl Gb3 on Gastrointestinal Epithelial Cell [3H]
Vinblastine Transport.
FIGURE 3.13. Disulfide Cross- linking of MDR1 Mutants Defines AdamantylGb3
Binding Site.
FIGURE 4.1. Accumulation of Gb3 in Fabry Mice By 10 Weeks of Age.
FIGURE 4.2. Effect of Cyclosporin A (CsA) on Cultured Gaucher and Fabry B-cell
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Line Glycosphingolipids (GSLs).
FIGURE 4.3. Accumulation of Gb3 in Livers of Fabry Neonatal Mice.
FIGURE 4.4. Comparison of VT1 Staining of Fabry Untreated Liver Tissue and Fabry,
CsA, OC144 and Tariquidar Treated Liver Tissue.
FIGURE 4.5. Comparison of VT1 Staining of Fabry Untreated Heart Tissue and Fabry,
CsA, OC144 and Tariquidar Treated Heart Tissue.
FIGURE 4.6. Glycolipid Profile of Liver Extractions of Fabry Untreated and Treated
Mice.
FIGURE 4.7. Liver Gb3 Levels Quantitated by HPLC.
FIGURE 4.8. Comparison of VT1 Staining, Cholesterol Depletion VT1 Staining, and
Anti-Gb3 Staining of Fabry Mice Liver Sections.
FIGURE 5.1. Restriction Analysis of CerS1 (uog1).
FIGURE 5.2. CerS1 (uog1) Transfection of HeLa Cells – Effect on Neutral and Acidic
GSLs Biosynthesis.
FIGURE 5.3. CsA Treatment of CerS1 (uog1) Transfected HeLa Cells. Effect of MDR1
Inhibition on CerS1.
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LIST OF SCHEMAS
SCHEME 1.1. Schematic Topological Structure of Representative ABC Transporters.
SCHEME 1.2. Proposed Structure of P-glycoprotein.
SCHEME 1.3. X-ray Structure of P-glycoprotein.
SCHEME 1.4. Proposed Model of P-glycoprotein Substrate Transport.
SCHEME 1.5. General Molecular Structure of a Glycosphingolipid.
SCHEME 1.6. Biosynthesis and Catabolism of Sphingolipids.
SCHEME 1.7. Structure of Globotriaosylceramide.
SCHEME 1.8. Glycosphingolipid (GSL) Catabolism and Disease.
SCHEME 2.1. Proposed Model for MDR1 Inhibition and Possible Therapeutic
Approaches.
SCHEME 3.1. Structural Models of Gb3 and Adamantyl Gb3 (adaGb3).
SCHEME 4.1. Protocol for Treatment of Neonatal Fabry Mice with MDR1 Inhibitors.
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CHAPTER ONE: INTRODUCTION
1.1. Multidrug Resistance
“Cross-resistance between different cytostatic agents which are structurally and
functionally dissimilar is a common phenomenon called multidrug resistance (MDR)” (Volm,
1998). MDR is a process characterized by the energy-dependent efflux of a wide variety of
structurally unrelated hydrophobic compounds, conferring cellular drug resistance. A group of
certain characteristic membrane proteins is responsible for this process (Sharom, 1997;
Gottesman et al., 2002; Kaur, 2002). The first evidence of this phenomenon was reported in
1970 (Bielder, 1970), when Chinese hamster ovarian cancer cells were able to survive not only
to increasing concentrations of actinomycin D, but also to different anticancer drugs of clinical
importance (Ferry and Kerr, 1994). Among these drugs were anthracyclines (doxorubicin and
daunomycin), vinca alkaloids (vincristine, vinblastine, and vindesine), etoposide, and colchicine
(Ferry and Kerr, 1994). This mechanism appeared to diminish the intracellular accumulation of
the drug and it was attributed to a 170 kDa membrane glycoprotein (Ferry and Kerr, 1994).
Overexpression of P-glycoprotein (P-gp), a 170 kDa plasma membrane glycoprotein, has been
found not only in different human and animal MDR cell lines, but also in transplantable
tumours. Victor Ling’s group in Toronto who first identified and cloned P-glycoprotein from a
MDR cell line CHRB30, proposed it as the diect or indirect mediator of this phenomenon
(Riordan et al., 1985).
MDR proteins are present in both eukaryotes and prokaryotes. All MDR proteins
isolated in eukaryotes, i.e. P-gp, belong to the ATP-binding cassette (ABC) family, ATP-
dependent transporters (Sharom, 1997; Gottesman et al., 2002; Kaur, 2002). Instead, prokaryotic
MDR proteins isolated from bacteria are not only ATP-dependent transporters but also proton-
motive force (PMF)-driven transporters (Paulsen and Skurray, 1993; Nikaido, 1994). The
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prokaryotic MDR proteins are classified into three families: a) the major facilitator superfamily
(i.e. Bmr and QacA); b) the small MDR family (EmrE and Smr); and c) the
resistance/nodulation/cell division family (MexB and AcrB) (Zheleznova et al., 2000; Kaur,
2002).
In order to understand the mechanism of action of these MDR proteins, it is necessary
to know their structures; but like any other membrane protein; they are difficult to crystallize
(Oldham et al., 2008). The Escherichia coli vitamin B12 importer was the first ABC transporter
whose high-resolution structure was reported in 2002 (Locher et al., 2002). Since then, the
crystal structures of five more importers and two exporters have also been reported (Oldham et
al., 2008). Dawson et al. published the first high resolution structure for an ABC exporter, the
MDR transporter Sav 1866 from Staphylococcus aureus (Dawson and Locher, 2006; Dawson
and Locher, 2007). Zolnericks et al. reported the first biochemical evidence that supports the
similar domain arrangement between Sav1866, one of the most important MDR exporters, and
P-gp (Zolnerciks et al., 2007). Until the beginning of 2009, the structure of P-gp was proposed
based on a homology model of the X-ray structure of Sav1866 3D structure, considered as an
appropriate template for the modeling of the human P-gp (Globisch et al., 2008); but
fortunately, the mouse P-gp’s X-ray crystal structure was recently reported (Aller et al., 2009).
P-gp crystal structure highlights and allows us to understand why it is able to transport such a
broad specificity of compounds (Aller et al., 2009). The crystal structures of the Escherichia
coli lipid flippase MsbA (two nucleotide-bound and two apo forms), recently reported, also
emphasized the dynamics of ABC exporters (Ward et al., 2007; Oldham et al., 2008). The
crystal structures of some MDR regulatory proteins already reported also helped in the structural
analysis of MDR transporters, since some of the substrates exported by these transporters also
bind to the MDR regulatory proteins (Zheleznova et al., 1999; Schumacher et al., 2001; Kaur,
2002). But more structural analysis of MDR transporters is necessary in order to compare the
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structure of the binding sites from the transporters with the ones from the regulators, to elucidate
the evolution of these binding sites (Kaur, 2002).
1.1.1. Multidrug Resistance in Cancer
Chemotherapy is the treatment of choice for several types of disseminated cancers
(Gottesman et al., 2002) and was shown to be the cure for leukemias, lymphomas, sarcomas,
choriocarcinoma, and testicular cancers in children and adults (Gottesman et al., 2002; Vassal,
2005; Kle in, 2006). In cases of breast cancer with no signs of metastasis, chemotherapy was
also shown to improve long-term survival (Gottesman and Pastan, 1993). But metastatic cancers
generally show resistance to chemotherapy from the very beginning (intrinsic resistance) or
when they recurred, after the first round of chemotherapy (acquired resistance) (Gottesman and
Pastan, 1993). MDR is the main cause of broad-spectrum resistance to chemotherapy in human
cancer. Thus, the causes of MDR have been the main focus of study of cancer researchers for
the last decades (Gottesman and Pastan, 1993).
This phenomenon in the treatment with anticancer drugs comes from the combination of
individual variations in patients and genetic differences in the somatic cells of the tumors, even
if they come from the same tissue (Gottesman et al., 2002). This resistance is usually intrinsic to
the cancer itself, but acquired resistance has also been reported due to the development of new
and more effective therapies. The most common cause of MDR to a broad range of anticancer
drugs is the expression of one or more energy-dependent transporters that sense and efflux
anticancer drugs from cells. The insensitivity to drug- induced apoptosis and the induction of
drug-detoxifying mechanisms might also play an important role in acquired anticancer drug
resistance (Gottesman et al., 2002). In progress studies are focused on how to elude drug
resistance in cancer cells in order to design new drugs not susceptible to the mechanisms of
resistance (Gottesman et al., 2002; Nobili et al., 2006; Pauwels et al., 2007; Yuan et al., 2008).
1.1.2. Cellular Mechanisms of Multidrug Resistance
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MDR (Gottesman et al., 1994) can be the result from changes that limit accumulation of
drugs within cells by limiting uptake, or enhancing their efflux, or affecting membrane lipids
such as ceramide (Liu, 2001; Gottesman et al., 2002). Another mechanism that has been
described as MDR is the expression of the lung resistance-related protein (LRP), that regulates
the entry of drugs to the nucleus of cells (Dalton and Scheper, 1999; Gottesman et al., 2002).
The best studied mechanisms of MDR are the ones that alter the accumulation of drugs
inside the cells (Ambudkar et al., 1999; Borst et al., 2000a; Gottesman et al., 2002). The net
impaired balance between the mechanisms of drug entry and exit is responsible for this
accumulation.
The first experiments on MDR mutant cell lines showed that the MDR mechanism is the
result of changes that reduced accumulation of drugs within the cell (Kessel et al., 1968; Biedler
and Reihm, 1970; Gottesman and Pastan, 1993), due to an increased drug efflux (Dano, 1973) or
a decrease in cell permeability (Ling and Thompson, 1974; Gottesman and Pastan, 1993).
Several reports on this topic suggest that for most of the cell lines studied and for most of the
drugs analyzed, an increased efflux and a decreased influx were both responsible for MDR
(Dano, 1973; Skovsgaard, 1978; Gottesman and Pastan, 1993). Immunofluorescence studies and
isotopic labeling studies on single cells revealed that this mechanism is energy dependent
(Willingham et al., 1986). Initial biochemistry analysis of MDR cell lines suggested that
alterations of one or more major glycoproteins might affect the plasma membrane proteins
(Juliano and Ling, 1976; Beck et al., 1979; Gottesman and Pastan, 1993). The most important
alteration found was the increased expression of a cell surface phospho-glycoprotein, called P-
glycoprotein by Ling and his colleagues (Juliano and Ling, 1976; Riordan and Ling, 1979),
encoded by the mdr gene in both rodents and humans (Gros et al., 1986; Ueda et al., 1986). The
mdr gene that encodes the multidrug transporter responsible for both increased drug efflux and
decreased influx was reported to be essential for the MDR phenotype in animals and cells in
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culture (Chen et al., 1986; Ueda et al., 1986; Gottesman and Pastan, 1993). Other reports that
use purified, reconstituted P-glycoprotein, further demonstrate that P-glycoprotein is the
transporter itself (Sharom et al., 1993; Shapiro and Ling, 1995; Ambudkar et al., 1998).
Additional studies on MDR mutant cells further demonstrated the existence of various
mechanisms of multidrug resistance with pheno typic variations according to the mechanism
(Gottesman and Pastan, 1993). Other mechanisms different from P-gp, were also reported to
alter the intracellular accumulation of drugs (McGrath and Center, 1987; Marquardt and Center,
1992). P-gp and the other proteins responsible for the MDR phenotype share DNA sequence and
structural homology; they all belong to the ABC transporters family (Gottesman et al., 1996;
Muller et al., 1998; Borst et al., 2000a; Linton and Higgins, 2007). The bioavailability of several
drugs can be affected by the MDR phenotype, including the vinca alkaloids (vinblastine and
vincristine), the anthracyclines (doxorubicin and daunorubicin), the RNA transcription inhibitor
actinomycin-D, and the microtubule-stabilizing drug paclitaxel (Gottesman and Pastan, 1993;
Ambudkar et al., 1999; Gottesman et al., 2002).
Water-soluble agents, such as antifolate methotrexate, or nucleotide analogues like 5-
fluorouracil and 8-azaguanine, and cisplatin also fail to accumulate inside the cells where there
is no evidence of increased efflux (Gottesman et al., 2002). This alternative mechanism of MDR
consists of a reduction in the drug uptake, and it has been reported not only for water-soluble
compounds but also for drugs that are internalized by endocytosis (Shen et al., 1998; Shen et
al., 2000.Gottesman, 2002 #5812; Gottesman et al., 2002).
When there is no evidence of drug accumulation, MDR can be promoted by the
activation of detoxifying systems, DNA repair and the cytochrome P450 (Wacher et al., 1995;
Muller et al., 1998; Benet et al., 1999; Gottesman, 2002). In fact, Schuetz et al. reported a
coordinate induction of the multidrug transporter P-gp and cytochrome P450 (Schuetz et al.,
1996).
6
In 2002, Dr. Gottesman explained the phenomenon of multifactorial MDR as follows:
“Although the process that results in uncontrolled cell growth in cancer favours clonal
expansion, tumour cells that are exposed to chemotherapeutic agents will be selected for their
ability to survive and grow in the presence of cytotoxic drugs. These cancer cells are likely to be
genetically heterogeneous because of the mutated phenotype. So, in any population of cancer
cells that is exposed to chemotherapy, more than one mechanism of multidrug resistance can be
present. This phenomenon has been called multifactorial multidrug resistance” (Gottesman et
al., 2002).
1.2. ATP-Binding Cassette Transporters
ABC transporters are integral membrane proteins that are able to transport structurally
diverse substrates across the membrane bilayer by using the energy generated by ATP binding
and hydrolysis (Oldham et al., 2008). Almost 300 proteins have been described as members of
the ABC superfamily, the largest family of transmembrane proteins (Biemans-Oldehinkel et al.,
2006; Szakacs et al., 2006; Higgins, 2007; Stavrovskaya and Stromskaya, 2008). ABC proteins
are present in all living organisms; fifty of them have been reported in man and another fifty, in
mouse (Dean, 2002; Stavrovskaya and Stromskaya, 2008). As many ABC proteins have been
discovered in the past few years, there is still not much information available about them. Some
eukaryotes ABC transporters are known for their key role in genetic diseases like cystic fibrosis
or Tangier disease (Gottesman and Ambudkar, 2001). The most well studied ABC transporters
are associated with the MDR phenotype by extruding chemotherapeutic drugs out of cancer
cells (Deeley et al., 2006; Hollenstein et al., 2007). Prokaryotic ABC transporters are
responsible for the uptake of essential nutrients or the extrusion of toxic substances, playing a
key role in drug and antibiotic resistance of microbial pathogens (Davidson and Chen, 2004;
Hollenstein et al., 2007; Davidson et al., 2008).
7
The human members of the ABC transporter family are classified into seven different
groups or subfamilies (Dean and Allikmets, 2001; Stavrovskaya and Stromskaya, 2008). This
classification is based on the number and combination of the transmembrane domains (TMDs)
and of the nucleotide-binding domains (NBDs, ATP-binding domains) (Stavrovskaya and
Stromskaya, 2008). A 30-40% homology between NBDs of all proteins of this family has been
reported regardless of species and substrate specificity (Sarkadi et al., 2006; Stavrovskaya and
Stromskaya, 2008).
P-glycoprotein and the multidrug resistance protein (MRP) belong to the ABC
transporter family, and are both associated with MDR (Gottesman et al., 1996; McKeegan et al.,
2003). Other transporters of this family associated with resistance to antimicrobial drugs are
present in some pathogenic fungi and parasitic protozoa like PgpA, an MRP homologue
responsible for resistance to an antimonial drug in Leishmania (Legare et al., 2001; McKeegan
et al., 2003)}. A few bacterial homologues have also been identified, such as the LmrA
multidrug transporter from Lactococcus lactis (van Veen et al., 1996) and the DrrAB
doxorubicin/daunorubicin transporter from the anthracyclines-producing actinomycete
Streptomyces (Kaur, 1997; McKeegan et al., 2003).
The ABCB subfamily, with P-gp as the most important member, is considered unique as
it contains four full transporters and seven half transporters (Dean, 2002; Szakacs et al., 2006).
P-gp/MDR1 is the first member of the ABCB family that has been cloned and its main
characteristic is that it confers the MDR phenotype to cancer cells (Riordan et al., 1985).
ABCB4 (MDR 2/3) and ABCB11 members of this family are both responsible for the secretion
of bile acids in the liver (Stavrovskaya and Stromskaya, 2008). ABCB2 and ABCB3 (TAP) half
transporters members of this family, transport peptides into the endoplasmic reticulum in order
to be presented as antigens by the Class I HLA molecules (Stavrovskaya and Stromskaya,
2008). ABCB9, another half transporter member of this family is located in the lysosomes
8
(Zhang et al., 2000; Stavrovskaya and Stromskaya, 2008). ABCB6, ABCB7, ABCB8 and
ABCB10, all half transporters are localized in the mitochondria, where they might be involved
in the transport of Fe/S protein precursors and in the metabolism of iron itself (Dean, 2002;
Stavrovskaya and Stromskaya, 2008).
Although the members of the ABC family were initially identified as drug transporters,
some members transport some other substrates including dyes, organic and inorganic ions,
peptides and proteins, heavy metals, lipids, steroids, antibiotics (Saurin et al., 1999; Young and
Holland, 1999; Gillet et al., 2007; Stavrovskaya and Stromskaya, 2008). Among the few
exceptions that have no transport functions is protein ABCC7 (CFTR), a channel that plays a
key role in the regulation of Cl- flow in epithelial cells (Riordan et al., 1989; Gadsby et al.,
1995; Stavrovskaya and Stromskaya, 2008).
1.2.1. Structural Organization and Mechanism of ABC Transporters
The common structure of the ABC transporters consists of four domains, essential for
their functioning (Klein et al., 1999; Pedersen, 2007). Two of these domains are cytoplasmic
nucleotide-binding domains (NBDs) that bind and hydrolyze ATP (Sharom, 2006). The NBDs
of all ABC proteins have two distinct motifs, Walker A and Walker B, common to other
proteins involve in ATP or GTP binding, and also a signature C motif, only present in the ABC
superfamily (Sharom, 2006). Several amino acid residues in these 3 motifs were shown to have
specific roles in the ATP catalysis (Loo and Clarke, 1998; Frelet and Klein, 2006; Sharom,
2006). As examples, a D residue at positions 555 and 1,200 in the Walker B motif of human P-
gp is important for the coordination of Mg2+ (Hrycyna et al., 1999) and an adjacent glutamate
residue, called the catalytic carboxylate base, is also critical for the ATP hydrolysis although its
role is still in dispute (Sauna et al., 2002; Zaitseva et al., 2005a; Zaitseva et al., 2005b; Sauna et
al., 2006). The structure of the two transmembrane domains (TMDs) is characterized by a
number of transmembrane a-helices, usually six (Stavrovskaya and Stromskaya, 2008).
9
Different combinations of NBDs and TMDs form the multidomain polypeptides of the ABC
family. Complete transporters, generally located at the cell plasma membrane, are characterized
by the (TMD-NBD)2 structure, while halftype transporters containing only one set of TMD-
NBD are mostly located in intracellular membranes (Klein et al., 1999) (i.e., TAPa and TAP2 in
the endoplasmic reticulum) (Stavrovskaya and Stromskaya, 2008). The only exception is the
half transporter ABCG2 (also BCRP or MXR) found in the cell plasma membrane (Rocchi et
al., 2000; Stavrovskaya and Stromskaya, 2008).
Bacterial transporters, like Sav1866, are usually homodimers with one NBD and one
TMD (two homodimers usually function together) (Dawson and Locher, 2007). ABCB1 (P-gp),
ABCB4 (P-gp 3 or MDR2/3), and ABCB11 (BCEP or SPGP) are characterized by the (TMD-
NBD)2 structure (Stavrovskaya and Stromskaya, 2008). Although proteins from the same
subfamily, ABCB2 (TAP1) and ABCB3 (TAP2) have the (TMD-NBD)1 structure. There are
also some transporters of the ABCC (MRP) subfamily that have a third TMD at the N-terminus
(designated as TMD0) with five transmembrane helices, such as ABCC1 (MRP1), ABCC2
(MRP2), ABCC3 (MRP3), ABCC6 (MRP6), ABCC8 (SUR1), and ABCC9 (SUR2) (Kruh and
Belinsky, 2003; Deeley and Cole, 2006; Stavrovskaya and Stromskaya, 2008). Some subfamily
homologues of the MRP proteins have no TMD0 (ABCC4 (MRP4) and ABCC5 (MRP5))
(Stavrovskaya and Stromskaya, 2008). CFTR (ABCC7) is also included in this same MRP
subfamily (ABCC subfamily), but its structure has two TMD-NBD motifs linked by a unique
domain, called the R (regulatory) domain, with multiple consensus phosphorylation sites and
many charged amino acids (Riordan et al., 1989; Sheppard and Welsh, 1999; Stavrovskaya and
Stromskaya, 2008). Members of the ABCE and ABCF subfamilies have only one NBD (Klein
et al., 1999; Stavrovskaya and Stromskaya, 2008). This diversity on the domain organization of
ABC proteins is shown on Scheme 1.1.
10
Scheme 1.1. Schematic topological structure of representative ABC transporters. The
structure of the ABC transporters consists of combinations of transmembrane (TMD) and
nucleotide-binding (NBD) domains. These topological structures shown have yet to be proven.
a) ABC transporters such as multidrug resistance (MDR1) and multidrug resistance associated
protein 4 (MRP4) have two TMDs and two NBDs. b) MRP1, 2, 3 and 6 also possess two TMDs
and two NBDs, and also an additional five transmembrane helices segment at the amino-
11
terminal end. c) The half- transporter ABCG2 contains of one TMD and one NBD. (Oval) N-
linked glycosylation sites for MDR1, (Polygons) phosphorylation sites for MDR1 (Zhang,
2007).
P-gp/MDR1 (ABCB1) and the cystic fibrosis transmembrane regulator (CFTR/ABCC7)
have very similar structures with a repeat of one TMD coupled to one NBD (Chen et al., 1986;
Gros et al., 1986; Hyde et al., 1990). An R domain of 332 amino acids with several PKA and
PKC consensus sites separates these repeats in CFTR (Riordan et al., 1989). In the case of P-gp,
the region that separates these repeats is a linker (L) domain of 46 amino acids, that also has
multiple PKA, PKC, and P-gp specific V1-kinase consensus sites (Chambers et al., 1995;
Vankeerberghen et al., 1999). CFTR phosphorylation by protein kinase A allows its proper
function as a chloride channel (Rich et al., 1990; Bear et al., 1992) and as a regulator of other
ion channels (Stutts et al., 1997; Schwiebert et al., 1998; Vankeerberghen et al., 1999;
Vennekens et al., 1999). P-gp’s efflux function does not depend on its phosphorylation although
there are reports that describe phosphorylation of its PKC sites (Vankeerberghen et al., 1999;
Vennekens et al., 1999). Vankeerberghen et al. studied the role of the CFTR R domain on its
maturation by using hybrid constructs in which different parts of the CFTR R domain were
replaced by the P-gp linker domain, and viceversa (Vankeerberghen et al., 1999). In
experiments where the C-terminal part or the complete R domain of CFTR was replaced by the
P-gp linker, no effect on the efficiency of CFTR maturation was reported compared to wild
type, suggesting that the R domain itself is not responsible for the inefficient maturation of
CFTR in the ER, but to the overall structure of the protein (Vankeerberghen et al., 1999). But
this research group was able to show that the length, and the presence and distribution of the
consensus sites for protein kinase A in the R domain play a role in the coupling between the
activation by cAMP and the chloride transport through the pore of the CFTR chloride channel
(Vankeerberghen et al., 1999).
12
Several structures of these ABC transporters have been recently solved (Davidson and
Chen, 2004; Moody and Thomas, 2005; Hollenstein et al., 2007), helping to understand their
functional mechanisms (Pedersen, 2007). Although all ABC transporters share the same
structure for the ATP binding domains, this structure is quite different for the TMD, responsible
for the binding and transport of specific physiological substrates. Conformational or positional
changes of these TMD induced by the binding/hydrolysis of ATP help with the transport of the
specific substrate from one side of the plasma membrane to the other (Pedersen, 2007).
ABC proteins are divided into two subtypes according to the direction of the transport:
prokaryotes ABC importers that require a binding protein that delivers the substrate to the
external face of the transporter; and eukaryotic ABC exporters, that recruit their substrates
directly from the cytoplasm or from the inner leaflet of the lipid bilayer (Hollenstein et al.,
2007).
Several different mechanisms have been proposed in the literature in order to understand
the ATP-driven import and export from ABC transporters (Davidson and Chen, 2004; Higgins
and Linton, 2004; Hollenstein et al., 2007). Crystal structures of full transporters allow to
understand how the ATP binding and hydrolysis coupled to the inward-facing or outward-facing
conformations of the TMDs (Hollenstein et al., 2007). Hollenstein et al. also proposed that the
key element in this unidirectional transport is the ‘closing’ of the NBDs, driven by the
hydrolysis of ATP, minimizing the distance between the coupling helices to 10–15 Å when
compared with the nucleotide-free state (Hollenstein et al., 2007). These coupling helices
produce a flipping of the TMDs from an inward-facing to an outward-facing conformation
(Hollenstein et al., 2007). This flipping justifies why ABC importers accept substrates from
their binding proteins, while ABC exporters extrude bound drugs to the environment. When
ADP and Pi are released, the transporters may turn back to an inward-facing conformation.
13
Then, the importers free their substrates into the cytoplasm, while the exporters bind new
substrates into their high-affinity binding sites (Hollenstein et al., 2007).
1.2.2. ABC Transporters in Normal Cells
All ABC transporters are expressed in normal tissues, despite being identified as drug-
resistance proteins (Borst et al., 2000a; Sparreboom et al., 2003; Gillet et al., 2007). According
with their distribution, ABC proteins transport both a variety of endogenous substrates as well
as exogenous drugs (Gottesman et al., 2002).
ABC transporters play a key role in the regulation of the permeability of the central
nervous system (Gottesman et al., 2002). The blood-brain barrier and the blood-cerebrospinal-
fluid barrier are responsible for protecting the brain against blood-borne toxins. P-gp is located
on the luminal surface of the blood-brain barrier formed by the endothelial cells of capillaries,
and thus, preventing the entrance of cytotoxins through the endothelium (Gottesman et al.,
2002; Zhou, 2008). MRP proteins are localized to the basolateral membrane of the choroid
plexus, where they pump the metabolic waste products of the cerebrospinal- fluid into the blood
(Girardin, 2006). ABC transporters are also present in other barriers such as the testis barrier for
the protection of the testicular tissue, and in the placenta to protect the developing fetus
(Gottesman et al., 2002). The role of P-gp in the blood-testes barrier as well as in the blood
brain barrier is to transport toxins into the capillary lumen (Schrader et al., 2007). MRP1
instead, is localized on the basolateral surface of Sertoli cells and its main function is to protect
sperm within the testicular tubules (Augustine et al., 2005). The role of P-gp in the placenta is to
protect the fetus from toxic cationic xenobiotics (Gottesman et al., 2002; Zhou, 2008). MRP
family members and the half-transporter ABCG2 are also localized in the placenta (Maliepaard
et al., 2001; Gottesman et al., 2002). Apparently, the role of MRP1 and other isoforms is to
protect the fetal blood from toxic organic anions and to excrete glutathione/glucuronide
metabolites into the maternal circulation (St-Pierre et al., 2000; Gottesman et al., 2002).
14
ABC transporters are also localized in the liver, the gastrointestinal tract and the kidney
to excrete toxins, to protect the entire organism (Gottesman et al., 2002). ABCB4, with 78%
amino acid sequence identity with MDR1, is localized in the apical membrane of hepatocytes,
where functions as a phosphatidylcholine translocase, reducing the toxicity of bile salts
(Gottesman et al., 2002; Dietrich et al., 2003). In humans, MRP3 is localized to the basolateral
surface of hepatocytes, where transports organic anions from the liver back into the bloodstream
(Scheffer et al., 2000). MRP6, also highly expressed in liver cells, share the same function as
MRP3 (Kool et al., 1999). MRP2 is also localized on the apical surface of hepatocytes, where
transports bilirubin-glucuronide and other organic anions into the bile (Gottesman et al., 2002;
Dietrich et al., 2003)..
P-gp is localized in apical membranes of mucosal cells in the gastrointestinal tract,
where it pumps out toxins as a mechanism of defence (Gottesman et al., 2002). Previous
reported studies suggested that P-gp might have a significant role in oral drug bioavailability
(Dietrich et al., 2003). On the contrary, MRP1 is located in the basolateral membrane of
mucosal cells, transporting substrates into the interstitium and the bloodstream (Gottesman et
al., 2002). The localization of MRP2 in the canalicular membrane of hepatocytes and the apical
surface of epithelial cells, like P-gp, confirm that MRP2 might also be involved in the regulation
of drug bioavailability (Gottesman et al., 2002; Dietrich et al., 2003).
1.2.3. ABC Transporters in Cancer Cells
Even though MDR in cancer cells is associated with a variety of ABC transporters, the
majority of the clinical studies target P-gp (Gottesman et al., 2002). First studies reported a high
level of P-gp expression in colon and kidneys, and also in adrenocortical and hepatocellular
cancers (Gottesman et al., 2002; Barbat et al., 2007). But the fact that these types of cancers did
not respond to drugs different from P-gp substrates suggested that other MDR mechanisms are
also involved (Gottesman, 2002).
15
When the expression of MRP1 was studied in clinical samples, it was found to be highly
expressed in leukaemias, oesophageal carcinoma and non-small-cell lung cancers (Gottesman et
al., 2002; Modok et al., 2006). Antibodies against MRP1 seem to be more specific than those
that recognize P-gp (Scheffer and Scheper, 2002). Expression levels of other ABC transporters
in human tissue still needs further studies (Gottesman, 2002).
1.2.4. ABC Lipid Transporters
The first evidence of a possible relationship between mammalian ABC transporters and
lipid transport was described by Smit and colleagues (Smit et al., 1993; van Meer et al., 2006).
They reported that when they knocked out the ABC transporter MDR2 (ABCB4), closely
related with P-gp (ABCB1) in mice (Gottesman and Ling, 2006), the animals became jaundiced
as they did not have phospholipid phosphatidylcholine (PC) in the bile. Mosser et al. also
reported that the accumulation of very long chain fatty acids found in a disease called
adrenoleukodystrophy was linked to deletions in the ABC transporter gene ABCD1 (Mosser et
al., 1993; van Meer et al., 2006). Another important finding was the MDR transporter ABCB1 is
able to transport a wide variety of membrane lipids analogs across the plasma membrane (van
Helvoort et al., 1996; van Meer et al., 2006). In the last years, many lipid- linked inherited
diseases have been reported associated with mutations in one of the ABC transporters, and
currently, nearly half of the 50 human ABC transporters were reported to be involved in lipid
transport (Borst et al., 2000b; Pohl et al., 2005; van Meer et al., 2006).
There is some evidence in the literature that the clinical disorders that involve impaired
lipid release from the plasma membrane have been associated with mutations in an ABC
transporter (van Meer et al., 2006). ABC transporters mutations have also been correlated with
impaired lipid transport into the lumen of organelles and consequent release via exocytosis or
endocytotic recycling (van Meer et al., 2006). Reported results in the literature show that ABC
transporters cover the whole range of hydrophobicity in lipid transport, from a short ether PC,
16
the platelet activating factor (PAF), extruded by ABCB1 (P-gp) (Ernest and Bello-Reuss, 1999;
Raggers et al., 2001; van Meer et al., 2006), to long-chain PCs, transported by ABCB4 (Smit et
al., 1993; van Meer et al., 2006).
1.3. P-glycoprotein – MDR1
P-glycoprotein (P-gp), encoded by the MDR1 gene, is a 170 kDa plasma membrane ATP
dependent transporter that belongs to the so-called ABC transporters, and it works as a pump to
extrude anticancer drugs and other compounds out of the cells (Gottesman and Pastan, 1993;
Germann, 1996). It was the first of the ABC transporter to be cloned (Riordan et al., 1985).
Since P-gp is in part responsible for conferring a multi-drug resistance (MDR) phenotype to
cancer cells that leads to resistance to chemotherapy drugs, it is the ABC transporter protein best
characterized (Romsicki and Sharom, 1998; Kwan and Brodie, 2005; Zhou, 2008). Cells with
MDR phenotype show cross resistance to a variety of structurally and functionally unrelated
drugs (Breier et al., 2005; Zhou, 2008). The MDR phenotype can be intrinsic or acquired during
chemotherapy (Zhou, 2008).
MDR1 is expressed on the apical membranes of many secretory cell types such as gut
epithelia, liver cells, kidney tubules, and the blood–brain barrier; consequently, MDR1 was
proposed to be involved in the extrusion of drugs and their metabolites outside the cells
(Thiebaut et al., 1987; Cordon-Cardo et al., 1990; Zhou, 2008). Thus, MDR1 plays a key role in
the digestive absorption, cerebral disposition, and biliary and urinary elimination of
pharmaceutical drugs (Schinkel, 1997; Zhou, 2008). Therefore, Mdr1a/1b knockout mice were
shown to have serious defects in drug distribution (Schinkel, 1997; Johnstone et al., 2000).
Other sites of MDR1 expression are the adrenal gland, hematopoietic stem cells, natural killer
cells, antigen-presenting dendritic cells, and T and B lymphocytes (Klimecki et al., 1994;
Randolph et al., 1998; Johnstone et al., 2000).
17
Localization of P-gp in intracellular membranes has also been reported (Willingham et
al., 1987; Gong et al., 2000; Solazzo et al., 2006). Its expression on the nuclear membrane has
been associated with extrusion of drugs fom the nucleus of resistant cells (Maraldi et al., 1999;
Solazzo et al., 2006). High levels of P-gp were also found in intracytoplasmic vesicles, as part
of the Golgi apparatus, suggesting that P-gp might also play its efflux role in this region
(Gervasoni et al., 1991; Rutherford and Willingham, 1993; Molinari et al., 1994; Meschini et al.,
2000; De Rosa et al., 2004). MDR1 is also expressed in the mitochondrial membrane of MDR-
positive cells, where it extrudes anticancer drugs from the mitochondria into the cytosol,
protecting the mitochondrial DNA from damage to antiproliferative drugs (Solazzo et al., 2006).
1.3.1. Structure of P-glycoprotein
Three groups in parallel have cloned and sequenced the cDNA for P-gp from hamster
(Riordan and Ling, 1979; Gros et al., 1986), mouse (Hsu et al., 1989), and human cell lines
(Chen et al., 1986). Chen et al. reported in 1986, the isolation of the human MDR1 cDNA from
the KB carcinoma cell line, selected for resistance to colchicine (Chen et al., 1986; Ueda et al.,
1987), that codes for P-gp (Ueda et al., 1986), a surface glycoprotein overexpressed in drug-
resistant Chinese hamster ovary cell mutants (Juliano and Ling, 1976). P-gp belongs to the
multigene pgp or mdr family, that include three members in rodents (hamster and mouse)
(MDR1a,b and MDR2) and two members in humans (ABCB1 /MDR1 and ABCB4/MDR3) (Ng
et al., 1989).
Reported cross linkage of mdr/pgp genes on the chromosome suggests that the P-gp
gene family came from one or more gene duplications (de Bruijn et al., 1986; Bell et al., 1987;
Van der Bliek et al., 1988; Loo and Clarke, 1999). However, Chen et al. suggested that P-gp
came from fusion of genes based on the intron-exon structure study on the human ABCB1
(Chen et al., 1990; Loo and Clarke, 1999). It has been reported that P-gp isoforms share the
same overall structure, although they have distinct functions (ABCB1 versus ABCB4 in humans;
18
Abcb1a and b versus Abcb4 in mice (Juranka et al., 1989; Loo and Clarke, 1999). Transfection
and overexpression of Abcb1a and b of mouse, or ABCB1 of human confers multidrug
resistance to drug-sensitive culture cells; while transfection of mouse Abcb4 or human ABCB4
do not confer drug resistance (Gros et al., 1986; Ueda et al., 1987; Loo and Clarke, 1999).
Human ABCB4 and mouse Abcb4 act as phospholipid translocases (Smit et al., 1993; Ruetz and
Gros, 1994a; Smith et al., 1994; Loo and Clarke, 1999). Published reports indicate that the drug
specificity of human P-gp differs from both mouse trasnporters; while the level of vinblastine
resistance does not vary more than 2.5 fold between mouse and human MDR transporters,
mouse MDR1 confers a level of colchicine resitance 13 fold more than its human counterpart
(Tang-Wai et al., 1995; Loo and Clarke, 1999).
The human ABCB1 and ABCB4 genes (designated also as MDR3) are located on
chromosome 7q21.1 (Lincke et al., 1991; Zhou, 2008)}. The gene product of ABCB1 has 1280
amino acids and the gene product of ABCB4 has 1279 amino acids, and both have a molecular
mass of approximately 170 kDa, and an 80% amino acid homology was reported between the
two proteins (Zhou, 2008). P-gp/MDR1 and MDR2 have a common tandemly duplicated
topologic structure with each half of the molecule containing a nucleotide binding domain
(NBD) and a TMD with six predicted and highly hydrophobic transmembrane helices (Borst
and Elferink, 2002; Shilling et al., 2006; Zhou, 2008). The TMDs are supposed to form the
pathway through which the drug substrate crosses the membrane (Zhou, 2008). The NBDs, and
the NH2- and COOH-termini of P-gp, are located intracellularly, and it was reported that the
first extracellular loop is N-glycosylated (Zhou, 2008). As it has been described before in
general for ABC transporters, each NBD of P-gp consists of two core consensus motifs referred
to as the Walker A and B motifs, and a C signature motif (Walker et al., 1982), and they are
directly involved in the binding and hydrolysis of ATP. The two 610 amino acids half molecules
are separated by a highly charged non-conserved 60 amino acid ‘linker region’, found to be
19
phosphorylated at several sites by protein kinase C (Higgins et al., 1997; Zhou, 2008). This
flexible linker region is necessary for an adequate interaction between the two halves of the
protein, key for the right functioning of the molecule, in particular for the communication
between the two ATP sites (Ambudkar et al., 2003; Zhou, 2008) (Scheme 1.2).
Scheme 1.2. Proposed Structure of P-glycoprotein (Zhou, 2008).
Several studies have reported the existence of different topological orientations for P-
gp/MDR1, and also conformational changes in P-gp structure have been registered after
nucleotide binding that cause changes in the epitope accessibility (Druley et al., 2001a; Druley
et al., 2001b; Ruth et al., 2001), in protease susceptibility (Julien et al., 2000), in drug binding
(Martin et al., 2000b), and also in fluorescence, and spectroscopic characteristics measurements
(Sonveaux et al., 1996; Sonveaux et al., 1999; Zhou, 2008). In order to determine P-gp
structural resolution, researchers were able to over-express, purify, and successfully reconstitute
it. Low resolution structures were obtained by using electron cryomicroscopy (Rosenberg et al.,
2001; Rosenberg et al., 2003). First reports described a 25-Å electron-microscopy structure with
a significant chamber that is associated with the NBDs at its inner surface level (Rosenberg et
20
al., 2001). Further studies done on P-gp trapped in distinct catalytic states revealed dramatic
rearrangements of P-gp TMDs (Rosenberg et al., 2003; Zhou, 2008).
Recently, Aller et al. reported the 3.8-Å x-ray structure of mouse P-gp with an inward
facing conformation, which has 87% homology with human P-gp (Aller et al., 2009). It reveals
a 6,000 Å internal cavity that can accommodate two compounds simultaneously. This large
cavity is formed by two groups of six transmembrane helices: TMs 1, 2, 3, 6, 10, and 11 in the
first group, and TMs 4, 5, 7, 8, 9, and 12 in the second group, and it opens both to the inner
leaflet of the membrane and to the cytoplasm. Hydrophobic substrates can enter directly from
the membrane through two portals, one formed by TMs 4 and 6, and the other formed by TMs
10 and 12. The two NBDs are separated by 30 Å. The two halves of the molecule span 136 Å
perpendiculars to the cell membrane and 70 Å in the same plane as the membrane bilayer (Aller
et al., 2009) (Scheme 1.3).
Scheme 1.3. X-ray Structure of P-glycoprotein. Front view of 3.8 Å x-rays P-gp structure.
Crossover of TMs 4 and 5, and TMs 10 and 11 stabilize the inward facing conformation.
Horizontal bars represent approximate position of the lipid bilayer. N: N-termini domain, C: C-
termini domain (Aller et al., 2009).
21
1.3.2. Mechanism of Action
It is still a dilemma that we do not understand how P-gp can recognize such a wide
variety of structurally diverse compounds as transport substrates, and efficiently pump them out
by coupling their transport to the ATP hydrolysis.
P-gp/MDR1 has been reported to have two types of binding sites, the ones involved in
transport and the ones involved in regulation (Martin et al., 2000a; Martin et al., 2000b; Shilling
et al., 2006; Zhou, 2008). It has been reported that P-gp/ MDR1 binding sites of the N- and C-
terminal halves may generate a single region in the whole P-gp protein structure (Morris et al.,
1994; Loo et al., 2003; Zhou, 2008). The presence of allosteric sites of P-gp/MDR1, different
from the transport ones, has also been reported for indolizin sulfone SR33557 and the 1,4-
dihydropyridines, with evidence that shows control of the P-gp binding site for transport of
vinblastine (Martin et al., 1997). P-gp/MDR1 multiple drug binding sites could account for the
wide range of compounds that can be transported by this protein (Zhou, 2008).
Previous studies have also determined that there are two major substrate binding sites on
P-gp/MDR1, one at the TMD helices 5 and 6, and the other one at TMD helices 11 and 12
(Wang et al., 2003b). All binding sites of P-gp/MDR1 appear to be able to switch between high-
and low-affinity conformations, along with their modulators that affect their action (Martin et
al., 2000a). This switch can be the result of substrate binding and/or ATP hydrolysis (Martin et
al., 2000a). Conformational changes in P-gp/MDR1 have been demonstrated using 2H/H-
exchange kinetics, proteolytic accessibility, and changes in antibody epitope recognition
(Mechetner et al., 1997; Sonveaux et al., 1999).
Although several studies reported that P-gp may have up to four distinct drug-binding
sites (Dey et al., 1997; Pascaud et al., 1998; Shapiro et al., 1999; Lugo and Sharom, 2005). On
the contrary, Loo et al. proposed a common drug-binding pocket that lies at the interface
between the TMDs, where substrates bind through a ‘substrate-induced fit’ mechanism (Loo and
22
Clarke, 2002). They proposed that the binding of a substrate would involve various
combinations of residues from different TMDs and structurally different substrates could share
the same residues during drug binding. As the common drug-binding pocket is relatively large
(Loo and Clarke, 2001; Sauna et al., 2004), it can accommodate different substrates
simultaneously (Loo et al., 2003; Lugo and Sharom, 2005). They initially proposed a model of
P-gp, where the last three TM segments of each TMD (TMs 4–6 and 10–12 respectively)
formed the drug binding pocket (Loo and Clarke, 2000), while TMs 5 and 8 and TMs 2 and 11
(Loo et al., 2004a; Loo et al., 2004b) formed the gates for entry of drug substrates (Loo and
Clarke, 2005). As a recent study from their group showed that TM1 also lines the drug-binding
pocket (Loo et al., 2006a), they modified the predicted model of Pgp such that TM1 was placed
close to TMs 4–6. The revised model of P-gp also predicts that the first TM segment in the
second TMD (TM7) is also close to the drug-binding pocket (Loo et al., 2006b). This model is
also consistent with the crystal structures of MsbA recently reported (Ward et al., 2007).
In a recent review, Sharom et al. (Sharom, 2006) proposed that the mechanism of
transport by ABC proteins involves 2 distinct, but coupled, cycles. The first cycle, called the
catalytic one where ATP is hydrolyzed; consists of the ATP binding, the formation of a putative
nucleotide sandwich dimer, followed by the hydrolysis of ATP, the dissociation of Pi, and
finally, the ADP dissociation (Sharom, 2006). The catalytic cycle provides the energy that will
be coupled to the transport of the substrate across the membrane, although there is still some
controversy reported if this energy comes from the ATP binding or its hydrolysis (Higgins and
Linton, 2004; Hanekop et al., 2006; Sharom, 2006). P-gp drug transport consists of three steps:
substrate entry into the binding pocket, generation of conformational changes, and finally the
release of the drug in the extracellular milieu (Sharom, 2006). When the drug is effluxed from
the outer membrane, a re-orientation of the drug-binding site has been reported, from the
cytosolic side of the membrane (likely in the inner membrane leaflet) to the extracellular side
23
(possibly the outer membrane leaflet), along with a change from high- to low drug-binding
affinity (Sharom, 2006). The reported conformational changes allow communication between
the drug-binding site and the NBD domains, in order to activate the ATP hydrolysis and initiate
the transport cycle (Sharom, 2006). Senior et al. (Senior et al., 1995a) suggested that the protein
operates by an alternating sites mechanism, in which only 1 catalytic site can be in the transition
state at any time, and the 2 sites alternate in catalysis (Senior et al., 1995b; Senior et al., 1995a).
There is still no information available on the co-operation between the two active sites works at
the molecular level, or how ATP hydrolysis is coupled to the drug transport process (Ambudkar
et al., 2006). It has been reported that the drug transport occurs during the relaxation of a high-
energy intermediate state from the hydrolysis of ATP, where one molecule of ATP is
hydrolyzed for each drug molecule translocated (Senior et al., 1995a; Sharom, 2006).
In terms of the ATP hydrolysis, it is still controversial if one or two rounds are required
for each transport cycle (Sharom, 2006). Sauna et al. proposed a model in which two ATP
molecules are hydrolyzed per cycle, where the first one is used for the transport of the substrate
itself while the second one is used to get P-gp ready for another transport cycle (Sauna and
Ambudkar, 2001; Sharom, 2006).
Recently, Sauna et al. (Sauna et al., 2007a) proposed the occluded nucleotide
conformation of P-gp for its transport model. This model proposed that ATP binding takes place
initially at both NBDs and leads to an asymmetric occluded state in which one of the ATP
molecules is bound tightly enough to be considered an occluded state (Sauna et al., 2007a),
proposed to be closely associated with the high affinity/low affinity switch at the drug-binding
site (Sauna et al., 2007a). If there is no occluded ATP state, there is no switch between high
affinity to low affinity at the drug-binding site (Sauna and Ambudkar, 2000; Sauna and
Ambudkar, 2001).
24
It has been described previously that P-gp substrates partition into the plasma membrane,
and that the ATPase activity stimulated by the drug binding requires the presence of lipids
(Callaghan et al., 1997). The inward facing conformation of P-gp recently described confirms
this finding, as the internal cavity is large enough and the two portals are open wide enough to
accommodate hydrophobic molecules and phospholipids at the same time (Aller et al., 2009).
Thus, both lipids and substrates stay together during the initial entry to the P-gp cavity and
promotes ATPase activity for their subsequent transport (Aller et al., 2009).
The most recent proposed model for P-gp transport, based on its recent structure (Aller
et al., 2009) and on the outward facing conformation of MsbA and Sav 1866 (Dawson and
Locher, 2006; Ward et al., 2007) states that the substrate partitions into the lipid bilayer from the
outside of the cell to the inner leaflet, and enters to the internal cavity through an open portal.
The consequent binding of ATP to NBDs causes a large conformational change that presents the
substrate and the drug binding site to the extracellular space. Depending on the substrate, it can
be released by decreased binding affinity due to a conformational change, or can be facilitated
by ATP hydrolysis. The release of the substrate to the inner leaflet is occluded according to this
conformation, producing only a unidirectional transport to the extracellular space (Scheme 1.4)
(Aller et al., 2009).
25
.
Scheme 1.4. Proposed Model of P-glycoprotein Substrate Transport. In red, substrate; in
blue, residues involved in drug binding pocket; in yellow, ATP binding molecules (Aller et al.,
2009).
1.3.3. P-glycoprotein Substrates and Inhibitors
P-gp substrates include a wide variety of structurally different compounds, from natural
products, chemotherapeutic drugs, steroids, fluorescent dyes, to linear and cyclic peptides, and
ionophores (Sharom, 2006). In terms of physicochemical properties, most of them are weakly
amphipathic and relatively hydrophobic, also some of them contain aromatic rings and are
positively charged (Sharom, 2006). Examples of physiological substrates are peptides, platelet-
activating factor, lipids, steroid hormones, and small cytokines, but there is not much
physiological evidence for the great majority of the substrates since they are they identified by
their cytotoxicity in in vitro experiments (Sharom, 2006).
Seelig has found that a common characteristic for most P-gp/MDR1 substrates is to have
two or three electron-donor groups with a fixed spatial separation of 2.5Å and 4.6Å,
respectively, and that an increased number of these elements, also increases the affinity for drug
binding (Seelig, 1998; Zhou, 2008). These data corresponds to some transmembrane sequences
26
of P-gp/MDR1, rich in amino acids with hydrogen bonding donor side-chains involved in
substrate recognition (Seelig, 1998; Zhou, 2008). Seelig et al. have further found that the
number and strength of the hydrogen bonds formed between the substrate and P-gp/MDR1
determine the dissociation of the P-gp–substrate complex (Seelig and Landwojtowicz, 2000).
Research scientists involved in the development of novel drugs have used various
techniques in evaluating permeability/absorption of drug candidates during the drug candidate
selection process (Balimane and Chong, 2005); but most of them only provide information on
permeability characteristics of test compounds but no information on their potential to interact
with P-gp (Balimane and Chong, 2005). Several in vitro and in vivo models are used for
assessing P-gp interactions with test compounds (Adachi et al., 2001; Polli et al., 2001;
Yamazaki et al., 2001; Perloff et al., 2003; Balimane et al., 2004). In vitro assays such as
ATPase assay, rhoadmine-123 uptake assay, calcein AM uptake assay, cell based bi-directional
assay, radio- ligand binding assay along with in vivo models such as transgenic (knockout mice)
and mutant animal models are the most commonly used (Balimane and Chong, 2005). But all
these models only provide information regarding one aspect of the P-gp interaction, if the
compound studied is a substrate or an inhibitor of P-gp (Balimane and Chong, 2005). More
recently, Balimane et al. developed a combined cell based method to identify P-gp substrates
and inhibitors in a single assay, although it needs to be optimized for each setting as it is a cell
based assay, and factors such as cell type might play a key role for its standarization (Balimane
and Chong, 2005).
Some physicochemical characteristics such as lipophilicity, hydrogen-bonding ability,
molecular weight, and surface area were reported to affect the ability of a certain drug to bind to
P-gp/MDR1 (Bain et al., 1997; Wang et al., 2003b). Some P-gp/MDR1 substrates are also
CYP3A substrates and they are called CYP/P-gp bi-substrates, and overlap in tissue distribution
has also been described for CYP3A and P-gp (Suzuki and Sugiyama, 2000; Wang et al., 2003b).
27
It has been reported that both P-gp/MDR1 and CYP3A proteins act synergistically as a
protective barrier in the bioavailability of orally administered drugs (Cummins et al., 2002;
Wang et al., 2003b).
Modulators, also called chemosensitizers, reversers, or inhibitors, comprise a group of
compounds capable of reversing MDR in intact cells by blocking Pgp drug efflux (Tan et al.,
2000; Sharom, 2006). The majority of modulators binds to Pgp at the substrate-binding pocket,
and competes with P-gp transport substrates (Sharom, 2006). Many modulators, such as
cyclosporin A and verapamil, are also known to be transported by Pgp (Sharom, 2006). Pgp
modulators also belong to many different structural classes, and have similar molecular features
as transport substrates (Wiese and Pajeva, 2001; Sharom, 2006).
Therapeutic uses of P-gp/MDR1 inhibitors consist of their co-administration with
existing chemotherapy drugs, which help to avoid potential drug resistance in cancer cells
(Dean, 2002). P-gp/MDR1 inhibitors, like substrates, also share some common chemical
features, i.e., aromatic ring structures, a tertiary or secondary amino group and high lipophilicity
(Wang et al., 2003b; Zhou, 2008). Some P-gp/MDR1 inhibitors are also referred to as
chemosensitizers as they help to reduce resistance to chemotherapy (Shiraga et al., 2001; Zhou,
2008). Novel potent P-gp/MDR1 reversing agents are necessary for inhibiting MDR in clinical
practice (Zhou, 2008).
Inhibition of P-gp/MDR1 can be achieved by binding to P-gp with very high-affinity as
a substrate and non-competitively prevent other drugs from binding, or by efficient inhibiting
ATP hydrolysis either at the ATP binding site or by inhibiting protein kinase C, involved in
ATP coupling to P-gp/MDR1 (Wang et al., 2003b). P-gp/MDR1 inhibition can also be achieved
by nonspecific protein kinase inhibitors that are active against protein kinase C (Chaudhary and
Roninson, 1993).
28
The use of modulators in the clinical practice is very important since they are
coadministered with Pgp substrate drugs and can improve their uptake in the gut and delivery to
the brain, and can also increase the cytotoxicity of anti-cancer drugs to tumour cells (Ambudkar
et al., 1999; Thomas and Coley, 2003). Some of the first generation of chemosensitizers
identified were also substrates for P-gp, and they compete with the cytotoxic compounds for P-
gp efflux, meaning that they must be used in high concentrations to achieve adequate
intracellular concentrations of the cytotoxic drug (Ambudkar et al., 1999). But many of these
chemosensitizers act also as substrates for other transporters and enzyme systems, provoking
unpredictable pharmacokinetic interactions in the presence of the chemotherapy agents. Second
generation of chemosensitizers were developed to overcome these limitations, some of them
were analogs of the first ones, but with less toxicity and greater potency (Krishna and Mayer,
2000; Thomas and Coley, 2003). Valspodar (PSC 833) is the best characterized and most
studied of these second generation agents; it is a non- immunosuppresive analog of cyclosporine
A and has an inhibitory capacity 10- to 20-fold greater than first generation cyclosporin A (te
Boekhorst et al., 1992; Thomas and Coley, 2003). The use of second-generation P-gp
modulators combined with chemotherapy agents in clinical trials has resulted in the reversal of
MDR but with limited success in treating refractory cancers (Advani et al., 1999; Chico et al.,
2001; Thomas and Coley, 2003). Although they have better pharmacologic features than the
first-generation compounds, they also have some disadvantages for their clinical use: they
inhibit the metabolism and excretion of cytotoxic agents leading to high toxicity levels that
cause to reduce the doses of chemotherapy in clinical trials (Thomas and Coley, 2003). The use
of these second-generation modulators has been limited due to their unpredictable effects on
their metabolism by cytochrome P450 (Thomas and Coley, 2003). Many of these second
generation modulators, although are more selective than first generation inhibitors, also act as
substrates for other ABC transporters, and the use of them as inhibitors could impair the ability
29
of normal cells and tissues to protect themselves from cytotoxic agents (Thomas and Coley,
2003).
The development of third-generation molecules that specifically inhibit P-gp with great
potency was possible thanks to structure-activity relationships and combinatorial chemistry that
help to overcome the limitations of the second-generation P-gp modulators (Krishna and Mayer,
2000; Thomas and Coley, 2003). These novel modulators have the advantage that do not affect
cytochrome P450 3A4 at relevant concentrations, and they also have no inhibitory effect on
other ABC transporters (Dantzig et al., 1999; Wandel et al., 1999; Thomas and Coley, 2003).
These characteristics practically assure high specificity for P-gp (Thomas and Coley, 2003).
Clinical trials with these third generation modulators have shown no relevant alterations in the
pharmacokinetics of the coadministered chemotherapeutic agent; thus, no chemotherapy dose
reduction was necessary (Thomas and Coley, 2003). Tariquidar is one of the most promising
members of this group, with high affinity for P-gp and high potency to inhibit its ATPase
activity (Martin et al., 1999; Mistry et al., 2001; Thomas and Coley, 2003). While phase I and II
clinical trials with third generation tariquidar showed promising results (Stewart et al., 2000;
Fracasso et al., 2004; Sandler et al., 2004; Morschhauser et al., 2007); the phase III trial with
tariquidar was performed in combination with first- line chemotherapy for patients with non-
small cell lung cancer but, due to toxicity problems, the trial was abandoned (Szakacs et al.,
2006). Further trials are ongoing and will provide more information in the future.
Third-generation P-gp inhibitors have shown to be promising as they have significant
advantages over the second-generation agents, in particular the lack of interaction with
cytochrome P450 3A4 (Thomas and Coley, 2003). The ongoing clinical trials with these new
agents will be able to show if treatment with these compounds are effective in increasing
survival of patients with cancer (Thomas and Coley, 2003). However, in general, this approach
30
has been disappointing, despite the fact that treatment fa ilure and patient survival are linked to
Pgp expression in several cancers (Thomas and Coley, 2003; Polgar and Bates, 2005).
1.3.4. P-glycoprotein Silent Polymorphisms
In the 1950s, clinical observations showed that patients have individual differences in
their responses to drugs and that these characteristics could be inherited (Evans and Johnson,
2001). Several studies reported that genetic factors influence the variety of individual responses
to medications regarded to toxicity and efficacy (Evans and Johnson, 2001). After the
completion of the human genome project, the development of functional genomics,
bioinformatics and high- throughput screening have shown that the extent of genetic variation in
the human general population is greater than had been estimated (Sachidanandam et al., 2001),
and the most common sequence variation is the single-nucleotide polymorphism (SNP). SNPs,
that by definition have a minor allele frequency of >1%, are major variations determinants in
disease susceptibility, response to medication, and toxicity (Risch, 2000). The frequency of
SNPs is approximately 1 per 100bp to 1000 bp (Sauna et al., 2007a).
Recently, Kimchi-Sarfaty et al. observed that SNPs in MDR1 result in a protein with
altered drug and inhibitor interactions but with its amino acid sequence intact (Kimchi-Sarfaty et
al., 2007; Sauna et al., 2007a). “Synonymous SNPs are silent mutations that, if occurring in
protein-coding regions, involve nucleotide substitutions still coding for the same amino acid”
(Tsai et al., 2008). Many types of such SNPs can be found due to the degeneration of the genetic
code (Tsai et al., 2008). Thus, the amino acid sequence has no change (Sauna et al., 2007a).
A synonymous mutation or SNP are named ‘‘silent’’ according to Anfinsen’s principle
that says that the amino acid sequence of a protein alone determines its three-dimensional
structure and, consequently, its function (Anfinsen, 1973). Sauna et al. reported that in the case
of synonymous codons, there are preferred codons that strongly correlate with the relative
abundance of the corresponding tRNAs, and that natural selection acts on these synonymous
31
mutations (Sauna et al., 2007a). A list of synonymous mutations correlates with human diseases
and new mutations are being added to this list regularly (Chamary et al., 2006). Several studies
also show ‘‘cotranslational folding’’ where proteins fold while being translated, leading to
different intrapeptide contacts during folding from those that occur during refolding of the
covalently intact but unfolded polypeptide (Batey et al., 2006). Protein folding is not only
controlled thermodynamically but also kinetically where translational pauses are needed for
proteins to fold correctly (Purvis et al., 1987; Sauna et al., 2007b). Few studies support the ides
that kinetics could also be influenced by codon bias because the rate of translation would be
rapid over mRNA stretches that use frequent codons and slower when rare codons are used
(Sauna et al., 2007b).
The human MDR1 or ABCB1 gene is located on chromosome 7 at q21.1 (Callen et al.,
1987), has over 120 kb and contains 28 exons and the coding region accounts for less than 5%
of the total (Sakaeda, 2005). Although SNPs have been reported in the MDR1 gene since 1989
(Kioka et al., 1989), the first systematic screening was done by Hoffmeyer et al. in 2000 when
15 SNPs were detected (Hoffmeyer et al., 2000). Since then, over 50 SNPs have been identified
in the human MDR1 gene (Ambudkar et al., 2003). Kimchi-Sarfaty et al. reported that certain
combinations of synonymous SNPs with frequent-to-rare codon substitutions alter the shape of
the transport substrate binding site and the substrate specificity of P-gp, despite the identical
protein sequence (Kimchi-Sarfaty et al., 2007). Szakacs et al. reported that taking into
consideration that some inhibitors bind to one of P-gp conformational states but not another, that
single residue substitutions can dramatically change substrate specificity, and that the P-gp
recognizes a large variety of substrates and inhibitors in a large pleomorphic substrate–inhibitor
interaction site, the conformational change between the Pgp states is limited (Szakacs et al.,
2006; Kimchi-Sarfaty et al., 2007). It has been reported that these polymorphisms constitute risk
factors for several diseases (Asano et al., 2003; Pawlik et al., 2005; Tan et al., 2005; Markova et
32
al., 2006) and may affect the progression of others (van den Heuvel-Eibrink et al., 2001; Goreva
et al., 2003; Jamroziak et al., 2004; Sills et al., 2005). Point mutations are known in mammalian
P-gp's to affect substrate specificity (Ambudkar et al., 1999), thus, pharmacokinetics of several
drugs, such as cyclosporin A (Hesselink et al., 2003), nelfinavir (Zhu et al., 2004), fexofenadine
(Kim et al., 1998) and digoxin (Kafka et al., 2003) are affected by these SNPs (Kimchi-Sarfaty
et al., 2007). All these observations demonstrate that MDR proteins can easily alter their
substrate specificity, even with subtle changes in the conformation of the drug-binding pocket
(Kimchi-Sarfaty et al., 2007).
1.3.5. P-glycoprotein Regulation
Regulation of P-glycoprotein can occur at the transcription and translation levels, and
also at the level of membrane retrieval and reinsertion (Trauner and Boyer, 2003). The main
attention has been given to regulation at the level of transcription (Zhou, 2008). The mechanism
of transcription of the MDR1 gene is very complex and only partially understood (Zhou, 2008).
MDR1 gene transcription can be activated by different factors. Induction of P-gp/MDR1 has
been reported for drugs like phenytoin, ritonavir, nelfinavir, and amprenavir (Zhou, 2008).
MDR1 can also be induced by a variety of chemical compounds such as inducers of
differentiation, phorbol esters and carcinogens (Stavrovskaya and Stromskaya, 2008), but also
by physical stress, such as X-irradiation, ultraviolet light radiation, and heat shock (Seelig,
1998). P-gp/MDR1 induction by drugs is clinically relevant when leads to altered drug
absorption and oral bioavailability, and to the deve lopment of MDR to chemotherapeutic agents
by cancer cells (Zhou, 2008). It has been described that induction of P-gp/MDR1 expression is
regulated by nuclear factors at the transcriptional level (Kuwano et al., 2004).
Several signalling cascades are involved in MDR1 transcriptional regulation
(Stavrovskaya and Stromskaya, 2008). The MDR1 gene promoter region has several regulatory
33
sequences for the binding of trans-acting proteins from different superfamilies (Scotto and
Johnson, 2001).
Retinoids control one of the signaling pathways associated with MDR1 transcription
(Stavrovskaya and Stromskaya, 2008). Retinoids are associated with the regulation of cell
proliferation, differentiation and programmed death. They are used for treatment of
hemoblastoses and solid tumors, and for therapy of acute promyelocytic leukemia (Altucci and
Gronemeyer, 2001). The effect of retinoids is the result of their interaction with nuclear
receptors of the RAR family and RXR family. Stromskaya et al. reported that MDR1 gene
constitutive (basal) expression is enhanced by the hyperexpression of the RARa gene in cells of
solid tumors and some hemoblastoses (Stromskaya et al., 1998; Stromskaya et al., 2005).
The signal transduction pathway of sphingomyelin has also been reported in the
regulation of MDR1 /Pgp activity (Stavrovskaya and Stromskaya, 2008). The sphingolipid or
sphingomyelin signal pathway has been described as one of the main signal cascades operating
in regulation of cell apoptosis, differentiation, and proliferation. Ceramide is the key molecule
associated with this pathway (Obeid and Hannun, 1995), and it is generated in response to some
cytokines or different types of stress, including some antitumor drugs (Obeid and Hannun, 1995;
Hannun and Luberto, 2000). It has been reported that treatment of human hemoblastosis cells
with ceramide C2, a synthetic analogue of ceramide, increases the amount of MDR1 gene
mRNA and enhances Pgp expression and its functional activity (Shtil et al., 2000; Ktitorova et
al., 2001).
Regulation of P-gp/MDR can be achieved by the Ras mediated pathway (Miltenberger et
al., 1995; Kim et al., 1996). The Ras family proteins are described as the most important
components of pathways regulated by receptor and non-receptor tyrosine kinases (Agapova et
al., 2004; Schubbert et al., 2007). Ras activates a number of effector proteins that regulate cell
proliferation, motility, transport of macromolecules, as well as some othe r functions
34
(Stavrovskaya and Stromskaya, 2008). It has been reported that introduction of N-rasAsp12
gene into human and rat cells (several lines) results in the expression of active Pgp and the
emergence of MDR phenotype in some of them (Kopnin et al., 1995). However, the induction of
the MDR1/mdr1 gene by mutant Ras is dependent at least on the cell species affiliation; the
enhancement of P-gp functional activity was only found in rat cells but not in human (Kopnin et
al., 1995; Stromskaya et al., 1995; Stavrovskaya and Stromskaya, 2008).
Phosphoinositol-3-kinase (PI3K) and the signal transduction pathways activated by this
enzyme are also regulators of MDR1 (Stavrovskaya and Stromskaya, 2008). PI3K is one of the
most important mediators in signal transduction protecting the cell against a broad spectrum of
the cell death inducers (Agapova et al., 2004; Vogt et al., 2006; McCubrey et al., 2007). PI3K
can be activated by its direct interaction with tyrosine kinases and by binding to Ras proteins.
The phosphatase PTEN is an inhibitor of this signal pathway. It has been shown that functional
PTEN status in prostate cancer cells correlates with the expression level of genes/proteins MRP1
and BCRP, but the MDR1/Pgp status in the investigated cells is independent of the Akt/PTEN
status (Sherbakova et al., 2008). Introduction of the PTEN gene into cells show inhibition of
MRP1 in KB3-1 and KB8-5 cells, and in epidermoid carcinoma cell lines. In conclusion,
hyperexpression of PTEN alter the cell sensitivity to drugs depend ing on the mechanism of
action of the drug and on the cells into which the transgene was introduced (McCubrey et al.,
2006).
Any malignant transformation and tumor progression that alter any of the signal
transduction pathways involved in regulation of P-gp/MDR1 can have serious effects on the
activity of this transporter (Stavrovskaya and Stromskaya, 2008).
Y-box binding protein 1 (YB-1) is the most important nuclear inducer of the MDR1
gene. YB-1 is a protein associated with cell growth, DNA synthesis and repairing cell damage,
among other functions (Shibahara et al., 2004). YB-1 activity has been shown to be increased in
35
cancerous cells by both chemotherapeutic drug induction and induction via the cell damage
caused by chemotherapy (Kuwano et al., 2003; Kuwano et al., 2004; Stavrovskaya and
Stromskaya, 2008). YB-1 activity strongly correlates with P-gp/MDR1 expression; introduction
of YB-1 inhibiting substances showed that decreased YB-1 expression led to decreased P-
gp/MDR1 expression (Kuwano et al., 2004). It has been suggested that YB-1 is a stress- induced
protein (Kuwano et al., 2003). YB-1 function as a transcription factor can be stimulated by
external factors such as DNA-damaging substances, UV radiation, and high temperatures (Chin
et al., 1990; Zhou, 2008).. The transfer of YB-1 from the cytoplasm of a cell into the nucleus
where it binds to deoxyribonucleic acid (DNA) regions (Ohga et al., 1996), serves as a signal for
increased MDR1 transcription (Kuwano et al., 2004).
Cellular damage can also induce expression of the human MDR1 promoter gene in
response to cytotoxic drugs regardless if the drugs are transported by P-gp/MDR1 (Chaudhary
and Roninson, 1993) and that is one of the reasons why P-gp/MDR1 is active in terms of
multidrug resistance. Activation of P-gp/MDR1 transcription is one of the primary stress
response mechanisms in cells, regardless if the stress comes from cellular damage, drug
response, or other means (Ledoux et al., 2003).
1.3.6.. P-glycoprotein Knockout Mice
Mice have two genes, in contrast to humans, that encode the drug transporting P-gps,
Abcb1a (also called mdr3) and Abcblb (also called mdr1), respectively (Hsu et al., 1989;
Devault and Gros, 1990). The mouse Abcb1a gene is mainly localized in the membranes of the
intestine, liver, and blood capillaries of brain and testis (Arceci et al., 1988; Croop et al., 1989;
Teeter et al., 1990; Schinkel et al., 1994). The Abcb1b gene is mainly located in the adrenal,
placenta, ovary and (pregnant) uterus (Arceci et al., 1988; Croop et al., 1989; Teeter et al., 1990;
Schinkel et al., 1994). Expression of Abcb1a and Abcb1b is similar in kidney (Arceci et al.,
1988; Croop et al., 1989; Teeter et al., 1990; Schinkel et al., 1994). All these previously reported
36
data suggest that Abcbla and Abcblb in the mouse has the same function as MDR1
(Stavrovskaya and Stromskaya, 2008)
Mdr knock out mice were generated for characterization of the normal physiological
function(s) of the mdr1-type P-glycoproteins (Schinkel et al., 1994). Mice have been generated
initially with a disruption of the Abcb1a gene (Schinkel et al., 1994). These Abcbla (-/-) mice
served as confirmation of the protective role of P-gp in extruding toxic compounds out of the
cells. Unexpectedly, these mice turned out to be 100-fold more sensitive to the centrally
neurotoxic pesticide, ivermectin (oral administration) (Schinkel et al., 1994). Further analysis
showed that wild-type mice have high levels of mdrla P-glycoprotein at the blood-brain barrier,
whereas Abcb1a (-/-) mice did not have detectable P-glycoprotein there. This resulted in
approximately 100-fold higher levels of the neurotoxin in the brain 24 h after administration,
thus explaining the increased sensitivity of these mice (Schinkel et al., 1994). The KO Abcb1a (-
/-) mice were also 3-fold more sensitive to the anticancer agent, vinblastine (intravenous
administration), and they accumulated much higher levels of this drug in their brains. They
displayed overall increased accumulation and decreased elimination of vinblastine in tissues and
plasma (Schinkel et al., 1994). Analysis of these KO mice allowed to confirm the important
pharmacological role of the Abcb1a P-gp in the blood-brain barrier, as a protective barrier of the
brain against the entry of a range of toxic xenobiotics and drugs, and also in the intestine, by
limiting the entry of these compounds from the intestinal lumen, and taking part in their active
excretion from the bloodstream (Schinkel et al., 1995; Mayer et al., 1996; Schinkel et al., 1996;
Sparreboom et al., 1997). The striking finding was that, beside the pharmacological effects,
Abcb1a (-/-) KO mice are completely healthy (Schinkel et al., 1994). This suggests that P-
glycoprotein activity, at least in some organs, is not essential for life. However, mdrlb P-
glycoprotein is still present in Abcb1a (-/-) mice.
37
Mice with a deficiency in the Abcb1b gene, and in both the mdr1a and mdr1b genes
were generated and characterized (Schinkel et al., 1997). Pharmacologically, Abcb1a/b (-/-)
mice behaved similarly to the previously analyzed Abcb1a (-/-) mice, with increased brain
penetration and reduced elimination of drugs (Schinkel et al., 1997). However, both Abcb1a and
Abcb1b P-gps contributed to the extrusion of rhodamine from haematopoietic progenitor cells,
suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow
against cytotoxic anticancer drugs (Schinkel et al., 1997).
All KO strains were healthy and fertile, with neither physiological abnormalities nor a
decreased life span (Schinkel et al., 1997). In consequence, mdr1-type P-gps are not essential
under laboratory conditions, for the basic physiological functioning of the organism, although it
cannot be excluded that human may respond differently from mice to the absence of mdr1-type
P-gp activity (Schinkel et al., 1997).
Another approach reported was the disruption of the rodent gene for the Abcb4 P-gp,
closely related to the Abcb1a and Abcb1b P-gps. These mice confirmed that this gene is
essential for phospholipid excretion in the liver (Mayer et al., 1996).
These KO mice models should provide a useful model system to further test the
pharmacological roles of drug-transporting P-gps, and allow predicting the consequences of
complete and specific inhibition of P-gp by administration of P-gp blockers.
1.3.7. Other Approaches in Modulation of Multidrug Resistance
In addition to the reversal of multidrug resistance by chemical compounds, promising
strategies for eradication of multidrug resistance have also been performed with experimental
therapeutics.
One of the approaches was the development of monoclonal antibodies directed against
extra cellular epitopes of MDR1/P-gp (Lage, 2008). Their high specificity against well-defined
epitopes makes them ideal for tumour therapy. Although not only several monoclonal antibodies
38
(Mabs) but also polyclonal antibodies were analyzed, only the ones directed against
extracellular determinants of membrane proteins are potentially useful in immunotherapy, such
as MRK16, HYB-612, or 265/F4 (Lage, 2008). Hamada and Tsuruo were the first to describe
the specific effects of P-gp reactive Mabs against MDR cells in vitro (Hamada and Tsuruo,
1986). UIC2 antibody was shown to be as strong as verapamil (well-known Pgp inhibitor) at its
highest clincally achievable concentrations in inhibiting the drug efflux activity of this ABC-
transporter (Mechetner and Roninson, 1992). Immunotoxins, a monoclonal antibody coupled to
a cytotoxic agent via a linker molecule, have also been analyzed as potential treatment for
MDR. The hybrid toxin (i.e., Pseudomonas coupled to mab MRK16) binds to a specific antigen
and exerts its cytotoxic effect after internalization (FitzGerald et al., 1987). Other approach used
for MDR1 modulation is the use of bispecific antibodies, chimeric proteins with each of the two
antigen binding sites that recognize different antigens (Van Dijk et al., 1989). As an example,
the CD3/MRK16 heteroconjugate was able to induce specific cytolysis of ovarian carcinoma
cells and melanoma cells both expressing P-gp where the cytolysis was mediated by activated
T- lymphocytes (Van Dijk et al., 1989). The results of these antibody-directed approaches appear
to be promising alternatives to conventional chemotherapy for the treatment of MDR cancer
cells in patients. But some problems limit their clinical application. The monoclonal antibodies,
immunotoxins and bispecific antibodies used are commonly of murine origin and are
immunogenic in humans, so the construction of chimeric mouse-human antibodies might reduce
the immune reactions against murine antibodies. Another problem is the failure to achieve
therapeutically effective antibody concentrations within solid tumours. The third problem is that
these treatments affect both tumour and normal cells due to the wide expression of P-gp
throughout the human body (Lage, 2008).
39
RNA-technology based strategies have also been evaluated to overcome drug resistance,
such as ribozyme and RNA interference (RNAi) approaches (Lage, 2006), with promising
preclinical data but there is no clinical trial planned yet (Lage, 2008; Stein et al., 2008).
With the advancement of the human genome project, there is a tendency to design
individually tailored treatment regimens for cancer patients, but unfortunately for the MDR
field, this will require more time and research since there are no reliable diagnostic tests
developed for MDR introduced into the clinical routine yet (Lage, 2008; Stein et al., 2008).
1.3.8. P-glycoprotein and Stem Cells
Stem cells are a unique population of cells capable of self renewal and differentiation
into different cell types (Lin et al., 2006). Nowadays, the efforts in regenerative medicine are
focused in the search for suitable renewable sources of cells that can be used to treat human
diseases (Priddle et al., 2006). Stem cells are able to evade the host’s immune system and can be
expanded in culture, making them a promising source of cells for therapeutic applications
(Priddle et al., 2006). Stem cell populations have been identified in adult tissues like the brain,
bone marrow, blood, liver, pancreas and others (Erlandsson and Morshead, 2006; Taupin,
2006). In particular, the hematopoietic stem cells (HSCs) from bone marrow can differentiate
into all the different immune cells and have been satisfactorily used to treat leukemia,
lymphoma and immune deficiencies (Chan and Yoder, 2004; Bonnet, 2005). A specific group of
HSCs from blood and bone marrow called the “side population” (SP), CD34-negative, are
probably among the best-characterized examples of pluripotent adult stem cells to date (Goodell
et al., 1996; Jackson et al., 2001; Wu and Wei, 2004; Challen and Little, 2006)}. Different types
of stem cells can be found but due to their very low numbers in the human body, unique
biologically stem cell markers are used to identify and isolate them (Goodell et al., 1996;
Jackson et al., 2001).
40
Researchers are focusing their interests in the regulation of stem cell biology by the
ATP-binding cassette (ABC) transporters (Lin et al., 2006). Dean et al. reported that P-
glycoprotein as well as other ABC transporters are hyperexpressed in stem cells (Dean et al.,
2005). It has already been shown more than 15 years ago that CD34+ cells hyperexpress P-gp
(Chaudhary and Roninson, 1991; Stavrovskaya and Stromskaya, 2008). The ABC transporter
BCRP (ABCG2) was also found to be expressed by stem hemopoietic cells and is considered as
a stem cell marker (Zhou et al., 2001; Stavrovskaya and Stromskaya, 2008). It was also reported
that overexpression of P-gp in bone marrow cells led to the proliferation of SP cells, that results
in prolonged survival in culture and enhanced repopulation after transplantation into mice
(Bunting et al., 2000; Lin et al., 2006). Thus, P-gp expression can be used as a marker of
proliferating stem cells, while BCRP expression can be used to identify quiescent ones (Lin et
al., 2006).
Functions of P-gp and other ABC transporters in stem cells are still not clear. It was
hypothesized that they protect stem cells (both normal and tumor) against damaging substances
(Dean et al., 2005; Raaijmakers, 2007). ABC transporters might also be involved in regulation
of self-renewal and differentiation of stem cells (Good and Kuspa, 2000; Dean et al., 2005).
As cancer stem cells express drug transporters that make them resistant to many
chemotherapy agents, new anticancer strategies are planned to target these cells with their
special properties (Dean et al., 2005). Clinical studies have attempted to overcome drug
resistance through combination therapies in which a cytotoxic drug was given along with an
ABC-transporter inhibitor without much luck (Dean et al., 2005). The targeting of the mdr1
gene to overcome multidrug resistance is one of the most promising gene therapeutic
approaches (Dean et al., 2005), where the MDR1 cDNA is transferred into hematopoietic stem
cells during high dose chemotherapy and stem cell transplantation. The ex vivo transfer of the
MDR1 gene into murine hematopoietic stem cell transplantation have been shown to protect
41
drug-sensitive normal hematopoietic stem cells from chemotherapy (Efferth et al., 2007). The
problem with this approach is the efficacy of gene transduction in human that is about two
orders of magnitude lower than in murine stem cells (Efferth et al., 2007); more efficient vector
systems are needed for clinical applications. Clinical trials of retroviral vectors containing drug
resistance genes have been shown that this approach is safe and are now being designed to
address the therapeutically relevant issues and to overcome the limitations (Nobili et al., 2006).
New lines of research address point mutations in the MDR1 gene that affect the drugs’ affinity
to bind to P-glycoprotein (Gottesman et al., 1995), trying to improve the success of the
treatment during high-dose therapy and stem cell transplantation using artifically mutated
“custom-made” P-gp species with higher affinities for certain drugs (Nobili et al., 2006; Efferth
et al., 2007).
There are other gene therapeutic approaches that target the mdr1 gene as well: the
antisense strategies to reduce P-glycoprotein expression in cancer cells that consist of
suppression of P-glycoprotein expression by antisense oligodeoxynucleotides, triplex-forming
oligonucleotides, or hammerhead ribozymes sensitizes cancer cells towards anti-tumor drugs
(Wang et al., 2003a; Qia et al., 2005; Kaszubiak et al., 2007).
1.4. Glycosphingolipids
Glycosphingolipids (GSLs), amphipathic compounds consisting of sugar and ceramide
moieties, are ubiquitous components of the plasma membrane of all vertebrate cells. GSLs are
considered to be receptors for microorganisms and their toxins, modulators of cell growth and
differentiation, and organizers of cellular attachment to matrices. The first glycosphingolipid
was discovered in 1874 by Johann Ludwig W. Thudichum in brain tissue; he called it
42
cerebroside. The “sphingosin” backbone of GSLs was so named by him too for its enigmatic
(“Sphinx- like”) properties (Hakomori et al., 1998).
1.4.1. Structure of Glycosphingolipids
The molecular structure of glycosphingolipids consists of a ceramide backbone and a
sugar headgroup. The hydrophobic ceramide part, consisting of a sphingoid base and a fatty
acid, is inserted in a cellular membrane whereas the sugar headgroup mostly faces the non-
cytosolic space (Kanfer and Hakomori, 1983) (Scheme 1.5).
SCHEME 1.5. General Molecular Structure of a Glycosphingolipid (Kanfer and Hakomori,
1983).
GSLs are very heterogenous molecules considering their backbone and headgroup
structures, with more than 60 different sphingoid bases and more than 300 different
oligosaccharide chains characterized, with the different combinations of both that create
thousands of different structures (Degroote et al., 2004). The different sphingoid bases can vary
in length, saturation, hydroxylation, and branching (Karlsson, 1970; Degroote et al., 2004).
Sphingosine is the most important sphingoid base described in mammals (Degroote et al.,
2004).
43
It was reported that the heterogeneity of the fatty acids in the ceramide group could
influence the localization and functions of GSLs on the plasma membrane, through direct
interaction with cholesterol, phospholipids, and the transmembrane domains of receptor
proteins, and could also mediate signal transduction (Hakomori and Igarashi, 1993).
1.4.2. Classification of Glycosphingolipids
Glycosphingolipids are classified as neutral, acidic (anionic), or basic (cationic)
(Hakomori, 2003). Most acidic GSLs are gangliosides containing sialic acid, while other acidic
GSLs contain a sulphate group. Cationic GSLs, such as plasmalopsychosine or
glyceroplasmalopsychosine, are very rare to be found and might be involved in intracellular
signalling (Nudelman et al., 1992; Hikita et al., 2001; Hikita et al., 2002; Hakomori, 2003).
The great majority of GSLs have one of the four basic core structures, defined in terms
of the identity and chemical binding sugars that are closest to the ceramide (Hakomori, 1986;
Hakomori, 2008). GSLs are also classified according to the core structure as ganglio-series,
globo-series, lacto-series type 1, and lacto-series type 2. In each of the four core structures, the
ceramide molecule is linked to glucose and the glucose is linked to galactose. In the ganglio-
series GSLs, the chain continues with N-acetylgalactosamine, with another galactose attached
(Hakomori, 1986; Hakomori, 2008). The internal galactose and the terminal galactose are each
linked to an acidic sugar, sialic acid. In the globo-series, the initial glucose-galactose chain is
continued with another galactose and N-acetylgalactosamine. In the lacto-series, the initial chain
is continued with N-acetylglucosamine and galactose (Hakomori, 1986; Hakomori, 2008). If the
position of the last bond (1-3 or 1-4) is considered between N-acetylglucosamine and galactose,
the lacto-series molecules are further classified into type 1 and type 2 molecules. The lacto-
series GSLs can be extended and branched to form a variety of molecules, i.e., the blood-group
antigens of the ABH system (Hakomori, 1986; Hakomori, 2008).
44
Gangliosides, sialic acid containing GSLs of the ganglio-series, are abundant on
neuronal cells and contribute to the function of the nervous system (Hakomori, 1981; Sandhoff
and Kolter, 2003). The GSLs patterns at the cell surface change with cell growth, cell
differentiation, viral transformation, ontogenesis and oncogenesis (Hakomori, 1981).
1.4.3. Biosynthesis and Degradation of Glycosphingolipids
Lipids are synthesized in different intracellular organelles, and then transported to the
appropriate compartments to be further metabolized or act as functional molecules. The
heterologous distribution of lipids is tightly regulated, which is determined by the localization of
lipid synthases and their topology in the membrane, and several types of trafficking, including
flip-flop, vesicular, and nonvesicular trafficking (Holthuis and Levine, 2005).
The first step in glycolipid biosynthesis in animals is the intracellular formation of the
membrane anchor, ceramide, to which single carbohydrate residues will be added (Kolter et al.,
2002; Merrill, 2002); at the same time, intracellular movement of metabolic intermediates and
final products to the plasma membrane occur (van Meer and Lisman, 2002).
The synthesis of ceramide as the initial step for de novo biosynthesis of GSLs occurs at
the membranes of the endoplasmic reticulum (ER) (Merrill, 2002; Sandhoff and Kolter, 2003).
The condensation of the amino acid L-serine with a fatty acyl coenzyme A, usually palmitoyl
coenzyme A, to 3-ketosphinganine, the precursor for all sphingoid bases, is catalyzed by the
enzyme serine palmitoyl transferase (Sandhoff and Kolter, 2003), followed by subsequent
acylation that produces ceramide (Guillas et al., 2001; Sandhoff and Kolter, 2003). Sphingosine,
the precursor of sphingolipids, is formed during sphingolipid degradation (Sandhoff and Kolter,
2003). All the enzymes that participate in the biosynthesis of ceramide are located on the
cytosolic leaflet of the ER membrane (Michel and van Echten-Deckert, 1997). Ceramide, the
common precursor of GSLs and sphingomyelin, is then translocated into the lumen of the ER.
Biosynthesis of sphingomyelin (SM) is mainly localized on the luminal surface of the Golgi
45
membranes (Futerman et al., 1990; Sandhoff and Kolter, 2003) but some other sites have also
been reported (Miro Obradors et al., 1997; Sandhoff and Kolter, 2003).
It has also been reported that ceramide can be converted into galactosylceramide
(GalCer) in certain cell types, as ceramide can cross the ER membrane spontaneously (Degroote
et al., 2004). GalCer based GSLs are the major lipid constituents of the myelin sheath around
the axons and neuronal cells in mammals (Degroote et al., 2004). In the ER, a Gal residue is
transferred from UDP-Gal to ceramide by UDP-Gal:ceramide galactosyltransferase or GalCer
synthase (Schulte and Stoffel, 1993; Kapitonov and Yu, 1997). GalCer passes through the
lumen of the Golgi apparatus, where it can be sulfated to sulfatide (Coetzee et al., 1998) en
route to the plasma membrane, and it can also be galactosylated in kidney epithelia (Degroote
et al., 2004).
Ceramide exits the ER and follows the vesicular pathway to early Golgi compartments
where it is converted to glucosylceramide, GlcCer (Degroote et al., 2004). GlcCer is present in
most eukaryotic cells and also in some bacteria, and serves as the major precursor for complex
GSLs. Hanada et al. (Hanada et al., 2003) reported that the synthesis of SM but not GlcCer
depended on ceramide transport to the trans-Golgi by the ceramide transport protein CERT, a
pathway that is regulated by phosphoinositides, sterols and CERT phosphorylation (Perry and
Ridgway, 2006; Halter et al., 2007). GlcCer synthase has been described to be present in the
pre-Golgi and trans-Golgi membranes, as well as in the cis-medial Golgi, together with SM
synthase (Futerman et al., 1990; Futerman and Pagano, 1991; Kohyama-Koganeya et al., 2004).
It is now currently accepted that GlcCer is synthesized on the cytosolic surface of the Golgi and
translocates across the Golgi membrane for higher GSL synthesis in the late Golgi (Lannert et
al., 1994; Lannert et al., 1998). This translocation is presumably mediated by the lipid flippase
function of P-glycoprotein/MDR1 (De Rosa et al., 2004; Eckford and Sharom, 2005), although
recent reports indicated that the cytoplasmic protein FAPP2 can transport GlcCer to the TGN
46
and flip it into the lumen by an unknown flippase (D'Angelo et al., 2007). A recent study shows
that FAPP2 can also mediate the retrograde transport of GlcCer from the Golgi complex to the
ER where it can spontaneously flip into the lumen and after vesicular traffic to the Golgi, be
processed to complex GSLs (Halter et al., 2007). Both reports show that FAPP2 knockdown
impairs neutral GSLs synthesis (Halter et al., 2007).
More complex GSLs are built by the stepwise addition of individual sugars from their
activated nucleotide precursors onto GlcCer (Degroote et al., 2004). The following
glycosylation steps are catalyzed by glycosyltransferases located in the lumen of the Golgi
apparatus (Maccioni et al., 2002; Giraudo and Maccioni, 2003; Halter et al., 2007). In mammals,
the first step is the conversion of GlcCer to lactosylceramide (LacCer) catalyzed by LacCer
synthase (Takizawa et al., 1999). The oligosaccharide chain of mammalian GSLs can be
elongated by galactosyl-, N-acetylgalactosaminyl-, N-acetylglucosaminyl-, sialyl-, and
fucosyltransferases (Lloyd and Furukawa, 1998; Kolter and Sandhoff, 2006). These enzymes
are responsible for the formation of the oligosaccharide backbone that defines the different
series of GSLs (ganglio, lacto, neolacto, globo, isoglobo) (Chester, 1998). Sialyltransferases and
fucosyltransferases are responsible for the synthesis of the peripheral sugars of the
oligosaccharide (Chester, 1998; Kolter and Sandhoff, 2006). All gangliosides, except GM4,
have LacCer as a precursor (Kolter and Sandhoff, 2006). In most cell types, de novo
biosynthesis constitutes only a minor pathway for GSLs synthesis ; the majority of GSLs are
formed by recycling of their building blocks within a salvage-pathway (Tettamanti, 2004; Kolter
and Sandhoff, 2006). After their biosynthesis, GSLs reach the plasma membrane through
exocytosis (Kolter and Sandhoff, 2006).
The degradation of membranes in the lysosomes requires transport and lipid sorting,
before GSLs and other membrane components can be degraded (Kolter and Sandhoff, 2005).
GSLs are hydrolyzed stepwise by lysosomal glycosidases from their non-reducing end (Kolter
47
and Sandhoff, 2005). GlcCer can be degraded not only by a glucocerebrosidase in lysosomes,
but also by a non- lysosomal glucocerebrosidase in the cytosol (van Weely et al., 1993; Kolter
and Sandhoff, 2006). The degradation products, monosaccharides, sialic acid, fatty acids, and
sphingosine, leave the lysosomes, and can be used by salvage processes or can be further
degraded. Transporters localized in the endosomal and lysosomal membranes are responsible
for this (Kolter and Sandhoff, 2006) (Scheme 1.6).
Scheme 1.6. Biosynthesis and Catabolism of Sphingolipids ; SPT=serine palmitoyl
transferase, KSR=3-ketosphinganine reductase, SAT=sphinganine/sphingosine N-acyl
transferase, DCD=dihydroceramide desaturase, CDase=ceramidase, SPP=sphingosine 1-
phosphate phosphatase, SK=sphingosine kinase, SPL=sphingosine 1-phosphate lyase,
SMS=sphingomyelin synthase, SMase=sphingomyelinase, SMD=sphingomyelin deacylase,
CK=ceramide kinase, CPP=ceramide 1-phosphate phosphatise (Nussbaumer, 2008).
48
1.4.4. Inhibitors of Glycosphingolipids Biosynthesis
The experimental use of specific inhibitors of GSL biosynthesis allows identification of
functions of endogenous GSLs. The length and degree of unsaturation of the acyl chain of
ceramide and its metabolites are important in the biological functions that these molecules play
in cells (Delgado et al., 2006). The type and level of the different GSLs species present in
different cells depend on the enzymes involved in their synthesis and degradation (Delgado et
al., 2006).
Two different metabolic pathways lead to the formation of ceramide, the lipid precursor
in GSLs biosynthesis (Delgado et al., 2006): the anabolic or de novo pathway, and the catabolic
pathway. The first forms ceramide from simple components, such as palmitoyl CoA and serine,
while in the catabolic pathway, ceramide results from the hydrolysis of complex sphingolipids,
mainly SM and GSLs (Delgado et al., 2006).
The first step in the GSLs biosynthesis is the condensation of serine and palmitoyl CoA
into 3-ketodihydrosphingosine by serine palmitoyltransferase (SPT) (Hanada, 2003). Natural
products such as sphingofungins, lipoxamycin and myriocin inhibit SPT with potent and highly
selective activity (Hanada, 2003; Delgado et al., 2006). The next enzyme in the pathway,
ceramide synthase (CerS), catalyses the acylation of the amino group of sphinganine and other
sphingoid bases, using acyl CoA esters of different chain lengths. Inhibition of CerS by
fumonisins, a group of fungi metabolites, has been reported (Desai et al., 2002; Delgado et al.,
2006). Fumonisin B1 (FB1) is the most important compound of this group. N-acylated forms of
FB1 are potent CerS inhibitors (Humpf et al., 1998) although some selective fatty acid forms of
CerS were reported to be resistant to FB-1 (Wang et al., 1991; Wang and Merrill, 2000;
Venkataraman et al., 2002; Delgado et al., 2006).
The last enzyme in the de novo pathway of biosynthesis of ceramide is the
dihydroceramide desaturase (DES) (Michel and van Echten-Deckert, 1997; Delgado et al.,
49
2006). The cyclopropane-containing sphingolipid GT11 is a competitive inhibitor of DES
(Triola et al., 2001; Triola et al., 2003). A dihydroceramde analogue with a cyclopropane ring at
C-5 and C-6 of the sphinganine backbone has been also reported as an irreversible inhibitor of
DES that leads to a covalent modification of the protein (Savile et al., 2001). Another inhibitor
of DES has also been recently reported, 4-hydroxifenretinide (Delgado et al., 2006; Schulz et
al., 2006).
The enzyme inhibitors of GSLs biosynthesis at the Cer level are limited to the
glucosylation step (catalyzed by GlcCerS), its catabolic counterparts (GlcCerase and non
lysosomal glucosylceramidase) and the galactosyltransferase leading to LacCer from the
initially formed GlcCer (Delgado et al., 2006). Delgado et al. referred to the enzyme
glucosylceramide synthase (GlcCerS) as one of the few glucosyltransferases for which efficient
inhibitors are known (Compain and Martin, 2003; Delgado et al., 2006). The reported inhibitors
can be classified into: (a) those structurally related to Cer, and (b) synthetic analogues of
naturally occurring iminosugars, such as the a-glycosidase inhibitor deoxynojirimycin (DNJ).
Vunnam and Radin described some ceramide analogues in which the unsaturated alkyl chain
and the amino moiety of ceramide were replaced by an aromatic ring and a heteocyclic system,
respectively (Vunnam and Radin, 1980). The most potent inhibitors described were 1-phenyl-2-
decanoylamino-3-morpholino-propanol (PDMP) and its N-palmitoyl homologue (PPMP). One
of the new analogues from this group, hydroxyl-P4, was shown to be more potent and more
selective for the ceramide-specific glucosyl transferase (Lee et al., 1999; Delgado et al., 2006).
The ethylenedioxy derivative of this group of compounds has recently been described as a
specific inhibitor (Hillig et al., 2005). The second group of inhibitors comprises the synthetic
analogues of ceramide, iminosugars and related compounds. Iminosugars are in general,
inhibitors of glycosidases (Winchester and Fleet, 1992; el Ashry et al., 2000). N-
butyldeoxynojirimycin (NBDNJ) and N-butyldeoxygalactonojirimycin (NBDGJ) have shown
50
inhibitory activity against GlcCerS (Platt et al., 1994a; Platt et al., 1994b). Butters et al. have
reported that inhibition of GlcCerS by NBDNJ is competitive for Cer and non-competitive for
UDP-glucose, showing a structural mimicry between NBDNJ and Cer (Butters et al., 2000).
Butters et al. have even shown that a chain length between 3 and 6 carbon atoms minimum is
required for inhibition of GlcCerS, and longer alkyl chains are better inhibitors but more toxic
(Butters et al., 2005). Some of these compounds are already in use for the treatment of certain
sphingolipidosis (Asano et al., 2000; Cox, 2005; Delgado et al., 2006; Lachmann, 2006).
1.4.5. Lipid Rafts
“Already early on, the tremendous variability in the three-dimensional structure of GSLs
that can be generated by combining various carbohydrates in different orders and with different
glycosidic bonds, suggested that the oligosaccharides on glycoproteins and glycosphingolipids
might serve to mediate highly specific interactions between cells, cells and matrix and between
cells and soluble signaling molecules” (Hakomori, 2002). The lipid raft hypothesis in the late
1980s showed an alternative role for sphingolipids (Simons and van Meer, 1988). GSLs self-
aggregate in the lumenal leaflet of the membrane of the trans Golgi network; these aggregates
were transported to the apical plasma membrane domains of epithelial cells in budding vesicles,
thus sorting GSLs with apical proteins (van Meer et al., 1987; Hoetzl et al., 2007). This
hypothesis also explains that SM and cholesterol are also included in these aggregates in the
anterograde secretory pathway, keeping low concentrations of these lipids in the endoplasmic
reticulum (ER) (Hoetzl et al., 2007). Thus, complex sphingolipids localize on the non-cytosolic
surface and they can reach other organelles by vesicular transfer only (van Meer, 1989). In the
case of mitochondria and peroxisomes, no GSLs or SM are present in these membranes since
there is no vesiclar transport to these organelles (van Meer, 1989). The cellular distibution of
cholesterol is determined by its high affinity for sphingolipids according to this model
(Wattenberg and Silbert, 1983; Hoetzl et al., 2007). Glycosphingolipid-based rafts have been
51
described to play key roles in the signaling by immune receptors (Gomez-Mouton et al., 2001;
Pierce, 2002; Holowka et al., 2005; Barbat et al., 2007), in glycosignaling platforms on the cell
surface (Hakomori, 2002), and in other signal transduction events (i.e., disruption of actin
polymerization in cardiac fibroblasts) (Mayor et al., 2006).
The identification and quantitation of lipid rafts was based on the detergent resistance,
hence the alternative name, detergent-resistant membranes (Brown and Rose, 1992; Edidin,
2003). According to physical studies, their existence in cells has been controversial but new
imaging techniques allow their optical resolution in cellular systems (Gaus et al., 2003; Parton
and Richards, 2003). The most accepted definition of lipid rafts came from the 2006 Keystone
Symposium: “membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol-
and sphingolipid-enriched domains that compartmentalize cellular processes” (Mayor et al.,
2006). Small rafts can sometimes be stabilized through protein-protein interactions.
Sphingolipids, GSLs and sphingomyelin, certain phospholipids, and cholesterol, as well as
scaffold and/or functional membrane proteins are the main constituents of lipid rafts (Simons
and Ikonen, 1997; Simons, 2002). Membrane proteins preferentially associated with lipid rafts
include glycosylphosphatidylinositol (GPI-anchored cell surface proteins attached to the outer
leaflet, palmitoylated and myristoylated proteins, and cholesterol- or phospholipid-binding
proteins (Rajendran and Simons, 2005).
Almeida et al. explained that the association between different lipids in rafts is not
sufficient to be physically stable; thus, mixtures of cholesterol, sphingomye lin and unsaturated
phospholipids at physiological temperatures will have a tendency to dissipate and reform
(Almeida et al., 2005). This confirms the very short half- lives of lipid rafts (100 nanoseconds or
less). Endocytosis and vesicular trafficking maintain lipid raft structure (Shaw, 2006). It has
been reported that the formation of larger raft structures with longer half- lives depend on the
52
perturbations of membrane proteins during cell-cell contact or receptor engagement processes
(Shaw, 2006).
Contrary to the lipid raft hypothesis, some consider the interactions between proteins
and not the lipid raft recruitment, as the main driving forces for the assembly of signaling
complexes in immune cells (Douglass and Vale, 2005; Larson et al., 2005; Shaw, 2006). In
conclusion, in the present model, proteins are recruited first to a specific location in the
membrane, and lipids are recruited due to their association with proteins, but when the raft
structure is formed, this recruits other proteins and lipids to the signaling complex (Shaw, 2006).
1.4.6. Biological Functions of Glycosphingolipids
GSLs are present in diverse organisms, such as yeast, insects, worms, fishes and
mammals. GSLs are present in all organs but sometimes, a specific type is more abundant in a
certain tissue (Sabourdy et al., 2008). GSLs are mostly membrane components, present in
different subcellular compartments, but can also be found in biological fluids (Sabourdy et al.,
2008). The first evidence of the importance of GlcCer-derived GSLs was reported by Yamashita
et al., when embryos from a GlcCerS KO mouse were not able to survive more than 7.5 days,
confirming a critical role for GSLs in embryonic development and differentiation of certain
tissues (Yamashita et al., 1999). GSLs have been found to play key roles in the regulation of
several cellular functions. First evidence comes from the studies of Hannun, Obeid and
Kolesnick who reported the role of simple sphingolipids such as sphingosine and ceramide in
signal transduction (Hannun and R.M., 1987; Kolesnick, 1991; Hannun and Linardic, 1993;
Kolesnick and Golde, 1994; Obeid and Hannun, 1995; Sabourdy et al., 2008). It has been
reported that GSLs can participate in diverse cellular mechanisms such as cell proliferation,
differentiation, migration, cell cycle arrest or apoptosis (Mathias et al., 1998; Spiegel and
Milstien, 2003; Ogretmen and Hannun, 2004; Sabourdy et al., 2008). Animal models with
53
specific mutations in many of the proteins that regulate GSLs metabolism help to elucidate all
the biological functions in which GSLs might be involved (Sabourdy et al., 2008).
Deletion of the GlcCer gene in mice, a key molecule in GSL metabolism, results in
embryonic lethality during gastrulation (Yamashita et al., 1999). Mice where GlcCer synthase
was conditionally knocked out in the nervous system developed severe ataxia and died within
24 days (Jennemann et al., 2005). Thus, GlcCer was determined to have a key role in neural
differentiation. It has also been reported that GlcCer plays a key role in the maintenance of the
skin barrier (Jennemann et al., 2007; Sabourdy et al., 2008). Other functions of GlcCer can be
revealed through the phenotype of humans or mice with inherited defects of GlcCer degrading
enzymes. In humans, Gaucher disease, i.e., the most frequent lysosomal lipid storage disorder
often manifests as a visceral, non-neuronopathic disorder. There are also more severe,
neuronopathic forms of Gaucher disease (Sabourdy et al., 2008). In conclusion, primary
accumulation of GlcCer in the acidic cell compartments induces neurodegeneration, possibly
through alterations in calcium homeostasis (Pelled et al., 2005; Sabourdy et al., 2008), and
altered skin permeability. Male infertility was also observed when degradation of GlcCer in a
non- lysosomal compartment is defective (Yildiz et al., 2006; Walden et al., 2007).
LacCer is the metabolic intermediate for all GlcCer containing GSLs. But no genetic
defect in LacCer synthase in mammals has been reported. Undegraded LacCer accumulates
when both acid ß-galactosidase and ß-galactosylceramidase are simultaneously deficient, but
this condition in mice results in a milder phenotype (galactosylceramidase deficient mouse)
(Tohyama et al., 2000).
Genetic defects in the biosynthesis of some GSLs can occur in humans without
detrimental consequences. This is the case in individuals of the minor blood group p, where the
synthesis of Gb3 is defective (Kojima et al., 2000; Sabourdy et al., 2008). The absence of any
overt phenotype in Gb3 synthase-null mice (Okuda et al., 2006) supports the idea that GSLs
54
from the globo-series are not essential for development. In contrast, defective breakdown of Gb3
as well as other GSLs with a terminal a-Gal, as seen in the human lysosomal disorder Fabry
disease, results in a pathological condition that leads to dysfunction of cells from blood vessels,
i.e., endothelial, perithelial (Sabourdy et al., 2008). It has recently been proposed that the
accumulation of Gb3, mainly in lysosomes but secondarily in caveolar regions of the plasma
membrane (Schwarz et al., 1993), could affect nitric oxide generation and endothelial signaling.
Other Gb3 functions will be further discussed later in this chapter.
Gangliosides are ligands for myelin-associated glycoprotein (MAG) which prevents
neuronal cell proliferation (Schwardt et al., 2009). That is why neurological impairment is
observed when ganglioside metabolism is impaired (Sabourdy et al., 2008). Inherited defects in
lysosomal breakdown of gangliosides, such as GM1- and GM2-gangliosidoses, are
characterized by lysosomal accumulation of undegraded gangliosides (Sabourdy et al., 2008).
These metabolic disorders are mainly caused by the deficient activity of the hydrolases that
cleave the oligosaccharide chain of gangliosides, such as ß-hexosaminidases or GM1-ß-
galactosidase; but they can also be caused by defects in ‘activators’ of such enzymes, i.e., GM2-
activator and saposins (Sabourdy et al., 2008). Defective GM3 synthesis has been described in
humans, associated with an epileptic syndrome starting early in infancy (Yamashita et al., 2003;
Sabourdy et al., 2008). GM3 also affects EGF function (Wang et al., 2000). It has also been
reported tha t gangliosides regulate other biological functions (Sabourdy et al., 2008), eg, the
lack of complex gangliosides results in male infertility (Liu et al., 1999; Kawai et al., 2001).
1.4.7. Globotriaosylceramide
1.4.7.1. Structure of Globotriaosylceramide. Globotriaosylceramide (Gb3) is a member of the
globo-series neutral GSLs. It is synthesized by a1,4-galactosyltransferase (a1,4Gal-T) from
LacCer (Taga et al., 1995; Kojima et al., 2000). The ceramide portion of Gb3 comprises a
sphingosine base and a fatty acid of various chain lengths. The characteristic sequence of
55
carbohydrate head group is Gal (a1-4) Gal (ß1-4) Glc (ß1-1)-Cer (Scheme 1.7). Gb3 shares
amphipathic characteristics of all GSLs and their fatty acid chain length, saturation and
hydroxylation, may vary yielding various Gb3 isoforms. Gb3 may also partition into lipid rafts
and interact with raft-associated proteins.
1.4.7.2. Biological Functions of Globotriaosylceramide.
Gb3 is widely expressed in a variety of tissues, but is a major GSL in human renal
cortex, heart, spleen, and placenta (Boyd and Lingwood, 1989; Kojima et al., 2000). It has also
been described in a number of epithelial and endothelial cell lines. Gb3 or CD77, is expressed as
a differentiation antigen on a subset of tonsillar B lymphocytes in the germinal centre, where
expression is very specific, and only occurs at a restricted stage (Mangeney et al., 1991; Wiels et
al., 1991). Human Burkitt lymphoma cells, derived from B cells, also express Gb3 (Wiels et al.,
1984). Gb3 is the PK antigen on human erythrocytes, which together with Gb4 or P, define the
antigens of the P/P1/Pk blood group (Hellberg et al., 2002; Hellberg et al., 2004). Gb3 also
serves as a receptor for the Escherichia coli elicited verotoxin (Lingwood et al., 1987).
56
SCHEME 1.7. Structure of Globotriaosylceramide (Gb3).
Globotriaosylceramide as a Cell Surface Antigen. Since a monoclonal antibody (mAb)
reactive with Burkitt’s lymphoma-associated antigen was reported by Wiels et al (Wiels et al.,
1981) and the recognized antigen was elucidated to be Gb3 (Nudelman et al., 1983), the
expression and biological significance of Gb3 have been vigorously studied (Balana et al., 1985;
Murray et al., 1985). Since Gb3 was clustered as CD77, it’s been called Gb3/CD77 (Knapp et al.,
1989). It was reported to be expressed in highly amounts on Burkitt’s lymphoma cells.
However, it is considered to be a differentiation antigen expressed on B cells and can also be
57
found in some malignant tumours of B cell lineage. Among normal leukocytes, it is only
expressed on a subset of tonsillar B cells in the germinal centers (Murray et al., 1985). The
observed rapid death of CD77+ germinal center B cells in vitro suggests that endogenous ligand
molecules interact with Gb3/CD77 to bring about the physiologic selection of immature B cells
(Mangeney et al., 1991) (Mangeney et al., 1995). It has also been reported that Gb3/CD77 with
the ganglioside GM3 may function as alternative cofactors for the entry of human
immunodeficiency virus type 1 in CD4-induced interactions between gp120 and GSL
microdomains (Puri et al., 1998b; Hammache et al., 1999). Cell surface GSLs also participate in
a critical V3 gp120 loop-mediated post-CD4-binding event, common for the entry of diverse
HIV strains (Nehete et al., 2002). Therefore, the regulation of Gb3/CD77 expression could be a
key target for the therapeutic approaches of viral infections such as HIV (Lund et al., 2006).
The human P blood group system consists of three antigens: P, P1 and Pk. The molecular
structures of P, P1 and Pk antigens have been found on Gb4 and Gb3 (Naiki and Marcus, 1974).
The combination of these three antigens defined five phenotypes of P blood group: P1, P2, P and
Pk1, and Pk
2. Lund et al have shown that Fabry disease, when Pk is accumulated due to reduced
activity of a-galactosidase A, is protective against R5 HIV-1 (Lund et al., 2005). In addition, a
soluble analogue of Pk inhibits HIV infection in vitro (Lund et al., 2006). It has also been shown
that pharmacological modulation of Pk expression in vitro in HIV infectable Pk-expessing non-T
cells implicate an important role for Pk in HIV infection (Lund et al., 2009). A recent report also
showed that P1k PBMCs were highly resistant to R5 and X4 HIV-1 infection, implicating Pk as a
new endogenous factor that may provide protection against HIV-1 infection (Lund et al., 2009).
Globotriaosylceramide as a Tumour Associated Antigen. The aberrant glycolipid
biosynthesis in cancer cells has been extensively reviewed (Hakomori, 1996; Hakomori, 2008;
Saddoughi et al., 2008), and many examples where Gb3 has been defined as a tumour selective
glycolipid have been reported (Nudelman et al., 1983; Mannori et al., 1990; Wenk et al., 1994;
58
Falguieres et al., 2008). As already mentioned, increased expression of Gb3 has been described
in Burkitt’s lymphoma cells. Increased expression of globo-series GSLs such as Gb3, Gb4 and
Gb5 are found to be associated with primary testicular germ cell tumours, especially seminomas
and embryonal carcinomas (Olie et al., 1996). Elevation of Gb3 content is associated with
human embryonal carcinomas (Wenk et al., 1994) and testicular tumours (Ohyama et al., 1990),
with both benign and malignant serous, mucous and endometroid tumours of the ovaries, and
significantly correlated with drug-resistant ovarian tumours and MDR ovarian cell lines (Arab et
al., 1997). The expression of Gb3 was strongly increased in colorectal adenocarcinomas and
their metastases compared with normal tissue, but not in benign adenomas (Falguieres et al.,
2008). Although normal human colonic epithelial cells lack the glycosphingolipid
globotriaosylceramide (Gb3), this molecule is highly expressed in metastatic colon cancer.
Transfection of Gb3 synthase, resulting in Gb3 expression in noncancerous polarized epithelial
cells lacking endogenous Gb3, induced cell invasiveness (Kovbasnjuk et al., 2005).
Furthermore, Gb3 knockdown by small inhibitory RNA in colon cancer epithelial cells inhibited
cell invasiveness (Kovbasnjuk et al., 2005).
Globotriaosylceramide Role in Signal Transduction. Gb3 can function as a signal
transduction molecule for the induction of apoptosis. As mentioned before, anti-Gb3 antibody,
immobilized on tissue culture dishes can induce apoptosis of Burkitt’s lymphoma cell lines,
which is preceded by cAMP-dependent protein kinase activation, increased intracellular cAMP
levels and increased intracellular Ca+2 concentration (Taga et al., 1997). Recently, it has been
shown that anti-Gb3 antibody induced apoptosis via a caspase-independent pathway that
involved partial depolarization of mitochondria (Tetaud et al., 2003). It has also been shown that
in human renal tubular cell line ACHN cells, Gb3 was only recovered in detergent- insoluble
microdomains (DIM) and was associated with Src family kinase Yes. Yes was assumed to be
59
activated and show increased Triton X-100 solubility in the early phase of retrograde
endocytosis of Stx-Gb3 complex (Katagiri et al., 1999).
The Gb3 dependent signal transduction of CD19 and the a2 interferon receptor
(IFNAR1) has also been demonstrated. Both the extracellular domain of the B-cell
differentiation antigen CD19 (Maloney and Lingwood, 1994) and the IFNAR1 chain of type I
interferon receptor (Lingwood and Yiu, 1992) show N-terminal amino acid similarity to
sequences within the VT1 B subunit involved in Gb3 binding, suggesting the possible lateral
association between these molecules and Gb3 on the cell surface. Since each molecule is a
member of a multi-molecular signal transduction complex, Gb3 may act as an accessory
molecule for the fully functional complete receptor complex. Cell surface expression of Gb3 has
been found to be associated with CD19-mediated B-cell homotypic adhesion (Maloney et al.,
1999), with CD19 mediated induction of B cell apoptosis (Khine et al., 1998), with a2
interferon induced growth inhibition (Ghislain et al., 1994) and, with a2 interferon antiviral
activity (Khine and Lingwood, 2000). CD19 targeting to the ER/nuclear envelope following cell
surface ligation is Gb3 dependent and necessary for induction of B cell apoptosis (Khine et al.,
1998). Thus, Gb3 mediates the intracellular trafficking of CD19 in the same way as for VT. VT
intracellular retrograde trafficking is preferentially mediated by shorter fatty acid Gb3 isoforms
(Arab et al., 1997) whereas, the longer fatty acid Gb3 isoforms are preferentially used to mediate
interferon/IFNAR1 antiviral signalling and this occurs at the cell surface (Khine and Lingwood,
2000).
Globotriaosylceramide as a Verotoxin Receptor. Gb3 has been recognized as the
receptor for verotoxins (VT) (Jacewicz et al., 1986; Lingwood et al., 1987), the Shiga- like
toxins from enterohemorrhagic Escherichia coli known to cause hemorrhagic enterocolitis and
haemolytic uremic syndrome (HUS) (O'Brien et al., 1983). VT specifically binds to Gb3, and the
terminal Gal (a1-4) Gal residue of the carbohydrate head group is critical for receptor
60
recognition. Removal of the terminal galactose or substitution with N-acetyl galactosamine
abolishes the toxin binding activity (Lingwood et al., 1987; Waddell et al., 1988). The ceramide
portion of Gb3 is also essential for VT specific recognition.
Although VT may bind to other Gal (a1-4) Gal containing GSLs such as galabiosyl
ceramide (Gb2) (Waddell et al., 1988), Gb3 is its functional receptor. Reconstitution of VT-
resistant cells with Gb3 alone is sufficient to sensitize the cells to VT induced cytotoxicity
(Waddell et al., 1990). The fatty acid chain length of Gb3 (Pellizzari et al., 1992; Boyd et al.,
1994; Kiarash et al., 1994a) and the chain length of the phospholipid bilayer that may contain
Gb3 (Arab and Lingwood, 1996) can have an important and dramatic effect on the ability of the
toxin to bind the carbohydrate moiety of Gb3. The effect can be different according to the
manner of glycolipid immobilization for ligand binding. Short chain-containing Gb3 species are
not efficient in VT binding. C6:0-containing Gb3 does not bind to VT (Pellizzari et al., 1992),
and C12 and C14-containing Gb3 have only minimal binding (Kiarash et al., 1994b). C16 to
C24-containing Gb3 homologues recognize VT effectively and Gb3 with unsaturated fatty acids
have higher binding capacity (Kiarash et al., 1994a). VT binding to Gb3 is increased as a
function of decreasing phosphatidylcholine acyl chain length of auxiliary
phospholipids/cholesterol bilayer in a microtiter well model system (Arab and Lingwood, 1996).
The lipid component and the lipid environment of Gb3 likely affect the surface exposure and
relative conformation of the carbohydrate moiety.
VT binding to cell surface Gb3 within lipid microdomains has been shown to activate
cystosolic raft-associated src-family kinases, indicating Gb3 can mediate transmembrane signals
(Katagiri et al., 1999; Falguieres et al., 2001). VT interactions with Gb3 within rafts results in
toxin internalization either by a clathrin-dependent or caveolae-dependent pathway, whereby
VT undergoes retrograde transport via the reverse of the secretory system to the
Golgi/ER/nuclear envelope (Khine and Lingwood, 1994; Sandvig et al., 1994; Arab and
61
Lingwood, 1998; Khine et al., 2004). Interestingly, the VT/Gb3 complex in the process of
retrograde transport to the ER is also present in lipid rafts (Smith et al., 2006). Cell cycle
dependent surface expression of Gb3 also determines the susceptibility of host cells to VT.
Although the total cellular level of Gb3 is the same, surface exposure of Gb3 and sensitivity to
VT is higher in log phase cell culture, and at the G1/S phases of the cell cycle (Pudymaitis and
Lingwood, 1992).
The intracellular targeting of VT and the sensitivity of the host cells to the toxin are also
influenced by the fatty acid chain length of the different isoforms of Gb3. Cells containing
higher levels of the shorter fatty acid Gb3 isoforms are more sensitive to VT and the toxin is
targeted to the ER, nuclear membrane and nucleus. In contrast, those with longer fatty acid Gb3
isoforms are less sensitive to VT and toxin targeting is only to the Golgi (Arab et al., 1998;
Lingwood, 1998).
Studies in a rabbit model have identified that localization of VT binding to Gb3
determines the VT-induced pathological changes. Clinical symptoms and microangiopathic
lesions in central nervous system, gastrointestinal tract and lungs have been described associated
with Gb3 expression in those organs. No lesions were reported in kidney (Zoja et al., 1992). In
human renal tissue, Gb3 is present in the distal convoluted tubules, adjacent to glomeruli, and in
collecting ducts in adults and infants specimens, but in infants is detected only in the glomeruli
(Lingwood, 1994). Gb3 is also present in some adult glomeruli (Chark et al., 2004; Khan et al.,
2009). The elevated level of local cytokines in the kidney during VTEC infection, in particular
TNF-a, increase the expression of Gb3 on endothelial cells by induction of galactosyltransferase
(van der Kar et al., 1995), could favour the development of VT-associated haemolytic uremic
syndrome (HUS) in infants, although Gb3 content of infant kidney is reduced. It was recently
reported that glomerular Gb3 is in detergent resistant membranes but tubular Gb3 is detergent
sensitive (Khan et al., 2009).
62
1.4.7.2.1. Antineoplastic Potential of Verotoxin
The antineoplastic effect of VT was actually described before the discovery of VT
(Farkas-Himsley and Cheung, 1976). In 1976, Farkas-Himsley and Cheung detected an anti-
neoplastic activity in extracts of one strain of E. coli (HSC10) and described it as a bacteriocin.
Later, the active component responsible for this activity was identified as VT1 (Farkas-Himsley
et al., 1995). This finding has introduced a new area of VT research as a new therapeutic for
cancer. In vitro and in vivo models of tumorigenicity and metastases to examine the biological
efficacy of this antineoplastic extract demonstrated that this activity was due to VT1 and that
purified VT1 was effective in the various models of antineoplasia previously studied (Farkas-
Himsley et al., 1995). Ovarian carcinoma cell lines in vitro were highly susceptible to anticancer
proteins and to VT1. Significantly, drug-resistant variants were more sensitive than their
parental counterpart (Farkas-Himsley et al., 1995). These results were explained afterwards
when it was reported that Gb3 expression was elevated in ovarian cancers relative to the normal
ovary and that, overall, the expression of the toxin receptor was inversely related to the degree
of differentiation of the tumour (Arab et al., 1997). In addition, blood vessels within the tumour
mass were positive for toxin binding, suggesting that Gb3 was up-regulated during tumour
angiogenesis. Therefore, VT1 treatment may offer dual targeting of both the tumour and the
blood supply. Consequently, VT could be used as an antineoplastic agent when standard
therapies for MDR ovarian tumours have failed.
Human astrocytoma cell lines are also Gb3-positive and VT-sensitive. Intra-tumour
injection of VT1 induced rapid apoptosis of human astrocytoma xenograft subcutaneous tumour
and blood vessels in nude mice and complete regression of tumour was achieved within 10 to 20
days (Arab et al., 1999). Recent publications also showed that a single intratumoral injection of
VT1 significantly improved survival in nude mice harbouring malignant meningioma
intracranial tumours (Salhia et al., 2002; Heath-Engel and Lingwood, 2003).
63
It has also been demonstrated that VT1 treatment of murine bone marrow ex vivo
effectively cures severe combined immunodeficient mice of human B-cell lymphoma xenograft
while sparing normal haematopoietic precursor cells (LaCasse et al., 1996). Therefore, VT1
may be potentially used for the purging of human bone marrow before autologous bone marrow
transplant in the case of Gb3-positive B-cell lymphomas (LaCasse et al., 1996; LaCasse et al.,
1999).
It was also reported that intratumoral VT1 injection of mice bearing renal cell tumours
produced a rapid reduction in the size of subcutaneous tumours with complete regression within
5 to 7 days (Ishitoya et al., 2004).
1.4.8. Lysosomal Storage Diseases
Human diseases caused by alterations in the metabolism of sphingolipids or
glycosphingolipids are mainly associated with disorders of the degradation of these compounds
(Kolter and Sandhoff, 2006). Sphingolipidoses, together with mucopolysaccharidoses,
mucolipidoses, glycoprotein and glycogen storage diseases, belong to the lysosomal storage
diseases (LSDs) (Futerman and Hannun, 2004).The LSDs are rare disorders with a frequency of
1 in 7,000-8,000 live births (Meikle et al., 1999). About 40 genetically different forms were
identified (Kolter and Sandhoff, 2006). The causes of LSDs vary from defects in enzymes,
cofactors (such as sphingolipid activator proteins), to defects in the targeting or transport
systems involved in the degradation process (Schuette et al., 2001; Kolter and Sandhoff, 2006).
The sphingolipidoses are a group of inherited diseases, associated with defects in genes
encoding proteins involved in the lysosomal degradation of sphingolipids, causing subsequent
accumulation of non-degradable storage material in one or more organs (Sillence and Platt,
2003; Raas-Rothschild et al., 2004) (Scheme 1.8). Most sphingolipidosis are associated with
high mortality. Some examples of glycosphingolipidoses include Fabry disease, Gaucher
disease, Sandhoff disease, Tay Sachs disease, and GM1 gangliosidosis. They are inherited as
64
either autosomal recessive or X-linked traits. (Desnick, 2004). Defects for almost every step in
the degradation pathway of GSLs have been identified. The only exception is the degradation of
lactosylceramide, which can be degraded by two different enzyme/activator systems (Zschoche
et al., 1994). Therefore, no single enzyme defect is known that leads to accumulation of
lactosylceramide. Most enzymes and cofactors deficient in the sphingolipidosis have been
characterized, their genes have been cloned, and animal models of most of the sphingolipidoses
have been created by targeted disruption of the respective genes in mice (Coetzee et al., 1998;
Kolter and Sandhoff, 2006).
1.4.8.1. Pathogenesis
Sphingolipidoses shows a high degree of phenotypic variability (Gieselmann, 2005).
Different sphingolipidosis have different onset, development, and symptoms but can also
drastically differ within the same disease. The cell- type specific pattern of glycosphingolipid
expression is the primary factor that determines the pathogenesis of these diseases (Kolter and
Sandhoff, 2006). Lipid storage in lipidosis patients occurs especially in those cells and organs in
which the lipid substrates of the corresponding deficient enzyme are mainly synthesized or
taken up by phagocytosis (Kolter and Sandhoff, 2006).
65
Scheme 1.8. Glycosphingolipid (GSL) Catabolism and Disease. Defects in lysosomal
catabolism lead to the accumulation of specific GSLs. These are the disease states that arise
because of mutations in the genes encoding the enzymes involved in GSL catabolism.
Abbreviations: Cer, ceramide; Gal, galactose; GalCer, galactosylceramide; GalNAc, N-
acetylgalactosamine; Glc, glucose; GlcCer, glucosylceramide; NeuAc, N-acetylneuraminic acid
(sialic acid) (Sillence and Platt, 2003).
Consequently, in Gaucher disease, the product accumulation is especially located in
macrophages, which have large amounts of sphingolipids to degrade after phagocytosis of cells
(Hein et al., 2007; Naito, 2008). The second important factor to be taken into consideration in
this type of diseases is the residual activity of the defective enzyme in many, although not in all
LSDs (Kolter and Sandhoff, 2006). The onset and the severity of the storage disease is in part
determined by the residual activity of the gene product in the lysosomes (Conzelmann and
66
Sandhoff, 1983; Leinekugel et al., 1992; Kolter and Sandhoff, 2006). If the lysosomal enzyme is
completely defective, there is an early onset of the disease and also a severe course of the
disease has been described; if on the contrary, only a few percent of residual activity is present,
there is a delay in the onset of the disease, with a mild course, that lead to the often
misdiagnosed adult forms of the diseases (Rapola, 1994; Kolter and Sandhoff, 2006). Genetic
and epigenetic factors also contribute to the expression of a disease in an individual patient. The
growing amount of accumulating material might initially lead to a mechanical damage of the
cell, and consequently, to apoptosis (Taniike et al., 1999; Finn et al., 2000). Downstream effects
of lysosomal storage also contribute to the pathogenesis of these diseases (Kolter and Sandhoff,
2006). Inflammatory responses like macrophage activation or cytokine release have also been
reported for patients with Gaucher disease (Orvisky et al., 2000), in the mouse model of
Sandhoff disease (Wada et al., 2000), in animal models of other gangliosidoses (Jeyakumar et
al., 2003) and in other LSDs (Futerman et al., 2004). Other secondary effects for these diseases
have been observed on lipid trafficking (Chen et al., 1999), phospholipid metabolism, calcium
ion homeostasis (Korkotian et al., 1999; Ginzburg et al., 2004; Jeyakumar et al., 2005), and
mitochondrial function (Lucke et al., 2004; Kolter and Sandhoff, 2006).
1.4.8.2. Fabry and Gaucher Diseases
Fabry disease is characterized by an inborn deficiency of lysosomal a- galactosidase A,
that catalyses the lysosomal hydrolysis of globotriaosylceramide, Gb3 (Desnick and
Wasserstein, 2001). Fabry disease is an X-chromosomal- linked inherited disorder with an
estimated frequency of 1:117,000 (Meikle et al., 1999) to 1:40,000 birth (Desnick and
Wasserstein, 2001) Hemizygous males have accumulation of Gb3 in the lysosomes of
endothelial, perithelial, and smooth-muscle cells of blood vessels (Kolter and Sandhoff, 2006).
Many cell types in the heart, kidneys, eyes, cornea, and the autonomous nervous system are also
affected. The first clinical symptoms usually occur during childhood or adolescence, and
67
include severe pain in the extremities, vascular cutaneous lesions, hypohidrosis, and corneal and
lenticular opacities (Kolter and Sandhoff, 2006). The disease develops to renal, cardiac, and/or
cerebral complications, which are the most common causes of death around 40 or 50 years of
age (Kolter and Sandhoff, 2006). A variant of the disease characterized by a milder progression
and a primary impairment of the heart muscle has been described when there is an enhanced
residual activity of more than 5% of normal of the defective enzyme (Kolter and Sandhoff,
2006). Heterozygous females have higher residual a-galactosidase A activity, with a mild form
of this disease, and are usually not as much affected as hemizygous males (Kolter and Sandhoff,
2006). The diagnosis of female patients is performed by determination of a- galactosidase A to
ß-glucuronidase ratio (Pedemonte et al., 2005). The symptoms result from accumulating
glycolipids in the affected tissues, the blockage of blood vessels, or both simultaneously. In the
kidney, the lesions are due to glycosphingolipid accumulation in several cell types, but renal
blood vessels are progressively and often the more extensively involved (Kolter and Sandhoff,
2006). On the other hand, the vascular involvement in the nervous system is the predominant
cause of this disease. About 180 different mutations have been associated with Fabry disease
including partial gene rearrangements, splice junction defects, and point mutations (Desnick and
Wasserstein, 2001). An animal model of this disease has also been created by Ohshima et al.
(Ohshima et al., 1997). Although it shows no clinical symptoms, it allows evaluation of
therapeutic strategies, as the use of adeno-associated virus mediated therapy, which leads to
long-term correction of storage (Platt et al., 2003). An enzyme replacement therapy using
recombinant a-galactosidase A derived from human skin fibroblasts (Sillence and Platt, 2003)
or CHO-cells (Eng et al., 2001) has been established and is now the treatment of choice (Kolter
and Sandhoff, 2006). A chemical chaperone approach with galactose was successful in a male
patient of the cardiac variant of the disease (Frustaci et al., 2001), and extensions of this
approach using inhibitors have been reported (Fan et al., 1999; Asano et al., 2000; Yamashita et
68
al., 2005). Deoxygalactonojirimycin treatment of knock- in mice that express the (R301Q)
variant of a-galactosidase A produces an increase of enzyme activity in the heart and the
consequent reduction of Gb3 storage (Ishii et al., 2004).
Gaucher disease is the most common form of the sphingolipidoses (Zhao and
Grabowski, 2002; Jmoudiak and Futerman, 2005). It is caused by the deficiency of
glucosylceramide-ß-glucosidase, also called glucocerebrosidase that results in accumulation of
glucosylceramide. Three different types of Gaucher disease have been characterized: the mild
form, Gaucher disease type I, has a nonneuropathic course and is the most frequent form of this
disease. It has a frequency of 1: 50000–200000 births. The life expectancies of these patients
range between 6 and 80 years. Gaucher disease type II, the acute form, is a very rare disease
characterized by the involvement of the nervous system with early onset and a life expectancy
of less than two years. The subacute or juvenile form, Gaucher disease type III, is an
intermediate variant of the other two types, mainly found in the Northern Swedish population.
In this case, the neurological symptoms have a later onset and a slower development than in
form II; the survival age of the patients is between a few years and four decades (Jmoudiak and
Futerman, 2005). In all variants, patients may show hepatosplenomegaly, anemia,
thrombocytopenia, and bone damage. The severity of these symptoms differs widely, but is
inversely correlated with the residual enzyme activity determined in skin fibroblasts of Gaucher
patients (Meivar-Levy et al., 1994). Even if the enzyme activity is reduced in all cell types, the
phenotype of type 1 of the disease is predominantly manifested in macrophages of the
reticuloendothelial system, since these cells have to degrade large amounts of glycolipids
derived from the phagocytosis of erythrocytes. Types II and III of the disease that affect the
CNS are characterized by a progressive loss of neuronal cells, due to the accumulation of
glucosylsphingosine, that produces neuronal toxicity (Denneberg et al., 1982; Orvisky et al.,
2000) and an inflammatory response (Mizukami et al., 2002). Around 200 mutations of the
69
glucosylceramide-ß-glucosidase locus have been identified in patients with Gaucher disease
(Christomanou et al., 1986; Schnabel et al., 1991; Rafi et al., 1993) but N370S accounts for
most . An animal model most resembling the type II form of the disease has been created by
targeted disruption of the glucosylceramide-ß-glucosidase gene in mice (Tybulewicz et al.,
1992). The animals store glucosylceramide in cells of the reticuloendothelial system and die
within 24 h after birth. Recently, additional viable models of Gaucher disease have been
developed by introduction of the point mutations N370S, V394L, D409H, or D409V into the
mouse glucosylceramide-ß-glucosidase (Elliott et al., 2003). Brady et al. have developed
enzyme therapy for the attenuated form of Gaucher disease (type I) (Brady, 2003). Enzyme
replacement is less promising for types 2 and 3 of the disease due to CNS involvement
(Campbell et al., 2004). Bone marrow transplants have been peformed as well with variable
results (Hobbs et al., 1987; Erikson et al., 1990; Ringden et al., 1995), since a serious problem is
the reversal of bone involvement, which has been overcome by peripheral blood stem cell
transplantation in the animal model (Yabe et al., 2005).
1.4.8.4. Therapeutic Approaches
The therapeutic approaches for sphingolipidosis aim to restore the defective degradation
capacity within the lysosomes (Kolter and Sandhoff, 2006). The current treatments that are in
use or under evaluation (Finn et al., 2000; Elleder, 2003) are enzyme replacement therapy
(ERT), cell-mediated therapy (CMT) including heterologous bone marrow transplantation
(BMT) and cell-mediated “cross correction”, gene therapy, enzyme-enhancement therapy with
chemical chaperones, and substrate reduction or substrate deprivation therapy (Fan, 2003;
Kolter and Wendeler, 2003; Desnick, 2004; Kolter and Sandhoff, 2006).
Enzyme replacement therapy (ERT). The principle of this therapeutic approach is to
reduce the accumulation of the substrate by administering an external supply of the defective
lysosomal enzyme (Desnick, 2004). This approach has been evaluated previously for many
70
LSDs both in cultured cells and in animal models, where the enzymes were taken up by cells by
receptor-mediated endocytosis (Kolter and Sandhoff, 2006). This therapeutic approach is
currently in use for patients with Gaucher disease type I and Fabry disease and it proves to be
quite successful (Desnick and Schuchman, 2002). One of the limitations of this method is that it
cannot be used for treatment of diseases of the CNS since the blood–brain barrier prevents the
access of the therapeutic enzymes to the neural cells (Kolter and Sandhoff, 2006).
Cell-mediated therapy (CMT). This therapeutic approach is based on the replacement or
compensation of the cells defective in the corresponding enzyme with normal cells, so as the
enzymes released by these normal cells can be uptaken by the deficient cells (“cross
correction”) (Kolter and Sandhoff, 2006). Bone marrow transplantation (BMT) or neural
progenitor cells are examples of this therapy (Kolter and Sandhoff, 2006). Promising results
from this therapeutic approach have been described in animal models of LSDs (Hoogerbrugge et
al., 1988; Birkenmeier et al., 1991), including an improvement of the neurological symptoms
and the regression of neuronal injury (Kolter and Sandhoff, 2006).
Gene therapy. In this approach, a functional copy of the mutated gene is introduced into
cells, making them produce the right enzyme (Kolter and Sandhoff, 2006). The deficient
enzyme needs to be stably over-expressed by a few cells, secreted in high levels, in order to
correct the phenotype of the adjacent cells (Kolter and Sandhoff, 2006). Animal models are
currently in use for the evaluation of retro- and lentiviral vectors (Ellinwood et al., 2004; Biffi
and Naldini, 2005). Limitations such as the efficiency in the delivery of the gene at the CNS, as
well as the limited time of the gene expression are being currently evaluated before this
approach can be applied in humans (Kolter and Sandhoff, 2006).
Enzyme-enhancement therapy. This method is based on the use of chemical chaperones
that can bind to partially defective variants of the enzyme in question preventing its degradation
by the quality control ystem of the ER (Fan, 2003; Jarosch et al., 2003; Desnick, 2004; Kolter
71
and Sandhoff, 2006). This approach has been used for Fabry disease (Fan et al., 1999), for
Gaucher disease (Sawkar et al., 2002), for GM1 gangliosidosis (Matsuda et al., 2003), and for
GM2 gangliosidosis (Tropak et al., 2004). Several substrates analogs and GSL enzyme
inhibitors are evaluated for this approach. Iminosugars of the nojirimycin type are perfect
candidates for this therapy and the substrate reduction approach (Butters et al., 2005; Kolter and
Sandhoff, 2006).
Substrate reduction therapy (SRT). This approach is based on the use of inhibitors of
GSL biosynthesis to avoid the continuous accumulation of the GSL substrate for degradation
into the lysosomes (Kolter and Sandhoff, 2006). Glucosyl ceramide synthase inhibitor N-
butyldeoxynojirimycin (miglustat, Zavesca®), has been initially investigated in the animal
model of Tay–Sachs disease (Platt et al., 1997). Currenty, ongoing clinical trials evaluate this
approach for the treatment of human patients with Gaucher disease, type I (Cox et al., 2000;
Elstein et al., 2004; Kolter and Sandhoff, 2006). Inhibitors of glucosyl ceramide synthase, N-
butylnojirimycins, are candidates for substrate reduction therapy of diseases caused by
accumulation of substances derived from glucosylceramide (Kolter and Sandhoff, 2006). This
approach is expected to be promising in the case of patients with some residual enzymatic
activity of the enzyme affected or to be used in combination with other methods, which restore
this activity (Kolter and Sandhoff, 2006).
1.5. Multidrug Resistance and Glycosphingolipids
Normal physiological functions of MDR1 have also been reported, apart from the
multidrug transporter function, (Mizutani et al., 2008). The following are examples of normal
functions of MDR1. MDR1 could be responsible for translocation of platelet-activating factor
(PAF) across the plasma membrane, as PAF inhibited MDR1 drug transport in cancer cells
(Raggers et al., 2001). High expression of MDR1 prevents stem-cell differentiation, leading to
the proliferation and amplification of the stem cell repertoire (Bunting et al., 2000), and MDR1
72
also plays a fundamental role in regulating programmed cell death, apoptosis (Smyth et al.,
1998; Mantovani et al., 2006). MDR1 might also be involved in the transport of cytokines, in
particular IL-1, IL-2, IL-4, and IFN-?, out of activated normal lymphocytes into the media
(Drach et al., 1996; Pawlik et al., 2005). MDR1 is also associated with a volume-activated
chloride channel (Marin et al., 2005), thus MDR1 is bifunctional with both transport and
channel regulators.
MDR1 has also a close relationship with lipids, and in particular, glycosphingolipids.
MDR1 can translocate both C6-NBD-PC and C(6)-NBD-PE across the apical membrane of
multidrug resistant cells (Abulrob and Gumbleton, 1999; Rothnie et al., 2001). MDR1 was also
shown to regulate the translocation of sphingomyelin (Come et al., 1999), and glucosylceramide
(Lala et al., 2000), and short chain C(6)-NBD-GlcCer was found in the apical medium of MDR1
cells exclusively, and not in the basolateral membrane (van IJzendoorn et al., 1997; van Meer et
al., 1999). Cells transfected with MDR1 were reported to esterify more cholesterol than their
sensitive parental counterparts (Gayet et al., 2005).
1.5.1. MDR1 and Ceramide Metabolism
It has been known for a long time that phospholipids (May et al., 1988), triglycerides
(Ramu et al., 1984) and cholesterol composition (Mountford and Wright, 1988; Mazzoni and
Trave, 1993) of MDR1 cells can differ from that of drug-sensitive cells. In addition,
observations in the early 1980s, reported differences in gangliosides composition of MDR1 cells
when compared to sensitive cells (Gascoyne and Van Heyningen, 1975). However, it was not
until the end of the 1990s, that MDR-related differences in simple sphingolipid composition
were further investigated.
Ceramide is one of the lipids constituting membrane microdomains, but is also present in
internal membranes, where it is synthesized. Ceramide is known to regulate anti-proliferative
responses, such apoptosis, growth arrest, differentiation and senescence in various human
73
cancer cell lines (Ogretmen and Hannun, 2001). In recent studies, metabolites of ceramide (Cer)
have emerged as important MDR regulators. Cabot et al. showed that the levels of GlcCer,
precursor of all higher glycosphingolipids and a simple glycosylated form of ceramide, was
consistently elevated in several MDR1 overexpressing cells (Lavie et al., 1996). Thus,
accumulation of glucosylceramide (GlcCer), has been shown to be a characteristic of some
MDR cells (Lavie et al., 1996; Lucci et al., 1998; Lala et al., 2000), and MDR1 cells are more
sensitive to depletion of GlcCer by various MDR1 reversing agents than their non-MDR
counterparts (Nicholson et al., 1999). Work on human ovarian carcinoma cells demonstrated
that, in addition to GlcCer, also sphingomyelin and GalCer levels were significantly enhanced in
MDR1 overexpressing cells, when compared to their drug sensitive counterpart (Veldman et al.,
2002). However, LacCer and all higher GSLs were substantially decreased in these cells, due to
altered intracellular localization of the biosynthesis enzyme LacCer synthase (Veldman et al.,
2002). In contrast, in MDR1-transfected HepG2 cells, LacCer is prominently elevated compared
with control cells, along with more moderate increases in GlcCer, ganglioside GM3 and SM
levels. These alterations were unaffected by cyclosporin A, and accompanied by upregulation of
LacCer synthase (Hummel et al., 2005). Nevertheless, MDR1-transfected MDCK cell line
showed dramatic accumulation of globotriaosylceramide (Gb3) and globo series GSLs,
correlated with MDR1 activity (Lala et al., 2000). Furthermore, observation of the requirement
in Gb3 expression for a cell to exhibit lectin- induced stimulation of MDR1 activity for both, PS
externalization and rhodamine 123 efflux, indicates a functional relationship between MDR1
and Gb3, even though this remains to be clarified (Sugawara et al., 2005). LacCer and Gb3
synthesis dependent on MDR1 functional activity was subsequently observed in various MDR1
cell lines, and it was proposed that MDR1 performs a translocase role for GlcCer at the level of
the Golgi membranes (De Rosa et al., 2004). The role of MDR1 as a lipid translocase will be
74
discussed in detail later in this chapter. This might be consistent with a general positive role for
P-gp in glycolipid biosynthesis and intracellular traffic.
On the other hand, the levels of the bioactive sphingolipids metabolites, ceramide and
sphingosine, were quite comparable in resistant cells and their sensitive counterpart (Veldman et
al., 2002). Furthermore, the deviations in GlcCer levels in multidrug resistant cells, are not
restricted to MDR1 overexpressing cells since MRP1-overexpressing cells, also show a 2- to 3-
fold increase in this lipid (Kok et al., 2000). Several metabolic mechanisms can be proposed to
underlie the observed differences in GlcCer levels. Not much data is reported on enzymatic
activities, but an increased glucosylceramide synthase (GCS) activity is probably an important
factor.
It has also been described that administration of a variety of chemotherapeutic drugs
leads to the production of ceramide, but this mechanism differs between agents, and possibly
between cell types (Strum et al., 1994; Suzuki et al., 1997; Maurer et al., 1999). Some agents,
such as daunorubicin, induce ceramide accumulation and subsequent cell death by either
activation of a neutral SMase or, alternatively, by an increased of de novo synthesis through
ceramide synthase (Bose et al., 1995; Jaffrezou et al., 1996). Upon administration of the MDR1
inhibitor etoposide, an increased activity of serine palmitoyltransferase, the first and rate-
limiting step in the synthesis of all sphingolipids, was observed (Perry et al., 2000). Modulators
of MDR1, such as verapamil, cyclosporin A and its analog SDZ PSC833, and tamoxifen, which
are well-known to inhibit the pump activity of P-gp and reverse resistance, have been shown to
retard the levels of GlcCer in various human cancer cells that do not express MDR1 or other
drug transporter proteins, implicating an additional mechanism of action of these agents
(Ogretmen and Hannun, 2001).
Inhibition of the Cer glycosylation pathway has been shown to increase MDR1 cell
sensitivity to cytotoxic drugs (Lavie et al., 1997; Czarny et al., 1999; Lucci et al., 1999).
75
Moreover, overexpression of GlcCer synthase confers doxorubicin (Adriamycin) resistance in
human breast cancer cells (Liu et al., 1999), whereas transfection of GlcCer synthase antisense
reverses doxorubicin resistance in MDR cells (Liu, 2000). P-gp inhibitors, such as PSC833 were
found to facilitate Cer accumulation by stimulating Cer synthase-mediated de novo Cer
synthesis (Liu et al., 1999; Lucci et al., 1999). Together these results suggest that Cer and Cer
metabolites may play an important role in regulating P-gp function. From this perspective,
mifepristone, one of the GlcCer synthase inhibitors used in these reports (Lucci et al., 1999),
had been previously reported in an independent study to inhibit P-gp activity in KG1a cells
(Fardel et al., 1996).
MDR1 transfected cells were observed to be sensitive to the pro-apoptotic action of the
GlcCer synthase inhibitor, PDMP, whereas control cells were not, and this seems to be related
to an effect on ceramide metabolism rather than a direct action on P-gp mediated cytotoxic drug
transport (Shabbits and Mayer, 2002). PDMP has been described to sensitize neuroblastoma
cells to paclitaxel (Taxol) and vincristine by reducing drug efflux presumably through P-gp
inhibition (Sietsma, 2001). Indeed, the pro-apoptotic effect on sensitive cells of GlcCer
synthesis inhibition depends on the cell type considered, varying from moderate in MCF-7 (Liu
et al., 2004), marginal in Hep-G2 (di Bartolomeo and Spinedi, 2001), to null on melanoma cells
(Veldman et al., 2003), and even to a cell-protective effect on leukemic cells (Grazide et al.,
2004), whereas the effect is marked on MDR cells (Liu, 2001). These observations strongly
support a role for endogenous Cer in regulating MDR1 capacity.
1.5.2. MDR1 as a Lipid Flippase
Higgins and Gottesman have initially proposed that MDR1 might function as a drug
flippase, moving hydrophobic molecules from the inner to the outer leaflet of the plasma
membrane (Higgins and Gottesman, 1992a). The fact that it was found that the P-glycoproteins
encoded by the ABCB4 (MDR3) gene in humans and the Abcb4 gene in mice are primarily
76
phosphatidylcholine translocators (Ruetz and Gros, 1994b; van Helvoort et al., 1996), and that
MRP1, the only member of the MRP family known to transport lipid analogues, is the main
transporter for the cellular excretion of the lipid metabolite leukotriene C4 (Wijnholds et al.,
1997), produced an increasing interest in the possibility that other ABC transporters were
involved in lipid transport (Borst et al., 2001). Further studies in intact cells by the group of van
Meer provided evidence that drug-transporting P-gps are also able to translocate short-chain
lipid analogs, phospholipids and glycosphingolipids, from the inner to the outer leaflet of the
plasma membrane (van Helvoort et al., 1996; van Helvoort et al., 1997; van Meer et al., 1999).
The range of lipid analogues translocated is remarkably large: not only C6-NBD-PC, but also
C6-NBD-phosphatidylethanolamine (PE) is transported. The MDR1 P-gp is able to transport C6-
NBD-SM, C6-NBD-GlcCer, C6-GlcCer and C8-GlcCer. Transport is inhibited by P-gp
inhibitors, such as verapamil and PSC833, and by energy depletion of the kidney cells.
Experiments using P-gp reconstituted in proteoliposomes also provided convincing evidence of
this flippase activity (Sharom et al., 2005). Romsicki and Sharom used a fluorescence
quenching technique to show that P-gp could flip a variety of NBD-labeled phospholipids and
sphingomyelin (Romsicki, 2001), and this work was extended by Eckford and Sharom, who
found that fluorescent analogues of the simple glycosphingolipids galactosyl- and
glucosylceramide were also flipped at high rates (Eckford and Sharom, 2005). Flipping of
lactosylceramide was substantially slower, suggesting that addition of a second polar sugar
residue to the headgroup presents a significant barrier to movement across the membrane
(Romsicki, 2001; Eckford and Sharom, 2005). The process of lipid flipping resembles drug
transport as it requires ATP hydrolysis and is inhibited by orthovanadate. Drugs and modulators
are able to compete with membrane lipids for flipping, and their inhibitory potency is highly
correlated with their MDR1 binding affinity (Romsicki, 2001; Eckford and Sharom, 2005),
suggesting that lipid translocation and drug transport share the same path in the protein.
77
The fluorescent lipids used in flippase studies in intact cells and model systems usually
(but not always) have one short acyl chain. It is still unknown whether P-gp can transport
natural membrane lipids, with two long acyl chains, as it is very difficult to test flipping of
normal unlabelled lipids in model systems (Sharom, 2006). However, P-gp was able to
translocate a fluorescent PE derivative with two 16- or 18-carbon acyl chains (Sharom, 2006).
In addition, a large number of ABC transporters appear to translocate natural membrane lipids
and sterols, and this may be a side activity of all the proteins in this family (Borst et al., 2000b;
Kalin et al., 2004; van Meer et al., 2006). Another approach reported was the use of a
microsome system instead of intact cells, showing that lactosylceramide and
globotriaosylceramide synthesis from endogenous or exogenously added liposomal
glucosylceramide was inhibited by the MDR1 inhibitor cyclosporin A, consistent with a direct
role for MDR1/glucosylceramide translocase activity in their synthesis (De Rosa et al., 2004).
Overexpression of MDR1 by retroviral transfection of MDCK cells with human MDR1
cDNA results in the accumulation of globotriaosylceramide (Gb3), the receptor for the
Escherichia coli-derived verotoxin, and increased sensitivity of cells (about 106-fold) to
verotoxin (VT) (Lala et al., 2000). These data show that P-gp plays a role in the synthesis of
glycolipids, and also support that verotoxin might be a potential anticancer agent for the
treatment of MDR1-expressing drug resistant cells. MDR1 inhibitors, e.g. ketoconazole or
cyclosporin A (CsA), prevented the increased Gb3 and VT sensitivity in various cell lines. In
contrast, cellular ganglioside synthesis in the same cells, was unaffected by MDR1 inhibition,
suggesting neutral and acid GSLs are synthesized from distinct precursor GSL pools (De Rosa
et al., 2004). Gb3 synthase was not significantly elevated in this MDR1-MDCK transfected
cells, and was not affected by CsA. Instead, synthesis of fluorescent analogs of LacCer (NBD-
LacCer) and NBD-Gb3 via NBD-GlcCer from exogenous NBD-C6-ceramide was prevented by
CsA (Lala et al., 2000). Immunoelectron microscopy showed that MDR1 drug efflux pump is,
78
in part, Golgi associated, and as a consequence, it is proposed MDR1 mediated flipping of
GlcCer within the Golgi as a primary mechanism by which GlcCer is provided as a substrate for
the various luminal glucosyltransferases involved in neutral GSL biosynthesis (De Rosa et al.,
2004).
1.5.2.1. Alternative Mechanisms for GlcCer transport into the Golgi
The fact that GlcCer is synthesized on the cytosolic surface of the Golgi but converted to
LacCer in the lumen (Lannert et al., 1994; Lannert et al., 1998) forced questions as whether and
how GlcCer crosses the Golgi membrane. This appeared to have been solved according to the
previous reports already mentioned in this chapter, and that can be summarized as follows:
short-chain GlcCer analogues were able to cross the Golgi membrane (Lannert et al., 1994;
Burger et al., 1996; Lannert et al., 1998), the identification of the multidrug transporters ABCB1
and –C1 as floppases for these molecules (van Meer et al., 2006), and a correlation between
MDR1 activity and complex GSLs synthesis in living cells (De Rosa et al., 2004).
However, the recent discoveries of two sphingolipid transfer proteins, CERT and
FAPP2, have brought controversy to the field of sphingolipid metabolism. This controversy is
based on the following, according to these new reports: glucosylceramide synthase (GCS) is
concentrated in the trans-Golgi, not in the cis-Golgi, and its activity was inhibited more than
twofold upon CERT knockdown (Hanada et al., 2003; Halter et al., 2007). Halter et al.
described that as natural GlcCer did not flop efficiently across the Golgi membrane and was not
a substrate for the multidrug transporter MDR1, instead newly synthesized GlcCer reached the
outside of the plasma membrane by a non vesicular transport pathway and was translocated by a
mechanism that involves a proton gradient (Halter et al., 2007). Finally, most GlcCer reached
LacCer synthase in the Golgi lumen via the ER, with a role from the trans-Golgi glycolipid-
binding protein FAPP2 in shuttling GlcCer to the ER (Halter et al., 2007). CERT transfers
ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, a crucial step for the
79
synthesis of sphingomyelin (Hanada et al., 1998; Fukasawa et al., 1999; Hanada, 2003), and
FAPP2 transfers GlcCer to appropriate sites for the synthesis of complex GSLs (D'Angelo et al.,
2007; Halter et al., 2007). These observations indicate that lipid transfer proteins, CERT and
FAPP2, spatially regulate lipid metabolism on the cytosolic side (Yamaji et al., 2008) of the
Golgi/ER. These latest studies only measured GM3 as the cells used I these experiments only
has GlcCer but no other neutral GSLs (D'Angelo et al., 2007; Halter et al., 2007).
1.5.3. MDR1, Cholesterol and Lipid Rafts
MDR1 has a special relationship with its membrane environment since it recognizes its
substrates within the lipid bilayer (Homolya et al., 1993. The transport substrates gain access to
the multiple drug binding sites which are located inside the cytoplasmic leaflet {Qu, 2002
#5762)after partitioning into the lipid phase of the membrane (Higgins and Gottesman, 1992a.
Strong evidence indicates, that the lipid composition of biological membranes is closely related
to MDR1 function {Modok, 2004 #5763). Surrounding lipids modulates the ATPase activity of
MDR1 (Romsicki and Sharom, 1998) and its interaction with its substrates (Romsicki and
Sharom, 1999).
Glucosylceramide and other glycosphingolipids are important constituents of detergent-
insoluble membrane domains termed DIGs (Parton and Simons, 1995) that are enriched also in
sphingomyelin and cholesterol (Harder and Simons, 1997). Lavie et al. reported that DIGs or
lipid rafts are related in their lipid composition and their insolubility in cold non- ionic
detergents, to nonclathrin-coated, plasma membrane vesicular invaginations termed caveolae
(Parton, 1996). Caveolin-1, a 21-kDa integral membrane protein, is a major caveolar coat
protein (Rothberg et al., 1992) that has the ability to engage in complex interactions with other
caveolin molecules, as well as other proteins (Okamoto et al., 1998). Lavie et al. first reported
that in MDR1 cells there was a dramatic increase in the number of caveolae and in the level of
caveolin-1 (Lavie et al., 1998). These findings may be related to the fact that a significant
80
fraction of cellular P-gp is associated with caveolin-rich membrane domains (Luker et al.,
2000). In contrast, Hinrichs et al. determined that MDR1 was localized in the non-caveolar
fraction of the plasma membrane (Hinrichs et al., 2004), while flow cytometry and confocal
microscopy showed that a substantial fraction of MDR1 was associated with lipid rafts and the
cytoskeleton in human colon carcinoma cells (Bacso et al., 2004). In 2005, it was reported that
MDR1 does not interact directly with caveolin-1, and is localized in intermediate-density
domains distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96
(Radeva et al., 2005). Barakat et al. reported the existence of two functional populations of P-
gp: the first one localized in rafts, that displays optimal ATPase activity almost completely
inhibited by orthovanadate and activated by verapamil; and the second one, located elsewhere in
the membrane, displays a lower ATPase activity, less sensitive to orthovanadate and inhibited
by verapamil (Barakat et al., 2005). Finally, after an exhaustive literature analysis, Orlowski et
al. concludes that P-gp exists in rafts and non-rafts domains, depending on the cell considered,
the experimental conditions and the method used; that when P-gp is present in rafts, interaction
with protein partners regulates its activity; and that P-gp handles the raft-constituting lipids with
particular efficiency, and it also influences membrane trafficking in the cell (Orlowski et al.,
2006).
Cholesterol seems to play an exceptional role in MDR1 function; the active cholesterol
redistribution across the membrane appears to be mediated by MDR1 (Luker et al., 2000).
MDR1 has been proposed to play a role in cholesterol esterification (Debry et al., 1997; Luker
et al., 1999). MDR1 has also been reported to regulate the translocation of sphingomyelin (SM)
(Bezombes et al., 1998), and it is possible that these two functions are interrelated. Depletion of
plasma membrane SM can induce cholesterol esterification by releasing cholesterol from the
plasma membrane into the intracellular pool (Slotte and Bierman, 1988). However, it cannot be
discounted that MDR1 somehow plays a direct role in trafficking cholesterol from the inner
81
plasma membrane to the endoplasmic reticulum (Johnstone et al., 2000). MDR1 may also be
involved in the relocation of cholesterol from the cytosolic to the exoplasmic leaflet of the
plasma membrane and in stabilization of the cholesterol-rich microdomains, rafts (Garrigues et
al., 2002; Orlowski et al., 2006). The ATPase activity of Pgp measured in the presence and
absence (basal ATPase activity) of Pgp substrates and modulators exhibit a strong dependence
on the amount of cholesterol incorporated into native membrane vesicles or proteoliposomes
(Rothnie et al., 2001; Garrigues et al., 2002; Kimura et al., 2007). Cholesterol depletion by
increasing concentrations of methyl-cyclodextrin decreased its ATPase activity (Garrigues et al.,
2002). Acute cholesterol depletion or saturation in different cell lines inhibited transport activity
(Bacso et al., 2004; Cai et al., 2004; Dos Santos et al., 2007), and in certain cell types
cholesterol saturation enhanced active drug efflux (Troost et al., 2004a; Troost et al., 2004b).
Thus, modulation of membrane cholesterol content can significantly alter Pgp function, but the
relationship between the cholesterol content and Pgp function may be complex. Enrichment of
membrane cholesterol changed lipid raft distribution but not the localization of P-gp, so MDR1
capacity depends on accurate lipid raft properties (Dos Santos et al., 2007).
It is well known that GSLs interact specifically with cholesterol, as demonstrated in both
artificial and cellular membranes (Brown, 1998; Brown and London, 2000). Pagano et al. have
thoroughly studied this interaction by using BODIPY-GSLs in normal and lysosomal storage
diseases (LSD) cell lines, in particular Niemann-Pick type-A and type-C (Chen et al., 1998;
Chen et al., 1999; Puri et al., 1999; Puri et al., 2001; Choudhury et al., 2002). In normal cells,
BIODIPY-LacCer is transported to the Golgi after endocytosis, while in multiple LSD cell
types, it accumulates in endosomal structures (Chen et al., 1998; Chen et al., 1999; Puri et al.,
1999; Choudhury et al., 2002). He concluded that GSL accumulation significantly alters
cholesterol distribution in LSD cells, and that cholesterol plays a major role in modulating the
intracellular targeting of GSLs. He also identified rab 7 and rab 9 as the main proteins
82
responsible in GSL transport and this suggests another possible therapeutic approach to LSD
treatment (Choudhury et al., 2002).
CHAPTER TWO: HYPOTHESIS AND SPECIFIC AIMS
2.1. Rationale
In multidrug resistance (MDR) cancer cells, higher levels of specific glycosphingolipids
have been reported with respect to their parental sensitive cells, such as glucosylceramide
(GlcCer), sphingomyelin (SM), and galactosylceramide (GalCer), whereas lactosylceramide
(LacCer) and all the more complex GSLs are present in lower amounts (Lavie et al., 1996;
Lucci et al., 1998; Kok et al., 2000; Morjani et al., 2001; Veldman et al., 2002). Cell
transfection with the MDR1 gene also results in elevated GSLs levels (Lala et al., 2000). Unlike
all other GSLs, GlcCer is made on the outer leaflet of the Golgi bilayer (Lannert et al., 1998),
but the active site of the glycosyltransferases responsible for further elongation of GSLs (to
LacCer first, and then to Gb3) are located within the Golgi lumen (Lannert et al., 1994; Burger
et al., 1996). Thus, GlcCer must be “flipped” into the Golgi lumen to access these
glycosyltransferases. MDR1 can function as a glycolipid flippase (van Helvoort et al., 1996;
Eckford and Sharom, 2005). We proposed MDR1 to be responsible for this translocation (De
Rosa et al., 2004).
Inhibition of GSLs biosynthesis results in the loss of drug resistance and diminished
expression of MDR1 (Lala et al., 2000; Gouaze et al., 2005), or even inhibition of MDR1
expression according to other report (Yang et al., 2004), suggesting that MDR1 requires GSLs
biosynthesis for its function and protein expression. In addition, the finding that MDR1 partially
colocalizes at the cell surface with Gb3 (Lala et al., 2000) suggests that Gb3 might be involved in
this requirement.
The hydrophobic character of GSLs makes them difficult to investigate in terms of their
function, and specifically in this case, the involvement of Gb3 in MDR1 expression and
83
function. Accordingly, there is a considerable interest in developing synthetic analogs of GSLs
which have sufficient water solubility and, at the same time, mimic the structural and functional
organization of GSLs in the plasma membrane. Replacement of the acyl chain of the ceramide
with a rigid adamantane frame (adamantyl-acyl ceramide) was chosen since the globular
structure of the adamantane group restricts the lateral interaction under aqueous conditions
(formation of lamellar structures). The resulting adaGb3 analog showed a markedly increased
solubility in water compared with natural Gb3, and a 1,000-fold enhanced inhibitory activity in a
Gb3-verotoxin binding assay (Mylvaganam and Lingwood, 1999) as compared to a lipid free
sugar derivative. Mahfoud et al. have reported that adaGb3 eliminates the cholesterol
requirement for high affinity HIV gp120/ Gb3 interaction providing a novel concept for
developing GSL-derived viral fusion inhibitors (Mahfoud et al., 2002b; Lund et al., 2006).
Various attempts have been made to overcome the phenomenon of multidrug resistance
in cancer. To date, third generation MDR inhibitors, more potent and specific, have overcome
toxicity side effects but they do not exhibit significant pharmacokinetic interaction with current
chemotherapeutic drugs in use (Krishna and Mayer, 2000; Thomas and Coley, 2003). The
development of new MDR1 inhibitors with higher selectivity and stronger potency remains a
primary research objective.
Lysosomal storage diseases, genetic deficiencies in glycoconjugate metabolism, each
due to a lack of a specific lysosomal sugar hydrolase or its activator protein, are often associated
with severe neurodegenerative pathology, caused by the intracellular accumulation of the
enzyme substrate (Futerman et al., 2004; Kolter and Sandhoff, 2006). Fabry disease is an X-
linked lysosomal storage disease resulting from the deficient activity of a-galactosidase A and
the progressive accumulation of Gb3. Despite its clinical success, the extraordinary cost of
Enzyme Replacement Therapy (ERT) has limited patient access and promoted the development
of alternative strategies (Wraith, 2006). Gene therapy is a candidate strategy for Fabry which
84
showed promising results for the future (Yoshimitsu et al., 2004). The third approach - substrate
reduction therapy (SRT)- has been to use inhibitors of glucosylceramide synthase, the first
enzyme required for the synthesis of most GSLs. But although it has proven effective in animal
storage disease models (Platt et al., 2003) and in clinical trials for Gaucher disease, another LSD
(Zimran and Elstein, 2003; Futerman et al., 2004), it inhibits glucosidase processing of N-linked
high mannose oligosaccharides (Tian et al., 2004) and glycogen breakdown (Andersson et al.,
2004). An effective inexpensive therapy for this and other lysosomal storage disorders needs to
be developed.
Ceramide is a key molecule in GSLs biosynthesis but also plays an important role as a
second messenger in a variety of intracellular signalling pathways (Hannun and Obeid, 2002;
Merrill, 2002; Futerman and Hannun, 2004). It has been reported that de novo ceramide
synthesis is regulated by members of the longevity assurance gene (LAG1) family, recently
renamed as ceramide synthases family (CerS) (Pewzner-Jung et al., 2006). Each member of the
CerS has a characteristic substrate preference base on a particular fatty acyl-CoA (Pewzner-Jung
et al., 2006). Thus, overexpression of CerS1 gene, previously known as UOG,1 selectively
increases C-18 ceramide synthesis in mammalian cells, but surprisingly this C18-ceramide is
further used to synthesize only neutral GSLs and not gangliosides (Venkataraman et al., 2002).
The finding in our previous study that CsA prevents neutral but not acidic GSL synthesis in a
variety of cell lines except HeLa cells (De Rosa et al., 2004), leads us to further investigate the
effect of MDR1 inhibition on specific acyl-CoA ceramide synthesis.
All these antecedents in the literature give support to propose the hypotheses for this
thesis.
2.2. Hypotheses
I – A soluble Gb3 analog, adamantyl Gb3, provides a probe of the colocalization of
MDR1 and Gb3, and a valuable tool to investigate the GSL biosynthesis required by MDR1.
85
II – MDR1 inhibition by third generation inhibitors can offer a potential and highly
selective alternative substrate reduction therapeutic approach for Fabry disease (and other
LSDs), given the fact that inhibition of MDR1 does not affect cellular ganglioside synthesis.
2.2.1. Specific Aims
1) Determine the effect of a water-soluble derivative of globotriaosyl ceramide
(Gb3), adamantyl Gb3, as possible inhibitor of MDR1.
2) Determine the effect of MDR1 inhibition to prevent the accumulation of
globotriaosyl ceramide (Gb3) in the Fabry mouse model of the human disease characterized by
lack of a-galactosidase A, responsible for Gb3 degradation.
3) Determine the effect of MDR1 on CerS genes, ceramide synthase genes with
characteristic substrate preference for a particular fatty acyl-CoA.
Scheme 2.1. Proposed Model for MDR1 Inhibition and Possible Therapeutic Approaches.
86
CHAPTER THREE: INHIBITION OF MULTIDRUG RESISTANCE BY
ADAMANTYLGb3, A GLOBOTRIAOSYLCERAMIDE ANALOG
Contents of this Chapter have been published in part in: De Rosa, M.F., C. Ackerley,
B.Wang, S. Ito, D.M. Clarke, and C. Lingwood. 2008. Inhibition of multidrug resistance by
adamantylGb3, a globotriaosylceramide analog. J Biol Chem. 283:4501-11.
3.1. Abstract
Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-
gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse
MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that
flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis.
We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co- localization, and GSL
removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3,
a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug
resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at
72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting
long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced
rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux,
restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human
intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to
the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-
linking of mutant MDR1 showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-
binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but
rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to
87
the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also upregulated total MDR1
and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1
provides a more physiologically based approach to MDR1 regulation to increase the
bioavailability of chemotherapeutic drugs.
3.2. Introduction
Multidrug resistance (MDR) is one of the major obstacles for successful
chemotherapy in patients with cancer. The MDR phenotype has been associated with
overexpression of P-glycoprotein in cancer cells (Germann, 1996). MDR1 is a 170-kDa plasma
membrane glycoprotein that uses ATP to pump hydrophobic molecules out of the cell (Riordan
et al., 1985; Gottesman and Pastan, 1993). Its efflux function decreases drug concentration in
tumor cells that results in chemotherapy failure. MDR1 is expressed in relatively high levels in
the apical membranes of epithelial cells of intestine, kidney, liver, and blood-brain/testes
barriers (Thiebaut et al., 1987). Thus, the presence of MDR1 within the endothelial cells’
epithelium can affect the bioavailability and the oral availability of therapeutic drugs (Oswald et
al., 2006). Various attempts have been made to reverse this resistance using MDR1 inhibitors
that interact with MDR1 and block drug efflux. First generation modulators such as calcium
channel blockers, calmodulin inhibitors, and cyclosporin were developed for pharmacological
uses other than reversal of MDR and were relatively nonspecific and weak inhibitors that were
also substrates of MDR1, and their deleterious toxicities with their use at required
concentrations to inhibit MDR1 have limited their clinical use (Ford et al., 1996; Sikic et al.,
1997). Clinical trials with second generation modulators such as the cyclosporin analog, PSC
833, or dexverapamil VX-710 have demonstrated some clinical benefits in terms of
enhancement of pharmacokinetics but also increased toxicity of cytotoxic drugs when
administered with these modulators (Fisher et al., 1996; Rowinsky et al., 1998). To date, a third
generation of more potent and specific modulators or inhibitors such as GG918 and LY335979
88
have overcome toxicity side effects but exhibited no significant pharmacokinetic interaction
with doxorubicin, etoposide, and paclitaxel in animal studies to make them suitable for co-
administration in cancer therapy (Hyafil et al., 1993; Dantzig et al., 1996). The development of
new MDR1 inhibitors with higher selectivity and stronger potency remains a major goal for this
field of research. In recent years, links have been established between MDR1 and
glycosphingolipids (GSL). Many cells expressing MDR1 show elevated levels of
glucosylceramide (GlcCer) and sphingomyelin (Lavie et al., 1996; Lucci et al., 1998; Morjani et
al., 2001; Veldman et al., 2002), and inhibitors of GlcCer synthase kill MDR cells (Nicholson et
al., 1999). Retroviral transfection of MDCK cells with human MDR1 gene results in a major
increase in neutral GSLs (Lala et al., 2000). Our previous studies showing that CsA inhibits
neutral GSL biosynthesis lead us to propose MDR1 as a Golgi glucosylceramide flippase that
enhances neutral GSL synthesis (De Rosa et al., 2004).We also observed partial MDR1 and
globotriaosylceramide (Gb3) cell surface co- localization and that inhibition of GSL biosynthesis
depleted cell surface MDR1. MDR1 may therefore interact with Gb3. In this study, the effect of
a water-soluble derivative of Gb3, adamantylGb3 (Mylvaganam and Lingwood, 1999) (Scheme
3.1), on MDCK-MDR1 and SK VLB was analyzed. Verotoxin was used to probe the potential
role of Gb3 in MDR1 function.
Scheme 3.1. Structural Models of Gb3 and Adamantyl Gb3 (adaGb3) (Mahfoud et al.,
2002b).
89
3.3. Materials and Methods
3.3.1. Materials
AdamantylGb3 (adaGb3) was prepared as described previously (Mylvaganam and
Lingwood, 1999). Dimethylformamide was added to a solution of oxalyl chloride in
dichloromethane. Adamantane acetic acid was then slowly added over 30 min. After stirring at
room temperature for 2 h, oxalyl chloride in excess and solvent were removed under a stream of
N2, and residual adamantane acetic acid was dissolved in dichloromethane. LysoGb3, prepared
by hydrolysis of Gb3, was suspended in dichloromethane and pyridine, and then 2 aliquots of the
adamantane acetic acid solution were added at 30-min intervals. After the reaction, the mixture
was dried under N2 (TLC; chloroform:methanol:water, 80:20:2 (v/v/v)) and the product purified
on a mini silica column. Adamantylgalactosylceramide (adaGalCer) was similarly made from
lysogalactosylceramide, prepared by hydrolysis of galactosylceramide. VT1 and VT2 were
purified as described (Rutjes et al., 2002). Some VT2 was affinity purified using an aminoGb4
(Nyholm et al., 1996) containing matrix (Boulanger et al., 1994), rather than a Cibachron blue
column in the standard procedure. Verotoxin 1 B subunit (VT1 B subunit) (Ramotar et al.,
1990) was purified by affinity chromatography as described (Boulanger et al., 1994). VT1B was
conjugated with fluorescein isothiocyanate (Invitrogen) as described (Khine and Lingwood,
1994). Rabbit polyclonal antiserum against purified VT1 B subunit was prepared as described
previously (Boyd et al., 1991). Cyclosporin A and vinblastine were purchased from Sigma.
Verapamil was a gift from A. Rasymas (Faculty of Pharmacy, Ohio State University).
[3H]Vinblastine (11.2 Ci/mmol) was purchased from Amersham Biosciences. [14C]Mannitol
(55.1 mCi/mmol) was purchased from DuPont.
3.3.2. Cell Culture
MDCK cells transfected with the human MDR1 cDNA were a gift from Dr. M.
Gottesman (National Institutes of Health, Bethesda) (Pastan et al., 1988). SK VLB ovarian
90
carcinoma cell line (MDR variant of the parental SK OV3) and C2BBe1 cell line (“brush
border-expressing”), subclone of human colorectal epithelial Caco-2 cell line, were obtained
from the American Type Culture Collection (Manassas, VA). MDR1-MDCK, Vero, and
SKVLB cells were maintained in a-minimal essential medium (Multicell, Wisent Inc., Quebec,
Canada) supplemented with 5%, for MDR1-MDCK and Vero, and 15% for SK VLB, of fetal
bovine serum, as well as 100 units penicillin/ml and 100 µg of streptomycin/ml (Multicell,
Wisent Inc., Quebec, Canada). MDR1-MDCK medium also contained 80 ng/ml colchicine, and
SKVLB medium contained 1 µg/ml vinblastine to maintain a continuous selection pressure to
express MDR1. C2BBe1 cell line is cultured in Dulbecco’s modified Eagle’s medium
(Multicell, Wisent Inc.) supplemented with 10% fetal bovine serum and 0.01 mg/ml human
transferrin (Sigma). All cell lines were incubated at 37°C in an atmosphere of 5% CO2, 95% air.
HEK 293 cell line is also cultured in Dulbecco’s modified Eagle’s medium (Multicell, Wisent
Inc.) supplemented with 10% fetal bovine serum with 100 units penicillin/ml and 100 µg of
streptomycin/ml (Multicell, Wisent Inc., Quebec, Canada)
3.3.3. Immunostaining of MDR1
MDR1-MDCK and SKVLB cells were grown on 12-mm diameter glass coverslips in a
24-well plate (BD Biosciences). For cell surface Gb3 localization, cells were exposed to 2.5
µg/ml of FITC-VT1B (Khine and Lingwood, 1994) in PBS for 1 h at 4 °C. For internalization,
cells were then incubated at 37 °C for 1 h and fixed. Costaining for human P-gp was performed
using the MRK-16 monoclonal antibody (5 mg/ml; Kamiya), followed by TRITC conjugated
goat anti-mouse secondary antibody (Sigma) also at 4 °C for 1 h. After washing, coverslips were
fixed, mounted with Dako Cytomation fluorescent mounting medium (Dako Cytomation, CA),
and examined by Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss Ltd., Toronto,
ON). For immunostaining of intracellular P-gp, cells were grown on glass coverslips to 50%
confluence, then washed, fixed in 4% paraformaldehyde in PBS, quenched with 50 mM NH4Cl,
91
and permeabilized for 15 min with 0.1% Triton X-100, 1% bovine serum albumin in PBS.
Antibody to P-gp (MRK-16) in permeabilization buffer was added to the cells for 1 h at 37 °C
and after washing, secondary TRTC-conjugated goat anti-mouse antibody was added to the cells
in permeabilization buffer for 1 h at 37 °C. Cells were washed, fixed again, mounted, and
examined as described before.
For inhibition of glycolipid biosynthesis, MDR1-MDCK and SKVLB cells were grown
in the presence of 5 µM of 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP;
Matreya Inc., Pleasant Gap, PA) for 5 days (Abe et al., 1992). For MDR1 inhibition studies,
MDR1-MDCK cells were grown in the presence of 4 µM CsA or 50 µM adaGb3 for 1h, 3h, 72 h
and 4 days. Fluorescent images were captured, stored, and analyzed with LSM software (Bolte
and Cordelieres, 2006). Digital images were transferred to Adobe Photoshop 8.0 for image
handling.
3.3.4. Post-embedding Immunogold Cryoelectron Microscopy
Logarithmic phase MDR1-MDCK and SKVLB cells were washed twice with PBS and
treated ± 5µg/ml VT1 B subunit at 4 °C for 1 h, or at 37°C for 1 h for internalization. Cells were
then harvested by scraping, pelleted by centrifugation at 1,000 X g for 5 min, and fixed in 4%
paraformaldehyde, 0.1% glutaraldehyde in 0.1 M Sorenson’s phosphate buffer, pH 7.4, for 2–4
h. Cells were then lightly pelleted and washed thoroughly in phosphate buffer. The cells were
then embedded in 15% gelatin, cut into mm3 pieces, and infused with 2.3 M sucrose for several
hours. The blocks were then mounted on aluminum cryo-ultramicrotomy pins and frozen in
liquid nitrogen. Ultrathin cryosections were then cut on a diamond knife at -95°C using a Leica
Ultracut R cryo-ultramicrotome (Leica Canada). Sections were transferred to Formvar-coated
nickel grids in a loop of molten sucrose and the grids washed thoroughly in PBS containing
0.15% glycine and 0.5% bovine serum albumin and PBS containing bovine serum albumin
alone. Sections were then incubated with a polyclonal antibody against VT1 B subunit (1:2000)
92
for 1 h. Following a thorough rinse in PBS/bovine serum albumin, samples were incubated in
goat anti-rabbit IgG 5 nm gold complex (Amersham Biosciences) for an hour. This procedure
was repeated on the same specimens except monoclonal antibody anti-MDR1 (MRK-16, 5
µg/ml) was used as the primary antibody and goat anti mouse IgG 10 nm gold complex as the
secondary antibody. Sections were then rinsed thoroughly with PBS followed by distilled water
and stabilized in a thin film of methylcellulose containing 0.2% uranyl acetate. Controls
included the omission of primary and secondary antisera and the use of an irrelevant antibody
(either poly- or monoclonal anti glial fibrillary acidic protein). Samples were then examined in a
JEOL JEM 1230 transmission electron microscope (JEOL USA) and images recorded either on
photographic plates or with a CCD camera (AMT Corp.). Image analysis was performed on a
minimum of 100 images from each group at a nominal magnification of X100,000 using an
image analysis program (Image Pro Plus, Media Cybernetics).
3.3.5. Neutral Glycolipids Extraction and Analysis
Vero cells were grown in medium supplemented with 50 µM of adaGalCer, adaLacCer
or adaGb3 for 4 days. Ten million cells from treated and untreated Vero cell line were scraped
from the culture dish, washed with phosphate buffer saline (PBS; CellgroTM, Mediatech Inc.,
Herndon, VA) and extracted with 20 volumes of chloroform/methanol (2:1) as described
previously (Pudymaitis et al., 1991). The extract was partitioned against water, and the lower
phase was dried, redissolved in chloroform/methanol (98:2) and separated by silica
chromatography (Boyd and Lingwood, 1989). The column was washed extensively with
chloroform, and the glycolipid fraction was eluted with acetone/methanol (9:1), evaporated, re-
dissolved in chloroform/methanol (2:1), and separated on silica plates (Macherey-Nagel Silica
gel 60 [40-63 µM]; Caledon, Georgetown, ON) by thin- layer chromatography (TLC) using the
solvent system chloroform/methanol/water (65:25:4). Glycosphingolipids (GSLs) were
visualized by staining with orcinol reagent, and Gb3 was detected by TLC overlay binding with
93
VT1. Purified glucosylceramide (GluCer), lactosylceramide (LacC), globotriaosylceramide
(Gb3), and globotetraosylceramide (Gb4) were run on the same plate as standards.
3.3.6. Verotoxin 1 Thin Layer Chromatography Overlay
Aliquots of the GSLs extracts were separated on silica plates (Macherey-Nagel Silica gel
60 [40-63 µM]; Caledon, Georgetown, ON) by TLC in chloroform/methanol/water (65:25:4).
The plates were dried and blocked with 1% gelatine in water at 37°C overnight. After being
washed three times with 50 mM TBS (50 mM Tris buffer, pH 7.4, with 0.9% NaCl) to eliminate
traces of gelatine, the plates were incubated with 0.1 µg/ml purified VT1 for 1 h at room
temperature with gentle shaking. They were washed again with TBS, and then plates were
incubated with mouse monoclonal anti-VT1 antibody (Boulanger et al., 1990b) (2 µg/ml) for 1 h
at room temperature with shaking, followed by incubation with goat anti-mouse IgG horseradish
peroxidase-conjugated antibody (diluted 1:2000 in TBS) (Sigma, St. Louis, MO), also for 1 h at
room temperature with shaking. Toxin binding was visualized using a 3 mg/ml solution of 4-
chloro-1-naphthol peroxidase substrate in methanol, freshly mixed with 5 volumes of TBS and
1:1000 dilution of 30% H2O2 (Lingwood et al., 1987). Colour development was stopped by
extensive but gentle washing under tap water, and the plates were finally dried under a blow
dryer.
3.3.7. Cytotoxicity Assay
For ada GSLs studies, Vero cells were grown in medium supplemented with 50 µM of
adaGalCer, adaLacCer or adaGb3 for 4 days, and tested for VT sensitivity. MDR1-MDCK and
SKVLB cells were cultured in the presence of either 4 µM CsA, 20 or 50 µM of adaGb3, or 20
µM of adaGalCer for 4 days, and tested for sensitivity to vinblastine over 3 days relative to
untreated cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (DDTB)
assay (Mosmann, 1983). The parental drug-sensitive cell line, MDCK-1, was also cultured in
the presence of either 4 µM CsA or 50 µM of adaGb3 for 4 days as a control. Dose-dependence
94
curves for each of the adaGSLs analogs were performed to determine the highest non-toxic
concentration of the analog that was used in the experiments. Briefly, log phase pretreated or
untreated cells plated in 96-well microtiter plates were incubated in triplicate with 200 µl of
tenfold dilutions of VT1, VT2, or vinblastine at 37ºC, 5% CO2 in a-minimal essential medium
with 5% or 15% fetal bovine serum and incubated at 37 °C in 5% CO2 with increasing dilutions
of vinblastine. After 72 h, 20 µl of a 5 mg/ml DDTB solution was added to the wells and
incubated at 37°C in 5% CO2 for 4 h. Supernatants were removed, and 100 µl of acidified
isopropyl alcohol was added and incubated in the dark at room temperature for 30 min. Plates
were agitated for 2–3 min, then read on an enzyme-linked immunosorbent assay reader at 490
nm. The concentration required to inhibit growth by 50% (IC50 values) was calculated from the
cytotoxicity curves using GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego).
The fold reversal of multidrug resistance was calculated by dividing the IC50 values in the
absence of the MDR1 inhibitors by those in the presence of the MDR1 inhibitors.
3.3.8. MDR1-MDCK Raft Isolation
Rafts were prepared as described previously (Brown and Rose, 1992; Kamau et
al., 2005). In brief, 2.5 x 108 MDCK-MDR1 untreated cells were lysed for 20 min on ice in 2 ml
TNE buffer containing 1% (v/v) Triton X-100 (Laemmeli, 1970; Brown and Rose, 1992). After
homogenization, the homogenate was mixed with an equal volume of 80% (w/v) sucrose
solution in TNE buffer and overlaid with a discontinuous sucrose gradient (2 ml each of 30, 20,
10%, and 1.5 ml of 5% [w/v] sucrose, all in TNE buffer without Triton X-100). After
centrifugation at 200,000 X g for 19 h at 48 C, in a SW41Ti rotor (Beckman Instruments,
Fullerton, CA), 1 ml fractions were collected from the top of the gradient (fractions 1–12)
without including the pellet at the bottom. Raft fractions 4–7 had higher optical densities (620
nm) than the rest of the fractions (Hooper, 1999). The distribution of protein along the gradient
was characteristically skewed, with most protein being concentrated within fractions 7–12
95
(density range 1.13–1.20 g/cm3). 30 µl of each fraction was boiled in sample buffer for SDS-
PAGE and immunoblotting.
3.3.9. Western Blot Analysis of MDR1
Untreated MDR1-MDCK cells or cells pretreated overnight with 10 µM CsA, or 4 µg/ml
VT1B, or for 1 h, 3 h, overnight, 3 days, or 4 days with 50 µM adaGb3, or for 5 days with PPMP
were pelleted by centrifuging at 500 X g for 5 min. Cell pellets were washed three times with
cold 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and solubilized in lysis buffer (50mM Tris-HCl,
pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.1% NaN3, 5% Nonidet P-40, 5 µg/ml aprotinin, 2 µg/ml
pepstatin, 1 mM phenylmethylsulfonyl fluoride) for 1 h with shaking at 4 °C. After spinning for
20 min at 12,000 X g at 4 °C, protein was determined in supernatants using a BCA-protein assay
(Pierce). All samples, including the ones obtained from raft isolation were denatured by heating
for 5 min at 95° C, before being loaded onto a 8% (w/v) gel for P-gp detection (4 mg
protein/slot) or onto a 15% (w/v) gel for caveolin detection (2 mg protein/slot) (rafts fractions
only). After SDS-PAGE, Western blotting followed (Hammerle et al., 2000). The transferred
nitrocellulose blot was blocked with 5% skim milk powder in 50mM Tris-HCl, pH 8, 150 mM
NaCl, 0.05% Tween 20 at 37 °C for 1 h. The membrane was then immunoblotted with
monoclonal antibody C219 (1 µg/ml) (DAKO, Glostrup, Denmark) in 1% skim milk overnight
or with rabbit anticaveolin antibody (1:5000), and was washed three times with 50 mM Tris-
buffered saline, 0.05% Tween 20. Following incubation with horseradish peroxidase-conjugated
goat anti-mouse IgG 1:1000 (Sigma) or anti-rabbit conjugated with alkaline phosphatase (Pierce
Biotechnology, Rockford, IL) (1:50,000) secondary antibodies for 2 h at room temperature, the
blots were developed using Supersignal West PICO enhanced chemiluminescence (ECL)
system (Pierce).
3.3.10. Rhodamine 123 Efflux Assay
96
For this assay, all the steps were performed at 37 °C on cells grown in chambers with
borosilicate coverglass system at a density of 5 x 104 cells/ml. MDR1-MDCK and SK VLB
cells were preincubated for 30 min with assay buffer (10 mM Hepes, 0.4 mM K2HPO4, 25 mM
NaHCO3, 3.0 mM KCl, 1.2 mM MgSO4, 1.4 mM CaCl2, 122 mM NaCl, 10 mM glucose)
(Fontaine et al., 1996; Hammerle et al., 2000). After the preincubation period, the assay buffer
was removed and replaced with 2.5 ml of assay buffer containing 100 µg/ml of rhodamine 123
(rho123) (Sigma). After 20 min, the wells were washed three times for 5 min with assay buffer.
For inhibition studies, 50 µM adaGb3 or 10 µM cyclosporin A for positive control in assay
buffer were added 5 min prior to the addition of rho123 and maintained in all the solutions until
visualization. MDR1-MDCK and SK VLB cells were also tested when treatment with 50 µM
adamantylGb3 was for 24 h. The efflux of rho123 was directly visualized in these chambers by
Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss Ltd.). Fluorescent images were
captured, stored, and analyzed with LSM software.
3.3.11. Drug Transport
Transport of 0.1 µM [3H] vinblastine, a P-glycoprotein substrate, was measured across a
C2BBe1 intestinal epithelial cell monolayer (a polarized subclone of Caco2 cells) grown on a
permeable membrane (Costar Transwell: 0.2 µm pore size; polycarbonate filter). After
preincubation for 45 min in the incubation media (Dulbecco’s modified Eagle’s media) without
vinblastine, transport experiments were conducted at 37°C. At time 0, labeled vinblastine was
added to either the basal or apical side of the monolayer in the presence or absence of an
inhibitor on both sides. Time course of basal-to-apical transport was examined by monitoring
the appearance of radioactivity in the apical side after adding vinblastine to the basal side.
Apical-to-basal transport was examined by monitoring appearance of radioactivity from the
apical to the basal side. As an extracellular marker, [14C] mannitol was used with [3H]
vinblastine to ensure integrity of the monolayer. The data obtained from the filter preparation,
97
which showed more than 4% of mannitol concentration over 2 h (relative to the initial
concentration in the opposite side at time 0), were excluded.
3.3.12. Disulfide Cross-linking Analysis
Construction of the human P-glycoprotein cross- linkable mutants F343C(TM6) /
F728C(TM7), F343C(TM6) / Q725C(TM7), and L339C(TM6) / F728C(TM7) was described
previously (Loo et al., 2006b; Wang et al., 2007). The mutant cDNAs were transiently
expressed in HEK 293 cells (25 10-cm plates). The cells were harvested and washed three times
with PBS, and membranes were prepared as described (Loo and Clarke, 1994b). The
membranes were suspended in 0.3 ml of Tris-buffered saline, pH 7.4, and preincubated for 15
min at 22 °C in the presence of different concentrations of the Gb3 analog, verapamil, or no
drug. Samples were then cross- linked by incubation with 0.2 mM of the methanethiosulfonate
cross- linker, 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate (M14M, 2.08 nm
spacer arm) (Toronto Research Chemicals, Downsview, Ontario, Canada) (a 1/10th volume of 2
mM M14M was added) for 3 min on ice. The cross- linking reactions were stopped by addition
of 2X SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS
containing 50mM EDTA) and no reducing agent. The reaction mixtures were then subjected to
SDS-PAGE (7.5% polyacrylamide) and immunoblot analysis with a rabbit polyclonal antibody
against MDR1 (Loo and Clarke, 1995). Intramolecular disulfide cross-linking between TMD1
and TMD2 can be detected because of slower mobility of the cross- linked product on SDS-
polyacrylamide gels (Loo et al., 2006a). The amount of cross- linking was quantitated by
scanning the gel lanes followed by analysis with the NIH Image Program Image J 1.34s
(available at www.rsb.info.nih.gov).
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3.4. Results
3.4.1. MDR1 in Part Co-localizes with Gb3
Our previous immunofluorescence study (Lala et al., 2000) had suggested that Gb3
and MDR1 were, in part, located in similar regions of the plasma membrane. Double
labeling of MDR1-MDCK and SK VLB cells using anti MDR1 and FITC-VT1B (Gb3
ligand) at 4oC shows considerable colocalization of these two cell surface antigens (Figure
3.1). Some cells however, lack Gb3 expression, indicating cell heterogeneity or variable cell
cycle expression. This is consistent with our earlier study (Lala et al., 2000). SKVLB MDR1
cell surface staining was less, but again, partial colocalization with Gb3 (VT1B) is seen
(Figure 3.1B).
Figure 3.1. Cell Surface Colocalization of MDR1 and FITC-VT1B in MDR1-
MDCK and SK VLB Cells. Cell surface double labelling with FITC-VT1B and anti-P-gp
(MRK16) detected using TRTC-conjugated goat anti-mouse antibody, at 4°C in MDR1-MDCK
(A) and SK VLB cells (B).
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To further study if this partial colocalization of MDR1 and Gb3 at cell surface also
corresponds intracellularly, SK VLB cells were permeabilized with MRK16 (anti-MDR1) and
internalization of VT1B was performed at 37°C. Strong intracellular colocalization between
VT1B and MDR1 confirms the association between Gb3 and MDR1 (Figure 3.2).
Figure 3.2. Intracellular Colocalization of MDR1 and FITC-VT1B in SK VLB
Cells. Internalization of FITC-VT1B by SK VLB cells at 37°C and permeabilization with anti-
MDR1 (MRK16).
The possible cell surface co-localization of MDR1 and Gb3 was therefore more
precisely defined by double labeling cryo-immunoelectron microscopy (Figure 3.3A).
Morphometry established that 33.4 ± 3.6 % of MDR1 was within 25 nm of VT1 in MDR1-
MDCK cells, while in SK VLB cells; this value was 10.0 ± 2.8 %. This is a conservative
100
estimate of co- localization, because the theoretical maximum distance for co- localized antigens
by indirect gold labeling is 50 nm. Prolonged inhibition of GSL synthesis by PPMP prevented
the cell surface detection of MDR1 by confocal immunomicroscopy (Figure 3.3B).
Figure 3.3. Plasma Membrane MDR1 and Glycosphingolipids. (A) Double labeling
cryo- immunoelectron microscopy of MDR1 and VT1B. Post-embedding cryo-double label
immunoelectron microscopy using monoclonal antibody MRK16 anti-MDR1 and rabbit anti-
VT1B with 10- and 5-nm gold antispecies antibodies, respectively, was performed on MDR1-
MDCK cells pretreated with VT1B for 1 h at 4 °C to define association of antibody-bound
MDR1 and Gb3, VT1B receptor, at cell surface level. Morphometric analysis showed MDR1
was co- localized with VT1 (arrows). Bar 500 nm. (Higher resolution image also included). Dr
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Cameron Ackerley and Ms. Aina Tulips contributed to this figure. B) Effect of inhibition of
GSL synthesis on cell surface MDR1 expression in MDR1-MDCK Cells. Cell surface labeling
with anti-MDR1 (MRK16) was detected by confocal microscopy using TRITC-conjugated goat
anti-mouse antibody, at 4 °C, prior to (panel i) or following (panel ii) 5 days of culture in the
presence of 5 µM of PPMP.
If we let VT1B internalize at 37ºC, co- localization of VT1B/Gb3 complex and
MDR1 with the same transport vesicle can be seen in the magnified area of the first panel, but
the proximity between both types of particles is not as close as at the cell surface (Figure 3.4.i).
The magnified area of the second panel, showed co-localization at the nuclear membrane,
consistent with the retrograde pathway of VT in MDR1-MDCK cells (Khine and Lingwood,
1994; Sandvig et al., 1994; Arab and Lingwood, 1998; Khine et al., 2004) (Figure 3.4.ii).
Morphometry established that 24.3 ± 5.7 of MDR1 was within 25 nm of VT1 in MDR1-MDCK
cells.
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Figure 3.4. Double Label Cryoimmunoelectronmicroscopy of Intracellular MDR1
and internalyzed VT1B. Post-embedding cryo-double label immunoelectron microscopy using
monoclonal antibody MRK16 anti-MDR1 and rabbit anti-VT1B with 10- and 5-nm gold
antispecies antibodies, anti-mouse and anti-rabbit respectively, was performed on MDR1-
MDCK cells pretreated with VT1B for 1 h at 37 °C to define intracellular association of
antibody-bound MDR1 and Gb3, VT1B receptor. Morphometric analysis showed MDR1 was
co-localized with VT1 (arrows). (Higher resolution images also included). Dr Cameron
Ackerley and Ms. Aina Tulips contributed to this figure.
3.4.2. AdaGb3 Prevents Multidrug Resistance in MDR1-MDCK and SKVLB Cells
In addition to the interaction of MDR1 with glucosylceramide (De Rosa et al.,
2004), taking into consideration the cell surface partial colocalization between MDR1 and Gb3
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and the advantages of the use of adamantyl GSLs analogs –water solubility, uptake and
intracellular trafficking as natural GSLs- we therefore, tested the effects of adaGb3 on known
MDR1 functions.
MDR1-MDCK cells are resistant to vinblastine compared with the parental cell line
MDCK. The ability of adaGb3 to reverse the drug resistance phenotype of MDR1-MDCK and
SKVLB was examined and compared with that of CsA, and with adaGalCer to test specificity.
Concentrations of 50 µM of either adaGb3 or adaGalCer had no cytotoxic effects on MDR1-
MDCK cells within the experimental period. AdaGb3 at 50 µM significantly reversed the
resistance of MDR1-MDCK cells to vinblastine (Figure 3.7.A) and reduced the IC50 values
similar to CsA (IC50 MDR1 = 12.6 µg/ml versus IC50 MDR1CsA = 8.8 x 10-6 µg/ml and IC50 MDR1adaGb3 -
50 = 7.2 x 10-6 µg/ml). Even treatment with adaGb3 at 20 µM reduced the IC50 value (IC50
MDR1adaGb3–20 = 9.6 x 10-2 µg/ml). Treatment with adaGalCer did not reverse the drug resistance
phenotype of MDR1-MDCK cells (IC50 MDR1adaGC = 13.9 µg/ml) showing a specific effect for
adaGb3 on the modulation of MDR1 function (Figure 3.5.A). Both CsA and adaGb3, if anything,
slightly reduced vinblastine sensitivity of the highly sensitive, parental untransfected MDCK-1
cells.
In SKVLB cells, naturally resistant to vinblastine (Figure 3.5.B), treatment with 50 µM
adaGb3, but not 50 µM adaGalCer, reversed the multidrug resistance phenotype by reducing the
IC50 for vinblastine by 10-fold (IC50 SKVLB = 79.2 µg/ml versus IC50 SKVLB adaGb3–50 = 7.8 µg/ml
and IC50 SKVLB adaGC = 76.9 µg/ml), similar to CsA (Figure 3.5.C). The effect of AdaGb3 and CsA
on vinblastine sensitivity was also tested in the parental drug-sensitive cell line MDCK1, with
no significant effect, as a control (Figure 3.5.B).
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105
Figure 3.5. Effect of AdamantylGb3 on Cell Resistance to Vinblastine. Vinblastine
sensitivity was monitored 72 h after addition to MDR1-MDCKcells (A) grown in 96-well plates
untreated (.___¨___) or pretreated either with 4 µMCsA (___¦ ___), 20 µM (- __? __ - ), or 50 µM
adaGb3 (--OF--O), or 50 µM adaGalCer (---? ---). Inset, structure of adaGb3. B, parental
MDCK-I cells grown in 96-well plates were untreated (.___¨___) or pretreated either with 4 µM
CsA (___¦ ___) or 50 µM adaGb3 (- __? __ -). C, SKVLB cells were grown in 96-well plates
untreated (.___¨___) or pretreated either with 4 µM CsA (___¦ ___), 50 µM adaGb3 (- __? __ -), or 50
µM adaGalCer (---? ---). AdaGb3 was as effective as CsA to increase cell sensitivity to
vinblastine. Each point in the graph represents the mean value of triplicate experiments,
repeated at least twice.
3.4.3. AdaGb3 Inhibits Cell Surface MDR1 Expression in the Long Term in MDR1-MDCK
and SKVLB Cells but Increases Intracellular MDR1 as PPMP, VT1B, and CsA
MDR1-MDCK cells pretreated with adaGb3 for 4 days and stained with anti-
MDR1 at 4°C showed complete loss of cell surface MDR1 expression (Figure 3.6.A, i, panel b)
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compared with the control with no pretreatment (Figure 3.6.A, i, panel a). Treatment with
another adamantyl GSL analog, adamantyl galactosylceramide (adaGalCer) had no effect on
MDR1 staining (Figure 3.6.A, i, panel d). This absence of MDR1 cell surface expression was
also observed when the cells were pretreated with CsA, a standard inhibitor of MDR1 (Figure
3.6.A, i, panel c). Similar prevention of cell surface MDR1 expression by adaGb3 and CsA was
seen for SKVLB cells (Figure 3.6.A, ii, panels a–d). A time course pretreatment of MDR1-
MDCK cells with adaGb3 and CsA for 1, 3, and 72 h showed that adaGb3 up-regulates MDR1
cell surface expression at 3 h, but by 72 h, it is completely lost compared with the untreated
control (Figure 3.6.B, i, panels b–d, versus panel a). This short term up-regulation has been
described for CsA (Herzog et al., 1993), and CsA up-regulation of MDR1 was seen within 1 h
as for adaGb3 (Figure 3.8.B, ii, panels b–d versus panel a). The total cellular MDR1 content was
monitored by Western blot after treatment of MDR1 MDCK cells with either CsA, adaGb3, or
VT1 B overnight, and compared with control (Figure 3.6.C, i, lanes 2–4 versus lane 1). Despite
or perhaps because of MDR1 inhibition, MDR1 synthesis is increased in the presence of CsA,
adaGb3, or VT1B (Figure 3.6.C, i, lane 2) (CsA < adaGb3 < VT1B). Total cellular MDR1
increased rapidly after inhibitor addition (2X-fold within 1 h) (Figure 3.6.C, ii, lane 3, and D) to
reach a maximum within 3 h (Figure 3.6.C, ii, lane 4, and D) that was maintained during
prolonged inhibitor exposure (Figure 3.6.C, ii, lanes 5 and 6). Cellular MDR1 is similarly
increased when GSL synthesis is prevented by PPMP (Figure 3.6.C, ii, lane 2). The loss of cell
surface MDR1 expression when intracellular MDR1 is increased suggests that cell surface
trafficking of MDR1 may be an unappreciated target of these inhibitors and GSL deficiency.
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Figure 3.6. Effect of AdamantylGb3 on MDR1 Expression in MDR1-MDCK and SKVLB
Cells. A. Cell surface labeling with monoclonal antibody anti-MDR1 (MRK16) of MDR1-
MDCK cells cultured for 4 days with PBS (control, i, panel a) or with 50 µM adaGb3 (i, panel
b), 4 µM CsA (i, panel c), or 50 µM adaGalCer (i, panel d), and SKVLB cells cultured for 4
days with PBS (control, ii, panel a), or with 50 µM adaGb3 (ii, panel b), 4 µM CsA (ii, panel
c), or 50 µM adaGalCer (ii, panel d). Images were recorded with a confocal laser microscope
using equal settings. MDR1 inhibitors prevent cell surface MDR1 expression. B. Cell surface
labeling with monoclonal antibody anti-MDR1 (MRK16) of MDR1-MDCK cells untreated
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(control, i, panel a) or pretreated with 50 µM adaGb3 for 1 h (i, panel b), for 3 h (i, panel c), or
for 72 h (i, panel d), and MDR1-MDCK cells untreated (control, ii, panel a), or pretreated with
4 µM CsA for 1 h (ii, panel b), for 3 h (ii, panel c), or for 72 h (ii, panel d). Images were
recorded with a confocal laser microscope using equal settings. AdaGb3 up-regulates cell
surface MDR1 expression for a short period of time (3 h) as CsA (1 and 3 h), but both prevents
expression at 72 h. C. MDR1 levels in CsA-, adamantylGb3-, VT1B-, and PPMP-treated
MDR1-MDCK cells. Cell SDS extracts were separated by SDS-PAGE and subjected to Western
blot using C219 anti-MDR1 antibody. MDR1-MDCK cells were treated with the following: i,
lane 1, untreated; lane 2, 10 µM CsA, lane 3, 50 µM adaGb3; or lane 4, 4 µg/ml VT1B
overnight (170-kDa marker for MDR1 was indicated); ii, lane 1, untreated cells; lane 2, 5 µM
PPMP for 5 days; lane 3, 50 µM adaGb3 for 1 h; lane 4, adaGb3 for 3 h; lane 5, adaGb3 for 3
days; and lane 6, adaGb3 for 4 days. D. Western blots (i and ii, from (C) were scanned and
quantitated by NIH Image J 1.34 relative to the untreated control (i and ii, 1). AdaGb3 induced a
rapid and sustained increase in the cellular MDR1 content. CsA, VT1B, or GSL depletion by
PPMP also induced MDR1 amplification.
3.4.4. Effect of Adamantyl Analogs on GSL Levels
Adamantyl-derivatives of several GSLs were synthesized in our lab by replacing
the heterogenous fatty acids with an adamantane frame. This procedure greatly increases the
water solubility and unlike the lipid free carbohydrate moiety, receptor function (VT1 binding to
adamantyl Gb3) and enzyme recognition as substrates are retained (Mylvaganam and Lingwood,
1999; Mylvaganam and Lingwood, 2003).Adamantyl-GSLs also retain membrane solubility and
intracellular trafficking characteristics when added to cells.
To study the effect of these adamantyl-GSLs derivatives on GSL biosynthesis,
analysis of Gb3 content, before and after Vero cells treatment with the GSLs analogs, was
performed on TLC plates, and Gb3 was identified by VT1 TLC overlay. The orcinol panel
(Figure 3.7.A), that stains all GSLs, shows that in the extract of Vero cells pretreated with ada
GalCer (line c), ada GalCer is converted to a new species (marked*) which is also VT1 reactive
as it can be seen in the VT1 overlay panel (Figure 3.7.B, line c) and Gb3 is concomitantly
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markedly reduced. This new species is probably adamantyl galabiosyl ceramide (ada Gb2). Gb2
has been described as an alternative receptor for VT (Cohen et al., 1987). Both ada LacCer and
ada Gb3 reduce cellular Gb3 levels, without any new product synthesized, but less significantly
than ada GalCer.
Figure 3.7. Treatment of Vero Cells with Ada-GSLs Analogs. A. Orcinol staining of
neutral GSL fraction from Vero cells extracts pretreated with ada GSLs analogs. B. VT1
overlay. Lane a of each panel corresponds to GSL standards, lane b to untreated Vero cells, lane
c to ada GalCer Vero treated cells, lane d to ada GalCer standard, lane e to ada LacCer Vero
treated cells, lane f to ada LacCer standard, lane g to ada Gb3 Vero treated cells, and lane h to
ada Gb3 standard.
VT1 and VT2 cytotoxicity assays were performed in triplicate to further analyze
if the reduced level of Gb3 and the synthesis of adaGb2 after ada GalCer treatment have any
effect on VT sensitivity. Pretreatment of Vero cells with P4, a potent GSL biosynthesis
inhibitor, for 5 days in the presence or abscence of ada GalCer was also analyzed. No effect on
VT1 or VT2 sensitivity was observed after treatment with ada GalCer, as the reduced Gb3 levels
were compensated with the synthesis of adaGb2, also a VT receptor. Although P4 efficiently
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inhibited VT sensitivity as a consequence of its inhibition of GSL biosynthesis, we expected to
see intermediate levels of VT sensitivity for the cells treated with both P4 and adaGalCer, as the
synthesis of ada Gb2 should not be affected by P4 as its site of action is UDP-glucose:N-
acylsphingosine glucosyltransferases in the synthesis of GlcCer (Lee et al., 1999) (Figure 3.8, A
and B).
Figure 3.8. Verotoxin Cytotoxicity Assays Before and After Treatment with
AdaGalCer and P4. A. Verotoxin 1 sensitivity, and B Verotoxin 2. Dose dependent VT1 and
VT2 cytotoxicity were assayed in triplicate, repeated at least twice.___¨___ Vero cells, ___¦ ___
Vero + 1 µM P4, ----? ---- Vero + 50 µM ada GalCer, and _ . _ X _ . _ Vero cells + 1 µM P4 + 50
µM ada GalCer.
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3.4.5. MDR1 Distribution in Lipid Rafts
Active MDR1 is predominantly localized in low-density caveolae enriched domains
(rafts) (Orlowski et al., 2006). To better understand the role of the lipid environment on
proteins, different methods have been developed to isolate specific membrane domains (Hooper,
1999; Pike, 2003; Pike, 2004; Blonder et al., 2006). Detergent-resistant rafts, prepared in cold
buffer containing 1% Triton (Brown and Rose, 1992), are enriched in cholesterol and GSLs
(Pike, 2003); hence, their role on the localization and function of P-gp is of interest. After the
lysis of treated and untreated cells, lipid rafts were prepared in cold buffer-containing 1% Triton
X-100, and the different fractions were analyzed by immunoblotting for MDR1 and for caveolin
(Figure 3.9).
In untreated MDR1-MDCK cells, there was an enrichment of P-gp in the rafts fractions
(4-7) (Figure 3.9), as it has already been described for this cell line (Kamau et al., 2005), and
also matches the distribution of caveolin 1 in the rafts fractions. Total P-gp and caveolin content
seems to be decreased after AdaGb3 treatment (data not shown), but the results were not
sufficiently clear for inclusion in this thesis. Apparently for both P-gp and caveolin, AdaGb3 has
little effect on their relative distribution in the membrane. The release or shedding of membrane
components, which includes transmembrane proteins such as P-gp, after cholesterol depletion, is
a phenomenon that was reported in several studies (Yunomae et al., 2003; Arima et al., 2004;
Kamau et al., 2005). This might be an explanation for the decrease level of P-gp in adaGb3
treated cells if it is confirmed. The role of adaGb3 in cholesterol redistribution might be further
studied as well as the analysis of cell free supernatants for evidence of P-gp.
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Figure 3.9. A. MDR1 Distribution in Raft Fractions. MDR1 contents in rafts (4-7) and non-
rafts fractions of untreated MDR1-MDCK cells were analyzed by immunoblotting. Caveolin
contents of untreated MDR1-MDCK cells in rafts and non-rafts fractions were also analyzed by
immunoblotting. Beth Binnington and Justyna Bartoszko contributed to this figure .
3.4.6. AdaGb3 Inhibits the Efflux of Rhodamine 123 in MDR1-MDCK and SKVLB Cells
AdaGb3 inhibition of MDR1 was tested in MDR1-MDCK cells and in SKVLB cells with
the fluorescent substrate rho123 in a confocal laser scanning microscope assay. After exposure
to rho123, cells were washed and incubated for 20 min at 37 °C before images were recorded.
Confocal microscopy showed that rho123 effluxed from MDR1-MDCK and SKVLB control
cells (Figure 3.10, i, panel a, and ii, panel a). Treatment of MDR1-MDCK cells with 10 µM
CsA or 50 µM adaGb3 for 1.5 h completely inhibited the rho123 efflux (Figure 3.10, i, panels b
and c). Although the efflux in SK VLB was also inhibited by 10 µM CsA during the 1.5-h
experiment, treatment with 50 µM adaGb3 for the same period of time showed partial inhibition,
with some of the rhodamine123 retained within the cells (Figure 3.10, ii, panels b and c). When
SK VLB cells were pretreated with 50 µM adaGb3 for 24 h, complete inhibition of the efflux
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was observed, as after CsA treatment (Figure 3.10, ii, panels b and d). In the control or non-
treated cells, most of the rho123 was pumped out and washed away, with little residual
intracellular fluorescent staining. Any fluorescence retained within the cells showed a patchy
distribution within the cytoplasm (Figure 3.10, i and ii, panels a and e) in these cells, whereas
both CsA-treated MDR1-MDCK cells and CsA-treated SK VLB cells were intensively and
homogeneously labeled (Figure 3.10, i and ii, panel b), as CsA blocked the pumping activity of
MDR1. This same inhibition pattern was observed when MDR-MDCK and SK VLB cells were
treated with adaGb3 (Figure 3.10, i and ii, panels c and d). Cell treatment with adaGalCer did
not prevent rho123 efflux (Figure 3.10, i and ii, panel e).
Figure 3.10. Inhibition of Efflux of Rho123 from Adamantyl Gb3-treated Cells. The
efflux of rho123 from control and drug-treated cells was monitored by confocal laser scanning
microscopy. For inhibition studies, the efflux assay was performed on MDR1-MDCK cells (i)
and on SKVLB cells (ii) in the presence of 10µM CsA (panel b), 50 µM adaGb3 added 30 min
(panel c) or 24h (panel d) prior to rho123 incubation, or 50 µM adaGalCer (panel e) also added
30 min before rho123 incubation. Untreated cells were used as controls (panel a).
3.4.7. Verotoxin Treatment Inhibits MDR1-mediated Rhodamine 123 Efflux
If the efficacy of adaGb3 to inhibit MDR1-dependent drug efflux was related to
alteration in the membrane organization of Gb3 containing lipid rafts, internalization of cell
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surfaceGb3 induced by verotoxin1 B subunit endocytosis might also affect MDR1-mediated
cellular drug efflux. Figure 3.11 shows that pretreatment of MDR1 MDCK or SK VLB cells
with VT1B (Figure 3.11, i and ii, panel d) or VT1 (Figure 3.11, i and ii, panel c) for 30 min at
37 °C inhibits rhodamine efflux. This was not observed for a non-Gb3-binding VT1B subunit
mutant (Bast et al., 1997). VT1 and VT1B were not as effective inhibitors as CsA or adaGb3 in
MDR1-MDCK cells but were as effective in SK VLB cells. VT2 had a similar but smaller
inhibitory effect than VT1 (not shown). The Gb3 content of SK VLB cells is significantly less
than that of MDR1-MDCK cells.
Figure 3.11. Verotoxin-mediated Internalization of Gb3 Inhibits MDR1-dependent
Rhodamine Efflux. MDR1-MDCK (i) and SKVLB (ii) cells were pretreated with 4 µM VT1
holotoxin or the pentamer of VT1B subunits at 37 °C for 30 min prior to analysis of rho123
efflux by confocal microscopy. Untreated control cells (panel a), 10 µM CsA treated cells
(panel b), 4 µM VT1 holotoxin (panel c), 4 µM VT1B subunit (panel d), and 4 µM VT1B
mutant (panel e) are shown.
3.4.8. AdaGb3 Inhibits MDR1-mediated Vinblastine Efflux in Polarized Gastrointestinal
Epithelial Cells
The ability of adaGb3 to inhibit MDR1 transport was analyzed by studying the flux of
radiolabeled vinblastine in the apical:basal (A:B) and in the basal:apical (B:A) direction, as
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compared with verapamil, a well known inhibitor of MDR1 (Figure 3.12). Apical/basolateral
dependence of inhibition was also evaluated by adding adaGb3 or verapamil, known MDR1
inhibtor, as positive control, either on the apical side only or on both sides. Polarized C2BBe1
cells grown on filters were used for these experiments. Vinblastine basal-to-apical flux was
higher than apical-to-basal flux, consistent with the unidirectional net transport mediated by
MDR1 into the gastrointestinal lumen. 20 µM verapamil, a positive inhibitory control, decreased
vinblastine basal-to-apical flux and increased apical to-basal flux, showing inhibition of MDR1.
5 µM and 50 µM adaGb3 showed effects similar to those of verapamil, indicating similar
inhibition of MDR1. A dose response was also apparent. When the inhibitors were added only
to the apical side, similar results were obtained. As shown in Figure 3.12.A, there was an
increase in the A:B transport of [3H] vinblastine across C2BBe1 cells in the presence of
inhibitor in the following order: 20 µM verapamil (3-fold) < 5 µM adaGb3 (3.4-fold) < 50 µM
adaGb3 (4.5-fold) (Table 3.1). Similar results are shown in Figure 3.12.B, where the inhibitor
was only added on the apical side as follows: 20 µM verapamil (1.5-fold) < 5 µM adaGb3 (3-
fold) < 50 µM adaGb3 (3.6-fold) (Table 3.1). There was also a decrease in the B:A transport of
[3H] vinblastine across C2BBe1 cells when they were treated with 50 µM adaGb3 (1.4-fold) and
similar to 20 µM verapamil (1.6-fold) treatment, even when adaGb3 was added to the apical side
only (Figure 3.12.C and 3.12.D, and Table 3.1). Pretreatment of C2BBe1 cells with 5 µM
adaGb3 showed no significant difference in the B:A transport of [3H] vinblastine (Figure 3.12.C
and 3.12.D, and Table 3.1). Results are expressed as a percentage of radioactivity accumulated
in the recipient side at each time point (100% is the initial concentration in the donor side of the
monolayer). C2BBe1cells are derived from the Gb3 expressing CaCo2 cell line (Kovbasnjuk et
al., 2001) and were confirmed to express Gb3.
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Figure 3.12. Effect of AdamantylGb3 on Gastrointestinal Epithelial Cell [3H]Vinblastine Transport. Polarized C2BBe1 cells were preincubated for 45 min with 5 µM (— —) or 50 µM (--- ---) adaGb3, or 20 µM verapamil (— —) as positive control in serum-free medium on both apical and basal sides, and apical side only. Untreated cells, — —. [3H] Vinblastine was added at time = 0 min to the apical side only for apical-to-basal (A:B) transport, or to the basal side only for basal-to-apical (B:A) transport. Each point is the mean ± S.D. (n = 3) expressed as a percentage (%) of flux over time. [14C] Mannitol was used as an extracellular marker. A and B, A:B [3H]vinblastine transport; C and D, B:A [3H] vinblastine transport. A and C, treatment with inhibitors (adaGb3 or verapamil) on both sides; B and D, treatment with inhibitors (adaGb3 or verapamil) on apical side only. Each point represents mean value and S.D. of three independent filter preparations. Dr Shinya Ito and Bernice Wang contributed to this figure and to the following Table.
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Table 3.1. Summary of effects of adaGb3 on MDR1-mediated [3H] vinblastine transport
Treatment on Apical and Basal Sides
(A+B)
Treatment on Apical Side Only
(A only)
Drug
Transport
Direction
20uM Ver
5uM AGb3
50uM AdaGb3
20uM Ver
5uM AdaGb3
50uM AdaGb3
A:B 3-fold increase
3.4-fold increase
4.5-fold increase
1.5-fold increase
3-fold increase
3.6-fold increase
[3H] Vinbl B:A 1.6-fold
decrease 1.1-fold decrease
1.4-fold decrease
1.4-fold decrease
1.2-fold decrease
1.3-fold decrease
3.4.9. AdaGb3 Differentially Binds the MDR1 Drug Binding Pocket as Compared with
Verapamil and Cyclosporin A
It was proposed (Loo and Clarke, 2002) that a common drug binding pocket lies at the
interface between the two six-member transmembrane domains of MDR1, and that the diverse
substrates bind through a “substrate- induced fit” mechanism. The common drug binding pocket
is relatively large (Loo and Clarke, 2001) and can accommodate different substrates
simultaneously. We evaluated the possible binding of adaGb3 to this common drug binding
pocket by cross- linking analysis (Loo et al., 2006a). If adaGb3 binds to this drug binding pocket,
cross- linking between cysteine residues of mutants prepared for binding analysis (Loo et al.,
2006a) would be inhibited. Membranes prepared from HEK 293 cells transfected with MDR1
expressing these cysteine substitution mutations (Loo et al., 2006a) were preincubated ±
adaGb3. These mutants contain the cross- linking cysteines at different “depths” within the 6th
and 7th TMDs. After the mixtures were treated with M14M crosslinker, they were subjected to
Western blot analysis. Figure 3.13 shows that pretreatment of the membranes with verapamil, a
known MDR1 inhibitor as positive control, completely prevented cross- linking in all three
mutations combinations. In contrast, no inhibition with adaGb3 pretreatment was seen for the
F343C containing mutants. However, when the mutation was earlier in TM6 (F339C), adaGb3
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prevented cross- linking, indicating that adaGb3 binding overlaps but is distinct from
CsA/verapamil within the common drug binding pocket.
Figure 3.13. Disulfide Cross-linking of MDR1 Mutants Defines AdamantylGb3-
Binding Site. Membranes prepared from HEK 293 cells expressing mutants
F343C(TM6)/F728C(TM7) (A, panel i) or F343C(TM6)/Q725C(TM7) (B, panel i) were
preincubated for 15 min at 22 °C with no drug (lane 2), 1 mM methanethiosulfonate-verapamil
(lane 3), or 0.11 mM adaGb3 (lane 4). C, panel i, membranes prepared from HEK 293 cells
expressing mutant L339C/F728C were preincubated for 15 min at 22 °C with different
concentrations of adaGb3 (0–500 µM) (lanes 2–6). The samples were cooled to 4 °C and then
treated with (A, panel i, and B, panel i, lanes 2–4, or C, panel i, lanes 2–6) or without (lane 1)
0.2 mM M14M cross- linker for 2 min at 4 °C. The reactions were stopped by addition of SDS
sampler buffer containing no reducing agent, and samples were subjected to immunoblot
analysis on 7.5% SDS-polyacrylamide gels. The positions of the cross- linked (arrow *) and
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mature (arrow) MDR1 are indicated. (A, panel ii, B, panel ii, and C, panel ii). The relative
extent of cross- linking was determined by scanning the gel lanes and pixel integration. The
amount of cross- linked product in the presence of drug substrate is compared with that without
drug substrate present (none; 100% (A, panel ii, and B, panel ii) or 0 µM; 100% (C, panel ii).
Dr David Clarke contributed to this figure.
3.5. Conclusions
MDR1 is the multimembrane spanning ATP-binding cassette (ABCB1)
transporter responsible for drug efflux in a variety of multiple drug resistant human tumours
(Lage, 2006). MDR1 is however, widely expressed in normal tissue, particularly kidney and GI
tract (Ambudkar et al., 2003). We have shown that the MDR1 drug efflux pump is, in part,
Golgi associated and is involved in neutral GSL biosynthesis (Lala et al., 2000). We propose
that MDR1 mediated glucosyl ceramide flipping within the Golgi is a primary mechanism by
which glucosyl ceramide is provided as a substrate for the various luminal glucosyl transferases
involved in neutral GSL biosynthesis (De Rosa et al., 2004). In addition to the interaction of
MDR1 with glucosyl ceramide, we found that cell surface MDR1 was partially co- localized
with Gb3 in MDR1 transfected cells (Lala et al., 2000). Inhibition of GSL biosynthesis results in
the loss of drug resistance and of cell surface MDR1. We speculated that an association of
MDR1 and cell surface GSLs, including Gb3, may be functional at the cell surface. MDR1 is
known to partition into plasma membrane lipid rafts and this has been implicated as a
mechanism for functional regulation of MDR1 (Lavie and Liscovitch, 2000). We therefore
tested the effect of adamantylGb3 on known MDR1 functions (De Rosa et al., 2008).
AdamantylGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1
expressing cells, in a manner similar to cyclosporine, a classical MDR1 inhibitor. In addition,
adamantylGb3 was able to reverse drug resistance to vinblastine in cell culture whereas
adamantyl galactosyl ceramide had no effect on drug resistance. Thus, adamantylGb3 is an
121
inhibitor of MDR1-mediated drug efflux. In addition to over expression in tumour cells
generating drug resistance, high expression of MDR1 in the apical membrane of gastrointestinal
mucosal epithelial cells can prove a significant restriction to the oral administration of a variety
of therapeutic drugs (Burger and Nooter, 2004). Vinblastine is an MDR1 substrate used as a
marker of MDR1 drug efflux and an index of oral drug bioavailability, since MDR1 is
expressed extensively within mucosal cells of the GI tract (Thiebaut et al., 1987). We found that
adamantylGb3 inhibited MDR1 mediated vinblastine efflux in polarized GI epithelial monolayer
cell culture systems as effective as verapamil, a standard MDR1 inhibitor (De Rosa et al., 2008).
AdamantylGb3 may provide a more physiologically-based approach to the inhibition of MDR1
function in cytotoxic drug resistance and restricted oral drug bioavailability. It is of interest to
note that Gb3 expression has been strongly associated with metastatic cancer within the GI tract
(Kovbasnjuk et al., 2005; Janssen et al., 2006) and Gb3 expression was markedly elevated in
biopsies of drug resistant ovarian carcinomas (Arab et al., 1997).
Further discussion of these results and future studies proposed are presented in Chapter
Six: Discussion.
Note: Ackerley, C. and Tilups, A. contributed to Figures 3.3.A and 3.4. Binnington, B.
and Bartoszko J. contributed to Figure 3.9. Ito, S. and Wang, B. contributed to Figure 3.12 and
Table 3.1. Clarke, D.M. contributed to Figure 3.13.
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CHAPTER FOUR: INHIBITION OF ABC DRUG TRANSPORTER, MDR1, AS
POSSIBLE THERAPY FOR FABRY DISEASE
Contents of this Chapter have been published in part in: Mattocks M, M. Bagovich, M.
De Rosa, S. Bond , B. Binnington, V. Rasaiah, and C.J. Medin, and C Lingwood . 2006.
Treatment of neutral glycosphingolipid storage disease via inhibition of the ABC Drug
Transporter, MDR1: Cyclosporin A can lower serum and some tissue globotriaosyl ceramide
levels in the Fabry’s mouse model. FASEB J. 273:2064-2075.
4.1. Introduction
Specific sugar hydrolases are the enzymes responsible for GSLs catabolism and are
located in the lysosomes together with their activator proteins. The lack of each of these
enzymes or activator proteins produced a distinct genetic deficiency. These diseases are grouped
together under the name of lysosomal storage diseases (LSDs) (Schuette et al., 1999; Kolter and
Sandhoff, 2006). The clinical symptoms of these diseases are mainly neurological and are
caused by the deposit of the enzyme substrate inside the cells that result mainly in lipid
inclusions that affect the normal physiological mechanisms of the cells. Different symptoms
were observed in patients according to the specific enzyme that is affected, the age at which the
first symptoms appeared and the residual activity of the enzyme that is present in their cells, as
only 10% of residual enzyme activity is enough to avoid clinical symptoms (Schuette et al.,
1999). One of the most used therapeutic approaches developed for treatment of these diseases is
enzyme-replacement therapy (ERT). This approach have been proved successful for two LSDs:
Gaucher characterized by glucosylceramide accumulation, and Fabry in which globotriaosyl
ceramide, Gb3, accumulates (Brady, 2003; Heukamp et al., 2003; Wilcox et al., 2004).
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Fabry patients treated with a-galactosidase, ERT, are able to reduce not only serum Gb3
levels by 50%, but also Gb3 levels in liver and kidney as well (Schiffmann et al., 2000;
Schiffmann et al., 2001). For a better understanding of this disease, Ohshima et al. developed an
animal model for Fabry disease, with complete lack of a-galactosidase (Ohshima et al., 1997).
Treatment of the Fabry animal model with ERT, depleted the elevated serum Gb3 levels,
compared to normal mice where the serum Gb3 levels are undetectable, but Gb3 levels in tissues
do not respond as well to ERT (Ohshima et al., 1997). In this last case, the administered enzyme
a-galactosidase needs to be taken up first by the specific cells in the tissue and then be directed
to the lysosomes. Chiba et al. have shown this in vitro where the replacement a-galactosidase
follows the mannose phosphate receptor pathway to get inside the cell (Chiba et al., 2002).
According to some data published by Ioannou’s group, a-galactosidase therapy seems to be
more effective in lowering liver Gb3 levels, some Gb3 lowering in spleen and heart, but it was
not effective at all for Gb3 in kidney (Ioannou, 2001).
One interesting particularity of the Fabry mouse model is that it does not develop the
clinical aspects of the disease (Eitzman et al., 2003), but in humans, Fabry disease pathology is
mainly evident in kidney (Schuette et al., 1999), major site of Gb3 synthesis in man (Boyd and
Lingwood, 1989), and in the heart, due to the association of Gb3 and the microvasculature. In
normal human kidney, Gb3 is present in the glomerulus, while in the mouse, is not (Lingwood
and Nutikka, 1994; Rutjes et al., 2002; Chark et al., 2004).
ERT seems to be very effective for Fabry disease but its extraordinary cost does not
allow every patient to afford it. Effective and more affordable treatment approaches need to be
developed. Gene therapy is a possible alternative for treatment of Fabry patients but it still needs
more work (Yoshimitsu et al., 2004). Another alternative therapeutic approach is the use of
glucosylceramide synthase inhibitors in order to block GSLs synthesis. As this enzyme is the
first one in the GSLs synthesis pathway, this strategy can be used for Fabry patients (restriction
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in Gb3 synthesis), and for Gaucher patients by restriction of GlcCer synthesis. Examples of
these inhibitors include PDMP, or P4 with improved selectivity on the inhibition of GlcCerS
(Lee et al., 1999), that helps with this approach (Abe et al., 2000; Abe et al., 2001). Other types
of glucosylceramide synthase inhibitors, like N-butyldeoxynojirimycin, were used effectively in
animal models of LSDs (Platt et al., 2003) and in clinical trials for Gaucher disease (Zimran and
Elstein, 2003; Futerman et al., 2004), but have other side effect such as the inhibition of
glucosidase processing of N-linked high mannose oligosaccharides (Tian et al., 2004) and
glycogen breakdown (Andersson et al., 2004).
One particular aspect of the synthesis of GlcCer is that it is made on the outer leaflet of
the Golgi bilayer (Lannert et al., 1998) but it needs to access the Golgi lumen to become further
elongated by glycosyltransferases that leads to the synthesis of more complex GSLs. There is
previous evidence in the literature that MDR1 can function as a glycolipid flippase (van
Helvoort et al., 1996; Eckford and Sharom, 2005). Dr Lingwood’s group has previously shown
evidence that MDR1 can be responsible for this translocation in the majority of cultured cells
(Lala et al., 2000; De Rosa et al., 2004). It has also been reported that increased levels of GlcCer
in MDR cells are a way to avoid ceramide- induced apoptosis (Lavie et al., 1996; Liu et al.,
2004), but some other reports do not agree (Norris-Cervetto et al., 2004). It has been proposed
that MDR1-mediated GlcCer Golgi translocation could be a component of this resistance.
However, we found that MDR1-translocated GlcCer is only associated with neutral GSL
synthesis (De Rosa et al., 2004) because inhibition of MDR1 does not affect cellular ganglioside
synthesis. Taking into consideration this last reported evidence, MDR1 inhibition provides a
much more selective treatment approach than the substrate reduction therapy as a clinical
management for Fabry disease. The vast experience in MDR1 modulation in clinical cancer
settings provides an additional advantage for this therapeutic approach since dosage and toxicity
for these inhibitors are very well known. Kidney and liver known MDR1 expression (Chin and
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Liu, 1994; Ernest et al., 1997) also matches the fact that these two sites are also the main sites
for Gb3 accumulation in the Fabry mouse model, making this approach feasible.
Third generation MDR1 inhibitors have been developed to improve specificity and to
minimize toxic side effects from first and second generation inhibitors. XR9576 (Tariquidar)
was discovered through a chemical program aimed to improve the potency and physiochemical
characteristics of MDR1 inhibitors (Mistry et al., 2001). Tariquidar was shown to potentiate the
cytotoxicity of clinical cancer drugs such as doxorubicin, paclitaxel, etoposide and vincristine
(Mistry et al., 2001). OC144-093, a novel substituted diacylimidazole, was synthesized
according to a solid phase combinatorial chemistry technology called OntoBLOCK system, a
(Newman et al., 2000). It was shown to be orally active, potent, and non toxic as MDR1
inhibitor (Newman et al., 2000).
We determined the effect of MDR1 inhibition on Gb3 synthesis in the Fabry mouse
model to evaluate this approach as a possible and reliable therapy for Fabry disease. A previous
study already characterized the abnormal Gb3 synthesis in this model, and has shown that
MDR1 inhibition could be a viable potential approach for the reduction of Gb3 in both liver and
heart of this model (Mattocks et al., 2006).
4.2. Materials and Methods
4.2.1. Materials
VT1 was purified as described (Rutjes et al., 2002). Verotoxin 1 B subunit (VT1 B
subunit) (Ramotar et al., 1990) was purified by affinity chromatography as described
(Boulanger et al., 1994). Rabbit polyclonal antiserum against purified VT1 B subunit was
prepared as described previously (Boyd et al., 1991). CsA was purchased from Sigma. MDR1
third generation inhibitors, XR9576 Tariquidar (free base and mesylate salt) was synthesized by
Xenova (Xenova Ltd., UK) and OC144-093 (free base and mesylate salt) was synthesized by
Ontogen Corporation (Carlsbad, CA, USA).
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4.2.2. Cell Culture
EBV-transformed B-lymphoblastoid cell lines from Gaucher type 1 and Fabry disease
(kindly supplied by J. Clarke, Hospital for Sick Children, Toronto, Canada) were cultured in
RPMI +15% fetal bovine serum (FBS) ± 4 µM CsA for 4 days. CsA induces a <10% reduction
in growth rate, compensated for in ana lyses.
4.2.3. Neutral Glycolipids Extraction and Analysis
Ten million cells from each cell line were scraped from the culture dish, washed with
phosphate buffer saline (PBS; CellgroTM, Mediatech Inc., Herndon, VA) and extracted with 20
volumes of chloroform/methanol (2:1) as described previously (Pudymaitis et al., 1991). The
extract was partitioned against water, and the lower phase was dried, redissolved in
chloroform/methanol (98:2) and separated by silica chromatography (Boyd and Lingwood,
1989). The column was washed extensively with chloroform, and the glycolipid fraction was
eluted with acetone/methanol (9:1), evaporated, re-dissolved in chloroform/methanol (2:1), and
separated on silica plates (Macherey-Nagel Silica gel 60 [40-63 µM]; Caledon, Georgetown,
ON) by thin- layer chromatography (TLC) using the solvent system chloroform/methanol/water
(65:25:4). Glycosphingolipids (GSLs) were visualized by staining with orcinol reagent, and Gb3
was detected by TLC overlay binding with VT1. Purified glucosylceramide (GluC),
lactosylceramide (LacC), globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4) were
run on the same plate as standards.
4.2.4. Gangliosides Extraction and Analysis
For ganglioside analysis, 107 cells from each Fabry cell line were harvested by scraping
and pelleted by centrifugation for 5 min at 300g. Cells were washed three times with PBS and
resuspended in distilled water. Total lipids were extracted with 20 volumes of
chloroform/methanol (2:1) as described previously (Pudymaitis et al., 1991). Gangliosides were
separated from pooled upper and lower phases by ion exchange chromatography on DEAE-
127
Sephadex A-25 (acetate form) (Yu and Ledeen, 1972; Yogeeswaran and Hakomori, 1975).
Gangliosides were eluted from the column with 0.4 M sodium acetate in methanol, and were
taken to dryness with a gentle stream of nitrogen. Phospholipids were degraded by mild alkaline
hydrolysis with 0.2 M NaOH in methanol for 1 h at 42ºC. The samples were neutralized with
0.2 mM HCl and then de-salted by running them through a silica-based C-18 cartridge (Sep-
PakTM; Waters Associates, Mississauga, ON). Individual gangliosides were separated on silica
plates (Macherey-Nagel Silica gel 60 [40-63 µM]; Caledon, Georgetown, ON) by TLC using
the solvent system CHCl3/CH3OH/0.2% CaCl2 (50:45:10), and visualized by staining with the
resorcinol-HCl reagent (Svennerholm, 1957). Purified GM3 was run on the same plate as
standard.
4.2.5. Verotoxin 1 Thin Layer Chromatography Overlay
Aliquots of the GSLs extracts were separated on silica plates (Macherey-Nagel Silica gel
60 [40-63 µM]; Caledon, Georgetown, ON) by TLC in chloroform/methanol/water (65:25:4).
The plates were dried and blocked with 1% gelatine in water at 37°C overnight. After being
washed three times with 50 mM TBS (50 mM Tris buffer, pH 7.4, with 0.9% NaCl) to eliminate
traces of gelatine, the plates were incubated with 0.1 µg/ml purified VT1 for 1 h at room
temperature with gentle shaking. They were washed again with TBS, and then plates were
incubated with mouse monoclonal anti-VT1 antibody (Boulanger et al., 1990a) (2 µg/ml) for 1 h
at room temperature with shaking, followed by incubation with goat anti-mouse IgG horseradish
peroxidase-conjugated antibody (diluted 1:2000 in TBS) (Sigma, St. Louis, MO), also for 1 h at
room temperature with shaking. Toxin binding was visualized using a 3 mg/ml solution of 4-
chloro-1-naphthol peroxidase substrate in methanol, freshly mixed with 5 volumes of TBS and
1:1000 dilution of 30% H2O2 (Lingwood et al., 1987). Colour development was stopped by
extensive but gentle washing under tap water, and the plates were finally dried under a blow
dryer.
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4.2.6. Metabolic Labelling of MDR1-MDCK Glycosphingolipids
Cellular GSLs from Fabry cell lines were radiolabeled by incubating cells with [14C]
serine (0.5 µCi/ml) for 72 h, with or without 4 µM CsA. After removal of medium, cells were
rinsed twice with PBS (pH 7.4) and harvested by gently scraping. Cellular lipids were extracted
and applied on DEAE-Sephadex A-25 (acetate form) as above (Yu and Ledeen, 1972;
Yogeeswaran and Hakomori, 1975). Neutral GSLs were unbound and gangliosides were eluted
with 0.4 M ammonium acetate (Yogeeswaran and Hakomori, 1975). Lipids were separated by
TLC, and labelled species detected by autoradiography scanned in a Phospho Imager and
quantitated by ImageQuant Version 1.2 software (Molecular Dynamics, 1997) (Amersham
Biosciences, CA, USA). Purified neutral glycolipids and gangliosides as standards were run on
the same plates and developed with orcinol staining.
4.2.7. Cytotoxicity Assay
MDR1-MDCK cells were cultured in the presence of either 4 µM CsA, 100 µM of
Tariquidar, or 2 µM of OC144 for 4 days, and tested for sensitivity to vinblastine or VT1 over 3
days relative to untreated cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (DDTB) assay (Mosmann, 1983). The parental drug-sensitive cell line, MDCK-1, was
also cultured in the presence of either 4 µM CsA, 100 µM of Tariquidar, or 2 µM of OC144 for
4 days as a control. Dose-dependence curves for each of the MDR1 inhibitors were performed
to determine the highest non-toxic concentration of the analog that was used in the experiments.
Briefly, log phase pretreated or untreated cells were plated in 96-well microtiter plates were
incubated in triplicate with 200 µl of tenfold dilutions of VT1, or vinblastine at 37ºC, 5% CO2.
in a-minimal essential medium with 5% fetal bovine serum and incubated at 37°C in 5% CO2.
After 72 h, 20 µl of a 5 mg/ml DDTB solution was added to the wells and incubated at 37°C in
5% CO2 for 4 h. Supernatants were removed, and 100 µl of acidified isopropyl alcohol was
added and incubated in the dark at room temperature for 30 min. Plates were agitated for 2–3
129
min, then read on an enzyme-linked immunosorbent assay reader at 490 nm. The concentration
required to inhibit growth by 50% (IC50 values) was calculated from the cytotoxicity curves
using GraphPad Prism 3.0 software (GraphPad Software Inc., San Diego). The fold reversal of
multidrug resistance was calculated by dividing the IC50 values in the absence of the MDR1
inhibitors by those in the presence of the MDR1 inhibitors.
4.2.8. Treatment of Neonatal Fabry Mice
Thirty-nine Fabry mice and 24 C57 Black 6 mice as controls divided into 4
groups were injected with vehicle (0.9% NaCl + 6% EtOH), 30 mg/kg of CsA or any of two 3rd
generation MDR1 inhibitors, 2 mg/kg tariquidar or 20 mg/kg OC144, twice a week since they
were 7 days old and until they were 10 weeks old (Scheme 4.1). All drugs were diluted in 0.9%
NaCl, also used as control. Then, they were sacrificed and organs such as heart, liver, spleen,
kidneys and brain were obtained for VT1 imunostaining of cryosections, and GSLs tissue
extraction for analysis of mainly Gb3 by TLC overlay and HPLC. Experimentation using the
Fabry mouse is necessary to demonstrate the in vivo potential of MDR1 inhibition as an
approach to treatment of Fabry disease in man and was carried out under ethical approval. Mice
were euthanized under conditions of minimized trauma (Anesthetic: 100 ug/g Ketalene + 10
ug/g Xylazine).
130
* vehicle: 0.9% NaCl + 6% EtOH
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
* vehicle: 0.9% NaCl + 6% EtOH
Qualitative monitoring of Gb3 levels in liver and heart by immnohistochemistryand Gb3 quantitative analysis in liver by HPLC
at 10 weeks old* vehicle: 0.9% NaCl + 6% EtOH
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
Fabry Mice (N=39) Black 6 Mice (N=24)
7 days old, 2X/week
Vehicle*(N=8)
CsA(N=11)
MDR1inhA
(N=11)
MDR1inhB(N=9)
Vehicle*(N=6)
CsA(N=6)
MDR1inhA(N=6)
MDR1inhB(N=6)
* vehicle: 0.9% NaCl + 6% EtOH
Qualitative monitoring of Gb3 levels in liver and heart by immnohistochemistryand Gb3 quantitative analysis in liver by HPLC
at 10 weeks old
Scheme 4.1. Protocol for Treatment of Neonatal Fabry Mice with MDR1 Inhibitors.
4.2.9. Tissue Extraction of Neutral GSLs
One hundred to 120 mg of tissue was homogenized in 9 volumes of cold PBS. Aliquots
were stored at -20°C for protein analysis and the rest extracted first in 5.5 volumes of isopropyl
alcohol (IPA) for 1h. Then the extraction continued in 3.5 volumes of chloroform for another
hour. The extracts were filtered through glass wool and an aliquot was separated for HPLC
analysis. They were dried under N2 and saponified overnight in 0.1N NaOH in MeOH at 37 °C
(Boyd and Lingwood, 1989). The glycolipid extract was neutralized, partitioned against water
and was used for VT1 TLC overlay and orcinol staining of lipids, as well as Gb3 HPLC analysis.
4.2.10. VT1 Tissue Staining
Five-micrometer frozen tissue sections were air-dried overnight at room temperature on
the lab bench. When dry, a PAP hydrophobic barrier pen was used to encircle sections.
Throughout all incubation steps, slides were kept in a humid chamber at room temperature.
Sections were blocked with endogenous peroxidase blocker (Universal Block, KPL Inc.,
131
Gaithersburg, MD) for 20 min. After extensive rinses with 1M NaCl / Pi solution, sections were
blocked with 1% normal goat serum / NaCl / Pi (NGS–NaCl / Pi) for 20 min. Without washing,
sections were then stained with VT1 (200 ng/mL) in NGS–NaCl / Pi) for 30 min. After five
vigorous rinses with NaCl / Pi, sections were incubated with rabbit anti-VT1B (Mattocks et al.,
2006). (1:1000 in NGS–NaCl / Pi) for 30 min, washed and then incubated with HRP-conjugated
goat anti-(rabbit IgG) (1 : 500 in NGS–NaCl / Pi) for 30 min. Following washing, sections were
developed using DAB substrate for 5 min. To stop the DAB reaction, sections were dipped in
distilled water for 4 min. Hematoxylin counterstain was applied for 30 s; excess staining was
removed by immersing sections in distilled water for 4 min and ‘blued’ by immersing in tap
water for 4 min. Sections were then dehydrated for 2 min in each of 70%, 95% and 100%
ethanol, cleared in xylene for 5 min and mounted in Permount.
4.2.11. Epitope Unmasking Treatment
Liver sections were either incubated with 10 mM methyl-ß-cyclodextrin in water
for 45 in at 37°C. After extensive washing with TBS, sections were blocked with endogenous
peroxidase blocker and 1% NGS/TBS as above, and successively treated with 1 µg/ml VT1,
rabbit anti-VT1 6869, and HRP-conjugated goat anti-rabbit in the same manner as described
before.
4.2.12. Anti-Gb3 Tissue Staining
Liver sections were treated with endogenous peroxidase blocker, Avidin/Biotin
blocker, and 1% NGS/TBS as above, with TBS washing between steps. After a 30 minute
incubation with mAb 38.13 anti-Gb3 (5 µg/ml), sections were successively treated wit biotin-
conjugated goat anti-rat IgM (1:1000), and Vecstatin ABC Elite reagent (Vector, CA, USA).
Sections were counterstained with Mayer’s Hematoxylin (Dako, CA, USA), washed with water
and then dehydrated, cleared and mounted with Permount (Sigma).
132
4.2.13. HPLC Quantitation of Gb3
Quantitative analysis of Gb3 was performed by normal-phase HPLC by using a Sedex
55 evaporative light scattering detector (ELSD) as previously described (Ohshima et al., 1999).
One to 5 g tissue was minced with a scalpel and then homogenized with a Polytron or hand
homogenizer in a known volume of PBS. More PBS may be required to rinse out the
homogenizer. To the total volume of PBS, 15 volumes of chloroform:methanol 2:1 were added.
For tissue extracts, saponification is required to obtain good HPLC data. The lipid residue is
resuspended in methanol for 2 to 24 hours. Then, it was neutralized with HCl, partitioned as
above then the lower phase was washd once with theoretical upper phase (C/M/W; 3:48:47).
Dry down the lower phase and it was dissolved in a minimum volume of chloroform:methanol
(2:1) and chromatographed on a Luna 3 Silica column (150 3 4.6 mm) from Phenomenex,
Belmont, CA. Glycolipids were eluted with a solvent containing
chloroform:methanol:water:NH4OH (66:30:3.5:0.5) at a flow rate of 1 ml per minute. The
evaporator on the ELSD was set at a temperature of 40°C, nitrogen pressure was 2.4 bar and the
gain at 10. Standard Gb3 was obtained from Matreya, Pleasant Gap, PA (Figure 4.1)
Standards Untreated 10 weeks old C57 Black 6 WT (liver)
Untreated 10 weeks old Fabry mice (liver)
Standards Untreated 10 weeks old C57 Black 6 WT (liver)
Untreated 10 weeks old Fabry mice (liver)
Figure 4.1. Accumulation of Gb3 in Fabry Mice by 10 weeks. Liver GSLs extractions of the
corresponding 10 weeks-old Black 6 WT and Fabry mice were analyzed by HPLC and
compared with the curve of Gb3 standards. Dr Jeffrey Medin and Vanessa Rasaiah
contributed to this figure.
133
4.3. Results
4.3.1. MDR1 inhibition in LSD cell lines
Epstein Barr virus (EBV) transformed B-cell lines from Gaucher and Fabry LSD
patients were cultured with 4 µM CsA for four days. The GSLs fractions were purified and
separated by thin layer chromatography (TLC). Figure 4.2.shows the accumulation of glucosyl
ceramide (GlcCer) was prevented in CsA-treated Gaucher B lymphoblasts. Three cell lines were
tested. Glc-Cer accumulated in each, but in one cell line, lactosyl ceramide accumulated also
(Figure 4.2, lane 6). In each case, CsA was found to delete GlcCer and reduce other neutral
GSLs present. Inhibition of MDR1-mediated GlcCer translocation results in increased access to
the cytosolic glucocerebrosidase (Forsyth et al., 1993) which is not defective in Gaucher LSD.
CsA treatment of a Fabry B-cell line (Figure 4.2.B–F) also showed significant inhibition of
accumulated Gb3, monitored by orcinol stain (Figure 4.2B) and VT1/TLC overlay (Figure 4.2C).
This indicates residual a-galactosidase activity in this cell line. Metabolic labeling of neutral
GSLs (including Gb3) within the Fabry cell line was prevented by CsA (Figure 4.2D),
confirming MDR1 inhibition reduces de novo Gb3 synthesis. Steady-state levels (Figure 4.2E)
and metabolically labeled (Figure 4.2F) gangliosides in this Fabry cell line were unaffected by
CsA. GM3 is the major ganglioside present but additional, more complex gangliosides were
detected by metabolic labeling.
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Figure 4.2. Effect of Cyclosporin A on Cultured Gaucher and Fabry B-cell Line
Glycosphingolipids The neutral GSL fraction (from 2 · 106 cells per lane) was separated by thin
layer chromatogram (TLC) (C /M/W 65 : 25 : 4 v/ v / v). The doublets corresponding to GlcCer
and Gb3 are shown by arrows. (A) Gaucher lymphoblastoid cell lines, detected using orcinol
spray. Lane 1, GSL standards, GlcCer, GalCer, LacCer, Gb3, Gb4, Gb5 (Forssman) as indicated.
Lanes 2, 4, 6, Neutral GSLs of untreated 5072, 5410, 5831 Gaucher cell lines. Lanes 3, 5, 7,
Neutral GSLs of CsA-treated 5072, 5410, 5831 cell lines. (B–F) Fabry lymphoblastoid cell line.
Cells were grown with 14C-serine. 14C-radiolabeled GSLs were detected by phosphoimaging.
(B) Orcinol detection of total neutral GSL fraction, (C) VT1 overlay of panel B to detect Gb3
only, (D) 14C-metabolic radiolabeled GSL phosphoimage of panel B. Lane 1, GSL standards as
in (A, lane 1); lane 2, untreated cells; lane 3, CsA-treated cells. The 14C-radiolabeled species
below Gb3 were not characterized. (E) The ganglioside fraction from 14C-labeled Fabry cells
was separated by TLC (C /M/W 60 : 25 : 10 0.2 M CaCl2 v / v / v) and detected using orcinol
(GM3), or (F) phosphoimaging of the 14C-metabolic labeled species. Lane 1, ganglioside
standards GM2 and GM1 as indicated; lane 2, untreated cells; lane 3, CsA-treated cells. The
accumulated lymphoid GlcCer in Gaucher cells was eliminated by CsA. The extent to which
more complex neutral GSLs were reduced varied between cell lines. CsA treatment of Fabry
cells significantly reduced the Gb3 and neutral GSL content without effect on the ganglioside
profile.
Asthe Fabry mouse has no a-galactosidase activity and already accumulated Gb3 cannot
therefore turnover, Mattocks et al. (Mattocks et al., 2006) designed a treatment protocol in our
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lab in which the effect of MDR1 inhibition by CsA on accumulation of Gb3 via de novo
synthesis was assessed. This protocol consisted of 12 adult Fabry mice treated with a-
galactosidase (enzyme replacement therapy, ERT) but 6 of them also received CsA. Serum Gb3
levels were monitored for nine weeks post ERT at wich time some organs were extracted for
Gb3 content. VT1/TLC overlay and histochemistry were used to determine Gb3 localization on
tissues cryosections. These adult Fabry mice, treated with both conventional enzyme
replacement therapy and CsA, showed not only long term serum Gb3 recovery to lower levels
but depletion of Gb3 in the liver as well (Mattocks et al., 2006).
From the results of this study, we decided to treat neonatal Fabry mice with CsA alone,
or with either one of two other MDR1 third-generation inhibitors, tariquidar and OC144, to
prevent tissue Gb3 buildup, as a way to assess if MDR1 inhibition could offer an alternative
therapeutical approach for Fabry disease.
4.3.2. Accumulation of Gb3 in Fabry mice by 10 weeks
We first analyzed the time course of Gb3 buildup in Fabry mice in order to
determine the period of time that the neonatal Fabry mice need to be treated with MDR1
inhibition to be able to see, if there is any, significant differences in Gb3 levels between treated
and untreated Fabry mice.
Liver GSLs extractions from groups of 3 Fabry mice and 3 C57 Black 6 WT
mice, at 4, 6, and 10 weeks after birth, were performed and analyzed by HPLC. In Figure 4.1.,
significant Gb3 buildup levels were identified in Fabry mice versus Black 6 WT mice at 10
weeks.
Accumulation of Gb3 is also shown within frozen sections of liver of neonatal Fabry
mice by VT1 immunostaining in Figure 4.3 at 10 weeks of age, compared to undetectable Gb3
levels in C57 Black 6 control mice at the same age.
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Figure 4.3.Accumulation of Gb3 in Livers of Fabry Neonatal Mice. VT1 immunostaining of
liver frozen sections from Fabry unt reated mice compared with liver frozen sections of Black 6
WT mice at 10 weeks of age. Arrows in the Fabry untreated panels show VT1 immunostaining.
Magnification x10.
4.3.3. Inhibition of MDR1 prevents Acumulation of Gb3 in Fabry mice livers
The localization of Gb3 within frozen sections of liver from MDR1 treated Fabry and
untreated Fabry mice monitored by VT1 binding is shown in Figure 4.4. Within the Fabry liver,
VT1 binding detected Gb3 in Kupffer cells, distributed throughout the section, and in cells lining
the portal veins. The elevated levels of Gb3 detected in untreated Fabry mice livers were
significantly reduced after treatment with any of the three inhibitors, CsA, tariquidar or OC144
(Figure 4.4).
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In the livers of treated Fabry mice, VT1 staining of Kupffer cells was greatly reduced.
Gb3 expression in central and portal vein endothelial cells was significantly reduced and many
vessels negative for Gb3 were observed in CsA-treated mice, but more dramatically in tariquidar
and OC144-treated mice. Portal triad staining was unaffected by MDR1 inhibition treatment as
has been described before (Mattocks et al., 2006).
Figure 4.4. Comparison of VT1 Staining of Fabry Untreated Liver Tissue and
Fabry CsA, OC144 and Tariquidar Treated Liver Tissue. Magnification: x10 for the first
two panels in each row and x20 for the last two panels in each row. Each row represents a single
animal. Arrows indicate specific VT1 immunostaining.
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4.3.4. Inhibition of MDR1 prevents buildup of Gb3 in Fabry mice hearts
The localization of Gb3 within heart frozen sections from MDR1 treated Fabry
mice and untreated Fabry mice monitored by VT1 binding is shown in Figure 4.5. VT1 staining
in untreated Fabry mice corresponds to a subpopulation of larger blood vessel endothelial cells
already described (Mattocks et al., 2006). Treatment with MDR1 inhibitors suppressed this
staining pattern (Figure 4.5).
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Figure 4.5. Comparison of VT1 staining of Fabry Untreated Heart tissue and Fabry
CsA, OC144 and Tariquidar treated heart tissue. The first three panels in each row were
counter stained with hematoxylin, while the last panel in each row was not counter stained. Each
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row represents a single animal. Magnification: x10. Arrows indicate specific VT1
immunostaining.
4.3.5. Quantitative liver Gb3 analysis by TLC and HPLC in treated vs. untreated mice
In order to quantify the difference in Gb3 levels in untreated and treated Fabry
liver tissues, liver GSL extractions were analyzed by thin layer chromatography TLC (Figure
4.6) and HPLC (Figure 4.7). Surprisingly, we found that liver Gb3 levels of treated Fabry mice
with any of the three MDR modulators, were equal or even higher than the Fabry untreated
animals (Figures 4.6 and 4.7).
Figure 4.6. Glycolipid profile of liver extractions of Fabry untreated and MDR1
Fabry treated mice. Total GSLs extractions were analyzed on TLC plates (as described in
Materials and Methods) and stained with iodine (first panel), with orcinol (second panel), and
141
immunostained with VT1 for Gb3 overlay (third panel). The corresponding GSLs standards
were included in each TLC plate and their position indicated on the side of the plate.
Figure 4.7. Liver Gb3 Levels Quantitated by HPLC. Liver GSLs extractions were
analyzed by HPLC in order to quantitate Gb3 in each group of animals. Standard Regression
analysis (bottom panel). Dr Jeffrey Medin and Xin Fan contributed to this figure.
4.3.6. Cholesterol Depletion and anti-Gb3 liver staining
In order to determine if the discrepancy between the VT livers staining results
and the quantitation TLC and HPLC results was associated to the effect that any of the
treatments may be masking Gb3 due to cholesterol redistribution or masking the Gb3 VT binding
epitope, we pretreated liver sections of each of the groups with mehyl-ß-cyclodextran to
sequester cholesterol, or immunostaining with mAb anti-Gb3. Cholesterol depletion has effect
Gb3 levels in Liver
0.56 0.57
16.9915.12 19.81
15.98
48.48
0
10
20
30
40
50
60
Gb
3 (n
mol
/mg
pro
tein
)
B6 MaleB6 FemaleFabry Nacl MaleFabry CsA MaleFabry Nacl FemaleFabry CsA FemaleFabry OC114 Male
Gb3 levels in Liver
0.56 0.57
16.9915.12 19.81
15.98
48.48
0
10
20
30
40
50
60
Gb
3 (n
mol
/mg
pro
tein
)
B6 MaleB6 FemaleFabry Nacl MaleFabry CsA MaleFabry Nacl FemaleFabry CsA FemaleFabry OC114 Male
Gb3 standards
y = 40157x 2 + 2E+06x + 50160R2 = 0.9997
0
2000000
4000000
6000000
8000000
10000000
12000000
0 1 2 3 4 5 6Concentration
142
only for CsA treated liver section but was not effective with the other treatments (Figure 4.8).
Anti Gb3 staining resembles VT1 staining on liver treated sections (Figure 4.8).
143
Figure 4.8. Comparison of VT1 Staining, Cholesterol Depletion VT1 Staining and
Anti-Gb3 Staining of Liver Fabry Mice Sections. The first two panels were counter stained
with hematoxylin while the third was not counter stained. Liver sections of one representative
animal of each group were shown. In the first row of each group, the sections were
immunostained with VT1. In the second row, sections were also immunostained with VT1 but
they were previously treated with methyl-ß-cyclodextran (MBCD) to remove cholesterol. In the
third row, the sections were immunostained with anti-Gb3 for comparison. Arrows indicate VT1
specific immunostaining in the first two rows or specific Gb3 immunostaining. Magnification:
10x.
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4.4. Conclusions
Our group has already shown that the MDR1 inhibitor, cyclosporin A (CsA) can deplete
Gaucher lymphoid cell lines of accumulated glucosylceramide and Fabry cell lines of
accumulated globotriaosylceramide (Gb3), by preventing de novo synthesis (Mattocks et al.,
2006).
Liver and heart sections of Fabry mice treated with MDR1 inhibitors (CsA, tariquidar,
OC144) showed significant less Gb3 than liver and heart sections of untreated Fabry mice, and
thus, MDR1 inhibition offers a potential alternative therapeutic approach for Fabry disease
given the extraordinary cost of conventional enzyme replacement therapy.
Quantitation of Gb3 levels in liver extractions of treated versus untreated Fabry mice by
TLC and HPLC showed discrepant results than the ones obtained by VT1 tissue
immunostaining. Studies in human kidney have shown VT undetectable in renal glomeruli, but
cholesterol extraction induced strong VT1/VT2 glomerular staining, suggesting that cholesterol
can mask Gb3 in lipid raft domains (Khan et al., 2009). Cholesterol depletion was used as an
approach to overcome this discrepancy, but changes in VT staining were only seen in CsA
treated samples, suggesting that CsA treatment affect lipid environment and thus, masks Gb3.
Further discussion of these results and future studies proposed are presented in Chapter
Six: Discussion.
Note: Medin, C.J. and Rasaiah, V. contributed to Figure 4.2. Medin, C.J. and Wang, X.
contributed to Figure 4.7.
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CHAPTER FIVE: MDR1 DEPENDENT AND INDEPENDENT MECHANISMS
FOR GOLGI GLUCOSYLCERAMIDE TRANSLOCATION IN HeLa CELLS
Contents of this Chapter have been published in: De Rosa, M.F., and Lingwood C. A.
2009. Multidrug Resistance Protein (MDR1) and Glycosphingolipids Biosynhesis. In
GLYCOBIOLOGY RESEARCH TRENDS. Powell, G.and McCabe O. (Eds.), Nova Publishers,
NY.
5.1. Introduction
Ceramide has been a focus of attention for research due to its key role in the
GSLs biosynthetic pathway (Merrill, 2002) and in many intracellular signalling pathways
(Hannun and Obeid, 2002; Kolesnick, 2002). It can be synthesized through sphingomyelin
hydrolysis or de novo, by ceramide synthase (CerS), located on the cytosolic side of the ER
(Merrill, 2002). Its structure is a sphingoid long chain base with a fatty acid attached via an
amide bond. Back in 1940’s, its chemical composition revealed that the most common fatty acid
attached to its sphingoid base was stearic acid (C18:0). But later, with technical advancement, it
was reported that mammalian GSLs can contain a variety of fatty acids in terms of different
carbon chain length, from C14 to C32, predominantly saturated and with a- or ? -hydroxyl
groups (van Meer, 2005; Pewzner-Jung et al., 2006).
Guillas et al. reported the identity of CerS in yeast in 2001, when they showed
that the products of the Lag1p (longevity assurance gene) and Lacp1 genes were necessary for
the synthesis of C26- ceramide (Guillas et al., 2001). Homologs of the two Lag1 and Lac1
genes, isolated in a screen for longevity-related genes (D'Mello N et al., 1994) and in a data base
screen (Jiang et al., 1998) respectively, were originally found in human, mouse, and
Caenorhabditis elegans, but now their presence was shown in almost all species. Human and
mouse homologs were put together in a family of genes called Lass (longevity assurance) genes.
146
All members of the Lass family encode multi-transmembrane (TM) spanning proteins
(Venkataraman et al., 2002). Specific functional roles for each of the members identified in the
Lass family in mammals were determined by overexpression of their corresponding genes in
mammalian cells in culture. Venkataraman et al. reported that overexpression of UOG1 or
LASS1 produced a selective increase in C18-ceramide (Venkataraman et al., 2002). While
overexpression of LASS4 (TRH1) showed an increase in C18- and C20-ceramide synthesis,
overexpression of LASS5 (TRH4) showed selectivity for C16 acyl-CoA and consequently,
increase in C16-ceramide synthesis (Riebeling et al., 2003). LASS6 produce shorter acyl chain
ceramides (C14 and C16) (Mizutani et al., 2005), and LASS3 was reported to produce C18- and
C24-ceramides (Mizutani et al., 2006). Lahiri et al. confirmed in 2005, that the members of the
LASS family are ceramide synthases themselves and not merely regulators of ceramide
synthases encoded by other genes, by showing that purified LASS5 has CerS activity per se
(Lahiri and Futerman, 2005). It is not known yet why mammals have multiple CerS genes,
while all the other enzymes involved in GSLs biosynthesis have only one or two isoforms
(Venkataraman et al., 2002), but it suggests that each specific fatty acid ceramide might have an
important and specific role in the cellular metabolism.
Venkataraman et al. have shown that human embryonic kidney 293T cells transfected
with the uog1 gene and metabolically labeled with [4,5-3H]sphinganine or L-3-[3H]serine
became resistant to fumonisin B1, a potent inhibitor of ceramide synthase. These transfected
cells continued to produce ceramide but surprisingly, this C18-ceramide was further used to
synthesize only neutral glycosphingolipids and not gangliosides (Venkataraman et al., 2002).
A previous study from our group showed that while MDR1 inhibitors suppressed neutral
GSLs biosynthesis in 11 cell lines of different origins, HeLa cells ws the only cell line in which
GSLs biosynthesis was not inhibited by an MDR1 inhibitor, CsA (De Rosa et al., 2004). We
hypothesize that HeLa cells might be a good example to show that alternative mechanisms for
147
GlcCer translocation must exist besides MDR1. To confirm this hypothesis, we further studied
the effect of MDR1 inhibition on GSLs made via ceramide synthase with characteristic substrate
preference for a particular fatty acyl-CoA.
5.2. Materials and Methods
5.2.1. Materials
Plasmids pcDNA-UOG1(5211) and pcDNA 3.0 vector were a gift from Dr Anthony
Futerman (Weizmann Institute of Science, Israel) (Venkataraman et al., 2002). CsA and FB1
were purchased from Sigma. (St. Louis, USA).
5.2.2. Purification and Amplification of Plasmids
The uog1 fragment (1.3 kilobases) was released by restriction digestion using EcoR1 and
Xba I sites, and subjected to run in a 1% agarose gel in 0.5X TAE buffer to verify purity and
integrity. A second restriction option with EcoRI and Apa I, instead of Xba I, was used to finally
release the right DNA fragment since according to the first gel, the Xba I site was probably
destroyed when subcloning (Venkataraman et al., 2002). Standard Maxiprep procedure was
used for amplification of the plasmids. After DNA concentrations of plasmid and vector, and
purity ratio were determined, plasmid DNAs were ready for transfection.
5.2.3. Cell Culture and Transfection
HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)
(CellgroTM, Mediatech Inc., Herndon, VA), with high glucose (4.5 gm/L) and supplemented
with 10% heat- inactivated FBS. Twenty-four hours before transfection, exponentially growing
HeLa cells were grown on 10 cm tissue culture dishes in 10% FBS DMEM. The dishes were
incubated for 20-24 h, at 37°C in a humidified incubator with an atmosphere of 5% CO2. HeLa
cells were transfected by the calcium phosphate method.
148
5.2.4. Neutral Glycolipids Extraction and Analysis
Ten million cells from uog1 transfected or non-transfected HeLa cell line, treated with
fumonisin B or 4 µM CsA for 24 h, or untreated, were scraped from the culture dish, washed
with phosphate buffer saline (PBS; CellgroTM, Mediatech Inc., Herndon, VA) and extracted with
20 volumes of chloroform/methanol (2:1) as described previously (Pudymaitis et al., 1991). The
extract was partitioned against water, and the lower phase was dried, redissolved in
chloroform/methanol (98:2) and separated by silica chromatography (Boyd and Lingwood,
1989). The column was washed extensively with chloroform, and the glycolipid fraction was
eluted with acetone/methanol (9:1), evaporated, re-dissolved in chloroform/methanol (2:1), and
separated on silica plates (Macherey-Nagel Silica gel 60 [40-63 µM]; Caledon, Georgetown,
ON) by thin- layer chromatography (TLC) using the solvent system chloroform/methanol/water
(65:25:4). Glycosphingolipids (GSLs) were visualized by staining with orcinol reagent, and Gb3
was detected by TLC overlay binding with VT1. Purified glucosylceramide (GluC),
lactosylceramide (LacC), globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4) were
run on the same plate as standards.
5.2.5. Gangliosides Extraction and Analysis
For ganglioside analysis, 107 cells from each HeLa variant cell line were harvested by
scraping and pelleted by centrifugation for 5 min at 300g. Cells were washed three times with
PBS and resuspended in distilled water. Total lipids were extracted with 20 volumes of
chloroform/methanol (2:1) as described previously (Pudymaitis et al., 1991). Gangliosides were
separated from pooled upper and lower phases by ion exchange chromatography on DEAE-
Sephadex A-25 (acetate form) (Yu and Ledeen, 1972; Yogeeswaran and Hakomori, 1975)
(Yogeeswaran and Hakomori, 1975). Gangliosides were eluted from the column with 0.4 M
sodium acetate in methanol, and were taken to dryness with a gentle stream of nitrogen.
Phospholipids were degraded by mild alkaline hydrolysis with 0.2 M NaOH in methanol for 1 h
149
at 42ºC. The samples were neutralized with 0.2 mM HCl and then de-salted by running them
through a silica-based C-18 cartridge (Sep-PakTM; Waters Associates, Mississauga, ON).
Individual gangliosides were separated on silica plates (Macherey-Nagel Silica gel 60 [40-63
µM]; Caledon, Georgetown, ON) by TLC using the solvent system CHCl3/CH3OH/0.2% CaCl2
(50:45:10), and visualized by staining with the resorcinol-HCl reagent (Svennerholm, 1957).
Purified GM3 was run on the same plate as standard.
5.2.6. Metabolic Labelling of MDR1-MDCK Glycosphingolipids
Cellular GSLs from Fabry cell lines were radiolabeled by incubating cells with [14C]
serine (0.5 µCi/ml) for 72 h, with or without 4 µM CsA. After removal of medium, cells were
rinsed twice with PBS (pH 7.4) and harvested by gently scraping. Cellular lipids were extracted
and applied on DEAE-Sephadex A-25 (acetate form) as above (Yu and Ledeen, 1972)
(Yogeeswaran and Hakomori, 1975). Neutral GSLs were unbound and gangliosides were eluted
with 0.4 M ammonium acetate (Yogeeswaran and Hakomori, 1975). Lipids were separated by
TLC, and labelled species detected by autoradiography scanned in a Phospho Imager and
quantitated by ImageQuant Version 1.2 software (Molecular Dynamics, 1997) (Amersham
Biosciences, CA, USA). Purified neutral glycolipids and gangliosides as standards were run on
the same plates and developed with orcinol staining.
5.3. Results
5.3.1. Restriction Analysis for CerS1 (uog1)
The filter paper with adsorbed plasmids was cut into small pieces, resuspended in
ultrapure distilled water and 5 µL of this suspension was used to transform DH5a competent
cells. Miniprep procedure for plasmid DNA pur ification was used and restriction analysis with
EcoRI and XbaI was performed to release the uog1 fragment (Figure 5.1.A). We were not able
to obtain the right size fragment from this restriction, apparently because the XbaI site was
damaged in the subcloning procedure. When we tried a second restriction attempt with EcoRI
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and ApaI, the next available site in the uog1 DNA sequence, we were able to release the 1.3 kb
fragment of interest (Figure 5.1).
.
Figure 5.1. Restriction Analysis of CerS1 (uog1). Each restriction product was run in a
1% agarose gel. Panel A: lane 1, pcDNA 3.0 + EcoR 1; lane 2, pcDNA 3.0 + Xba 1; lane 3,
puog1 + EcoR 1; lane 4, puog1 + Xba 1; lane 5, puog1 + EcoR 1 + Xba 1; lane 6, 1 kb Ladder.
Panel B: lane 1, pcDNA 3.0 + EcoR 1; lane 2, pcDNA 3.0 + Apa 1; lane 3, puog1 + EcoR 1;
lane 4, puog1 + Apa 1; lane 5, puog1 + EcoR 1 + Apa 1; lane 6, 1 kb Ladder (New England
Biolabs). Arrow indicates fragment (1.3 kb).
5.3.2. C18 Ceramide Synthesis (CerS1 – uog1) is channeled into Neutral GSLs but not into
Gangliosides in HeLa Cells
The effect of CerS1 (uog1) transfection on GSL biosynthesis was analyzed. In Figure
5.2, the orcinol stained TLC showed reduced GSLs levels when HeLa cells were pretreated with
FB-1, a potent CerS inhibitor. Metabolic labeling of neutral GSLs, in the central panel, shows
that de novo synthesized Gb3 levels in HeLa CerS1 transfected cells were increased (arrow), and
that this increase is resistant to GSLs biosyntheis inhibition by FB-1. In contrast, ganglioside
levels were not affected by C18 CerS1 transfection. These results indicate that C18 ceramide
synthesis in HeLa cells due to transfection with CerS1 gene, is channeled into neutral GSLs but
not into gangliosides.
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Figure 5.2. CerS1 (uog1) Transfection of HeLa cells – Effect on Neutral and Acidic
GSLs biosynthesis. The neutral GSL fraction (from 2 · 106 cells per lane) was separated by thin
layer chromatogram (TLC) (C /M/W 65 : 25 : 4 v/ v / v). Left Panel, GSLs detected using
orcinol spray. Lane 1, GSL standards, GlcCer, GalCer, LacCer, Gb3, Gb4, Gb5 (Forssman) as
indicated. Lanes 2, neutral GSLs of untreated HeLa cells. Lanes 3, neutral GSLs of FB-1-
treated HeLa cells. Central and Right Panels, cells were grown with 14C-serine. 14C-
radiolabeled GSLs were detected by phosphoimaging. Central, Lane 1, GSL standards (orcinol
detection); lane 2, untreated HeLa cells; lane 3, FB-1-treated HeLa cells; lane 3, CerS1 HeLa
transfected cells; lane 4, CerS1 HeLa transfected cells treated with FB-1. Right, the ganglioside
fraction from 14C-labeled HeLa cells was separated by TLC (C /M/W 60 : 25 : 10 0.2 M CaCl2 v
/ v / v) and detected using phosphoimaging of the 14C-metabolic labeled species. Lane 1,
ganglioside standards GM3,GM2 and GM1 as indicated (orcinol detection); lane 2, untreated
HeLa cells; lane 3, FB-1-treated HeLa cells; lane 3, CerS1 HeLa transfected cells; lane 4,
CerS1 HeLa transfected cells treated with FB-1. Arrows show uog1 based Gb3 is insensitive to
FB-1 while endogenous Gb3 is sensitive to FB-1 (De Rosa, 2009).
5.3.3. C18 Ceramide Synthesis (CerS1 - uog1) is inhibited by CsA in HeLa cells
The effect of CsA, MDR1 inhibitor, was analyzed on uog1 transfected HeLa cells.
Although GSL biosynthesis in HeLa cells is not susceptible to CsA, de novo C18 neutral
GSLs synthesis from the uog1 C18 ceramide is inhibited.
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Figure 5.3. CsA Treatment of CerS1 (uog1) Transfected HeLa cells. Effect of
MDR1 inhibition on CerS1. The neutral GSL fraction (from 2 · 106 cells per lane) was separated
by thin layer chromatogram (TLC) (C /M/W 65 : 25 : 4 v/ v / v). Left Panel, GSLs detected
using orcinol spray. Lane 1, GSL standards, GlcCer, GalCer, LacCer, Gb3, Gb4, Gb5 (Forssman)
as indicated. Lanes 2, neutral GSLs of untreated HeLa cells. Lanes 3, neutral GSLs of CsA-
treated HeLa cells. Central and Right Panels, cells were grown with 14C-serine. 14C-
radiolabeled GSLs were detected by phosphoimaging. Central, Lane 1, GSL standards (orcinol
detection); lane 2, untreated HeLa cells; lane 3, CsA-treated HeLa cells; lane 3, CerS1 HeLa
transfected cells; lane 4, CerS1 HeLa transfected cells treated with CsA. Right, the ganglioside
fraction from 14C-labeled HeLa cells was separated by TLC (C /M/W 60 : 25 : 10 0.2 M CaCl2 v
/ v / v) and detected using phosphoimaging of the 14C-metabolic labeled species. Lane 1,
ganglioside standards GM3,GM2 and GM1 as indicated (orcinol detection); lane 2, untreated
HeLa cells; lane 3, CsA-treated HeLa cells; lane 3, CerS1 HeLa transfected cells; lane 4, CerS1
HeLa transfected cells treated with CsA. Arrows show uog1 based Gb3 is sensitive to MDR1
inhibitor CsA, while endogenous HeLa Gb3 is resitant to CsA (De Rosa, 2009).
5.4. Conclusions
It has been reported that MDR1 inhibitors, CsA or ketoconazole, inhibit GSL
biosynthesis in various cell lines studied but not in HeLa cells, as they do not express MDR1
(De Rosa et al., 2004). When these cells were transfected with uog1 gene, a lass gene (longevity
153
assurance gene family, recently renamed as CerS family) encoding a C18 ceramide synthase,
subsequent C18 GSL synthesis is fumonisin B resistant as previously reported (Venkataraman et
al., 2002) but CsA sensitive, indicating that both MDR1 dependent and independent
mechanisms for GlcCer translocation are present in these cells (Figures 5.2 and 5.3).
Future studies on HeLa cells and other cell lines transfected with other CerS - lass genes
will help to determine if any other fatty acyl-CoA, specific for ceramide synthesis is MDR
dependent, and will also help to elucidate different mechanisms for GlcCer Golgi translocation
in HeLa cells.
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CHAPTER SIX: DISCUSSION
Adamantyl Gb3 as an MDR1 Inhibitor
MDR1 is associated with drug failure in various cancers and reduced oral drug
bioavailability because the intestinal epithelial cell apical membrane is a prominent MDR1
expression site (Thiebaut et al., 1987). Therefore, an understanding of multidrug resistance is of
wide clinical importance. Although defining the mechanism of MDR1 remains a major
challenge, many newer MDR1 inhibitors have been developed. However, few of these achieve
clinical success because of intrinsic toxicities and effects on normal tissues expressing MDR1.
MDR1 inhibitors based on physiological substrates have not been reported. Although it is not
established that Gb3 is a substrate for MDR1, its precursor GSL, glucosylceramide, is a substrate
(De Rosa et al., 2004; Eckford and Sharom, 2005).
AdaGb3, a semi-synthetic analog of Gb3 with a marked increased solubility in water
which retains its membrane solubility characteristics, was originally designed to inhibit
verotoxin binding to its GSL receptor, Gb3, and achieved a 1000-fold enhanced inhibitory
activity as compared with the lipid-free sugar (Mylvaganam and Lingwood, 1999). Gb3 also
plays a role in human immunodeficiency virus infection (Puri et al., 1998a), within cholesterol-
enriched lipid rafts (Rawat et al., 2006). Cholesterol enhanced the interaction of gp120 and Gb3,
but adaGb3 proved an even more effective receptor (Mahfoud et al., 2002a). AdaGb3 inhibits
human immunodeficiency virus infection in vitro and prevents gp120-mediated host cell fusion,
opening a new therapeutic route (Lund et al., 2006; Lund et al., 2009)
The prevention of MDR1 cell surface expression by GSL depletion, partial VT1/MDR1
cell surface co-localization, and the inhibitory effect of VT1/VT1B on MDR1 infer a link
between Gb3 and MDR1, in addition to its flippase role in GSL biosynthesis already reported
(Lala et al., 2000; De Rosa et al., 2004; Mattocks et al., 2006).
155
Our findings that adaGb3 abrogated cell surface expression of MDR1 in both MDR1-
MDCK and SK VLB cells, increased intracellular MDR1, sensitized MDR1-MDCK and SK
VLB cells to vinblastine cytotoxicity similarly to CsA, increased apical-to basal transport of
vinblastine, even more effectively than verapamil, in C2BBe1 cells, and blocked rhodamine 123
efflux, similarly to CsA, show that adaGb3 is an effective inhibitor of MDR1.
Because MDR1 function is dependent on cholesterol-enriched lipid rafts (Barakat et al.,
2005; Orlowski et al., 2006), which are also GSL-enriched, and Gb3 is in rafts (Katagiri et al.,
1999), modulation of the MDR1/raft/GSL environment may be a feature of adaGb3 MDR1
inhibition. Our data show that adaGb3 or CsA treatment prevents cell surface MDR1
immunostaining, an important overlooked aspect of the mechanism of MDR1 inhibition,
whereas intracellular MDR1 accumulates. Thus, Gb3-containing lipid rafts may be important for
intracellular MDR1 surface trafficking. Inhibitor-induced loss of plasma membrane MDR1
expression has not been reported previously, but the increased intracellular accumulation of
MDR1 after CsA treatment, although counterintuitive, is often found (Jette et al., 1996). The
binding of the inhibitor to the drug site between TMD6 and TMD7 to prevent MDR1 surface
expression, and the similar effect of GSL depletion, implies that this drug-binding site may be a
lipid (GSL)-binding site, involved in MDR1 trafficking. GSLs can play roles in protein
intracellular transport (Sillence et al., 2002). The lipid flippase activity of MDR1 (van Helvoort
et al., 1996) may provide a basis for drug efflux (Higgins and Gottesman, 1992b) and surface
expression.
The effect of VT1B/VT1 to prevent rhodamine123 efflux further highlights the
importance of Gb3 containing lipid rafts for plasma membrane MDR1 function. Unlike adaGb3,
VT1B internalization/cell surface Gb3 depletion did not result in loss of cell surface MDR1
staining, although intracellular MDR1 synthesis was nevertheless increased (but less than for
adaGb3). A significant fraction of surface MDR1 is not co- localized with Gb3, and this could
156
therefore be VT1B-insensitive. This would be consistent with the more temporary VT1/VT1B
inhibition of MDR1-mediated drug efflux. VT1 internalization selectively depletes cell surface
Gb3 (Schapiro et al., 1998), and this can affect other cholesterol dependent cell surface antigens
(Jarvis et al., 2007). After internalization of the toxin-Gb3 complex, the plasma membrane Gb3
raft complement is reduced for at least 1 h (Falguieres and Johannes, 2006). The eventual
recovery of cell surface Gb3 rafts after VT1 treatment may explain the lack of effect of VT1B
treatment on long term MDR1-MDCK vinblastine resistance we observed. Treatment of mice
with VT2 has been shown to compromise the MDR1-dependent blood/brain barrier and to
induce MDR1 overexpression (Zhao et al., 2002). Our studies may provide a cellular
counterpart to these results.
The increase in cell surface expression of MDR1 seen in short time course with MDR1
inhibitors has been described, but this is nonfunctional because rhodamine efflux is inhibited at
this time. The down-regulation of surface MDR1 expression following more prolonged cell
treatment with an MDR1 inhibitor has not been generally reported, but this could be a
significant component in the mechanism of inhibitor action. This clearly occurs while total
cellular MDR1 is markedly elevated, implying a differential effect on cell surface MDR1
trafficking. This may be a self-amplification phenomenon. If GSL/cholesterol-enriched lipid
microdomains are a key component in MDR1 trafficking/maturation/function, inhibitors will
prevent Golgi MDR1-mediated GlcCer translocation to inhibit neutral GSL biosynthesis (De
Rosa et al., 2004) and thus further compromise MDR1 membrane organization/function. If
MDR1 is also involved in cholesterol homeostasis (Luker et al., 1999), MDR1 inhibition could
also restrict raft function to further deplete MDR1 activity.
The interface between the TM6 and TM7, which are the C-terminal and N-terminal
membrane spanning sections of the 1st and 2nd TMD hexamers, has been implicated as the
position of the MDR1 common drug-binding site (Loo and Clarke, 1994a). Cross- linking
157
binding analysis of adaGb3 within this common drug binding pocket at this interface between
the transmembrane domains of the two homologous halves of MDR1 using Cys mutants in TM6
and 7 (Loo and Clarke, 2001) surprisingly showed that adaGb3 (unlike CsA or verapamil) did
not inhibit disulfide cross- linking when the TM6 cysteine was close to the bilayer surface and
the TM7 in the exofacial region. However, when the TM6 cysteine was moved toward the
cytosolic face by one a-helix turn, adaGb3 prevented cross- linking (IC50 ~ 5 µM). Only this
central region of TM6 (Leu-339) is directly involved in the drug stimulated conformational
change required for the NBD-mediated ATP hydrolysis-dependent MDR1 function (Rothnie et
al., 2001). Aller et al. described three areas of binding in the large internal cavity of the recently
reported X-ray 3.8 Å structure of P-gp (Aller et al., 2009). There is an “upper” site mainly
formed by hydrophobic aromatic residues of TMs 1, 2, 6, 7, 11, and 12; a “middle”one mostly
formed by hydrophobic residues of TMs 1, 5 6, 7, 11, and 12; and a “lower” one mainly lined
with more polar and charged residues from TMs 1, 5, 6, 7, 8, 9, 11, and 12 (Aller et al., 2009).
Thus, adaGb3 might bind to the “middle” or “lower” sites of P-gp internal cavity. Therefore,
while CsA/verapamil/vinblastine bind to the upper or middle sites, adaGb3 is more restricted to
a functional domain deeper within the bilayer (“middle” and “lower” binding sites), It is
proposed that charged hydrophobic molecules bind to the hydrophylic residues of the lower site
(Aller et al., 2009), and although adaGb3 is still hydrophobic, its particular property of being
water soluble make it a candidate for binding the lower site. Modification of the L339C residue
altered signal transduction from several distinct MDR1 drug binding sites (Rothnie et al., 2001),
suggesting adaGb3 should prove effective against many MDR1 substrates. AdaGb3/MDR1
binding may be similar to colchicine, demecolcine, Hoescht 3342, or flupentixol that similarly
do not inhibit TM6 F343C disulfide cross- linking (Loo et al., 2006a).
It is clear that MDR1 can be expressed in cells lacking Gb3. In such cases, other GSLs
may substitute (Plo et al., 2002). However, drug resistant metastatic ovarian tumor cells have a
158
particularly high Gb3 content (Arab et al., 1997), and Gb3 is highly expressed in metastatic
colon carcinoma (Kovbasnjuk et al., 2005).
The strong MDR1 reversal effects of adaGb3 as well as its favourable in vivo features
make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the
intestinal epithelium.
AdaGb3 MDR1 inhibition is selective, because adaGalCer was unable to sensitize either
MDR1-MDCK or SK VLB cell lines to vinblastine, or to abrogate cell surface expression of
MDR1, or to inhibit rhodamine efflux. However, hexanoyl derivatives of GalCer, GlcCer and
LacCer have been shown to inhibit rhodamine efflux in a drug-resistant ovarian cancer cell line,
while only the hexanoyl derivative of GlcCer has been shown to reverse MDR (Veldman et al.,
1999). As there are no reports on direct binding of these hexanoyl derivatives to P-gp, we agree
with Veldman et al that these hexanoyl derivatives may be acting as modulators of P-gp
activity, more than substrates or inhibitors, through interactions of their hexanoyl group with
domains of the protein at the plasma bilayer, promoting ATPase activity for drug transport
(Veldman et al., 1999). This concept is also confirmed in the recently developed X-ray structure
of P-gp, where Aller et al. reaffirm the requirement of lipids along with specific substrates for
P-gp transport activity, with lipids playing a key role in stimulating ATPase hydrolysis required
for the transport to occur (Aller et al., 2009). Apparently, this is not the case for the adamantane
group, since preliminary data show that neither adaGalCer nor adaGlcCer are able to bind the
same binding site as adaGb3 by disulfide cross linking studies (Clarke, DM, personal
communication). In this case, we can confirm selectivity of adaGb3 versus adaGalCer and
adaGlcCer. The new reported structure for P-gp also addresses selectivity since it was shown
that P-gp can distinguish between two stereoisomers of a cyclic hexapeptide inhibitor, QZ59,
with different binding locations, orientation and stoichiometry ratios (Aller et al., 2009).
Interestingly, GlcCer has been implicated in the maintenance of Gb3 within lipid rafts (Smith et
159
al., 2006) which could provide an indirect mechanism for P-gp modulation. Thus, specific GSL
analogs provide a new approach to MDR reversal and are valuable probes of the mechanism of
action.
Inhibition of MDR1 as Possible Therapy for Fabry Disease
The fact that inhibitors of MDR1 deplete the synthesis of neutral GSLs but do not affect
ganglioside synthesis suggests a viable and selective therapeutic approach for LSDs in which
neutral GSLs accumulate (De Rosa et al., 2004). The other approaches based on substrate
reduction therapy were shown to have several side effects due to poor selectivity. The
therapeutic use of glucosylceramide synthase inhibitors lacks selectivity as it inhibits both the
neutral and acidic GSLs synthetic pathway. Moreover, knockout mice for GlcCer synthase are
not viable (Yamashita et al., 2002), even though cell lines in culture under this condition are
able to survive (Ichikawa et al., 1996). The use of imino sugars as another possibility for
therapeutics for LSDs also shows inhibition of both types of GSLs, neutral and acidic and also
affects amylase (Andersson et al., 2004; Tian et al., 2004).
The in vivo role of MDR1 in GSL synthesis has not been established. Knockout mice for
MDR1 are viable but it was already reported that skin fibroblasts from these mice show
defective synthesis of neutral GSLs (De Rosa et al., 2004). We have previously shown that an
alternative mechanism for GlcCer translocation of the Golgi membrane must exist in HeLa cells
(De Rosa et al., 2004) because their neutral GSLs were shown to be unaffected by MDR1
inhibition, specifically CsA. But, as shown in Chapter Five of this thesis, when these cells were
transfected with CerS1 gene, a CerS (ceramide synthase gene family) gene encoding a C18
ceramide synthase, subsequent C18 GSL synthesis is fumonisin B resistant as previously
reported (Venkataraman et al., 2002) but CsA sensitive, indicating that both MDR1 dependent
and independent mechanisms for GlcCer translocation are present in these cells. Our in vitro
studies in Gaucher’s lymphoblasts where CsA treatment completely abolishes GSL
160
accumulation, support MDR1 inhibition as possible treatment for neutral LSDs. This hypothesis
is further confirmed by the significant reduction of Gb3 that we also observed in CsA treated
Fabry lymphoblasts, with no effect on ganglioside synthesis. The study reported by our group
on adult Fabry mice treated with both ERT and MDR1 inhibitor CsA, effectively showed serum
Gb3 recovered to lower levels and depleted Gb3 levels in liver indicated that MDR1 should be
considered as potential therapy for Fabry disease (Mattocks et al., 2006). But in Fabry patients
that have no residual a-galactosidase activity or even in Fabry KO mice, MDR1 inhibition needs
to act preventively on Gb3 synthesis to be fully effective. MDR1 inhibition has no effect on the
Gb3 already accumulated, as for other substrate reduction alternatives. A protocol using neonatal
Fabry mice was designed to test the efficacy of MDR1 inhibition on de novo Gb3 synthesis, in
which animals were treated as early as 7 days after birth with CsA or either one of two MDR1
third generation inhibitors. Our immunohistochemical demonstration that the effect of CsA, and
even more of tariquidar and OC144 on the reduction of Fabry mouse liver and heart Gb3 levels,
approaches proof of concept. Unexpectedly, Gb3 extracted from livers shows no reduction by
TLC and HPLC analysis in any of the three treated groups. This might indicate that Gb3 was not
really reduced but might be relocated or masked within the cell membrane after MDR1
inhibition. Cholesterol might be playing a role in this redistribution or accessibility of detection
by VT in the case of CsA as shown after treatment with ß-methyl cyclodextrin, a cholesterol
sequester, as seen for human renal glomerular Gb3 (Khan et al., 2009) but not for the other
inhibitors. This will be an approach that will need further study as well as the in vivo
implications.
In the liver sections, VT1 staining of Kupffer cells and endothelial cells within the
central vein showed significantly less Gb3 accumulation after MDR1 inhibition treatment as
previously reported (Mattocks et al., 2006). One possible explanation of these results is that as
Kupffer cells are major reticulo-endothelial degradative sites, the Gb3 content of these cells may
161
come from serum or red blood cells degradation. In our previous study, it was suggested that
this decrease seen after CsA could result from the reduced serum Gb3 levels (Mattocks et al.,
2006). Kupffer cells are modified monocytes that share a common origin with endothelial cells.
The advantage of treating neonatal mice can be seen in the great reduction of Gb3 not only in
liver but also in the heart, not seen in our previous study with adult Fabry mice (Mattocks et al.,
2006). This finding is especially important since heart and other tissues than liver are less
sensitive to ERT (Ioannou et al., 2001), emphasizing MDR inhibition therapy much more
effective than ERT.
Our results further strengthen the use of MDR1 inhibition as a therapeutic approach for
Fabry disease. The use of third generation MDR1 inhibitors ruled out the side effects ascribed to
CsA treatment. This approach can also be used in other GSL storage diseases like Gaucher,
where inhibition of the translocation of GlcCer into the Golgi apparatus increase exposure to the
cytosolic glucosidase (not deficient in Gaucher disease) to reduce GSL accumulation. ERT is
reported to be clinically effective in Fabry patients (Wilcox et al., 2004) but the neurological
symptoms are still not solved, added to the extreme cost of this approach that makes it quite
unavailable for the great majority of the patients. Although new alternative or complementary
strategies are continuously developing (Siatskas and Medin, 2001; Platt et al., 2003; Kolter and
Sandhoff, 2006), MDR1 inhibition represents a potential novel alternative to the current
treatment of neutral GSL storage diseases.
6.1. Conclusions
The interplay between glycosphingolipids and MDR1 could provide a whole new
spectrum of innovative therapeutic options. On one side, verotoxin may provide a new approach
to cancer treatment (Arab et al., 1997; Arab et al., 1998; Arab et al., 1999)as it was observed
that drug resistant tumor cells are hypersensitive to verotoxin, based on altered expression of its
162
glycolipid receptor, Gb3, on multidrug resistant cells (Farkas-Himsley et al., 1995; Arab and
Lingwood, 1998).
The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo
features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs
across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a
new approach to MDR reversal.
Cyclosporin A treatment has been found to reduce the recovery of serum Gb3
levels in Fabry mice following a-galactosidase treatment. In such mice, the expression of Gb3
within the liver is also reduced in comparison with Fabry mice allowed to recover from ERT
without MDR1 inhibition (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated
with third generation MDR1 inhibitors showed significant less Gb3 than liver and heart sections
of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic
approach not only for Fabry disease given the extraordinary cost of conventional enzyme
replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher.
6.2. Future Directions
AdaGb3 was proven as a MDR1 inhibitor whose binding to MDR1 in part overlaps that
of CsA/verapamil/vinblastine but is more restricted to a functional domain deeper within the
bilayer. It will be interesting to study if there is any evidence of direct binding of these hexanoyl
GSLs derivatives by disulfide cross- linking studies to confirm their role as modulators or
inhibitors. Metabolic labeling studies on adaGb3 treated-MDR1 cells will be performed to
analyze and quantitate GSL levels newly synthesized. It will be interesting to study the effect of
these adaGSLs/MDR1 inhibitors on lipid raft distribution of MDR1 as the lipid environment
plays a key role in MDR1 function and regulation. Adamantyl derivatives of other receptor
glycolipids, for which the lipid moiety is necessary for receptor function (Suzuki et al., 1996;
Mamelak and Lingwood, 1997), such as adaSGC will also be tested.
163
A previous study from our group showed that CsA inhibition of GSL biosynthesis in a
cell- free microsomal assay supporting a direct role for MDR1 (De Rosa et al., 2004). This
complex assay requires transfer of GlcCer from the exogenous liposome to the outer
microsomal membrane for MDR1 translocation, to provide the necessary energy for MDR1
activity (Berninsone and Hirschberg, 2000). The microsomal inhibitory effect of CsA is faster
than in cell culture, consistent with a more immediate effect on GlcCer translocation. The CsA-
sensitive increase of microsomal LacCer synthesis after addition of liposomal GlcCer further
supports a GlcCer translocase activity. Future MDR1 studies on microsomes exposed to GlcCer
variants synthesized with different lengths in their fatty acid chains will also be performed to
analyze whether fatty acid content has an effect on GlcCer translocation to modulate this
particular role of MDR1 as a Golgi- lipid flippase.
In terms of the Fabry disease neonatal mice model, quantitative analysis of Gb3 levels by
extraction of GSLs from the corresponding treated and untreated Fabry hearts should be
performed by TLC, Gb3 overlay and HPLC, in order to match it with the immunohistochemistry
results and see if the discrepancy seen between these results for liver is the same in other tissues
or it is just a liver effect. Studies in human kidney have shown VT undetectable renal
glomerular Gb3 that become detectable when cholesterol is removed form the samples (Khan et
al., 2009). Immunohistochemistry and quantitative analysis of Gb3 levels in other tissues such as
spleen and brain from treated and untreated Fabry mice should be performed to study the
efficacy of MDR1 inhibition as a potential treatment for Fabry disease. The presence of ligand
undetectable Gb3 is an important new finding. In order to further prove MDR1 inhibition as an
approach to GSL storage disease therapy and the importance of MDR1 in Gb3 synthesis, MDR1
a/b KO mice (Schinkel et al., 1997) will be cross bred with the Fabry mice. It is expected that
the homozygous progeny will be ‘cured ‘of the Gb3 accumulation characteristic of the Fabry
mice. It has already been shown that in MDR 1a -/- 1b -/- skin fibroblasts, neural GSL synthesis
164
was ablated (De Rosa et al., 2004). Treatment of pregnant Gaucher mice with MDR1 inhibitors
to overcome neonatal lethality will be tested as another approach.
165
REFERENCES
Abe, A., L.J. Arend, L. Lee, C.A. Lingwood, R.O. Brady, and J.A. Shayman. 2000.
Glycosphingolipid depletion in Fabry disease lymphoblasts with potent inhibitors of
glucosylceramide synthase. Kid Intl. 57:446-454.
Abe, A., J. Inokuchi, M. Jimbo, H. Shimeno, A. Nagamatsu, J.A. Shayman, G.S. Shukla, and
N.S. Radin. 1992. Improved inhibitors of glucosylceramide synthase. J Biochem
(Tokyo). 111:191-6.
Abe, A., S.R. Wild, W.L. Lee, and J.A. Shayman. 2001. Agents for the treatment of
glycosphingolipid storage disorders. Curr Drug Metab. 2:331-8.
Abulrob, A.G., and M. Gumbleton. 1999. Transport of phosphatidylcholine in MDR3-negative
epithelial cell lines via drug- induced MDR1 P-glycoprotein. Biochem Biophys Res
Commun. 262:121-6.
Adachi, Y., H. Suzuki, and Y. Sugiyama. 2001. Comparative studies on in vitro methods for
evaluating in vivo function of MDR1 P-glycoprotein. Pharm Res. 18:1660-8.
Advani, R., H.I. Saba, M.S. Tallman, J.M. Rowe, P.H. Wiernik, J. Ramek, K. Dugan, B. Lum, J.
Villena, E. Davis, E. Paietta, M. Litchman, B.I. Sikic, and P.L. Greenberg. 1999.
Treatment of refractory and relapsed acute myelogenous leukemia with combination
chemotherapy plus the multidrug resistance modulator PSC 833 (Valspodar). Blood.
93:787-95.
Agapova, L.S., J.L. Volodina, P.M. Chumakov, and B.P. Kopnin. 2004. Activation of Ras-Ral
pathway attenuates p53- independent DNA damage G2 checkpoint. J Biol Chem.
279:36382-9.
Aller, S.G., J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P.M. Harrell, Y.T. Trinh, Q.
Zhang, I.L. Urbatsch, and G. Chang. 2009. Structure of P-glycoprotein reveals a
molecular basis for poly-specific drug binding. Science. 323:1718-22.
166
Almeida, P.F., A. Pokorny, and A. Hinderliter. 2005. Thermodynamics of membrane domains.
Biochim Biophys Acta. 1720:1-13.
Altucci, L., and H. Gronemeyer. 2001. The promise of retinoids to fight against cancer. Nat Rev
Cancer. 1:181-93.
Ambudkar, S.V., S. Dey, C.A. Hrycyna, M. Ramachandra, I. Pastan, and M.M. Gottesman.
1999. Biochemical, cellular, and pharmacological aspects of the multidrug transporter.
Annu Rev Pharmacol Toxicol. 39:361-98.
Ambudkar, S.V., I.W. Kim, and Z.E. Sauna. 2006. The power of the pump: mechanisms of
action of P-glycoprotein (ABCB1). Eur J Pharm Sci. 27:392-400.
Ambudkar, S.V., C. Kimchi-Sarfaty, Z.E. Sauna, and M.M. Gottesman. 2003. P-glycoprotein:
from genomics to mechanism. Oncogene. 22:7468-85.
Ambudkar, S.V., I.H. Lelong, J. Zhang, and C. Cardarelli. 1998. Purification and reconstitution
of human P-glycoprotein. Methods Enzymol. 292:492-504.
Andersson, U., G. Reinkensmeier, T.D. Butters, R.A. Dwek, and F.M. Platt. 2004. Inhibition of
glycogen breakdown by imino sugars in vitro and in vivo. Biochem Pharmacol. 67:697-
705.
Anfinsen, C.B. 1973. Principles that govern the folding of protein chains. Science. 181:223-30.
Arab, S., and C. Lingwood. 1998. Intracellular targeting of the endoplasmic reticulum/nuclear
envelope by retrograde transport may determine cell hypersensitivity to Verotoxin:
sodium butyrate or selection of drug resistance may induce nuclear toxin targeting via
globotriaosyl ceramide fatty acid isoform traffic. J Cell Physiol. 177:646-660.
Arab, S., and C.A. Lingwood. 1996. Influence of phospholipid chain length on
verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a surface
bilayer film and liposomes. Glycoconj. J. 13:159-166.
167
Arab, S., M. Murakami, P. Dirks, B. Boyd, S. Hubbard, C. Lingwood, and J. Rutka. 1998.
Verotoxins inhibit the growth of and induce apoptosis in human astrocytoma cells. J.
Neuro. Oncol. 40:137-150.
Arab, S., E. Russel, W. Chapman, B. Rosen, and C. Lingwood. 1997. Expression of the
Verotoxin receptor glycolipid, globotriaosylceramide, in Ovarian Hyperplasias. Oncol
Res. 9:553-563.
Arab, S., J. Rutka, and C. Lingwood. 1999. Verotoxin induces apoptosis and the complete,
rapid, long-term elimination of human astrocytoma xenografts in nude mice. Oncol Res.
11:33-39.
Arceci, R.J., J.M. Croop, S.B. Horwitz, and D. Housman. 1988. The gene encoding multidrug
resistance is induced and expressed at high levels during pregnancy in the secretory
epithelium of the uterus. Proc Natl Acad Sci U S A. 85:4350-4.
Arima, H., K. Yunomae, T. Morikawa, F. Hirayama, and K. Uekama. 2004. Contribution of
cholesterol and phospholipids to inhibitory effect of dimethyl-beta-cyclodextrin on
efflux function of P-glycoprotein and multidrug resistance-associated protein 2 in
vinblastine-resistant Caco-2 cell monolayers. Pharm Res. 21:625-34.
Asano, N., S. Ishii, H. Kizu, K. Ikeda, K. Yasuda, A. Kato, O.R. Martin, and J.Q. Fan. 2000. In
vitro inhibition and intracellular enhancement of lysosomal alpha-galactosidase A
activity in Fabry lymphoblasts by 1-deoxygalactonojirimycin and its derivatives. Eur J
Biochem. 267:4179-86.
Asano, T., K.A. Takahashi, M. Fujioka, S. Inoue, M. Okamoto, N. Sugioka, H. Nishino, T.
Tanaka, Y. Hirota, and T. Kubo. 2003. ABCB1 C3435T and G2677T/A polymorphism
decreased the risk for steroid- induced osteonecrosis of the femoral head after kidney
transplantation. Pharmacogenetics. 13:675-82.
168
Augustine, L.M., R.J. Markelewicz, Jr., K. Boekelheide, and N.J. Cherrington. 2005. Xenobiotic
and endobiotic transporter mRNA expression in the blood-testis barrier. Drug Metab
Dispos. 33:182-9.
Bacso, Z., H. Nagy, K. Goda, L. Bene, F. Fenyvesi, J. Matko, and G. Szabo. 2004. Raft and
cytoskeleton associations of an ABC transporter: P-glycoprotein. Cytometry A. 61:105-
16.
Bain, L.J., J.B. McLachlan, and G.A. LeBlanc. 1997. Structure-activity relationships for
xenobiotic transport substrates and inhibitory ligands of P-glycoprotein. Environ Health
Perspect. 105:812-8.
Balana, A., J. Wiels, C. Tetaud, Z. Mishal, and T. Tursz. 1985. Induction of cell differentiation
in Burkitt lymphoma lines. BLA: A glycolipid marker of B-cell differentiation. Int J
Cancer. 36:453-460.
Balimane, P.V., and S. Chong. 2005. A combined cell based approach to identify P-glycoprotein
substrates and inhibitors in a single assay. Int J Pharm. 301:80-8.
Balimane, P.V., K. Patel, A. Marino, and S. Chong. 2004. Utility of 96 well Caco-2 cell system
for increased throughput of P-gp screening in drug discovery. Eur J Pharm Biopharm.
58:99-105.
Barakat, S., L. Gayet, G. Dayan, S. Labialle, A. Lazar, V. Oleinikov, A.W. Coleman, and L.G.
Baggetto. 2005. Multidrug-resistant cancer cells contain two populations of P-
glycoprotein with differently stimulated P-gp ATPase activities: evidence from atomic
force microscopy and biochemical analysis. Biochem J. 388:563-71.
Barbat, C., M. Trucy, M. Sorice, T. Garofalo, V. Manganelli, A. Fischer, and F. Mazerolles.
2007. p56lck, LFA-1 and PI3K but not SHP-2 interact with GM1- or GM3-enriched
microdomains in a CD4-p56lck association-dependent manner. Biochem J. 402:471-81.
169
Bast, D.J., J.L. Brunton, M.A. Karmali, and S.E. Richardson. 1997. Toxicity and
immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor
binding in rabbits. Infect. Immun. 65:2019-2028.
Batey, S., K.A. Scott, and J. Clarke. 2006. Complex folding kinetics of a multidomain protein.
Biophys J. 90:2120-30.
Bear, C.E., C.H. Li, N. Kartner, R.J. Bridges, T.J. Jensen, M. Ramjeesingh, and J.R. Riordan.
1992. Purification and functional reconstitution of the cystic fibrosis transmembrane
conductance regulator (CFTR). Cell. 68:809-18.
Beck, W.T., T.J. Mueller, and L.R. Tanzer. 1979. Altered surface membrane glycoproteins in
Vinca alkaloid-resistant human leukemic lymphoblasts. Cancer Res. 39:2070-6.
Bell, D.R., J.M. Trent, H.F. Willard, J.R. Riordan, and V. Ling. 1987. Chromosomal location of
human P-glycoprotein gene sequences. Cancer Genet Cytogenet. 25:141-8.
Benet, L.Z., T. Izumi, Y. Zhang, J.A. Silverman, and V.J. Wacher. 1999. Intestinal MDR
transport proteins and P-450 enzymes as barriers to oral drug delivery. J Control
Release. 62:25-31.
Berninsone, P., and C. Hirschberg. 2000. Nucleotide sugar transporters of the Golgi apparatus.
Curr Opin Struct Biol. 10:542-7.
Bezombes, C., N. Maestre, G. Laurent, T. Levade, A. Bettaieb, and J.P. Jaffrezou. 1998.
Restoration of TNF-alpha- induced ceramide generation and apoptosis in resistant human
leukemia KG1a cells by the P-glycoprotein blocker PSC833. Faseb J. 12:101-9.
Biedler, J.L., and H. Reihm. 1970. Cellular resistance to actinomycin D in Chinese hamster cells
in vitro: cross resistance, radiographic, and cytogenetic studies. Cancer Res. 30:1174-
1184.
Bielder, J.a.R., H. 1970. Cellular resistance to Chinese hamster cells in vitro: cross resistance,
radioautographic studies and cytogenetic studies. Cancer Res. 30:1174-84.
170
Biemans-Oldehinkel, E., M.K. Doeven, and B. Poolman. 2006. ABC transporter architecture
and regulatory roles of accessory domains. FEBS Lett. 580:1023-35.
Biffi, A., and L. Naldini. 2005. Gene therapy of storage disorders by retroviral and lentiviral
vectors. Hum Gene Ther. 16:1133-42.
Birkenmeier, E.H., J.E. Barker, C.A. Vogler, J.W. Kyle, W.S. Sly, B. Gwynn, B. Levy, and C.
Pegors. 1991. Increased life span and correction of metabolic defects in murine
mucopolysaccharidosis type VII after syngeneic bone marrow transplantation. Blood.
78:3081-92.
Blonder, J., L.R. Yu, G. Radeva, K.C. Chan, D.A. Lucas, T.J. Waybright, H.J. Issaq, F.J.
Sharom, and T.D. Veenstra. 2006. Combined chemical and enzymatic stable isotope
labeling for quantitative profiling of detergent-insoluble membrane proteins isolated
using Triton X-100 and Brij-96. J Proteome Res. 5:349-60.
Bolte, S., and F.P. Cordelieres. 2006. A guided tour into subcellular colocalization analysis in
light microscopy. J Microsc. 224:213-32.
Bonnet, D. 2005. Normal and leukaemic stem cells. Br J Haematol. 130:469-79.
Borst, P., and R.O. Elferink. 2002. Mammalian ABC transporters in health and disease. Annu
Rev Biochem. 71:537-92.
Borst, P., R. Evers, M. Kool, and J. Wijnholds. 2000a. A family of drug transporters: the
multidrug resistance-associated proteins. J Natl Cancer Inst. 92:1295-302.
Borst, P., N. Zelcer, and A. van Helvoort. 2000b. ABC transporters in lipid transport. Biochim
Biophys Acta. 1486:128-44.
Bose, R., M. Vereheij, A. Haimovitz-Friedman, K. Scotto, Z. Fuks, and R. Kolesnick. 1995.
Ceramide synthase mediates daunorubicin- induced apoptosis: an alternative mechanism
for generating death signals. Cell. 82:405-414.
171
Boulanger, J., M. Huesca, S. Arab, and C.A. Lingwood. 1994. Universal method for the facile
production of glycolipid/lipid matrices for the affinity purification of binding ligands.
Anal Biochem. 217:1-6.
Boulanger, J., M. Petric, C. Lingwood, H. Law, M. Roscoe, and M. Karmali. 1990a.
Neutralization receptor-based immunoassay for detection of neutralizing antibodies to
Escherichia coli verocytotoxin 1. J Clin Microbiol. 28:2830-3.
Boulanger, J., M. Petric, C.A. Lingwood, H. Law, M. Roscoe, and M. Karmali. 1990b.
Neutralization receptor-based immunoassay (NeutrELISA) for detection of neutralizing
antibodies to Escherichia coli verocytotoxin 1. J Clin Micro. 28:2830-2833.
Boyd, B., and C.A. Lingwood. 1989. Verotoxin receptor glycolipid in human renal tissue.
Nephron. 51:207-210.
Boyd, B., S. Richardson, and J. Gariepy. 1991. Serological responses to the B subunit of Shiga-
like toxin 1 and its peptide fragments indicate that the B subunit is a vaccine candidate to
counter the action of the toxin. Infect Immun. 59:750-757.
Boyd, B., Z. Zhiuyan, G. Magnusson, and C.A. Lingwood. 1994. Lipid modulation of glycolipid
receptor function: Presentation of galactose a1-4 galactose disaccharide for Verotoxin
binding in natural and synthetic glycolipids. Eur. J. Biochem. 223:873-878.
Brady, R.O. 2003. Gaucher and Fabry diseases: from understanding pathophysiology to rational
therapies. Acta Paediatr Suppl. 92:19-24.
Breier, A., M. Barancik, Z. Sulova, and B. Uhrik. 2005. P-glycoprotein--implications of
metabolism of neoplastic cells and cancer therapy. Curr Cancer Drug Targets. 5:457-68.
Brown, D.A., and E. London. 2000. Structure and function of sphingolipid- and cholesterol-rich
membrane rafts. J Biol Chem. 275:17221-4.
Brown, D.A., and J.K. Rose. 1992. Sorting of GPI-anchored proteins to glycolipid-enriched
membrane subdomains during transport to the apical cell surface. Cell. 68:533-544.
172
Brown, R.E. 1998. Sphingolipid organization in biomembranes: what physical studies of model
membranes reveal. J Cell Sci. 111 ( Pt 1):1-9.
Bunting, K.D., S. Zhou, T. Lu, and B.P. Sorrentino. 2000. Enforced P-glycoprotein pump
function in murine bone marrow cells results in expansion of side population stem cells
in vitro and repopulating cells in vivo. Blood. 96:902-9.
Burger, H., and K. Nooter. 2004. Pharmacokinetic resistance to imatinib mesylate: role of the
ABC drug pumps ABCG2 (BCRP) and ABCB1 (MDR1) in the oral bioavailability of
imatinib. Cell Cycle. 3:1502-5.
Burger, K., P. van der Bijl, and G. van Meer. 1996. Topology of sphingolipid galactosyl
transferase in ER and Golgi: transbilayer movement of monohexyl sphingolipids is
required for higher glycosphingolipid biosynthesis. J Cell Biol. 133:15-28.
Butters, T.D., R.A. Dwek, and F.M. Platt. 2000. Inhibition of glycosphingolipid biosynthesis:
application to lysosomal storage disorders. Chem Rev. 100:4683-96.
Butters, T.D., R.A. Dwek, and F.M. Platt. 2005. Imino sugar inhibitors for treating the
lysosomal glycosphingolipidoses. Glycobiology. 15:43R-52R.
Cai, C., H. Zhu, and J. Chen. 2004. Overexpression of caveolin-1 increases plasma membrane
fluidity and reduces P-glycoprotein function in Hs578T/Dox. Biochem Biophys Res
Commun. 320:868-74.
Callaghan, R., G. Berridge, D.R. Ferry, and C.F. Higgins. 1997. The functional purification of
P-glycoprotein is dependent on maintenance of a lipid-protein interface. Biochim
Biophys Acta. 1328:109-24.
Callen, D.F., E. Baker, R.N. Simmers, R. Seshadri, and I.B. Roninson. 1987. Localization of the
human multiple drug resistance gene, MDR1, to 7q21.1. Hum Genet. 77:142-4.
Campbell, P.E., C.M. Harris, and A. Vellodi. 2004. Deterioration of the auditory brainstem
response in children with type 3 Gaucher disease. Neurology. 63:385-7.
173
Challen, G.A., and M.H. Little. 2006. A side order of stem cells: the SP phenotype. Stem Cells.
24:3-12.
Chamary, J.V., J.L. Parmley, and L.D. Hurst. 2006. Hearing silence: non-neutral evolution at
synonymous sites in mammals. Nat Rev Genet. 7:98-108.
Chambers, T.C., U.A. Germann, M.M. Gottesman, I. Pastan, J.F. Kuo, and S.V. Ambudkar.
1995. Bacterial expression of the linker region of human MDR1 P-glycoprotein and
mutational analysis of phosphorylation sites. Biochemistry. 34:14156-62.
Chan, R.J., and M.C. Yoder. 2004. The multiple facets of hematopoietic stem cells. Curr
Neurovasc Res. 1:197-206.
Chark, D., A. Nutikka, N. Trusevych, J. Kuzmina, and C. Lingwood. 2004. Differential
carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and
monoclonal antibody: Role in human renal glomerular binding. Eur. J. Biochem. 271:1-
13.
Chaudhary, P.M., and I.B. Roninson. 1991. Expression and activity of P-glycoprotein, a
multidrug efflux pump, in human hematopoietic stem cells. Cell. 66:85-94.
Chaudhary, P.M., and I.B. Roninson. 1993. Induction of multidrug resistance in human cells by
transient exposure to different chemotherapeutic drugs. J Natl Cancer Inst. 85:632-9.
Chen, C., M. Patterson, C. Wheatley, J. O'Brien, and R. Pagano. 1999. Broad screening test for
sphingolipid-storage diseases. Lancet. 354:901-5.
Chen, C.J., J.E. Chin, K. Ueda, D.P. Clark, I. Pastan, M.M. Gottesman, and I.B. Roninson.
1986. Interna l duplication and homology with bacterial transport proteins in the mdr1 (P-
glycoprotein) gene from multidrug-resistant human cells. Cell. 47:381-9.
Chen, C.J., D. Clark, K. Ueda, I. Pastan, M.M. Gottesman, and I.B. Roninson. 1990. Genomic
organization of the human multidrug resistance (MDR1) gene and origin of P-
glycoproteins. J Biol Chem. 265:506-14.
174
Chen, C.S., G. Bach, and R.E. Pagano. 1998. Abnormal transport along the lysosomal pathway
in mucolipidosis, type IV disease. Proc Natl Acad Sci U S A. 95:6373-8.
Chester, M.A. 1998. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN).
Nomenclature of glycolipids--recommendations 1997. Eur J Biochem. 257:293-8.
Chiba, Y., H. Sakuraba, M. Kotani, R. Kase, K. Kobayashi, M. Takeuchi, S. Ogasawara, Y.
Maruyama, T. Nakajima, Y. Takaoka, and Y. Jigami. 2002. Production in yeast of alpha-
galactosidase A, a lysosomal enzyme applicable to enzyme replacement therapy for
Fabry disease. Glycobiology. 12:821-8.
Chico, I., M.H. Kang, R. Bergan, J. Abraham, S. Bakke, B. Meadows, A. Rutt, R. Robey, P.
Choyke, M. Merino, B. Goldspiel, T. Smith, S. Steinberg, W.D. Figg, T. Fojo, and S.
Bates. 2001. Phase I study of infusional paclitaxel in combination with the P-
glycoprotein antagonist PSC 833. J Clin Oncol. 19:832-42.
Chin, K.V., and B. Liu. 1994. Regulation of the multidrug resistance (MDR1) gene expression.
In Vivo. 8:835-41.
Chin, K.V., S. Tanaka, G. Darlington, I. Pastan, and M.M. Gottesman. 1990. Heat shock and
arsenite increase expression of the multidrug resistance (MDR1) gene in human renal
carcinoma cells. J Biol Chem. 265:221-6.
Choudhury, A., M. Dominguez, V. Puri, D.K. Sharma, K. Narita, C.L. Wheatley, D.L. Marks,
and R.E. Pagano. 2002. Rab proteins mediate Golgi transport of caveola-internalized
glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells. J Clin Invest.
109:1541-50.
Christomanou, H., A. Aignesberger, and R.P. Linke. 1986. Immunochemical characterization of
two activator proteins stimulating enzymic sphingomyelin degradation in vitro. Absence
of one of them in a human Gaucher disease variant. Biol Chem Hoppe Seyler. 367:879-
90.
175
Coetzee, T., K. Suzuki, and B. Popko. 1998. New perspectives on the function of myelin
galactolipids. Trends Neurosci. 21:126-30.
Cohen, A., G.E. Hannigan, B.R.G. Williams, and C.A. Lingwood. 1987. Roles of globotriosyl-
and galabiosylceramide in verotoxin binding and high affinity interferon receptor. J.
Biol. Chem. 262:17088-17099.
Come, M.G., A. Bettaieb, A. Skladanowski, A.K. Larsen, and G. Laurent. 1999. Alteration of
the daunorubicin-triggered sphingomyelin-ceramide pathway and apoptosis in MDR
cells: influence of drug transport abnormalities. Int J Cancer. 81:580-7.
Compain, P., and O.R. Martin. 2003. Design, synthesis and biological evaluation of iminosugar-
based glycosyltransferase inhibitors. Curr Top Med Chem. 3:541-60.
Conzelmann, E., and K. Sandhoff. 1983. Partial enzyme deficiencies: residual activities and the
development of neurological disorders. Dev Neurosci. 6:58-71.
Cordon-Cardo, C., J.P. O'Brien, J. Boccia, D. Casals, J.R. Bertino, and M.R. Melamed. 1990.
Expression of the multidrug resistance gene product (P-glycoprotein) in human normal
and tumor tissues. J Histochem Cytochem. 38:1277-87.
Cox, T., R. Lachmann, C. Hollak, J. Aerts, S. van Weely, M. Hrebicek, F. Platt, T. Butters, R.
Dwek, C. Moyses, I. Gow, D. Elstein, and A. Zimran. 2000. Novel oral treatment of
Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate
biosynthesis. Lancet. 355:1481-5.
Cox, T.M. 2005. Substrate reduction therapy for lysosomal storage diseases. Acta Paediatr
Suppl. 94:69-75; discussion 57.
Croop, J.M., M. Raymond, D. Haber, A. Devault, R.J. Arceci, P. Gros, and D.E. Housman.
1989. The three mouse multidrug resistance (mdr) genes are expressed in a tissue-
specific manner in normal mouse tissues. Mol Cell Biol. 9:1346-50.
176
Cummins, C.L., W. Jacobsen, and L.Z. Benet. 2002. Unmasking the dynamic interplay between
intestinal P-glycoprotein and CYP3A4. J Pharmacol Exp Ther. 300:1036-45.
Czarny, M., Y. Lavie, G. Fiucci, and M. Liscovitch. 1999. Localization of phospholipase D in
detergent insoluble caveolin rich membrane domains. J Biol Chem. 274:2717-2724.
Dalton, W.S., and R.J. Scheper. 1999. Lung resistance-related protein: determining its role in
multidrug resistance. J Natl Cancer Inst. 91:1604-5.
D'Angelo, G., E. Polishchuk, G. Di Tullio, M. Santoro, A. Di Campli, A. Godi, G. West, J.
Bielawski, C.C. Chuang, A.C. van der Spoel, F.M. Platt, Y.A. Hannun, R. Polishchuk, P.
Mattjus, and M.A. De Matteis. 2007. Glycosphingolipid synthesis requires FAPP2
transfer of glucosylceramide. Nature. 449:62-7.
Dano, K. 1973. Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells.
Biochim Biophys Acta. 323:466-83.
Dantzig, A.H., R.L. Shepard, J. Cao, K.L. Law, W.J. Ehlhardt, T.M. Baughman, T.F. Bumol,
and J.J. Starling. 1996. Reversal of P-glycoprotein-mediated multidrug resistance by a
potent cyclopropyldibenzosuberane modulator, LY335979. Cancer Res. 56:4171-9.
Dantzig, A.H., R.L. Shepard, K.L. Law, L. Tabas, S. Pratt, J.S. Gillespie, S.N. Binkley, M.T.
Kuhfeld, J.J. Starling, and S.A. Wrighton. 1999. Selectivity of the multidrug resistance
modulator, LY335979, for P-glycoprotein and effect on cytochrome P-450 activities. J
Pharmacol Exp Ther. 290:854-62.
Davidson, A.L., and J. Chen. 2004. ATP-binding cassette transporters in bacteria. Annu Rev
Biochem. 73:241-68.
Davidson, A.L., E. Dassa, C. Orelle, and J. Chen. 2008. Structure, function, and evolution of
bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev. 72:317-64, table of
contents.
177
Dawson, R.J., and K.P. Locher. 2006. Structure of a bacterial multidrug ABC transporter.
Nature. 443:180-5.
Dawson, R.J., and K.P. Locher. 2007. Structure of the multidrug ABC transporter Sav1866 from
Staphylococcus aureus in complex with AMP-PNP. FEBS Lett. 581:935-8.
de Bruijn, M.H., A.M. Van der Bliek, J.L. Biedler, and P. Borst. 1986. Differential amplification
and disproportionate expression of five genes in three multidrug-resistant Chinese
hamster lung cell lines. Mol Cell Biol. 6:4717-22.
De Rosa, M.a.L., CA. 2009. Multidrug Resistance Protein 1 (MDR1) and Glycosphingolipids
Biosynthesis. In Glycobiology Research Trends. G.P.a.O. McCabe, editor. Nova Science
Publishers, Inc., New York, USA. 1-26.
De Rosa, M.F., C. Ackerley, B. Wang, S. Ito, D.M. Clarke, and C. Lingwood. 2008. Inhibition
of multidrug resistance by adamantylgb3, a globotriaosylceramide analog. J Biol Chem.
283:4501-11.
De Rosa, M.F., D. Sillence, C. Ackerley, and C. Lingwood. 2004. Role of Multiple Drug
Resistance Protein 1 in neutral but not acidic glycosphingolipid biosynthesis. J. Biol.
Chem. 279:7867-7876.
Dean, M. 2002. The human ATP-binding cassette (ABC) transporter superfamily. National
Center for
Biotechnology Information (NCBI), National library of Medicine, Bethesda, MD, USA.
Dean, M., and R. Allikmets. 2001. Complete characterization of the human ABC gene family. J
Bioenerg Biomembr. 33:475-9.
Dean, M., T. Fojo, and S. Bates. 2005. Tumour stem cells and drug resistance. Nat Rev Cancer.
5:275-84.
Debry, P., E.A. Nash, D.W. Neklason, and J.E. Metherall. 1997. Role of multidrug resistance P-
glycoproteins in cholesterol esterification. J Biol Chem. 272:1026-31.
178
Deeley, R.G., and S.P. Cole. 2006. Substrate recognition and transport by multidrug resistance
protein 1 (ABCC1). FEBS Lett. 580:1103-11.
Deeley, R.G., C. Westlake, and S.P. Cole. 2006. Transmembrane transport of endo- and
xenobiotics by mammalian ATP-binding cassette multidrug resistance proteins. Physiol
Rev. 86:849-99.
Degroote, S., J. Wolthoorn, and G. van Meer. 2004. The cell biology of glycosphingolipids.
Semin Cell Dev Biol. 15:375-87.
Delgado, A., J. Casas, A. Llebaria, J.L. Abad, and G. Fabrias. 2006. Inhibitors of sphingolipid
metabolism enzymes. Biochim Biophys Acta. 1758:1957-77.
Denneberg, T., M. Friedberg, L. Homberg, C. Mathiasen, K.O. Nilsson, R. Takolander, and M.
Walder. 1982. Combined plasmophoresis and hemodialysis treatment for severe
hemolytic uremic syndrome following campylobacter colitis. 71:243-245.
Desai, K., M.C. Sullards, J. Allegood, E. Wang, E.M. Schmelz, M. Hartl, H.U. Humpf, D.C.
Liotta, Q. Peng, and A.H. Merrill, Jr. 2002. Fumonisins and fumonisin analogs as
inhibitors of ceramide synthase and inducers of apoptosis. Biochim Biophys Acta.
1585:188-92.
Desnick, R.J. 2004. Enzyme replacement and enhancement therapies for lysosomal diseases. J
Inherit Metab Dis. 27:385-410.
Desnick, R.J., and E.H. Schuchman. 2002. Enzyme replacement and enhancement therapies:
lessons from lysosomal disorders. Nat Rev Genet. 3:954-66.
Desnick, R.J., and M.P. Wasserstein. 2001. Fabry disease: clinical features and recent advances
in enzyme replacement therapy. Adv Nephrol Necker Hosp. 31:317-39.
Devault, A., and P. Gros. 1990. Two members of the mouse mdr gene family confer multidrug
resistance with overlapping but distinct drug specificities. Mol Cell Biol. 10:1652-63.
179
Dey, S., M. Ramachandra, I. Pastan, M.M. Gottesman, and S.V. Ambudkar. 1997. Evidence for
two nonidentical drug- interaction sites in the human P-glycoprotein. Proc Natl Acad Sci
U S A. 94:10594-9.
di Bartolomeo, S., and A. Spinedi. 2001. Differential chemosensitizing effect of two
glucosylceramide synthase inhibitors in hepatoma cells. Biochem Biophys Res Commun.
288:269-74.
Dietrich, C.G., A. Geier, and R.P. Oude Elferink. 2003. ABC of oral bioavailability: transporters
as gatekeepers in the gut. Gut. 52:1788-95.
D'Mello N, P., A.M. Childress, D.S. Franklin, S.P. Kale, C. Pinswasdi, and S.M. Jazwinski.
1994. Cloning and characterization of LAG1, a longevity-assurance gene in yeast. J Biol
Chem. 269:15451-9.
Dos Santos, S.M., C.C. Weber, C. Franke, W.E. Muller, and G.P. Eckert. 2007. Cholesterol:
Coupling between membrane microenvironment and ABC transporter activity. Biochem
Biophys Res Commun. 354:216-21.
Douglass, A.D., and R.D. Vale. 2005. Single-molecule microscopy reveals plasma membrane
microdomains created by protein-protein networks that exclude or trap signaling
molecules in T cells. Cell. 121:937-50.
Drach, J., A. Gsur, G. Hamilton, S. Zhao, J. Angerler, M. Fiegl, N. Zojer, M. Raderer, I. Haberl,
M. Andreeff, and H. Huber. 1996. Involvement of P-glycoprotein in the transmembrane
transport of interleukin-2 (IL-2), IL-4, and interferon-gamma in normal human T
lymphocytes. Blood. 88:1747-54.
Druley, T.E., W.D. Stein, and I.B. Roninson. 2001a. Analysis of MDR1 P-glycoprotein
conformational changes in permeabilized cells using differential immunoreactivity.
Biochemistry. 40:4312-22.
180
Druley, T.E., W.D. Stein, A. Ruth, and I.B. Roninson. 2001b. P-glycoprotein-mediated
colchicine resistance in different cell lines correlates with the effects of colchicine on P-
glycoprotein conformation. Biochemistry. 40:4323-31.
Eckford, P.D., and F.J. Sharom. 2005. The reconstituted P-glycoprotein multidrug transporter is
a flippase for glucosylceramide and other simple glycosphingolipids. Biochem J.
389:517-26.
Edidin, M. 2003. The state of lipid rafts: from model membranes to cells. Annu Rev Biophys
Biomol Struct. 32:257-83.
Efferth, T., Y.J. Fu, Y.G. Zu, G. Schwarz, V.S. Konkimalla, and M. Wink. 2007. Molecular
target-guided tumor therapy with natural products derived from traditional Chinese
medicine. Curr Med Chem. 14:2024-32.
Eitzman, D.T., P.F. Bodary, Y. Shen, C.G. Khairallah, S.R. Wild, A. Abe, J. Shaffer-Hartman,
and J.A. Shayman. 2003. Fabry disease in mice is associated with age-dependent
susceptibility to vascular thrombosis. J Am Soc Nephrol. 14:298-302.
el Ashry, E.S., N. Rashed, and A.H. Shobier. 2000. Glycosidase inhibitors and their
chemotherapeutic value, Part 2. Pharmazie. 55:331-48.
Elleder, M. 2003. Sequelae of storage in Fabry disease--pathology and comparison with other
lysosomal storage diseases. Acta Paediatr Suppl. 92:46-53; discussion 45.
Ellinwood, N.M., C.H. Vite, and M.E. Haskins. 2004. Gene therapy for lysosomal storage
diseases: the lessons and promise of animal models. J Gene Med. 6:481-506.
Elliott, S.P., M. Yu, H. Xu, and D.B. Haslam. 2003. Forssman synthetase expression results in
diminished shiga toxin susceptibility: a role for glycolipids in determining host-microbe
interactions. Infect Immun. 71:6543-52.
Elstein, D., C. Hollak, J.M. Aerts, S. van Weely, M. Maas, T.M. Cox, R.H. Lachmann, M.
Hrebicek, F.M. Platt, T.D. Butters, R.A. Dwek, and A. Zimran. 2004. Sustained
181
therapeutic effects of oral miglustat (Zavesca, N-butyldeoxynojirimycin, OGT 918) in
type I Gaucher disease. J Inherit Metab Dis. 27:757-66.
Eng, C.M., N. Guffon, W.R. Wilcox, D.P. Germain, P. Lee, S. Waldek, L. Caplan, G.E.
Linthorst, and R.J. Desnick. 2001. Safety and efficacy of recombinant human a-
galactosidase a replacement therapy in Fabry's disease. New Engl J Med. 341:9-16.
Erikson, A., C.G. Groth, J.E. Mansson, A. Percy, O. Ringden, and L. Svennerholm. 1990.
Clinical and biochemical outcome of marrow transplantation for Gaucher disease of the
Norrbottnian type. Acta Paediatr Scand. 79:680-5.
Erlandsson, A., and C.M. Morshead. 2006. Exploiting the properties of adult stem cells for the
treatment of disease. Curr Opin Mol Ther. 8:331-7.
Ernest, S., and E. Bello-Reuss. 1999. Secretion of platelet-activating factor is mediated by
MDR1 P-glycoprotein in cultured human mesangial cells. J Am Soc Nephrol. 10:2306-
13.
Ernest, S., S. Rajaraman, J. Megyesi, and E.N. Bello-Reuss. 1997. Expression of MDR1
(multidrug resistance) gene and its protein in normal human kidney. Nephron. 77:284-
289.
Evans, W.E., and J.A. Johnson. 2001. Pharmacogenomics: the inherited basis for interindividual
differences in drug response. Annu Rev Genomics Hum Genet. 2:9-39.
Falguieres, T., and L. Johannes. 2006. Shiga toxin B-subunit binds to the chaperone BiP and the
nucleolar protein B23. Biol Cell. 98:125-34.
Falguieres, T., M. Maak, C. von Weyhern, M. Sarr, X. Sastre, M.F. Poupon, S. Robine, L.
Johannes, and K.P. Janssen. 2008. Human colorectal tumors and metastases express Gb3
and can be targeted by an intestinal pathogen-based delivery tool. Mol Cancer Ther.
7:2498-508.
182
Falguieres, T., F. Mallard, C. Baron, D. Hanau, C. Lingwood, B. Goud, J. Salamero, and L.
Johannes. 2001. Targeting of Shiga toxin b-subunit to retrograde transport route in
association with detergent-resistant membranes. Mol. Biol. Cell. 12:2453-68.
Fan, J.Q. 2003. A contradictory treatment for lysosomal storage disorders: inhibitors enhance
mutant enzyme activity. Trends Pharmacol Sci. 24:355-60.
Fan, J.Q., S. Ishii, N. Asano, and Y. Suzuki. 1999. Accelerated transport and maturation of
lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat
Med. 5:112-5.
Fardel, O., A. Courtois, B. Drenou, T. Lamy, V. Lecureur, P.Y. le Prise, and R. Fauchet. 1996.
Inhibition of P-glycoprotein activity in human leukemic cells by mifepristone.
Anticancer Drugs. 7:671-7.
Farkas-Himsley, H., and R. Cheung. 1976. Bacterial proteinaceous products (bacteriocins) as
cytotoxic agents of neoplasia. Cancer Res. 36:3561-3567.
Farkas-Himsley, H., B. Rosen, R. Hill, S. Arab, and C.A. Lingwood. 1995. Bacterial colicin
active against tumour cells in vitro and in vivo is verotoxin 1. Proc Natl Acad Sci.
92:6996-7000.
Ferry, D.R., and D.J. Kerr. 1994. Multidrug resistance in cancer. Bmj. 308:148-9.
Finn, L.S., M. Zhang, S.H. Chen, and C.R. Scott. 2000. Severe type II Gaucher disease with
ichthyosis, arthrogryposis and neuronal apoptosis: molecular and pathological analyses.
Am J Med Genet. 91:222-6.
Fisher, G.A., B.L. Lum, J. Hausdorff, and B.I. Sikic. 1996. Pharmacological considerations in
the modulation of multidrug resistance. Eur J Cancer. 32A:1082-8.
FitzGerald, D.J., M.C. Willingham, C.O. Cardarelli, H. Hamada, T. Tsuruo, M.M. Gottesman,
and I. Pastan. 1987. A monoclonal antibody-Pseudomonas toxin conjugate that
specifically kills multidrug-resistant cells. Proc Natl Acad Sci U S A. 84:4288-92.
183
Fontaine, M., W.F. Elmquist, and D.W. Miller. 1996. Use of rhodamine 123 to examine the
functional activity of P-glycoprotein in primary cultured brain microvessel endothelial
cell monolayers. Life Sci. 59:1521-31.
Ford, J.M., J.M. Yang, and W.N. Hait. 1996. P-glycoprotein-mediated multidrug resistance:
experimental and clinical strategies for its reversal. Cancer Treat Res. 87:3-38.
Forsyth, G., K. Romero, J. Alverson, D. VanderJagt, and R. Glew. 1993. Variable expression of
leukocyte cytosolic broad-specificity beta-glucosidase activity. Clin Chim Acta. 216:11-
21.
Fracasso, P.M., L.J. Goldstein, D.P. de Alwis, J.S. Rader, M.A. Arquette, S.A. Goodner, L.P.
Wright, C.L. Fears, R.J. Gazak, V.A. Andre, M.F. Burgess, C.A. Slapak, and J.H.
Schellens. 2004. Phase I study of docetaxel in combination with the P-glycoprotein
inhibitor, zosuquidar, in resistant malignancies. Clin Cancer Res. 10:7220-8.
Frelet, A., and M. Klein. 2006. Insight in eukaryotic ABC transporter function by mutation
analysis. FEBS Lett. 580:1064-84.
Frustaci, A., C. Chimenti, R. Ricci, L. Natale, M.A. Russo, M. Pieroni, C.M. Eng, and R.J.
Desnick. 2001. Improvement in cardiac function in the cardiac variant of Fabry's disease
with galactose- infusion therapy. N Engl J Med. 345:25-32.
Fukasawa, M., M. Nishijima, and K. Hanada. 1999. Genetic evidence for ATP-dependent
endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin
synthesis in Chinese hamster ovary cells. J Cell Biol. 144:673-85.
Futerman, A.H., and Y.A. Hannun. 2004. The complex life of simple sphingolipids. EMBO Rep.
5:777-82.
Futerman, A.H., and R.E. Pagano. 1991. Determination of the intracellular sites and topology of
glucosylceramide synthesis in rat liver. Biochem J. 280 ( Pt 2):295-302.
184
Futerman, A.H., B. Stieger, A.L. Hubbard, and R.E. Pagano. 1990. Sphingomyelin synthesis in
rat liver occurs predominantly at the cis and medial cisternae of the Golgi apparatus. J
Biol Chem. 265:8650-7.
Futerman, A.H., J.L. Sussman, M. Horowitz, I. Silman, and A. Zimran. 2004. New directions in
the treatment of Gaucher disease. Trends Pharmacol Sci. 25:147-51.
Gadsby, D.C., G. Nagel, and T.C. Hwang. 1995. The CFTR chloride channel of mammalian
heart. Annu Rev Physiol. 57:387-416.
Garrigues, A., A.E. Escargueil, and S. Orlowski. 2002. The multidrug transporter, P-
glycoprotein, actively mediates cholesterol redistribution in the cell membrane. Proc
Natl Acad Sci U S A. 99:10347-52.
Gascoyne, N., and W.E. Van Heyningen. 1975. Binding of cholera toxin by various tissues.
Infect Immun. 12:466-9.
Gaus, K., E. Gratton, E.P. Kable, A.S. Jones, I. Gelissen, L. Kritharides, and W. Jessup. 2003.
Visualizing lipid structure and raft domains in living cells with two-photon microscopy.
Proc Natl Acad Sci U S A. 100:15554-9.
Gayet, L., G. Dayan, S. Barakat, S. Labialle, M. Michaud, S. Cogne, A. Mazane, A.W.
Coleman, D. Rigal, and L.G. Baggetto. 2005. Control of P-glycoprotein activity by
membrane cholesterol amounts and their relation to multidrug resistance in human CEM
leukemia cells. Biochemistry. 44:4499-509.
Germann, U.A. 1996. P-glycoprotein--a mediator of multidrug resistance in tumour cells. Eur J
Cancer. 32A:927-44.
Gervasoni, J.E., Jr., S.Z. Fields, S. Krishna, M.A. Baker, M. Rosado, K. Thuraisamy, A.A.
Hindenburg, and R.N. Taub. 1991. Subcellular distribution of daunorubicin in P-
glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal
microscopy. Cancer Res. 51:4955-63.
185
Ghislain, J., C.A. Lingwood, and E.N. Fish. 1994. Evidence for glycosphingolipid modification
of the type 1 IFN receptor. J Immunol. 153:3655-3663.
Gieselmann, V. 2005. What can cell biology tell us about heterogeneity in lysosomal storage
diseases? Acta Paediatr Suppl. 94:80-6; discussion 79.
Gillet, J.P., T. Efferth, and J. Remacle. 2007. Chemotherapy- induced resistance by ATP-binding
cassette transporter genes. Biochim Biophys Acta. 1775:237-62.
Ginzburg, L., Y. Kacher, and A.H. Futerman. 2004. The pathogenesis of glycosphingolipid
storage disorders. Semin Cell Dev Biol. 15:417-31.
Girardin, F. 2006. Membrane transporter proteins: a challenge for CNS drug development.
Dialogues Clin Neurosci. 8:311-21.
Giraudo, C.G., and H.J. Maccioni. 2003. Ganglioside glycosyltransferases organize in distinct
multienzyme complexes in CHO-K1 cells. J Biol Chem. 278:40262-71.
Globisch, C., I.K. Pajeva, and M. Wiese. 2008. Identification of putative binding sites of P-
glycoprotein based on its homology model. ChemMedChem. 3:280-95.
Gomez-Mouton, C., J.L. Abad, E. Mira, R.A. Lacalle, E. Gallardo, S. Jimenez-Baranda, I. Illa,
A. Bernad, S. Manes, and A.C. Martinez. 2001. Segregation of leading-edge and uropod
components into specific lipid rafts during T cell polarization. Proc Natl Acad Sci U S A.
98:9642-7.
Gong, Y., Y. Wang, F. Chen, J. Han, J. Miao, N. Shao, Z. Fang, and R. Ou Yang. 2000.
Identification of the subcellular localization of daunorubicin in multidrug-resistant K562
cell line. Leuk Res. 24:769-74.
Good, J.R., and A. Kuspa. 2000. Evidence that a cell-type-specific efflux pump regulates cell
differentiation in Dictyostelium. Dev Biol. 220:53-61.
186
Goodell, M.A., K. Brose, G. Paradis, A.S. Conner, and R.C. Mulligan. 1996. Isolation and
functional properties of murine hematopoietic stem cells that are replicating in vivo. J
Exp Med. 183:1797-806.
Goreva, O.B., A.Y. Grishanova, O.V. Mukhin, N.P. Domnikova, and V.V. Lyakhovich. 2003.
Possible prediction of the efficiency of chemotherapy in patients with
lymphoproliferative diseases based on MDR1 gene G2677T and C3435T
polymorphisms. Bull Exp Biol Med. 136:183-5.
Gottesman, M.M. 2002. Mechanisms of cancer drug resistance. Annu Rev Med. 53:615-27.
Gottesman, M.M., and S.V. Ambudkar. 2001. Overview: ABC transporters and human disease.
J Bioenerg Biomembr. 33:453-8.
Gottesman, M.M., S.V. Ambudkar, B. Ni, J.M. Aran, Y. Sugimoto, C.O. Cardarelli, and I.
Pastan. 1994. Exploiting multidrug resistance to treat cancer. Cold Spring Harb Symp
Quant Biol. 59:677-83.
Gottesman, M.M., T. Fojo, and S.E. Bates. 2002. Multidrug resistance in cancer: role of ATP-
dependent transporters. Nat Rev Cancer. 2:48-58.
Gottesman, M.M., C.A. Hrycyna, P.V. Schoenlein, U.A. Germann, and I. Pastan. 1995. Genetic
analysis of the multidrug transporter. Annu Rev Genet. 29:607-49.
Gottesman, M.M., and V. Ling. 2006. The molecular basis of multidrug resistance in cancer: the
early years of P-glycoprotein research. FEBS Lett. 580:998-1009.
Gottesman, M.M., and I. Pastan. 1993. Biochemistry of multidrug resistance mediated by the
multidrug transporter. Annu Rev Biochem. 62:385-427.
Gottesman, M.M., I. Pastan, and S.V. Ambudkar. 1996. P-glycoprotein and multidrug
resistance. Curr Opin Genet Dev. 6:610-7.
187
Gouaze, V., Y.Y. Liu, C.S. Prickett, J.Y. Yu, A.E. Giuliano, and M.C. Cabot. 2005.
Glucosylceramide synthase blockade down-regulates P-glycoprotein and resensitizes
multidrug-resistant breast cancer cells to anticancer drugs. Cancer Res. 65:3861-7.
Grazide, S., A.D. Terrisse, S. Lerouge, G. Laurent, and J.P. Jaffrezou. 2004. Cytoprotective
effect of glucosylceramide synthase inhibition against daunorubicin- induced apoptosis in
human leukemic cell lines. J Biol Chem. 279:18256-61.
Gros, P., J. Croop, and D. Housman. 1986. Mammalian multidrug resistance gene: complete
cDNA sequence indicates strong homology to bacterial transport proteins. Cell. 47:371-
80.
Guillas, I., P.A. Kirchman, R. Chuard, M. Pfefferli, J.C. Jiang, S.M. Jazwinski, and A.
Conzelmann. 2001. C26-CoA-dependent ceramide synthesis of Saccharomyces
cerevisiae is operated by Lag1p and Lac1p. Embo J. 20:2655-65.
Hakomori, S. 1986. Glycosphingolipids. Scientific American. 254:40-53.
Hakomori, S. 1996. Tumor malignancy defined by aberrant glycosylation and
sphingo(glyco)lipid metabolism. Cancer Res. 56:5309-5318.
Hakomori, S. 2003. Structure, organization, and function of glycosphingolipids in membrane.
Curr Opin Hematol. 10:16-24.
Hakomori, S., K. Handa, K. Iwabuchi, S. Yamamura, and A. Prinetti. 1998. New insights in
glycosphingolipid function: "glycosignaling domain," a cell surface assembly of
glycosphingolipids with signal transducer molecules,involved in cell adhesion coupled
with signaling. Glycobiology. 8:xi-xix.
Hakomori, S., and Y. Igarashi. 1993. Gangliosides and glycosphingolipids as modulators of cell
growth, adhesion, and transmembrane signaling. Adv Lipid Res. 25:147-62.
Hakomori, S.I. 2002. The glycosynapse. Proc Natl Acad Sci U S A. 99:225-32.
188
Hakomori, S.I. 2008. Structure and function of glycosphingolipids and sphingolipids:
recollections and future trends. Biochim Biophys Acta. 1780:325-46.
Hakomori, S.-I. 1981. Glycosphingolipid in cellular interaction, differentiations, and
oncogenesis. Ann Rev Biochem. 150:733-764.
Halter, D., S. Neumann, S.M. van Dijk, J. Wolthoorn, A.M. de Maziere, O.V. Vieira, P. Mattjus,
J. Klumperman, G. van Meer, and H. Sprong. 2007. Pre- and post-Golgi translocation of
glucosylceramide in glycosphingolipid synthesis. J Cell Biol. 179:101-15.
Hamada, H., and T. Tsuruo. 1986. Functional role for the 170- to 180-kDa glycoprotein specific
to drug-resistant tumor cells as revealed by monoclonal antibodies. Proc Natl Acad Sci
U S A. 83:7785-9.
Hammache, D., N. Yahi, M. Maresca, G. Pieroni, and J. Fantini. 1999. Human erythrocyte
glycosphingolipids as alternative cofactors for human immunodeficiency virus type 1
(HIV-1) entry: Evidence for CD4-induced interactions between HIV-1 gp120 and
reconstituted membrane microdomains of glycosphingolipids (Gb3 and GM3). J Virol.
73:5244-5248.
Hammerle, S.P., B. Rothen-Rutishauser, S.D. Kramer, M. Gunthert, and H. Wunderli-
Allenspach. 2000. P-Glycoprotein in cell cultures: a combined approach to study
expression, localisation, and functionality in the confocal microscope. Eur J Pharm Sci.
12:69-77.
Hanada, K. 2003. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism.
Biochim Biophys Acta. 1632:16-30.
Hanada, K., T. Hara, M. Fukasawa, A. Yamaji, M. Umeda, and M. Nishijima. 1998.
Mammalian cell mutants resistant to a sphingomyelin-directed cytolysin. Genetic and
biochemical evidence for complex formation of the LCB1 protein with the LCB2 protein
for serine palmitoyltransferase. J Biol Chem. 273:33787-94.
189
Hanada, K., K. Kumagai, S. Yasuda, Y. Miura, M. Kawano, M. Fukasawa, and M. Nishijima.
2003. Molecular machinery for non-vesicular trafficking of ceramide. Nature. 426:803-
9.
Hanekop, N., J. Zaitseva, S. Jenewein, I.B. Holland, and L. Schmitt. 2006. Molecular insights
into the mechanism of ATP-hydrolysis by the NBD of the ABC-transporter HlyB. FEBS
Lett. 580:1036-41.
Hannun, Y.A., and C.M. Linardic. 1993. Sphingolipid breakdown products: anti-proliferative
and tumor-suppressor lipids. Biochem Biophys Acta. 1154:223-236.
Hannun, Y.A., and C. Luberto. 2000. Ceramide in the eukaryotic stress response. Trends Cell
Biol. 10:73-80.
Hannun, Y.A., and L.M. Obeid. 2002. The ceramide-centric universie of lipid-mediated cell
regulation: stress encounters of the lipid kind. Journal of Biological Chemistry.
277:25847-50.
Hannun, Y.A., and B. R.M. 1987. Lysosphingolipids inhibit protein kinase C: Implications for
the sphingolipodoses. Science. 235:670-674.
Harder, T., and K. Simons. 1997. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol
microdomains. Curr Opin Cell Biol. 9:534-42.
Heath-Engel, H.M., and C.A. Lingwood. 2003. Verotoxin sensitivity of ECV304 cells in vitro
and in vivo in a xenograft tumour model: VT1 as a tumour neovascular marker.
Angiogenesis. 6:129-41.
Hein, L.K., P.J. Meikle, J.J. Hopwood, and M. Fuller. 2007. Secondary sphingolipid
accumulation in a macrophage model of Gaucher disease. Mol Genet Metab. 92:336-45.
Hellberg, A., J. Poole, and M.L. Olsson. 2002. Molecular basis of the globoside-deficient P(k)
blood group phenotype. Identification of four inactivating mutations in the UDP-N-
190
acetylgalactosamine: globotriaosylceramide 3-beta-N-acetylgalactosaminyltransferase
gene. J Biol Chem. 277:29455-9.
Hellberg, A., A. Ringressi, V. Yahalom, J. Safwenberg, M.E. Reid, and M.L. Olsson. 2004.
Genetic heterogeneity at the glycosyltransferase loci underlying the GLOB blood group
system and collection. Br J Haematol. 125:528-36.
Herzog, C.E., M. Tsokos, S.E. Bates, and A.T. Fojo. 1993. Increased mdr-1/P-glycoprotein
expression after treatment of human colon carcinoma cells with P. glycoprotein
antagonists. J Biol Chem. 268:2946-2952.
Hesselink, D.A., R.H. van Schaik, I.P. van der Heiden, M. van der Werf, P.J. Gregoor, J.
Lindemans, W. Weimar, and T. van Gelder. 2003. Genetic polymorphisms of the
CYP3A4, CYP3A5, and MDR-1 genes and pharmacokinetics of the calcineurin
inhibitors cyclosporine and tacrolimus. Clin Pharmacol Ther. 74:245-54.
Heukamp, L.C., D.W. Schroder, D. Plassmann, J. Homann, and R. Buttner. 2003. Marked
clinical and histologic improvement in a patient with type-1 Gaucher's disease following
long-term glucocerebroside substitution. A case report and review of current diagnosis
and management. Pathol Res Pract. 199:159-63.
Higgins, C., and M. Gottesman. 1992a. Is the multidrug transporter a flippase? Trends Biochem
Sci. 17:18-21.
Higgins, C.F. 2007. Multiple molecular mechanisms for multidrug resistance transporters.
Nature. 446:749-57.
Higgins, C.F., R. Callaghan, K.J. Linton, M.F. Rosenberg, and R.C. Ford. 1997. Structure of the
multidrug resistance P-glycoprotein. Semin Cancer Biol. 8:135-42.
Higgins, C.F., and M.M. Gottesman. 1992b. Comparison of two stage epidermal carcinogenesis
initiated by 7,12-dimethylbenz(a)antracene or N-Methyl-N'-nitro-N-nitrosoguanidine in
newborn and adult SENCAR and BALB/c mice. Cancer Res. 41:770-773.
191
Higgins, C.F., and K.J. Linton. 2004. The ATP switch model for ABC transporters. Nat Struct
Mol Biol. 11:918-26.
Hikita, T., K. Tadano-Aritomi, N. Iida-Tanaka, J.K. Anand, I. Ishizuka, and S. Hakomori. 2001.
A novel plasmal conjugate to glycerol and psychosine ("glyceroplasmalopsychosine"):
isolation and characterization from bovine brain white matter. J Biol Chem. 276:23084-
91.
Hikita, T., K. Tadano-Aritomi, N. Iida-Tanaka, S.B. Levery, I. Ishizuka, and S. Hakomori.
2002. Cationic glycosphingolipids in neuronal tissues and their possible biological
significance. Neurochem Res. 27:575-81.
Hillig, I., D. Warnecke, and E. Heinz. 2005. An inhibitor of glucosylceramide synthase inhibits
the human enzyme, but no t enzymes from other organisms. Biosci Biotechnol Biochem.
69:1782-5.
Hinrichs, J.W., K. Klappe, I. Hummel, and J.W. Kok. 2004. ATP-binding cassette transporters
are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane
domains (DIGs) in human multidrug-resistant cancer cells. J Biol Chem. 279:5734-8.
Hobbs, J.R., K.H. Jones, P.J. Shaw, I. Lindsay, and M. Hancock. 1987. Beneficial effect of pre-
transplant splenectomy on displacement bone marrow transplantation for Gaucher's
syndrome. Lancet. 1:1111-5.
Hoetzl, S., H. Sprong, and G. van Meer. 2007. The way we view cellular (glyco)sphingolipids. J
Neurochem. 103 Suppl 1:3-13.
Hoffmeyer, S., O. Burk, O. von Richter, H.P. Arnold, J. Brockmoller, A. Johne, I. Cascorbi, T.
Gerloff, I. Roots, M. Eichelbaum, and U. Brinkmann. 2000. Functional polymorphisms
of the human multidrug-resistance gene: multiple sequence variations and correlation of
one allele with P-glycoprotein expression and activity in vivo. Proc Natl Acad Sci U S A.
97:3473-8.
192
Hollenstein, K., R.J. Dawson, and K.P. Locher. 2007. Structure and mechanism of ABC
transporter proteins. Curr Opin Struct Biol. 17:412-8.
Holowka, D., J.A. Gosse, A.T. Hammond, X. Han, P. Sengupta, N.L. Smith, A. Wagenknecht-
Wiesner, M. Wu, R.M. Young, and B. Baird. 2005. Lipid segregation and IgE receptor
signaling: a decade of progress. Biochim Biophys Acta. 1746:252-9.
Holthuis, J.C., and T.P. Levine. 2005. Lipid traffic: floppy drives and a superhighway. Nat Rev
Mol Cell Biol. 6:209-20.
Homolya, L., Z. Hollo, U.A. Germann, I. Pastan, M.M. Gottesman, and B. Sarkadi. 1993.
Fluorescent cellular indicators are extruded by the multidrug resistance protein. J Biol
Chem. 268:21493-6.
Hoogerbrugge, P.M., B.J. Poorthuis, A.E. Romme, J.J. van de Kamp, G. Wagemaker, and D.W.
van Bekkum. 1988. Effect of bone marrow transplantation on enzyme levels and clinical
course in the neurologically affected twitcher mouse. J Clin Invest. 81:1790-4.
Hooper, N. 1999. Detergent- insoluble glycosphingolipid/cholesterol-rich membrane domains,
lipid rafts and caveolae. Molr Membr Biol. 16:145-156.
Hrycyna, C.A., M. Ramachandra, U.A. Germann, P.W. Cheng, I. Pastan, and M.M. Gottesman.
1999. Both ATP sites of human P-glycoprotein are essential but not symmetric.
Biochemistry. 38:13887-99.
Hsu, S.I., L. Lothstein, and S.B. Horwitz. 1989. Differential overexpression of three mdr gene
family members in multidrug-resistant J774.2 mouse cells. Evidence that distinct P-
glycoprotein precursors are encoded by unique mdr genes. J Biol Chem. 264:12053-62.
Hummel, I., K. Klappe, and J.W. Kok. 2005. Up-regulation of lactosylceramide synthase in
MDR1 overexpressing human liver tumour cells. FEBS Lett. 579:3381-4.
Humpf, H.U., E.M. Schmelz, F.I. Meredith, H. Vesper, T.R. Vales, E. Wang, D.S. Menaldino,
D.C. Liotta, and A.H. Merrill, Jr. 1998. Acylation of naturally occurring and synthetic 1-
193
deoxysphinganines by ceramide synthase. Formation of N-palmitoyl-aminopentol
produces a toxic metabolite of hydrolyzed fumonisin, AP1, and a new category of
ceramide synthase inhibitor. J Biol Chem. 273:19060-4.
Hyafil, F., C. Vergely, P. Du Vignaud, and T. Grand-Perret. 1993. In vitro and in vivo reversal
of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res.
53:4595-602.
Hyde, S.C., P. Emsley, M.J. Hartshorn, J. Mimmach, U. Gileadi, S.R. Pearce, M.P. Gallagher,
D.R. Gill, R.E. Hubbard, and C.F. Higgins. 1990. Structural model of ATP binding
proteins associated with cystic fibrosis, multi drug resistance and bacterial transport.
Nature. 346:362-365.
Ichikawa, S., H. Sakiyama, G. Suzuki, K.I. Hidari, and Y. Hirabayashi. 1996. Expression
cloning of a cDNA for human ceramide glucosyltransferase that catalyzes the first
glycosylation step of glycosphingolipid synthesis. Proc Natl Acad Sci U S A. 93:4638-
43.
Ioannou, Y.A. 2001. Multidrug permeases and subcellular cholesterol transport. Nat Rev Mol
Cell Biol. 2:657-668.
Ioannou, Y.A., K.M. Zeidner, R.E. Gordon, and R.J. Desnick. 2001. Fabry disease: preclinical
studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzyme-
deficient mice. Am J Hum Genet. 68:14-25.
Ishii, S., H. Yoshioka, K. Mannen, A.B. Kulkarni, and J.Q. Fan. 2004. Transgenic mouse
expressing human mutant alpha-galactosidase A in an endogenous enzyme defic ient
background: a biochemical animal model for studying active-site specific chaperone
therapy for Fabry disease. Biochim Biophys Acta. 1690:250-7.
194
Ishitoya, S., H. Kurazono, H. Nishiyama, E. Nakamura, T. Kamoto, T. Habuchi, A. Terai, O.
Ogawa, and S. Yamamoto. 2004. Verotoxin induces rapid elimination of human renal
tumor xenografts in SCID mice. J Urol. 171:1309-13.
Jacewicz, M., H. Clausen, E. Nudelman, A. Donohue-Rolfe, and G.T. Keusch. 1986.
Pathogenesis of Shigella diarrhea. XI. Isolation of a shigella toxin-binding glycolipid
from rabbit jejunum and HeLa cells and its identification as globotriaosylceramide. J.
Exp. Med. 163:1391-1404.
Jackson, K.A., S.M. Majka, H. Wang, J. Pocius, C.J. Hartley, M.W. Majesky, M.L. Entman,
L.H. Michael, K.K. Hirschi, and M.A. Goodell. 2001. Regeneration of ischemic cardiac
muscle and vascular endothelium by adult stem cells. J Clin Invest. 107:1395-402.
Jaffrezou, J.P., T. Levade, A. Bettaieb, N. Andrieu, C. Bezombes, N. Maestre, S. Vermeersch,
A. Rousse, and G. Laurent. 1996. Daunorubicin- induced apoptosis: triggering of
ceramide generation through sphingomyelin hydrolysis. Embo J. 15:2417-24.
Jamroziak, K., W. Mlynarski, E. Balcerczak, M. Mistygacz, J. Trelinska, M. Mirowski, J.
Bodalski, and T. Robak. 2004. Functiona l C3435T polymorphism of MDR1 gene: an
impact on genetic susceptibility and clinical outcome of childhood acute lymphoblastic
leukemia. Eur J Haematol. 72:314-21.
Janssen, K.P., D. Vignjevic, R. Boisgard, T. Falguieres, G. Bousquet, D. Decaudin, F. Dolle, D.
Louvard, B. Tavitian, S. Robine, and L. Johannes. 2006. In vivo tumor targeting using a
novel intestinal pathogen-based delivery approach. Cancer Res. 66:7230-6.
Jarosch, E., U. Lenk, and T. Sommer. 2003. Endoplasmic reticulum-associated protein
degradation. Int Rev Cytol. 223:39-81.
Jarvis, R.M., A. Chamba, M.J. Holder, A. Challa, D.C. Smith, M.N. Hodgkin, J.M. Lord, and J.
Gordon. 2007. Dynamic interplay between the neutral glycosphingolipid CD77/Gb3 and
195
the therapeutic antibody target CD20 within the lipid bilayer of model B lymphoma
cells. Biochem Biophys Res Commun. 355:944-9.
Jennemann, R., R. Sandhoff, L. Langbein, S. Kaden, U. Rothermel, H. Gallala, K. Sandhoff, H.
Wiegandt, and H.J. Grone. 2007. Integrity and barrier function of the epidermis critically
depend on glucosylceramide synthesis. J Biol Chem. 282:3083-94.
Jennemann, R., R. Sandhoff, S. Wang, E. Kiss, N. Gretz, C. Zuliani, A. Martin-Villalba, R.
Jager, H. Schorle, M. Kenzelmann, M. Bonrouhi, H. Wiegandt, and H.J. Grone. 2005.
Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural
defects after birth. Proc Natl Acad Sci U S A. 102:12459-64.
Jette, L., E. Beaulieu, J.M. Leclerc, and R. Beliveau. 1996. Cyclosporin A treatment induces
overexpression of P-glycoprotein in the kidney and other tissues. Am J Physio.
270:F756-765.
Jeyakumar, M., R.A. Dwek, T.D. Butters, and F.M. Platt. 2005. Storage solutions: treating
lysosomal disorders of the brain. Nat Rev Neurosci. 6:713-25.
Jeyakumar, M., R. Thomas, E. Elliot-Smith, D.A. Smith, A.C. van der Spoel, A. d'Azzo, V.H.
Perry, T.D. Butters, R.A. Dwek, and F.M. Platt. 2003. Central nervous system
inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2
gangliosidosis. Brain. 126:974-87.
Jiang, J.C., P.A. Kirchman, M. Zagulski, J. Hunt, and S.M. Jazwinski. 1998. Homologs of the
yeast longevity gene LAG1 in Caenorhabditis elegans and human. Genome Res. 8:1259-
72.
Jmoudiak, M., and A.H. Futerman. 2005. Gaucher disease: pathological mechanisms and
modern management. Br J Haematol. 129:178-88.
Johnstone, R.W., A.A. Ruefli, and M.J. Smyth. 2000. Multiple physiological functions for
multidrug transporter P-glycoprotein. Trends Biochem Sci. 25:1-6.
196
Juliano, R.L., and V. Ling. 1976. A surface glycoprotein modulating drug permeability in
chinese hamster ovary cell mutants. Biochem Biophys Acta. 455:152-162.
Julien, M., S. Kajiji, R.H. Kaback, and P. Gros. 2000. Simple purification of highly active
biotinylated P-glycoprotein: enantiomer-specific modulation of drug-stimulated ATPase
activity. Biochemistry. 39:75-85.
Juranka, P.F., R.L. Zastawny, and V. Ling. 1989. P-glycoprotein: multidrug-resistance and a
superfamily of membrane-associated transport proteins. Faseb J. 3:2583-92.
Kafka, A., G. Sauer, C. Jaeger, R. Grundmann, R. Kreienberg, R. Zeillinger, and H. Deissler.
2003. Polymorphism C3435T of the MDR-1 gene predicts response to preoperative
chemotherapy in locally advanced breast cancer. Int J Oncol. 22:1117-21.
Kalin, N., J. Fernandes, S. Hrafnsdottir, and G. van Meer. 2004. Natural phosphatidylcholine is
actively translocated across the plasma membrane to the surface of mammalian cells. J
Biol Chem. 279:33228-36.
Kamau, S.W., S.D. Kramer, M. Gunthert, and H. Wunderli-Allenspach. 2005. Effect of the
modulation of the membrane lipid composition on the localization and function of P-
glycoprotein in MDR1-MDCK cells. In Vitro Cell Dev Biol Anim. 41:207-16.
Kanfer, J., and S. Hakomori. 1983. Sphingolipid Biochemistry. Plenum Press, New York.
Kapitonov, D., and R.K. Yu. 1997. Cloning, characterization, and expression of human
ceramide galactosyltransferase cDNA. Biochem Biophys Res Commun. 232:449-53.
Karlsson, K.A. 1970. Sphingolipid long chain bases. Lipids. 5:878-91.
Kaszubiak, A., A. Kupstat, U. Muller, R. Hausmann, P.S. Holm, and H. Lage. 2007. Regulation
of MDR1 gene expression in multidrug-resistant cancer cells is independent from YB-1.
Biochem Biophys Res Commun. 357:295-301.
Katagiri, Y., T. Mori, H. Nakajima, C. Katagiri, T. Taguchi, T. Takeda, N. Kiyokawa, and J.
Fujimoto. 1999. Activation of Src family kinase induced by Shiga toxin binding to
197
globotriaosyl ceramide (Gb3/CD77) in low density, detergent- insoluble microdomains.
J. Biol. Chem. 274:35278-35282.
Kaur, P. 1997. Expression and characterization of DrrA and DrrB proteins of Streptomyces
peucetius in Escherichia coli: DrrA is an ATP binding protein. J Bacteriol. 179:569-75.
Kaur, P. 2002. Multidrug resistance: can different keys open the same lock? Drug Resist Updat.
5:61-4.
Kawai, H., M.L. Allende, R. Wada, M. Kono, K. Sango, C. Deng, T. Miyakawa, J.N. Crawley,
N. Werth, U. Bierfreund, K. Sandhoff, and R.L. Proia. 2001. Mice expressing only
monosialoganglioside GM3 exhibit lethal audiogenic seizures. J Biol Chem. 276:6885-8.
Kessel, D., V. Botterill, and I. Wodinsky. 1968. Uptake and retention of daunomycin by mouse
leukemic cells as factors in drug response. Cancer Res. 28:938-41.
Khan, F., F. Proulx, and C.A. Lingwood. 2009. Detergent-resistant globotriaosyl ceramide may
define verotoxin/glomeruli-restricted hemolytic uremic syndrome pathology. Kidney Int.
75:1209-16.
Khine, A.A., M. Firtel, and C.A. Lingwood. 1998. CD77-dependent retrograde transport of
CD19 to the nuclear membrane: Functional Relationship between CD77 and CD19
during germinal center B-cell apoptosis. J. Cell. Physiol. 176:281-292.
Khine, A.A., and C.A. Lingwood. 1994. Capping and receptor mediated endocytosis of cell
bound verotoxin(Shiga- like toxin) 1; Chemical identification of an amino acid in the B
subunit necessary for efficient receptor glycolipid binding and cellular internalization. J.
Cell. Physiol. 161:319-332.
Khine, A.A., and C.A. Lingwood. 2000. Functional significance of globotriaosylceramide in a2
interferon/ type I interferon receptor mediated anti viral activity'. J. Cell. Physiol.
182:97-108.
198
Khine, A.A., P. Tam, A. Nutikka, and C.A. Lingwood. 2004. Brefeldin A and filipin distinguish
two Globotriaosyl ceramide/ Verotoxin-1 intracellular trafficking pathways involved in
Vero cell cytotoxicity. Glycobiology. 14:701-712.
Kiarash, A., B. Boyd, and C.A. Lingwood. 1994a. Glycosphingolipid receptor function is
modified by fatty acid content: Verotoxin 1 and Verotoxin 2c preferentially recognize
different globotriaosyl ceramide fatty acid homologues. J. Biol. Chem. 269:11138-
11146.
Kiarash, A., B. Boyd, and C.A. Lingwood. 1994b. The role of the lipid moiety in glycolipid
receptor function. Model studies of verotoxin binding to fatty acid homologues of
globotriaosylceramide demonstrate lipid dependent alterations in carbohydrate receptor
function. In Recent Advances in Verocytotoxin-Producing Eshcerichia Coli Infections.
M.A. Karmali and A.G. Goglio, editors. Elsevier Science, B.V., Bergamo, Italy. 175-
187.
Kim, R.B., M.F. Fromm, C. Wandel, B. Leake, A.J. Wood, D.M. Roden, and G.R. Wilkinson.
1998. The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-
1 protease inhibitors. J Clin Invest. 101:289-94.
Kim, S.H., S.H. Lee, N.H. Kwak, C.D. Kang, and B.S. Chung. 1996. Effect of the activated Raf
protein kinase on the human multidrug resistance 1 (MDR1) gene promoter. Cancer
Lett. 98:199-205.
Kimchi-Sarfaty, C., J.M. Oh, I.W. Kim, Z.E. Sauna, A.M. Calcagno, S.V. Ambudkar, and M.M.
Gottesman. 2007. A "silent" polymorphism in the MDR1 gene changes substrate
specificity. Science. 315:525-8.
Kimura, Y., N. Kioka, H. Kato, M. Matsuo, and K. Ueda. 2007. Modulation of drug-stimulated
ATPase activity of human MDR1/P-glycoprotein by cholesterol. Biochem J. 401:597-
605.
199
Kioka, N., J. Tsubota, Y. Kakehi, T. Komano, M.M. Gottesman, I. Pastan, and K. Ueda. 1989.
P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries
Gly185 with an altered pattern of multidrug resistance. Biochem Biophys Res Commun.
162:224-31.
Klein, E.A. 2006. Chemoprevention of prostate cancer. Annu Rev Med. 57:49-63.
Klein, I., B. Sarkadi, and A. Varadi. 1999. An inventory of the human ABC proteins. Biochim
Biophys Acta. 1461:237-62.
Klimecki, W.T., B.W. Futscher, T.M. Grogan, and W.S. Dalton. 1994. P-glycoprotein
expression and function in circulating blood cells from normal volunteers. Blood.
83:2451-8.
Knapp, W., P. Dörken, P. Rieber, R.E. Schmidt, H. Stein, and A.E. von dem Borne. 1989. CD
antigens 1989 [letter]. Int J Cancer. 44:190-191.
Kohyama-Koganeya, A., T. Sasamura, E. Oshima, E. Suzuki, S. Nishihara, R. Ueda, and Y.
Hirabayashi. 2004. Drosophila glucosylceramide synthase: a negative regulator of cell
death mediated by proapoptotic factors. J Biol Chem. 279:35995-6002.
Kojima, Y., S. Fukumoto, K. Furukawa, O. T., J. Wiels, K. Yokoyama, Y. Suzuki, T. Urano,
and M. Ohta. 2000. Molecular cloning of globotriaosylceramide/CD77 synthase, a
glycosyltransfease that initiates the synthesis of globo series glycosphingolipids. J Biol
Chem. 275:15152-15156.
Kok, J.W., R.J. Veldman, K. Klappe, H. Koning, C.M. Filipeanu, and M. Muller. 2000.
Differential expression of sphingolipids in MRP1 overexpressing HT29 cells. Int J
Cancer. 87:172-8.
Kolesnick, R. 2002. The therapeutic potential of modulating the ceramide/sphingomyelin
pathway. J Clin Invest. 110:3-8.
200
Kolesnick, r., and D. Golde. 1994. The Sphingomyelin Pathway in Tumour Necrosis factor and
Interleukin-1 Signalling. Cell. 77:325-326.
Kolesnick, R.N. 1991. Sphingomyelin and derivatives as cellular signals. Prog Lipid Res. 30:1-
38.
Kolter, T., R.L. Proia, and K. Sandhoff. 2002. Combinatorial ganglioside biosynthesis. J Biol
Chem. 277:25859-62.
Kolter, T., and K. Sandhoff. 2005. Principles of lysosomal membrane digestion: stimulation of
sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.
Annu Rev Cell Dev Biol. 21:81-103.
Kolter, T., and K. Sandhoff. 2006. Sphingolipid metabolism diseases. Biochim Biophys Acta.
1758:2057-79.
Kolter, T., and M. Wendeler. 2003. Chemical chaperones--a new concept in drug research.
Chembiochem. 4:260-4.
Kool, M., M. van der Linden, M. de Haas, G.L. Scheffer, J.M. de Vree, A.J. Smith, G. Jansen,
G.J. Peters, N. Ponne, R.J. Scheper, R.P. Elferink, F. Baas, and P. Borst. 1999. MRP3,
an organic anion transporter able to transport anti-cancer drugs. Proc Natl Acad Sci U S
A. 96:6914-9.
Kopnin, B.P., T.P. Stromskaya, R.V. Kondratov, V.S. Ossovskaya, E.N. Pugacheva, E.Y.
Rybalkina, O.A. Khokhlova, and P.M. Chumakov. 1995. Influence of exogenous ras and
p53 on P-glycoprotein function in immortalized rodent fibroblasts. Oncol Res. 7:299-
306.
Korkotian, E., A. Schwarz, D. Pelled, G. Schwarzmann, M. Segal, and A. Futerman. 1999.
Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic
reticulum density and in functional calcium stores in cultured neurons. J Biol Chem.
274:21673-21678.
201
Kovbasnjuk, O., M. Edidin, and M. Donowitz. 2001. Role of lipid rafts in Shiga toxin 1
interaction with the apical surface of Caco-2 cells. J Cell Sci. 114:4025-4031.
Kovbasnjuk, O., R. Mourtazina, B. Baibakov, T. Wang, C. Elowsky, M.A. Choti, A. Kane, and
M. Donowitz. 2005. The glycosphingolipid globotriaosylceramide in the metastatic
transformation of colon cancer. Proc Natl Acad Sci U S A. 102:19087-92.
Krishna, R., and L.D. Mayer. 2000. Multidrug resistance (MDR) in cancer. Mechanisms,
reversal using modulators of MDR and the role of MDR modulators in influencing the
pharmacokinetics of anticancer drugs. Eur J Pharm Sci. 11:265-83.
Kruh, G.D., and M.G. Belinsky. 2003. The MRP family of drug efflux pumps. Oncogene.
22:7537-52.
Ktitorova, O.V., E.C. Kakpakova, M.M. Vinogradova, E.N. Il'ina, V.M. Govorun, T.N.
Zabotina, A.A. Stavrovskaia, and A.A. Shtil. 2001. [Relationship between the induction
of MDR1, a multidrug resistance gene in tumor cells, and apoptosis]. Ontogenez.
32:295-301.
Kuwano, M., Y. Oda, H. Izumi, S.J. Yang, T. Uchiumi, Y. Iwamoto, M. Toi, T. Fujii, H.
Yamana, H. Kinoshita, T. Kamura, M. Tsuneyoshi, K. Yasumoto, and K. Kohno. 2004.
The role of nuclear Y-box binding protein 1 as a global marker in drug resistance. Mol
Cancer Ther. 3:1485-92.
Kuwano, M., T. Uchiumi, H. Hayakawa, M. Ono, M. Wada, H. Izumi, and K. Kohno. 2003. The
basic and clinical implications of ABC transporters, Y-box-binding protein-1 (YB-1) and
angiogenesis-related factors in human malignancies. Cancer Sci. 94:9-14.
Kwan, P., and M.J. Brodie. 2005. Potential role of drug transporters in the pathogenesis of
medically intractable epilepsy. Epilepsia. 46:224-35.
LaCasse, E.C., M.R. Bray, B. Patterson, W.-M. Lim, S. Perampalam, L.G. Radvanyi, A.
Keating, A.K. Stewart, R. Buckstein, J.S. Sandhu, N. Miller, D. Banderjee, D. Singh,
202
A.R. Belch, L.M. Pilarski, and J. Gariépy. 1999. Shiga- like toxin I receptor on human
breast cancer, lymphoma, and myeloma and absence from CD34+ hematopoietic stem
cells: Implications for ex vivo tumor purging and autologous stem cell transplantation.
Blood. 94:1-12.
LaCasse, E.C., M.T. Saleh, B. Patterson, M.D. Minden, and J. Gariépy. 1996. Shiga- like toxin
purges human lymphoma from bone marrow of severe combined immunodeficient mice.
Blood. 88:1561-1567.
Lachmann, R.H. 2006. Miglustat: substrate reduction therapy for glycosphingolipid lysosomal
storage disorders. Drugs Today (Barc). 42:29-38.
Laemmeli, U.K. 1970. Cleavage of structural proteins during the assembly of head of
bacteriophage T4. Nature. 227:680-685.
Lage, H. 2006. MDR1/P-glycoprotein (ABCB1) as target for RNA interference-mediated
reversal of multidrug resistance. Curr Drug Targets. 7:813-21.
Lage, H. 2008. An overview of cancer multidrug resistance: a still unsolved problem. Cell Mol
Life Sci. 65:3145-67.
Lahiri, S., and A.H. Futerman. 2005. LASS5 is a bona fide dihydroceramide synthase that
selectively utilizes palmitoyl-CoA as acyl donor. J Biol Chem. 280:33735-8.
Lala, P., S. Ito, and C.A. Lingwood. 2000. Transfection of MDCK cells with the MDR1 gene
results in a major increase in globotriaosyl ceramide and cell sensitivity to
verocytotoxin: role of P-gp in glycolipid biosynthesis. J Biol Chem. 275:6246-6251.
Lannert, H., C. Bunning, D. Jeckel, and F. Wieland. 1994. Lactosyl ceramide is synthesized in
the lumen of the Golgi apparatus. FEBS Lett. 342:91-96.
Lannert, H., K. Gorgas, I. Meißner, F.T. Wieland, and D. Jeckel. 1998. Functional organization
of the Golgi apparatus in glycosphingolipid biosynthesis. Lactosylceramide and
203
subsequent glycosphingolipids are formed in the lumen of the late Golgi. J Biol Chem.
273:2939-2946.
Larson, D.R., J.A. Gosse, D.A. Holowka, B.A. Baird, and W.W. Webb. 2005. Temporally
resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living
cells. J Cell Biol. 171:527-36.
Lavie, Y., H. Cao, S.L. Bursten, A.E. Giuliano, and M.C. Cabot. 1996. Accumulation of
glucosylceramides in multidrug-resistant cancer cells. J Biol Chem. 271:19530-19536.
Lavie, Y., H. Cao, A. Volner, A. Lucci, T.-Y. Han, V. Geffen, A.E. Giuliano, and M.C. Cabot.
1997. Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin
A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human
cancer cells. J Biol Chem. 272:1682-1687.
Lavie, Y., G. Fiucci, and M. Lisovitch. 1998. Up-regulation of calveolae and calveolar
constituents in Multidrug-resistant cancer cells. J Biol Chem. 273:32380-32383.
Lavie, Y., and M. Liscovitch. 2000. Changes in lipid and protein constituents of rafts and
caveolae in multidrug resistant cancer cells and their functional consequences. Glycoconj
J. 17:253-9.
Ledoux, S., R. Yang, G. Friedlander, and D. Laouari. 2003. Glucose depletion enhances P-
glycoprotein expression in hepatoma cells: role of endoplasmic reticulum stress
response. Cancer Res. 63:7284-90.
Lee, L., A. Abe, and J.A. Shayman. 1999. Improved inhibitors of glucosylceramide synthase. J
Biol Chem. 274:14662-14669.
Legare, D., S. Cayer, A.K. Singh, D. Richard, B. Papadopoulou, and M. Ouellette. 2001. ABC
proteins of Leishmania. J Bioenerg Biomembr. 33:469-74.
204
Leinekugel, P., S. Michel, E. Conzelmann, and K. Sandhoff. 1992. Quantitative correlation
between the residual activity of beta-hexosaminidase A and arylsulfatase A and the
severity of the resulting lysosomal storage disease. Hum Genet. 88:513-23.
Lin, T., O. Islam, and K. Heese. 2006. ABC transporters, neural stem cells and neurogenesis--a
different perspective. Cell Res. 16:857-71.
Lincke, C.R., J.J. Smit, T. van der Velde-Koerts, and P. Borst. 1991. Structure of the human
MDR3 gene and physical mapping of the human MDR locus. J Biol Chem. 266:5303-10.
Ling, V., and L.H. Thompson. 1974. Reduced permeability in CHO cells as a mechanism of
resistance to colchicine. J Cell Physiol. 83:103-16.
Lingwood, C.A. 1994. Verotoxin-binding in human renal sections. Nephron. 66:21-28.
Lingwood, C.A. 1998. Oligosaccharide receptors for bacteria: a view to a kill. Current Biology.
2:695-700.
Lingwood, C.A., H. Law, S. Richardson, M. Petric, J.L. Brunton, S. DeGrandis, and M.
Karmali. 1987. Glycolipid binding of purified and recombinant Escherichia coli-
produced verotoxin in vitro. J. Biol. Chem. 262:8834-8839.
Lingwood, C.A., and A. Nutikka. 1994. A novel chemical procedure for the selective removal of
nonreducing terminal N-acetyl residues from glycolipids. Anal. Biochem. 217:119-123.
Lingwood, C.A., and S.C.K. Yiu. 1992. Glycolipid modification of a2- interferon binding:
Sequence similarity between a2- interferon receptor and the verotoxin (Shiga- like toxin)
B-subunit. Biochem. J. 283:25-26.
Linton, K.J., and C.F. Higgins. 2007. Structure and function of ABC transporters: the ATP
switch provides flexible control. Pflugers Arch. 453:555-67.
Liu, Y., Han, TY, Giuliano, AE, Cabot, MC. 2001. Ceramide glycosylation potentiates cellular
multidrug resistance. FASEB J. 15:719-30.
205
Liu, Y., Han, TY, Giuliano, AE, Hansen, N, Cabot, MC. 2000. Uncoupling ceramide
glycosylation by transfection of glucosylceramide synthase antisense reverses
adriamycin resistance. J Biol Chem. 275:7138-43.
Liu, Y.-Y., T.-Y. Han, A.E. Giuliano, and M.C. Cabot. 1999. Expression of glucosylceramide
synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in
human breast cancer cells. J Biol Chem. 274:1140-1146.
Liu, Y.Y., T.Y. Han, J.Y. Yu, A. Bitterman, A. Le, A.E. Giuliano, and M.C. Cabot. 2004.
Oligonucleotides blocking glucosylceramide synthase expression selectively reverse
drug resistance in cancer cells. J Lipid Res. 45:933-40.
Lloyd, K.O., and K. Furukawa. 1998. Biosynthesis and functions of gangliosides: recent
advances. Glycoconj J. 15:627-36.
Locher, K.P., A.T. Lee, and D.C. Rees. 2002. The E. coli BtuCD structure: a framework for
ABC transporter architecture and mechanism. Science. 296:1091-8.
Loo, T.W., M.C. Bartlett, and D.M. Clarke. 2003. Simultaneous binding of two different drugs
in the binding pocket of the human multidrug resistance P-glycoprotein. J Biol Chem.
278:39706-10.
Loo, T.W., M.C. Bartlett, and D.M. Clarke. 2004a. Disulfide cross-linking analysis shows that
transmembrane segments 5 and 8 of human P-glycoprotein are close together on the
cytoplasmic side of the membrane. J Biol Chem. 279:7692-7.
Loo, T.W., M.C. Bartlett, and D.M. Clarke. 2004b. Val133 and Cys137 in transmembrane
segment 2 are close to Arg935 and Gly939 in transmembrane segment 11 of human P-
glycoprotein. J Biol Chem. 279:18232-8.
Loo, T.W., M.C. Bartlett, and D.M. Clarke. 2006a. Transmembrane segment 1 of human P-
glycoprotein contributes to the drug-binding pocket. Biochem J. 396:537-45.
206
Loo, T.W., M.C. Bartlett, and D.M. Clarke. 2006b. Transmembrane segment 7 of human P-
glycoprotein forms part of the drug-binding pocket. Biochem J. 399:351-9.
Loo, T.W., and D.M. Clarke. 1994a. Mutations to amino acids located in predicted
transmembrane segment 6 (TM6) modulate the activity and substrate specificity of
human P-glycoprotein. Biochemistry. 33:14049-57.
Loo, T.W., and D.M. Clarke. 1994b. Prolonged association of temperature-sensitive mutants of
human P-glycoprotein with calnexin during biogenesis. J Biol Chem. 269:28683-9.
Loo, T.W., and D.M. Clarke. 1995. P-glycoprotein. Associations between domains and between
domains and molecular chaperones. J Biol Chem. 270:21839-44.
Loo, T.W., and D.M. Clarke. 1998. Mutational analysis of human P-glycoprotein. Methods
Enzymol. 292:480-92.
Loo, T.W., and D.M. Clarke. 1999. The human multidrug resistance P-glycoprotein is inactive
when its maturation is inhibited: potentia l for a role in cancer chemotherapy. Faseb J.
13:1724-32.
Loo, T.W., and D.M. Clarke. 2000. The packing of the transmembrane segments of human
multidrug resistance P-glycoprotein is revealed by disulfide cross- linking analysis. J Biol
Chem. 275:5253-6.
Loo, T.W., and D.M. Clarke. 2001. Determining the dimensions of the drug-binding domain of
human P-glycoprotein using thiol cross-linking compounds as molecular rulers. J Biol
Chem. 276:36877-80.
Loo, T.W., and D.M. Clarke. 2002. Location of the rhodamine-binding site in the human
multidrug resistance P-glycoprotein. J Biol Chem. 277:44332-8.
Loo, T.W., and D.M. Clarke. 2005. Do drug substrates enter the common drug-binding pocket
of P-glycoprotein through "gates"? Biochem Biophys Res Commun. 329:419-22.
207
Lucci, A., W. Cho, T. Han, A. Giuliano, D. Morton, and M. Cabot. 1998. Glucosyl ceramide: a
marker for multiple-drug resistant cancers. Anticancer Res. 18:475-480.
Lucci, A., T.Y. Han, Y.Y. Liu, A.E. Giuliano, and M.C. Cabot. 1999. Multidrug resistance
modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in
drug-resistant cancer cells. Cancer. 86:300-11.
Lucke, T., W. Hoppner, E. Schmidt, S. Illsinger, and A.M. Das. 2004. Fabry disease: reduced
activities of respiratory chain enzymes with decreased levels of energy-rich phosphates
in fibroblasts. Mol Genet Metab. 82:93-7.
Lugo, M.R., and F.J. Sharom. 2005. Interaction of LDS-751 and rhodamine 123 with P-
glycoprotein: evidence for simultaneous binding of both drugs. Biochemistry. 44:14020-
9.
Luker, G., C. Pica, A. Kumar, D. Covey, and D. Piwnica-Worms. 2000. Effects of cholesterol
and enantiomeric cholesterol on P-glycoprotein localization and function in low-density
membrane domains. Biochem. 39:7651-61.
Luker, G.D., K.R. Nilsson, D.F. Covey, and D. Piwnica-Worms. 1999. Multidrug resistance
(MDR1) P-glycoprotein enhances esterification of plasma membrane cholesterol. J Biol
Chem. 274:6979-91.
Lund, N., D. Branch, M. Mylvaganam, D. Chark, X. Ma, D. Sakac, B. Binnington, J. Fantini, A.
Puri, R. Blumenthal, and C. Lingwood. 2006. A novel soluble mimic of the glycolipid
globotriaosylceramide inhibits HIV infection. AIDS. 20:1-11.
Lund, N., D.R. Branch, D. Sakac, C. Lingwood, C. Siatskas, C. Robinson, R. Brady, and J.
Medin. 2005. Lack of Susceptibility of Cells from Patients with Fabry Disease to
Infection with R5 Human Immunodeficiency Virus. AIDS. 19:1543-1546.
208
Lund, N., M.L. Olsson, S. Ramkumar, D. Sakac, V. Yahalom, C. Levene, A. Hellberg, X.Z. Ma,
B. Binnington, D. Jung, C.A. Lingwood, and D.R. Branch. 2009. The human P(k) histo-
blood group antigen provides protection against HIV-1 infection. Blood. 113:4980-91.
Maccioni, H.J., C.G. Giraudo, and J.L. Daniotti. 2002. Understanding the stepwise synthesis of
glycolipids. Neurochem Res. 27:629-36.
Mahfoud, R., N. Garmy, M. Maresca, N. Yahi, A. Puigserver, and J. Fantini. 2002a.
Identification of a common sphingolipid-binding domain in Alzheimer, prion and HIV-1
proteins. J Biol Chem. 277:11292-6.
Mahfoud, R., M. Mylvaganam, C.A. Lingwood, and J. Fantini. 2002b. A novel soluble analog
of the HIV-1 fusion cofactor, globotriaosylceramide(Gb3), eliminates the cholesterol
requirement for high affinity gp120/Gb3 interaction. J. Lipid Res. 43:1670-1679.
Maliepaard, M., G.L. Scheffer, I.F. Faneyte, M.A. van Gastelen, A.C. Pijnenborg, A.H.
Schinkel, M.J. van De Vijver, R.J. Scheper, and J.H. Schellens. 2001. Subcellular
localization and distribution of the breast cancer resistance protein transporter in normal
human tissues. Cancer Res. 61:3458-64.
Maloney, M.D., B. Binnington-Boyd, and C.A. Lingwood. 1999. Globotriaosyl ceramide
modulates interferon-a-induced growth inhibition and CD19 expression in Burkitt's
lymphoma cells. Glycoconj J. 16:821-828.
Maloney, M.D., and C.A. Lingwood. 1994. CD19 has a potential CD77 (globotriaosyl
ceramide)-binding site with sequence similarity to verotoxin B-subunits: Implications of
molecular mimicry for B cell adhesion and enterohemorrhagic Escherichia coli
pathogenesis. J. Exp. Med. 180:191-201.
Mamelak, D., and C. Lingwood. 1997. Expression and sulfogalactolipid binding of the
recombinant testis-specific cognate heat shock protein 70. Glycoconj J. 14:715-722.
209
Mangeney, M., Y. Richard, D. Coulaud, T. Tursz, and J. Wiels. 1991. CD77: an antigen of
germinal center B cells entering apoptosis. Eur. J. Immunol. 21:1131-1140.
Mangeney, M., G. Rousselet, S. Taga, T. Tursz, and J. Wiels. 1995. The fate of human CD77+
germinal center B lymphocytes after rescue from apoptosis. Mol Immunol. 32:333-339.
Mannori, G., O. Cecconi, G. Mugnai, and S. Ruggieri. 1990. Role of glycolipids in the
metastatic process: Characteristics neutral glycolipids in clones with different metastatic
potentials isolated from a murine fibrosarcoma cell line. Int J Cancer. 45:984-988.
Mantovani, I., A. Cappellini, P.L. Tazzari, V. Papa, L. Cocco, and A.M. Martelli. 2006.
Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-
lymphoblastoid CEM cells. J Cell Physiol. 207:836-44.
Maraldi, N.M., N. Zini, S. Santi, K. Scotlandi, M. Serra, and N. Baldini. 1999. P-glycoprotein
subcellular localization and cell morphotype in MDR1 gene-transfected human
osteosarcoma cells. Biol Cell. 91:17-28.
Marin, M., A. Poret, G. Maillet, F. Leboulenger, and F. Le Foll. 2005. Regulation of volume-
sensit ive Cl- channels in multi-drug resistant MCF7 cells. Biochem Biophys Res
Commun. 334:1266-78.
Markova, S., T. Nakamura, T. Sakaeda, H. Makimoto, H. Uchiyama, N. Okamura, and K.
Okumura. 2006. Genotype-dependent down-regulation of gene expression and function
of MDR1 in human peripheral blood mononuclear cells under acute inflammation. Drug
Metab Pharmacokinet. 21:194-200.
Marquardt, D., and M.S. Center. 1992. Drug transport mechanisms in HL60 cells isolated for
resistance to adriamycin: evidence for nuclear drug accumulation and redistribution in
resistant cells. Cancer Res. 52:3157-63.
210
Martin, C., G. Berridge, C.F. Higgins, and R. Callaghan. 1997. The multi-drug resistance
reversal agent SR33557 and modulation of vinca alkaloid binding to P-glycoprotein by
an allosteric interaction. Br J Pharmacol. 122:765-71.
Martin, C., G. Berridge, C.F. Higgins, P. Mistry, P. Charlton, and R. Callaghan. 2000a.
Communication between multiple drug binding sites on P-glycoprotein. Mol Pharmacol.
58:624-32.
Martin, C., G. Berridge, P. Mistry, C. Higgins, P. Charlton, and R. Callaghan. 1999. The
molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein. Br
J Pharmacol. 128:403-11.
Martin, C., G. Berridge, P. Mistry, C. Higgins, P. Charlton, and R. Callaghan. 2000b. Drug
binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide
hydrolysis. Biochemistry. 39:11901-6.
Mathias, S., L.A. Pena, and R.N. Kolesnick. 1998. Signal transduction of stress via ceramide.
Biochem J. 335 ( Pt 3):465-80.
Matsuda, J., O. Suzuki, A. Oshima, Y. Yamamoto, A. Noguchi, K. Takimoto, M. Itoh, Y.
Matsuzaki, Y. Yasuda, S. Ogawa, Y. Sakata, E. Nanba, K. Higaki, Y. Ogawa, L.
Tominaga, K. Ohno, H. Iwasaki, H. Watanabe, R.O. Brady, and Y. Suzuki. 2003.
Chemical chaperone therapy for brain pathology in G(M1)-gangliosidosis. Proc Natl
Acad Sci U S A. 100:15912-7.
Mattocks, M., M. Bagovich, M. De Rosa, S. Bond, B. Binnington, V. Rasaiah, J. Medin, and C.
Lingwood. 2006. Treatment of neutral glycosphingolipid storage disease via inhibition
of the ABC Drug Transporter, MDR1: Cyclosporin A can lower serum and some tissue
globotriaosyl ceramide levels in the Fabry’s mouse model. FASEB J. 273:2064-2075.
211
Maurer, B.J., L.S. Metelitsa, R.C. Seeger, M.C. Cabot, and C.P. Reynolds. 1999. Increase of
ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)-
retinamide in neuroblastoma cell lines. J Natl Cancer Inst. 91:1138-46.
May, G.L., L.C. Wright, M. Dyne, W.B. Mackinnon, R.M. Fox, and C.E. Mountford. 1988.
Plasma membrane lipid composition of vinblastine sensitive and resistant human
leukaemic lymphoblasts. Int J Cancer. 42:728-33.
Mayer, U., E. Wagenaar, J.H. Beijnen, J.W. Smit, D.K. Meijer, J. van Asperen, P. Borst, and
A.H. Schinkel. 1996. Substantial excretion of digoxin via the intestinal mucosa and
prevention of long-term digoxin accumulation in the brain by the mdr 1a P-glycoprotein.
Br J Pharmacol. 119:1038-44.
Mayor, S., A. Viola, R.V. Stan, and M.A. del Pozo. 2006. Flying kites on slippery slopes at
Keystone. Symposium on Lipid Rafts and Cell Function. EMBO Rep. 7:1089-93.
Mazzoni, A., and F. Trave. 1993. Cytoplasmic membrane cholesterol and doxorubicin
cytotoxicity in drug-sensitive and multidrug-resistant human ovarian cancer cells. Oncol
Res. 5:75-82.
McCubrey, J.A., L.S. Steelman, S.L. Abrams, J.T. Lee, F. Chang, F.E. Bertrand, P.M.
Navolanic, D.M. Terrian, R.A. Franklin, A.B. D'Assoro, J.L. Salisbury, M.C. Mazzarino,
F. Stivala, and M. Libra. 2006. Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT
pathways in malignant transformation and drug resistance. Adv Enzyme Regul. 46:249-
79.
McCubrey, J.A., L.S. Steelman, W.H. Chappell, S.L. Abrams, E.W. Wong, F. Chang, B.
Lehmann, D.M. Terrian, M. Milella, A. Tafuri, F. Stivala, M. Libra, J. Basecke, C.
Evangelisti, A.M. Martelli, and R.A. Franklin. 2007. Roles of the Raf/MEK/ERK
pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys
Acta. 1773:1263-84.
212
McGrath, T., and M.S. Center. 1987. Adriamycin resistance in HL60 cells in the absence of
detectable P-glycoprotein. Biochem Biophys Res Commun. 145:1171-6.
McKeegan, K.S., M.I. Borges-Walmsley, and A.R. Walmsley. 2003. The structure and function
of drug pumps: an update. Trends Microbiol. 11:21-9.
Mechetner, E.B., and I.B. Roninson. 1992. Efficient inhibition of P-glycoprotein-mediated
multidrug resistance with a monoclonal antibody. Proc Natl Acad Sci U S A. 89:5824-8.
Mechetner, E.B., B. Schott, B.S. Morse, W.D. Stein, T. Druley, K.A. Davis, T. Tsuruo, and I.B.
Roninson. 1997. P-glycoprotein function involves conformational transitions detectable
by differential immunoreactivity. Proc Natl Acad Sci U S A. 94:12908-13.
Meikle, P.J., J.J. Hopwood, A.E. Clague, and W.F. Carey. 1999. Prevalence of lysosomal
storage disorders. Jama. 281:249-54.
Meivar-Levy, I., M. Horowitz, and A.H. Futerman. 1994. Analysis of glucocerebrosidase
activity using N-(1-[14C]hexanoyl)-D-erythroglucosylsphingosine demonstrates a
correlation between levels of residual enzyme activity and the type of Gaucher disease.
Biochem J. 303 ( Pt 2):377-82.
Merrill, A.H., Jr. 2002. De novo sphingolipid biosynthesis: a necessary, but dangerous,
pathway. J Biol Chem. 277:25843-6.
Meschini, S., A. Calcabrini, E. Monti, D. Del Bufalo, A. Stringaro, E. Dolfini, and G. Arancia.
2000. Intracellular P-glycoprotein expression is associated with the intrinsic multidrug
resistance phenotype in human colon adenocarcinoma cells. Int J Cancer. 87:615-28.
Michel, C., and G. van Echten-Deckert. 1997. Conversion of dihydrocermamide to ceramide
occurs at the cytosolic face of the endoplasmic reticulum. FEBS Lett. 416:153-155.
Miltenberger, R.J., P.J. Farnham, D.E. Smith, J.M. Stommel, and M.M. Cornwell. 1995. v-Raf
activates transcription of growth-responsive promoters via GC-rich sequences that bind
the transcription factor Sp1. Cell Growth Differ. 6:549-56.
213
Miro Obradors, M.J., D. Sillence, S. Howitt, and D. Allan. 1997. The subcellular sites of
sphingomyelin synthesis in BHK cells. Biochim Biophys Acta. 1359:1-12.
Mistry, P., A.J. Stewart, W. Dangerfield, S. Okiji, C. Liddle, D. Bootle, J.A. Plumb, D.
Templeton, and P. Charlton. 2001. In vitro and in vivo reversal of P-glycoprotein-
mediated multidrug resistance by a novel potent modulator, XR9576. Cancer Res.
61:749-58.
Mizukami, H., Y. Mi, R. Wada, M. Kono, T. Yamashita, Y. Liu, N. Werth, R. Sandhoff, K.
Sandhoff, and R.L. Proia. 2002. Systemic inflammation in glucocerebrosidase-deficient
mice with minimal glucosylceramide storage. J Clin Invest. 109:1215-21.
Mizutani, T., M. Masuda, E. Nakai, K. Furumiya, H. Togawa, Y. Nakamura, Y. Kawai, K.
Nakahira, S. Shinkai, and K. Takahashi. 2008. Genuine functions of P-glycoprotein
(ABCB1). Curr Drug Metab. 9:167-74.
Mizutani, Y., A. Kihara, and Y. Igarashi. 2005. Mammalian Lass6 and its related family
members regulate synthesis of specific ceramides. Biochem J. 390:263-71.
Mizutani, Y., A. Kihara, and Y. Igarashi. 2006. LASS3 (longevity assurance homologue 3) is a
mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate
specificity. Biochem J. 398:531-8.
Modok, S., H.R. Mellor, and R. Callaghan. 2006. Modulation of multidrug resistance efflux
pump activity to overcome chemoresistance in cancer. Curr Opin Pharmacol. 6:350-4.
Molinari, A., M. Cianfriglia, S. Meschini, A. Calcabrini, and G. Arancia. 1994. P-Glycoprotein
expression in the Golgi apparatus of multidrug-resistant cells. Int J. Cancer. 59:789-795.
Moody, J.E., and P.J. Thomas. 2005. Nucleotide binding domain interactions during the
mechanochemical reaction cycle of ATP-binding cassette transporters. J Bioenerg
Biomembr. 37:475-9.
214
Morjani, H., N. Aouali, R. Belhoussine, R.J. Veldman, T. Levade, and M. Manfait. 2001.
Elevation of glucosylceramide in multidrug-resistant cancer cells and accumulation in
cytoplasmic droplets. Int J Cancer. 94:157-65.
Morris, D.I., L.M. Greenberger, E.P. Bruggemann, C. Cardarelli, M.M. Gottesman, I. Pastan,
and K.B. Seamon. 1994. Localization of the forskolin labeling sites to both halves of P-
glycoprotein: similarity of the sites labeled by forskolin and prazosin. Mol Pharmacol.
46:329-37.
Morschhauser, F., P.L. Zinzani, M. Burgess, L. Sloots, F. Bouafia, and C. Dumontet. 2007.
Phase I/II trial of a P-glycoprotein inhibitor, Zosuquidar.3HCl trihydrochloride
(LY335979), given orally in combination with the CHOP regimen in patients with non-
Hodgkin's lymphoma. Leuk Lymphoma. 48:708-15.
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods. 65:55-63.
Mosser, J., A.M. Douar, C.O. Sarde, P. Kioschis, R. Feil, H. Moser, A.M. Poustka, J.L. Mandel,
and P. Aubourg. 1993. Putative X-linked adrenoleukodystrophy gene shares unexpected
homology with ABC transporters. Nature. 361:726-30.
Mountford, C.E., and L.C. Wright. 1988. Organization of lipids in the plasma membranes of
malignant and stimulated cells: a new model. Trends Biochem Sci. 13:172-7.
Muller, M.R., J. Thomale, M.F. Rajewsky, and S. Seeber. 1998. Drug resistance and DNA
repair in leukaemia. Cytotechnology. 27:175-185.
Murray, L.J., J.A. Habeshaw, J. Wiels, and M.F. Greaves. 1985. Expression of Burkitt
lymphoma-associated antigen (defined by the monoclonal antibody 38.13) on both
normal and malignant germinal-centre B cells. Int. J. Cancer. 36:561-565.
215
Mylvaganam, M., and C. Lingwood. 1999. Adamantyl globotriaosyl ceramide- a monovalent
soluble glycolipid mimic which inhibits verotoxin binding to its glycolipid receptor.
Biochem. Biophys. Res. Commun. 257:391-394.
Mylvaganam, M., and C.A. Lingwood. 2003. A preamble to aglycone reconstruction for
membrane-presented glycolipids. In Carbohydrate-based Drug Discovery. Vol. 2. C.-H.
Wong, editor. Wiley-VCH Press, Weinheim, Germany. 761-780.
Naiki, M., and D.M. Marcus. 1974. Human erythrocyte P and Pk blood group antigens:
Indentification as glycosphingolipids. Biochem. Biophys. Res. Comm. 60:1105-1111.
Naito, M. 2008. Macrophage differentiation and function in health and disease. Pathol Int.
58:143-55.
Nehete, P.N., E.M. Vela, M.M. Hossain, A.K. Sarkar, N. Yahi, J. Fantini, and K.J. Sastry. 2002.
A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host
cell surface glycosphingolipids is common to entry and infection by diverse HIV-1
strains. Antiviral Res. 56:233-51.
Newman, M.J., J.C. Rodarte, K.D. Benbatoul, S.J. Romano, C. Zhang, S. Krane, E.J. Moran,
R.T. Uyeda, R. Dixon, E.S. Guns, and L.D. Mayer. 2000. Discovery and characterization
of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance.
Cancer Res. 60:2964-72.
Ng, W.F., F. Sarangi, R.L. Zastawny, L. Veinot-Drebot, and V. Ling. 1989. Identification of
members of the P-glycoprotein multigene family. Mol Cell Biol. 9:1224-32.
Nicholson, K., D. Quinn, G. Kellett, and J. Warr. 1999. Preferential killing of multidrug
resistant KB cells by inhibitors of glucosylceramide synthase. Br J Cancer. 81:423-430.
Nikaido, H. 1994. Maltose transport system of Escherichia coli: an ABC-type transporter. FEBS
Lett. 346:55-8.
216
Nobili, S., I. Landini, B. Giglioni, and E. Mini. 2006. Pharmacological strategies for
overcoming multidrug resistance. Curr Drug Targets. 7:861-79.
Norris-Cervetto, E., R. Callaghan, F.M. Platt, R.A. Dwek, and T.D. Butters. 2004. Inhibition of
glucosylceramide synthase does not reverse drug resistance in cancer cells. J Biol Chem.
279:40412-8.
Nudelman, E., R. Kannagi, S. Hakomori, M. Parsons, M. Lipinski, J. Wiels, M. Fellows, and T.
Tursz. 1983. A glycolipid antigen associated with Burkitt lymphoma defined by a
monoclonal antibody. Science. 220:509.
Nudelman, E.D., S.B. Levery, Y. Igarashi, and S. Hakomori. 1992. Plasmalopsychosine, a novel
plasmal (fatty aldehyde) conjugate of psychosine with cyclic acetal linkage. Isolation
and characterization from human brain white matter. J Biol Chem. 267:11007-16.
Nussbaumer, P. 2008. Medicinal chemistry aspects of drug targets in sphingolipid metabolism.
ChemMedChem. 3:543-51.
Nyholm, P.G., G. Magnusson, Z. Zheng, R. Norel, B. Binnington-Boyd, and C.A. Lingwood.
1996. Two distinct binding sites for globotriaosyl ceramide on verotoxins: molecular
modelling and confirmation by analogue studies and a new glycolipid receptor for all
verotoxins. Chem. Biol. 3:263-275.
Obeid, L.M., and Y.A. Hannun. 1995. Ceramide: a stress signal and mediator of growth
suppression and apoptosis. J Cell Biochem. 58:191-8.
O'Brien, A.D., T.A. Lively, M.E. Chen, S.W. Rothman, and S.B. Formal. 1983. Escherichia coli
O157:H7 strains associated with hemorrhagic colitis in the United States produce a
Shigella dysenteriae 1 (Shiga)- like cytotoxin. Lancet. i:702.
Ogretmen, B., and Y.A. Hannun. 2001. Updates on functions of ceramide in chemotherapy-
induced cell death and in multidrug resistance. Drug Resist Updat. 4:368-77.
217
Ogretmen, B., and Y.A. Hannun. 2004. Biologically active sphingolipids in cancer pathogenesis
and treatment. Nat Rev Cancer. 4:604-16.
Ohga, T., K. Koike, M. Ono, Y. Makino, Y. Itagaki, M. Tanimoto, M. Kuwano, and K. Kohno.
1996. Role of the human Y box-binding protein YB-1 in cellular sensitivity to the DNA-
damaging agents cisplatin, mitomycin C, and ultraviolet light. Cancer Res. 56:4224-8.
Ohshima, T., G.J. Murray, W.D. Swaim, G. Longenecker, J.M. Quirk, C.O. Cardarelli, Y.
Sugimoto, I. Pastan, M.M. Gottesman, R.O. Brady, and A.B. Kulkarni. 1997. alpha-
Galactosidase A deficient mice: a model of Fabry disease. Proc Natl Acad Sci U S A.
94:2540-4.
Ohshima, T., R. Schiffmann, G.J. Murray, J. Kopp, J.M. Quirk, S. Stahl, C.C. Chan, P. Zerfas,
J.H. Tao-Cheng, J.M. Ward, R.O. Brady, and A.B. Kulkarni. 1999. Aging accentuates
and bone marrow transplantation ameliorates metabolic defects in Fabry disease mice.
Proc Natl Acad Sci U S A. 96:6423-7.
Ohyama, C., Y. Fukushi, M. Satoh, S. Saitoh, S. Orikasa, E. Nudelman, M. Straud, and S.-I.
Hakomori. 1990. Changes in glycolipid expression in human testicular tumors. Int J
Cancer. 45:1040-1044.
Okamoto, T., A. Schlegel, P.E. Scherer, and M.P. Lisanti. 1998. Caveolins, a family of
scaffolding proteins for organizing "preassembled signaling complexes" at the plasma
membrane. J Biol Chem. 273:5419-22.
Okuda, T., N. Tokuda, S. Numata, M. Ito, M. Ohta, K. Kawamura, J. Wiels, T. Urano, O.
Tajima, and K. Furukawa. 2006. Targeted disruption of Gb3/CD77 synthase gene
resulted in the complete deletion of globo-series glycosphingolipids and loss of
sensitivity to verotoxins. J Biol Chem. 281:10230-5.
Oldham, M.L., A.L. Davidson, and J. Chen. 2008. Structural insights into ABC transporter
mechanism. Curr Opin Struct Biol. 18:726-33.
218
Olie, R.A., B. Fenderson, K. Daley, J.W. Oosterhuis, J. Murphy, and L.H.J. Looijenga. 1996.
Glycolipids of human primary testicular germ cell tumours. Br J Cancer. 74:133-140.
Orlowski, S., S. Martin, and A. Escargueil. 2006. P-glycoprotein and 'lipid rafts': some
ambiguous mutual relationships (floating on them, building them or meeting them by
chance?). Cell Mol Life Sci. 63:1038-59.
Orvisky, E., E. Sidransky, C.E. McKinney, M.E. Lamarca, R. Samimi, D. Krasnewich, B.M.
Martin, and E.I. Ginns. 2000. Glucosylsphingosine accumulation in mice and patients
with type 2 Gaucher disease begins early in gestation. Pediatr Res. 48:233-7.
Oswald, S., S. Haenisch, C. Fricke, T. Sudhop, C. Remmler, T. Giessmann, G. Jedlitschky, U.
Adam, E. Dazert, R. Warzok, W. Wacke, I. Cascorbi, H.K. Kroemer, W. Weitschies, K.
von Bergmann, and W. Siegmund. 2006. Intestinal expression of P-glycoprotein
(ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphate-
glucuronosyltransferase 1A1 predicts the disposition and modulates the effects of the
cholesterol absorption inhibitor ezetimibe in humans. Clin Pharmacol Ther. 79:206-17.
Parton, R.G. 1996. Caveolae and caveolins. Curr Opin Cell Biol. 8:542-8.
Parton, R.G., and A.A. Richards. 2003. Lipid rafts and caveolae as portals for endocytosis: new
insights and common mechanisms. Traffic. 4:724-38.
Parton, R.G., and K. Simons. 1995. Digging into caveolae. Science. 269:1398-9.
Pascaud, C., M. Garrigos, and S. Orlowski. 1998. Multidrug resistance transporter P-
glycoprotein has distinct but interacting binding sites for cytotoxic drugs and reversing
agents. Biochem J. 333 ( Pt 2):351-8.
Pastan, I., M. Gottesman, K. Ueda, E. Lovelace, A. Rutherford, and M. Willingham. 1988. A
retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized
expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci. 85:4486-4490.
219
Paulsen, I.T., and R.A. Skurray. 1993. Topology, structure and evolution of two families of
proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes--
an analysis. Gene. 124:1-11.
Pauwels, E.K., P. Erba, G. Mariani, and C.M. Gomes. 2007. Multidrug resistance in cancer: its
mechanism and its modulation. Drug News Perspect. 20:371-7.
Pawlik, A., M. Baskiewicz-Masiuk, B. Machalinski, K. Safranow, and B. Gawronska-Szklarz.
2005. Involvement of P-glycoprotein in the release of cytokines from peripheral blood
mononuclear cells treated with methotrexate and dexamethasone. J Pharm Pharmacol.
57:1421-5.
Pedemonte, N., G.L. Lukacs, K. Du, E. Caci, O. Zegarra-Moran, L.J. Galietta, and A.S.
Verkman. 2005. Small-molecule correctors of defective DeltaF508-CFTR cellular
processing identified by high-throughput screening. J Clin Invest. 115:2564-71.
Pedersen, P.L. 2007. Transport ATPases into the year 2008: a brief overview related to types,
structures, functions and roles in health and disease. J Bioenerg Biomembr. 39:349-55.
Pelled, D., S. Trajkovic-Bodennec, E. Lloyd-Evans, E. Sidransky, R. Schiffmann, and A.H.
Futerman. 2005. Enhanced calcium release in the acute neuronopathic form of Gaucher
disease. Neurobiol Dis. 18:83-8.
Pellizzari, A., H. Pang, and C.A. Lingwood. 1992. Binding of verocytotoxin 1 to its receptor is
influenced by differences in receptor fatty acid content. Biochem. 31:1363-1370.
Perloff, M.D., E. Stormer, L.L. von Moltke, and D.J. Greenblatt. 2003. Rapid assessment of P-
glycoprotein inhibition and induction in vitro. Pharm Res. 20:1177-83.
Perry, D.K., J. Carton, A.K. Shah, F. Meredith, D.J. Uhlinger, and Y.A. Hannun. 2000. Serine
palmitoyltransferase regulates de novo ceramide generation during etoposide- induced
apoptosis. J Biol Chem. 275:9078-84.
220
Perry, R.J., and N.D. Ridgway. 2006. Oxysterol-binding protein and vesicle-associated
membrane protein-associated protein are required for sterol-dependent activation of the
ceramide transport protein. Mol Biol Cell. 17:2604-16.
Pewzner-Jung, Y., S. Ben-Dor, and A.H. Futerman. 2006. When do Lasses (longevity assurance
genes) become CerS (ceramide synthases)?: Insights into the regulation of ceramide
synthesis. J Biol Chem. 281:25001-5.
Pierce, S.K. 2002. Lipid rafts and B-cell activation. Nat Rev Immunol. 2:96-105.
Pike, L.J. 2003. Lipid Rafts: Bringing Order to Chaos. J Lip Res. 44:655-67.
Pike, L.J. 2004. Lipid rafts: heterogeneity on the high seas. Biochem J. 378:281-92.
Platt, F.M., M. Jeyakumar, U. Andersson, T. Heare, R.A. Dwek, and T.D. Butters. 2003.
Substrate reduction therapy in mouse models of the glycosphingolipidoses. Philos Trans
R Soc Lond B Biol Sci. 358:947-54.
Platt, F.M., G.R. Neises, R.A. Dwek, and T.D. Butters. 1994a. N-butyldeoxynojirimycin is a
novel inhibitor of glycolipid biosynthesis. J Biol Chem. 269:8362-5.
Platt, F.M., G.R. Neises, G.B. Karlsson, R.A. Dwek, and T.D. Butters. 1994b. N-
butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-
linked oligosaccharide processing. J Biol Chem. 269:27108-14.
Platt, F.M., G.R. Neises, G. Reinkensmeier, M.J. Townsend, V.H. Perry, R.L. Proia, B.
Winchester, R.A. Dwek, and T.D. Butters. 1997. Prevention of lysosomal storage in
Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science. 276:428-31.
Plo, I., G. Lehne, K.J. Beckstrom, N. Maestre, A. Bettaieb, G. Laurent, and D. Lautier. 2002.
Influence of ceramide metabolism on P-glycoprotein function in immature acute
myeloid leukemia KG1a cells. Mol Pharmacol. 62:304-12.
Pohl, A., P.F. Devaux, and A. Herrmann. 2005. Function of prokaryotic and eukaryotic ABC
proteins in lipid transport. Biochim Biophys Acta. 1733:29-52.
221
Polgar, O., and S.E. Bates. 2005. ABC transporters in the balance: is there a role in multidrug
resistance? Biochem Soc Trans. 33:241-5.
Polli, J.W., S.A. Wring, J.E. Humphreys, L. Huang, J.B. Morgan, L.O. Webster, and C.S.
Serabjit-Singh. 2001. Rational use of in vitro P-glycoprotein assays in drug discovery. J
Pharmacol Exp Ther. 299:620-8.
Priddle, H., D.R. Jones, P.W. Burridge, and R. Patient. 2006. Hematopoiesis from human
embryonic stem cells: overcoming the immune barrier in stem cell therapies. Stem Cells.
24:815-24.
Pudymaitis, A., G. Armstrong, and C.A. Lingwood. 1991. Verotoxin resistant clones are
deficient in the glycolipid globotriosyl ceramide: Differential basis of mutant phenotype.
Arch. Biochem. Biophys. 286:448-452.
Pudymaitis, A., and C.A. Lingwood. 1992. Susceptibility to verotoxin as a function of the cell
cycle. J Cell Physiol. 150:632-639.
Puri, A., P. Hug, K. Jernigan, J. Barchi, H.-Y. Kim, J. Hamilton, J. Wiels, G.J. Murray, R.O.
Brady, and R. Blumenthal. 1998a. The neutral glycosphingolipid globotriaosylceramide
promotes fusion mediated by a CD4-dependent CXCR4-utilizing HIV type 1 envelope
glycoprotein. Proc. Natl. Acad. Sci. 95:14435-14440.
Puri, A., P. Hug, I. Munoz-Berroso, and R. Blumenthal. 1998b. Human erythrocyte glycolipids
promote HIV-1 envelope glycoprotein mediated fusion of CD4+ cells. Biochem Biophys
Res Commun. 242:219-225.
Puri, V., R. Watanabe, M. Dominguez, X. Sun, C.L. Wheatley, D.L. Marks, and R.E. Pagano.
1999. Cholesterol modulates membrane traffic along the endocytic pathway in
sphingolipid-storage diseases. Nat Cell Biol. 1:386-388.
222
Puri, V., R. Watanabe, R.D. Singh, M. Dominguez, J.C. Brown, C.L. Wheatley, D.L. Marks,
and R.E. Pagano. 2001. Clathrin-dependent and -independent internalization of plasma
membrane sphingolipids initiates two Golgi targeting pathways. J Cell Biol. 154:535-47.
Purvis, I.J., A.J. Bettany, T.C. Santiago, J.R. Coggins, K. Duncan, R. Eason, and A.J. Brown.
1987. The efficiency of folding of some proteins is increased by controlled rates of
translation in vivo. A hypothesis. J Mol Biol. 193:413-7.
Qia, S., H. Wang, and X. Chen. 2005. Reversal of HCC drug resistance by using hammerhead
ribozymes against multidrug resistance 1 gene. J Huazhong Univ Sci Technolog Med
Sci. 25:662-4.
Raaijmakers, M.H. 2007. ATP-binding-cassette transporters in hematopoietic stem cells and
their utility as therapeutical targets in acute and chronic myeloid leukemia. Leukemia.
21:2094-102.
Raas-Rothschild, A., I. Pankova-Kholmyansky, Y. Kacher, and A.H. Futerman. 2004.
Glycosphingolipidoses: beyond the enzymatic defect. Glycoconj J. 21:295-304.
Radeva, G., J. Perabo, and F.J. Sharom. 2005. P-Glycoprotein is localized in intermediate-
density membrane microdomains distinct from classical lipid rafts and caveolar domains.
Febs J. 272:4924-37.
Rafi, M.A., G. de Gala, X.L. Zhang, and D.A. Wenger. 1993. Mutational analysis in a patient
with a variant form of Gaucher disease caused by SAP-2 deficiency. Somat Cell Mol
Genet. 19:1-7.
Raggers, R., I. Vogels, and G. van Meer. 2001. Multidrug-resistance P-glycoprotein (MDR1)
secretes platelet-activating factor. Biochem J. 357:859-65.
Rajendran, L., and K. Simons. 2005. Lipid rafts and membrane dynamics. J Cell Sci. 118:1099-
102.
223
Ramotar, K., B. Boyd, G. Tyrrell, J. Gariepy, C. Lingwood, and J. Brunton. 1990.
Characterization of Shiga-like toxin I B subunit purified from overproducing clones of
the SLT-I B cistron. Biochem J. 272:805-11.
Ramu, A., D. Glaubiger, and H. Weintraub. 1984. Differences in lipid composition of
doxorubicin-sensitive and -resistant P388 cells. Cancer Treat Rep. 68:637-41.
Randolph, G.J., S. Beaulieu, M. Pope, I. Sugawara, L. Hoffman, R.M. Steinman, and W.A.
Muller. 1998. A physiologic function for p-glycoprotein (MDR-1) during the migration
of dendritic cells from skin via afferent lymphatic vessels. Proc Natl Acad Sci U S A.
95:6924-9.
Rapola, J. 1994. Lysosomal storage diseases in adults. Pathol Res Pract. 190:759-66.
Rawat, S.S., M. Viard, S.A. Gallo, R. Blumenthal, and A. Puri. 2006. Sphingolipids, cholesterol,
and HIV-1: A paradigm in viral fusion. Glycoconj J. 23:189-97.
Rich, D.P., M.P. Anderson, R.J. Gregory, S.H. Cheng, S. Paul, D.M. Jefferson, J.D. McCann,
K.W. Klinger, A.E. Smith, and M.J. Welsh. 1990. Expression of cystic fibrosis
transmembrane conductance regulator corrects defective chloride channel regulation in
cystic fibrosis airway epithelial cells. Nature. 347:358-63.
Riebeling, C., J.C. Allegood, E. Wang, A.H. Merrill, Jr., and A.H. Futerman. 2003. Two
mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate
dihydroceramide synthesis using different fatty acyl-CoA donors. J Biol Chem.
278:43452-9.
Ringden, O., C.G. Groth, A. Erikson, S. Granqvist, J.E. Mansson, and E. Sparrelid. 1995. Ten
years' experience of bone marrow transplantation for Gaucher disease. Transplantation.
59:864-70.
Riordan, J., K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling. 1985. Amplification of P-
glycoprotein genes in multidrug-resistant mammalian cell lines. Nature. 316:817-9.
224
Riordan, J.R., and V. Ling. 1979. Purification of P-glycoprotein from plasma membrane
vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J
Biol Chem. 254:12701-5.
Riordan, J.R., J.M. Rommens, B. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S.
Lok, N. Plavsic, J.L. Chou, and et al. 1989. Identification of the cystic fibrosis gene:
cloning and characterization of complementary DNA. Science. 245:1066-73.
Risch, N.J. 2000. Searching for genetic determinants in the new millennium. Nature. 405:847-
56.
Rocchi, E., A. Khodjakov, E.L. Volk, C.H. Yang, T. Litman, S.E. Bates, and E. Schneider.
2000. The product of the ABC half- transporter gene ABCG2 (BCRP/MXR/ABCP) is
expressed in the plasma membrane. Biochem Biophys Res Commun. 271:42-6.
Romsicki, Y., and F.J. Sharom. 1998. The ATPase and ATP-binding functions of P-
glycoprotein--modulation by interaction with defined phospholipids. Eur J Biochem.
256:170-8.
Romsicki, Y., and F.J. Sharom. 1999. The membrane lipid environment modulates drug
interactions with the P-glycoprotein multidrug transporter. Biochem. 38:6887-6896.
Romsicki, Y., Sharom, FJ. 2001. Phospholipid flippase activity of the reconstituted P-
glycoprotein multidrug transporter. Biochemistry. 40:6937-47.
Rosenberg, M.F., A.B. Kamis, R. Callaghan, C.F. Higgins, and R.C. Ford. 2003. Three-
dimensional structures of the mammalian multidrug resistance P-glycoprotein
demonstrate major conformational changes in the transmembrane domains upon
nucleotide binding. J Biol Chem. 278:8294-9.
Rosenberg, M.F., G. Velarde, R.C. Ford, C. Martin, G. Berridge, I.D. Kerr, R. Callaghan, A.
Schmidlin, C. Wooding, K.J. Linton, and C.F. Higgins. 2001. Repacking of the
225
transmembrane domains of P-glycoprotein during the transport ATPase cycle. Embo J.
20:5615-25.
Rothberg, K.G., J.E. Heuser, W.C. Donzell, Y. Ying, J.R. Glenney, and R.G.W. Anderson.
1992. Caveolin, a protein component of caveolae membrane coats. Cell. 68:673-682.
Rothnie, A., D. Theron, L. Soceneantu, C. Martin, M. Traikia, G. Berridge, C.F. Higgins, P.F.
Devaux, and R. Callaghan. 2001. The importance of cholesterol in maintenance of P-
glycoprotein activity and its membrane perturbing influence. Eur Biophys J. 30:430-42.
Rowinsky, E.K., L. Smith, Y.M. Wang, P. Chaturvedi, M. Villalona, E. Campbell, C.
Aylesworth, S.G. Eckhardt, L. Hammond, M. Kraynak, R. Drengler, J. Stephenson, Jr.,
M.W. Harding, and D.D. Von Hoff. 1998. Phase I and pharmacokinetic study of
paclitaxel in combination with biricodar, a novel agent that reverses multidrug resistance
conferred by overexpression of both MDR1 and MRP. J Clin Oncol. 16:2964-76.
Ruetz, S., and P. Gros. 1994a. Functional expression of P-glycoprotein on secretory vesicles. J
Biol Chem. 269:12277-12284.
Ruetz, S., and P. Gros. 1994b. Phosphatidylcholine translocase: a physiological role for the
mdr2 gene. Cell. 77:1071-1081.
Ruth, A., W.D. Stein, E. Rose, and I.B. Roninson. 2001. Coordinate changes in drug resistance
and drug-induced conformational transitions in altered-function mutants of the multidrug
transporter P-glycoprotein. Biochemistry. 40:4332-9.
Rutherford, A.V., and M.C. Willingham. 1993. Ultrastructural localization of daunomycin in
multidrug-resistant cultured cells with modulation of the multidrug transporter. J
Histochem Cytochem. 41:1573-7.
Rutjes, N., B. Binnington, C. Smith, M. Maloney, and C. Lingwood. 2002. Differential tissue
targeting and pathogenesis of Verotoxins 1 and 2 in the mouse animal model. Kid. Intl.
62:832-845.
226
Sabourdy, F., B. Kedjouar, S.C. Sorli, S. Colie, D. Milhas, Y. Salma, and T. Levade. 2008.
Functions of sphingolipid metabolism in mammals--lessons from genetic defects.
Biochim Biophys Acta. 1781:145-83.
Sachidanandam, R., D. Weissman, S.C. Schmidt, J.M. Kakol, L.D. Stein, G. Marth, S. Sherry,
J.C. Mullikin, B.J. Mortimore, D.L. Willey, S.E. Hunt, C.G. Cole, P.C. Coggill, C.M.
Rice, Z. Ning, J. Rogers, D.R. Bentley, P.Y. Kwok, E.R. Mardis, R.T. Yeh, B. Schultz,
L. Cook, R. Davenport, M. Dante, L. Fulton, L. Hillier, R.H. Waterston, J.D.
McPherson, B. Gilman, S. Schaffner, W.J. Van Etten, D. Reich, J. Higgins, M.J. Daly,
B. Blumenstiel, J. Baldwin, N. Stange-Thomann, M.C. Zody, L. Linton, E.S. Lander,
and D. Altshuler. 2001. A map of human genome sequence variation containing 1.42
million single nucleotide polymorphisms. Nature. 409:928-33.
Saddoughi, S.A., P. Song, and B. Ogretmen. 2008. Roles of bioactive sphingolipids in cancer
biology and therapeutics. Subcell Biochem. 49:413-40.
Sakaeda, T. 2005. MDR1 genotype-related pharmacokinetics: fact or fiction? Drug Metab
Pharmacokinet. 20:391-414.
Salhia, B., J.T. Rutka, C. Lingwood, A. Nutikka, and W.R. Van Furth. 2002. The treatment of
malignant meningioma with verotoxin. Neoplasia. 4:304-11.
Sandhoff, K., and T. Kolter. 2003. Biosynthesis and degradation of mammalian
glycosphingolipids. Philos Trans R Soc Lond B Biol Sci. 358:847-61.
Sandler, A., M. Gordon, D.P. De Alwis, I. Pouliquen, L. Green, P. Marder, A. Chaudhary, K.
Fife, L. Battiato, C. Sweeney, C. Jordan, M. Burgess, and C.A. Slapak. 2004. A Phase I
trial of a potent P-glycoprotein inhibitor, zosuquidar trihydrochloride (LY335979),
administered intravenously in combination with doxorubicin in patients with advanced
malignancy. Clin Cancer Res. 10:3265-72.
227
Sandvig, K., M. Ryd, Ø. Garred, E. Schweda, and P.K. Holm. 1994. Retrograde transport from
the Golgi complex to the ER of both Shiga toxin and the nontoxic Shiga B-fragment is
regulated by butyric acid and cAMP. J. Cell. Biol. 126:53-64.
Sarkadi, B., L. Homolya, G. Szakacs, and A. Varadi. 2006. Human multidrug resistance ABCB
and ABCG transporters: participation in a chemoimmunity defense system. Physiol Rev.
86:1179-236.
Sauna, Z.E., and S.V. Ambudkar. 2000. Evidence for a requirement for ATP hydrolysis at two
distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein.
Proc Natl Acad Sci U S A. 97:2515-20.
Sauna, Z.E., and S.V. Ambudkar. 2001. Characterization of the catalytic cycle of ATP
hydrolysis by human P-glycoprotein. The two ATP hydrolysis events in a single
catalytic cycle are kinetically similar but affect different functional outcomes. J Biol
Chem. 276:11653-61.
Sauna, Z.E., M.B. Andrus, T.M. Turner, and S.V. Ambudkar. 2004. Biochemical basis of
polyvalency as a strategy for enhancing the efficacy of P-glycoprotein (ABCB1)
modulators: stipiamide homodimers separated with defined- length spacers reverse drug
efflux with greater efficacy. Biochemistry. 43:2262-71.
Sauna, Z.E., I.W. Kim, and S.V. Ambudkar. 2007a. Genomics and the mechanism of P-
glycoprotein (ABCB1). J Bioenerg Biomembr. 39:481-7.
Sauna, Z.E., C. Kimchi-Sarfaty, S.V. Ambudkar, and M.M. Gottesman. 2007b. The sounds of
silence: synonymous mutations affect function. Pharmacogenomics. 8:527-32.
Sauna, Z.E., M. Muller, X.H. Peng, and S.V. Ambudkar. 2002. Importance of the conserved
Walker B glutamate residues, 556 and 1201, for the completion of the catalytic cycle of
ATP hydrolysis by human P-glycoprotein (ABCB1). Biochemistry. 41:13989-4000.
228
Sauna, Z.E., K. Nandigama, and S.V. Ambudkar. 2006. Exploiting reaction intermediates of the
ATPase reaction to elucidate the mechanism of transport by P-glycoprotein (ABCB1). J
Biol Chem. 281:26501-11.
Saurin, W., M. Hofnung, and E. Dassa. 1999. Getting in or out: early segregation between
importers and exporters in the evolution of ATP-binding cassette (ABC) transporters. J
Mol Evol. 48:22-41.
Savile, C.K., G. Fabrias, and P.H. Buist. 2001. Dihydroceramide delta(4) desaturase initiates
substrate oxidation at C-4. J Am Chem Soc. 123:4382-5.
Sawkar, A.R., W.C. Cheng, E. Beutler, C.H. Wong, W.E. Balch, and J.W. Kelly. 2002.
Chemical chaperones increase the cellular activity of N370S beta -glucosidase: a
therapeutic strategy for Gaucher disease. Proc Natl Acad Sci U S A. 99:15428-33.
Schapiro, F., C.A. Lingwood, W. Furuya, and S. Grinstein. 1998. pH-independent targeting of
glycolipids to the Golgi complex. Am. J. Physiol. 274:319-332.
Scheffer, G.L., M. Kool, M. Heijn, M. de Haas, A.C. Pijnenborg, J. Wijnholds, A. van Helvoort,
M.C. de Jong, J.H. Hooijberg, C.A. Mol, M. van der Linden, J.M. de Vree, P. van der
Valk, R.P. Elferink, P. Borst, and R.J. Scheper. 2000. Specific detection of multidrug
resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a
panel of monoclonal antibodies. Cancer Res. 60:5269-77.
Scheffer, G.L., and R.J. Scheper. 2002. Drug resistance molecules: lessons from oncology.
Novartis Found Symp. 243:19-31; discussion 31-7, 180-5.
Schiffmann, R., J.B. Kopp, H.A. Austin, 3rd, S. Sabnis, D.F. Moore, T. Weibel, J.E. Balow, and
R.O. Brady. 2001. Enzyme replacement therapy in Fabry disease: a randomized
controlled trial. Jama. 285:2743-9.
Schiffmann, R., G.J. Murray, D. Treco, P. Daniel, M. Sellos-Moura, M. Myers, J.M. Quirk,
G.C. Zirzow, M. Borowski, K. Loveday, T. Anderson, F. Gillespie, K.L. Oliver, N.O.
229
Jeffries, E. Doo, T.J. Liang, C. Kreps, K. Gunter, K. Crutchfield, R.F. Selden, and R.O.
Brady. 2000. Infusion of a-galactosidase A reduces tissue globotriaosylceramide storage
in patients with Fabry disease. Proc Nat Acad Sci, USA. 97:365-370.
Schinkel, A.H. 1997. The physiological function of drug-transporting P-glycoproteins. Semin
Cancer Biol. 8:161-70.
Schinkel, A.H., U. Mayer, E. Wagenaar, C.A. Mol, L. van Deemter, J.J. Smit, M.A. van der
Valk, A.C. Voordouw, H. Spits, O. van Tellingen, J.M. Zijlmans, W.E. Fibbe, and P.
Borst. 1997. Normal viability and altered pharmacokinetics in mice lacking mdr1-type
(drug-transporting) P-glycoproteins. Proc Natl Acad Sci U S A. 94:4028-33.
Schinkel, A.H., J.J. Smit, O. van Tellingen, J.H. Beijnen, E. Wagenaar, L. van Deemter, C.A.
Mol, M.A. van der Valk, E.C. Robanus-Maandag, H.P. te Riele, and et al. 1994.
Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-
brain barrier and to increased sensitivity to drugs. Cell. 77:491-502.
Schinkel, A.H., E. Wagenaar, C.A. Mol, and L. van Deemter. 1996. P-glycoprotein in the blood-
brain barrier of mice influences the brain penetration and pharmacological activity of
many drugs. J Clin Invest. 97:2517-24.
Schinkel, A.H., E. Wagenaar, L. van Deemter, C.A. Mol, and P. Borst. 1995. Absence of the
mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of
dexamethasone, digoxin, and cyclosporin A. J Clin Invest. 96:1698-705.
Schnabel, D., M. Schroder, and K. Sandhoff. 1991. Mutation in the sphingolipid activator
protein 2 in a patient with a variant of Gaucher disease. FEBS Lett. 284:57-9.
Schrader, A.J., M. Seger, L. Konrad, P. Olbert, A. Hegele, R. Hofmann, and A. Heidenreich.
2007. Clinical impact of MDR1-expression in testicular germ cell cancer. Exp Oncol.
29:212-6.
230
Schubbert, S., K. Shannon, and G. Bollag. 2007. Hyperactive Ras in developmental disorders
and cancer. Nat Rev Cancer. 7:295-308.
Schuette, C.G., T. Doering, T. Kolter, and K. Sandhoff. 1999. The glycosphingolipidoses-from
disease to basic principles of metabolism. Biol Chem. 380:759-66.
Schuette, C.G., B. Pierstorff, S. Huettler, and K. Sandhoff. 2001. Sphingolipid activator
proteins: proteins with complex functions in lipid degradation and skin biogenesis.
Glycobiology. 11:81R-90R.
Schuetz, E.G., W.T. Beck, and J.D. Schuetz. 1996. Modulators and substrates of P-glycoprotein
and cytochrome P4503A coordinately up-regulate these proteins in human colon
carcinoma cells. Mol Pharmacol. 49:311-8.
Schulte, S., and W. Stoffel. 1993. Ceramide UDPgalactosyltransferase from myelinating rat
brain: purification, cloning, and expression. Proc Natl Acad Sci U S A. 90:10265-9.
Schulz, A., T. Mousallem, M. Venkataramani, D.A. Persaud-Sawin, A. Zucker, C. Luberto, A.
Bielawska, J. Bielawski, J.C. Holthuis, S.M. Jazwinski, L. Kozhaya, G.S. Dbaibo, and
R.M. Boustany. 2006. The CLN9 protein, a regulator of dihydroceramide synthase. J
Biol Chem. 281:2784-94.
Schumacher, M.A., B.K. Hurlburt, and R.G. Brennan. 2001. Crystal structures of SarA, a
pleiotropic regulator of virulence genes in S. aureus. Nature. 409:215-9.
Schwardt, O., H. Gathje, A. Vedani, S. Mesch, G.P. Gao, M. Spreafico, J. von Orelli, S. Kelm,
and B. Ernst. 2009. Examination of the biological role of the alpha(2-->6)-linked sialic
acid in gangliosides binding to the myelin-associated glycoprotein (MAG). J Med Chem.
52:989-1004.
Schwarz, A., G. Offermann, F. Keller, I. Bennhold, J. L'Age-Stehr, P.H. Krause, and M.J.
Mihatsch. 1993. The effect of cyclosporine on the progression of human
231
immunodeficiency virus type 1 infection transmitted by transplantation--data on four
cases and review of the literature. Transplantation. 55:95-103.
Schwiebert, E.M., M.M. Morales, S. Devidas, M.E. Egan, and W.B. Guggino. 1998. Chloride
channel and chloride conductance regulator domains of CFTR, the cystic fibrosis
transmembrane conductance regulator. Proc Natl Acad Sci U S A. 95:2674-9.
Scotto, K.W., and R.A. Johnson. 2001. Transcription of the multidrug resistance gene MDR1: a
therapeutic target. Mol Interv. 1:117-25.
Seelig, A. 1998. A general pattern for substrate recognition by P-glycoprotein. Eur J Biochem.
251:252-61.
Seelig, A., and E. Landwojtowicz. 2000. Structure-activity relationship of P-glycoprotein
substrates and modifiers. Eur J Pharm Sci. 12:31-40.
Senior, A.E., M.K. al-Shawi, and I.L. Urbatsch. 1995a. ATP hydrolysis by multidrug-resistance
protein from Chinese hamster ovary cells. J Bioenerg Biomembr. 27:31-6.
Senior, A.E., M.K. al-Shawi, and I.L. Urbatsch. 1995b. The catalytic cycle of P-glycoprotein.
FEBS Lett. 377:285-9.
Shabbits, J.A., and L.D. Mayer. 2002. P-glycoprotein modulates ceramide-mediated sensitivity
of human breast cancer cells to tubulin-binding anticancer drugs. Mol Cancer Ther.
1:205-13.
Shapiro, A.B., K. Fox, P. Lam, and V. Ling. 1999. Stimulation of P-glycoprotein-mediated drug
transport by prazosin and progesterone. Evidence for a third drug-binding site. Eur J
Biochem. 259:841-50.
Shapiro, A.B., and V. Ling. 1995. Reconstitution of drug transport by purified P-glycoprotein. J
Biol Chem. 270:16167-75.
Sharom, F.J. 1997. The P-glycoprotein efflux pump: How does it transport drugs? J Membrane
Biol. 160:161-175.
232
Sharom, F.J. 2006. Shedding light on drug transport: structure and function of the P-
glycoprotein multidrug transporter (ABCB1). Biochem Cell Biol. 84:979-92.
Sharom, F.J., M.R. Lugo, and P.D. Eckford. 2005. New insights into the drug binding, transport
and lipid flippase activities of the p-glycoprotein multidrug transporter. J Bioenerg
Biomembr. 37:481-7.
Sharom, F.J., X. Yu, and C.A. Doige. 1993. Functional reconstitution of drug transport and
ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J Biol
Chem. 268:24197-202.
Shaw, A.S. 2006. Lipid rafts: now you see them, now you don't. Nat Immunol. 7:1139-42.
Shen, D., I. Pastan, and M.M. Gottesman. 1998. Cross-resistance to methotrexate and metals in
human cisplatin-resistant cell lines results from a pleiotropic defect in accumulation of
these compounds associated with reduced plasma membrane binding proteins. Cancer
Res. 58:268-75.
Shen, D.W., S. Goldenberg, I. Pastan, and M.M. Gottesman. 2000. Decreased accumulation of
[14C]carboplatin in human cisplatin-resistant cells results from reduced energy-
dependent uptake. J Cell Physiol. 183:108-16.
Sheppard, D.N., and M.J. Welsh. 1999. Structure and function of the CFTR chloride channel.
Physiol Rev. 79:S23-45.
Sherbakova, E.A., T.P. Stromskaia, E. Rybalkina, O.V. Kalita, and A.A. Stavrovskaia. 2008.
[Role of PTEN protein in multidrug resistance of prostate cancer cells]. Mol Biol (Mosk).
42:487-93.
Shibahara, K., T. Uchiumi, T. Fukuda, S. Kura, Y. Tominaga, Y. Maehara, K. Kohno, Y.
Nakabeppu, T. Tsuzuki, and M. Kuwano. 2004. Targeted disruption of one allele of the
Y-box binding protein-1 (YB-1) gene in mouse embryonic stem cells and increased
sensitivity to cisplatin and mitomycin C. Cancer Sci. 95:348-53.
233
Shilling, R.A., H. Venter, S. Velamakanni, A. Bapna, B. Woebking, S. Shahi, and H.W. van
Veen. 2006. New light on multidrug binding by an ATP-binding-cassette transporter.
Trends Pharmacol Sci. 27:195-203.
Shiraga, K., K. Sakaguchi, T. Senoh, T. Ohta, S. Ogawa, T. Sawayama, H. Mouri, A. Fujiwara,
and T. Tsuji. 2001. Modulation of doxorubicin sensitivity by cyclosporine A in
hepatocellular carcinoma cells and their doxorubicin-resistant sublines. J Gastroenterol
Hepatol. 16:460-6.
Shtil, A.A., O.V. Ktitorova, E.S. Kakpakova, and O. Holian. 2000. Differential effects of the
MDR1 (multidrug resistance) gene-activating agents on protein kinase C: evidence for
redundancy of mechanisms of acquired MDR in leukemia cells. Leuk Lymphoma.
40:191-5.
Siatskas, C., and J.A. Medin. 2001. Gene therapy for Fabry disease. J Inherit Metab Dis. 24
Suppl 2:25-41.
Sikic, B.I., G.A. Fisher, B.L. Lum, J. Halsey, L. Beketic-Oreskovic, and G. Chen. 1997.
Modulation and prevention of multidrug resistance by inhibitors of P-glycoprotein.
Cancer Chemother Pharmacol. 40 Suppl:S13-9.
Sillence, D.J., and F.M. Platt. 2003. Storage diseases: new insights into sphingolipid functions.
Trends Cell Biol. 13:195-203.
Sillence, D.J., V. Puri, D.L. Marks, T.D. Butters, R.A. Dwek, R.E. Pagano, and F.M. Platt.
2002. Glucosylceramide modulates membrane traffic along the endocytic pathway. J
Lipid Res. 43:1837-1845.
Sills, G.J., R. Mohanraj, E. Butler, S. McCrindle, L. Collier, E.A. Wilson, and M.J. Brodie.
2005. Lack of association between the C3435T polymorphism in the human multidrug
resistance (MDR1) gene and response to antiepileptic drug treatment. Epilepsia. 46:643-
7.
234
Simons, K., Ehehalt, R. 2002. Cholesterol, lipid rafts, and disease. J Clin Invest. 110:597-603.
Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature. 387:569-572.
Simons, K., and G. van Meer. 1988. Lipid sorting in epithelial cells. Biochemistry. 27:6197-202.
Skovsgaard, T. 1978. Mechanisms of resistance to daunorubicin in Ehrlich ascites tumor cells.
Cancer Res. 38:1785-91.
Slotte, J.P., and E.L. Bierman. 1988. Depletion of plasma-membrane sphingomyelin rapidly
alters the distribution of cholesterol between plasma membranes and intracellular
cholesterol pools in cultured fibroblasts. Biochem J. 250:653-8.
Smit, J., A. Schinkel, R. Oude Elferink, A. Groen, E. Wagenaar, L. van Deemter, C. Mol, R.
Ottenhoff, N. van der Lugt, and M. van Roon. 1993. Homozygous disruption of the
murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile
and to liver disease. Cell. 75:451-62.
Smith, A.J., J.L. Timmermans-Hereijgers, B. Roelofsen, K.W. Wirtz, W.J. van Blitterswijk, J.J.
Smit, A.H. Schinkel, and P. Borst. 1994. The human MDR3 P-glycoprotein promotes
translocation of phosphatidylcholine through the plasma membrane of fibroblasts from
transgenic mice. FEBS Lett. 354:263-6.
Smith, D.C., D.J. Sillence, T. Falguieres, R.M. Jarvis, L. Johannes, J.M. Lord, F.M. Platt, and
L.M. Roberts. 2006. The Association of Shiga- like Toxin with Detergent-resistant
Membranes Is Modulated by Glucosylceramide and Is an Essential Requirement in the
Endoplasmic Reticulum for a Cytotoxic Effect. Mol Biol Cell. 17:1375-87.
Smyth, M.J., E. Krasovskis, V.R. Sutton, and R.W. Johnstone. 1998. The drug efflux protein, P-
glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of
caspase-dependent apoptosis. Proc Natl Acad Sci U S A. 95:7024-9.
235
Solazzo, M., O. Fantappie, N. Lasagna, C. Sassoli, D. Nosi, and R. Mazzanti. 2006. P-gp
localization in mitochondria and its functional characterization in multiple drug-resistant
cell lines. Exp Cell Res. 312:4070-8.
Sonveaux, N., A.B. Shapiro, E. Goormaghtigh, V. Ling, and J.M. Ruysschaert. 1996. Secondary
and tertiary structure changes of reconstituted P-glycoprotein. A Fourier transform
attenuated total reflection infrared spectroscopy analysis. J Biol Chem. 271:24617-24.
Sonveaux, N., C. Vigano, A.B. Shapiro, V. Ling, and J.M. Ruysschaert. 1999. Ligand-mediated
tertiary structure changes of reconstituted P-glycoprotein. A tryptophan fluorescence
quenching analysis. J Biol Chem. 274:17649-54.
Sparreboom, A., R. Danesi, Y. Ando, J. Chan, and W.D. Figg. 2003. Pharmacogenomics of
ABC transporters and its role in cancer chemotherapy. Drug Resist Updat. 6:71-84.
Sparreboom, A., J. van Asperen, U. Mayer, A.H. Schinkel, J.W. Smit, D.K. Meijer, P. Borst,
W.J. Nooijen, J.H. Beijnen, and O. van Tellingen. 1997. Limited oral bioavailability and
active epithelial excretion of paclitaxel (Taxol) caused by P-glycoprotein in the intestine.
Proc Natl Acad Sci U S A. 94:2031-5.
Spiegel, S., and S. Milstien. 2003. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat
Rev Mol Cell Biol. 4:397-407.
Stavrovskaya, A.A., and T.P. Stromskaya. 2008. Transport proteins of the ABC family and
multidrug resistance of tumor cells. Biochemistry (Mosc). 73:592-604.
Stein, U., W. Walther, A. Stege, A. Kaszubiak, I. Fichtner, and H. Lage. 2008. Complete in vivo
reversal of the multidrug resistance phenotype by jet- injection of anti-MDR1 short
hairpin RNA-encoding plasmid DNA. Mol Ther. 16:178-86.
Stewart, A., J. Steiner, G. Mellows, B. Laguda, D. Norris, and P. Bevan. 2000. Phase I trial of
XR9576 in healthy volunteers demonstrates modulation of P-glycoprotein in CD56+
lymphocytes after oral and intravenous administration. Clin Cancer Res. 6:4186-91.
236
St-Pierre, M.V., M.A. Serrano, R.I. Macias, U. Dubs, M. Hoechli, U. Lauper, P.J. Meier, and
J.J. Marin. 2000. Expression of members of the multidrug resistance protein family in
human term placenta. Am J Physiol Regul Integr Comp Physiol. 279:R1495-503.
Stromskaya, T.P., I.A. Grigorian, V.S. Ossovskaya, E.Y. Rybalkina, P.M. Chumakov, and B.P.
Kopnin. 1995. Cell-specific effects of RAS oncogene and protein kinase C agonist TPA
on P-glycoprotein function. FEBS Lett. 368:373-6.
Stromskaya, T.P., E.Y. Rybalkina, A.A. Shtil, T.N. Zabotina, N.A. Filippova, and A.A.
Stavrovskaya. 1998. Influence of exogenous RAR alpha gene on MDR1 expression and
P-glycoprotein function in human and rodent cell lines. Br J Cancer. 77:1718-25.
Stromskaya, T.P., E.Y. Rybalkina, T.N. Zabotina, A.A. Shishkin, and A.A. Stavrovskaya. 2005.
Influence of RARalpha gene on MDR1 expression and P-glycoprotein function in
human leukemic cells. Cancer Cell Int. 5:15.
Strum, J.C., G.W. Small, S.B. Pauig, and L.W. Daniel. 1994. 1-beta-D-
Arabinofuranosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells.
J Biol Chem. 269:15493-7.
Stutts, M.J., B.C. Rossier, and R.C. Boucher. 1997. Cystic fibrosis transmembrane conductance
regulator inverts protein kinase A-mediated regulation of epithelial sodium channel
single channel kinetics. J Biol Chem. 272:14037-40.
Sugawara, S., M. Hosono, Y. Ogawa, M. Takayanagi, and K. Nitta. 2005. Catfish egg lectin
causes rapid activation of multidrug resistance 1 P-glycoprotein as a lipid translocase.
Biol Pharm Bull. 28:434-41.
Suzuki, A., M. Iwasaki, M. Kato, and N. Wagai. 1997. Sequential operation of ceramide
synthesis and ICE cascade in CPT-11-initiated apoptotic death signaling. Exp Cell Res.
233:41-7.
237
Suzuki, H., and Y. Sugiyama. 2000. Role of metabolic enzymes and efflux transporters in the
absorption of drugs from the small intestine. Eur J Pharm Sci. 12:3-12.
Suzuki, T., A. Sometani, Y. Yamazaki, G. Horiki, Y. Mitzutani, H. Masuda, M. Yamada, H.
Tahara, G. Xu, D. Miyamoto, N. Oku, S. Okada, M. Kisio, A. Hasagawa, T. Ito, Y.
Kawaoka, and Y. Suzuki. 1996. Sulphatide binds to human and animal influenza A
viruses, and inhibits the viral infection. Biochem J. 318:389-393.
Svennerholm, L. 1957. Biochem Biophys Acta. 24:604-611.
Szakacs, G., J.K. Paterson, J.A. Ludwig, C. Booth-Genthe, and M.M. Gottesman. 2006.
Targeting multidrug resistance in cancer. Nat Rev Drug Discov. 5:219-34.
Taga, S., K. Carlier, Z. Mishal, C. Capoulade, M. Mangeney, Y. Lécluse, D. Coulaud, c. Tétaud,
L.L. Pritchard, T. Tursz, and J. Wiels. 1997. Intracellular signaling events in CD77-
mediated apoptosis of Burkitt's lymphoma cells. Blood. 90:2757-2767.
Taga, S., M. Manganey, T. Tursz, and J. Wiels. 1995. Differential regulation of
glycosphingolipid biosynthesis in phenotypically distinct Burkitt's lymphoma cell lines.
Int J Cancer. 61:261-267.
Takizawa, M., T. Nomura, E. Wakisaka, N. Yoshizuka, J. Aoki, H. Arai, K. Inoue, M. Hattori,
and N. Matsuo. 1999. cDNA cloning and expression of human lactosylceramide
synthase. Biochim Biophys Acta. 1438:301-4.
Tan, B., D. Piwnica-Worms, and L. Ratner. 2000. Multidrug resistance transporters and
modulation. Curr Opin Oncol. 12:450-8.
Tan, E.K., D.K. Chan, P.W. Ng, J. Woo, Y.Y. Teo, K. Tang, L.P. Wong, S.S. Chong, C. Tan, H.
Shen, Y. Zhao, and C.G. Lee. 2005. Effect of MDR1 haplotype on risk of Parkinson
disease. Arch Neurol. 62:460-4.
238
Tang-Wai, D.F., S. Kajiji, F. DiCapua, D. de Graaf, I.B. Roninson, and P. Gros. 1995. Human
(MDR1) and mouse (mdr1, mdr3) P-glycoproteins can be distinguished by their
respective drug resistance profiles and sensitivity to modulators. Biochemistry. 34:32-9.
Taniike, M., I. Mohri, N. Eguchi, D. Irikura, Y. Urade, S. Okada, and K. Suzuki. 1999. An
apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell
leukodystrophy. J Neuropathol Exp Neurol. 58:644-53.
Taupin, P. 2006. Neurogenesis in the adult central nervous system. C R Biol. 329:465-75.
te Boekhorst, P.A., J. van Kapel, M. Schoester, and P. Sonneveld. 1992. Reversal of typical
multidrug resistance by cyclosporin and its non- immunosuppressive analogue SDZ PSC
833 in Chinese hamster ovary cells expressing the mdr1 phenotype. Cancer Chemother
Pharmacol. 30:238-42.
Teeter, L.D., F.F. Becker, F.V. Chisari, D.J. Li, and M.T. Kuo. 1990. Overexpression of the
multidrug resistance gene mdr3 in spontaneous and chemically induced mouse
hepatocellular carcinomas. Mol Cell Biol. 10:5728-35.
Tetaud, C., T. Falguieres, K. Carlier, Y. Lecluse, J. Garibal, D. Coulaud, P. Busson, R.
Steffensen, H. Clausen, L. Johannes, and J. Wiels. 2003. Two distinct Gb3/CD77
signaling pathways leading to apoptosis are triggered by anti-Gb3/CD77 mAb and
verotoxin-1. J Biol Chem. 278:45200-8.
Tettamanti, G. 2004. Ganglioside/glycosphingolipid turnover: new concepts. Glycoconj J.
20:301-17.
Thiebaut, F., T. Tsuruo, H. Hamada, M.M. Gottesman, I. Pastan, and M.C. Willingham. 1987.
Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal
human tissues. Proc Natl Acad Sci U S A. 84:7735-8.
Thomas, H., and H.M. Coley. 2003. Overcoming multidrug resistance in cancer: an update on
the clinical strategy of inhibiting p-glycoprotein. Cancer Control. 10:159-65.
239
Tian, G., D. Wilcockson, V.H. Perry, P.M. Rudd, R.A. Dwek, F.M. Platt, and N. Platt. 2004.
Inhibition of alpha-glucosidases I and II increases the cell surface expression of
functional class A macrophage scavenger receptor (SR-A) by extending its half- life. J
Biol Chem. 279:39303-9.
Tohyama, J., M.T. Vanier, K. Suzuki, T. Ezoe, and J. Matsuda. 2000. Paradoxical influence of
acid beta-galactosidase gene dosage on phenotype of the twitcher mouse (genetic
galactosylceramidase deficiency). Hum Mol Genet. 9:1699-707.
Trauner, M., and J.L. Boyer. 2003. Bile salt transporters: molecular characterization, function,
and regulation. Physiol Rev. 83:633-71.
Triola, G., G. Fabrias, J. Casas, and A. Llebaria. 2003. Synthesis of cyclopropene analogues of
ceramide and their effect on dihydroceramide desaturase. J Org Chem. 68:9924-32.
Triola, G., G. Fabrias, and A. Llebaria. 2001. Synthesis of a Cyclopropene Analogue of
Ceramide, a Potent Inhibitor of Dihydroceramide Desaturase This work was supported
by the Direccion General de Ensenanza Superior e Investigacion Cientifica (grant PB97-
1171) and the Departament d'Universitats, Recerca i Societat de la Informacio,
Generalitat de Catalunya (grant 1999-SGR 00187 and a Predoctoral fellowship to G.T.).
We thank Dr. J. Casas, Dr. A. Delgado, and Dr. J. Joglar for their help in different
aspects of this work. Angew Chem Int Ed Engl. 40:1960-1962.
Troost, J., N. Albermann, W. Emil Haefeli, and J. Weiss. 2004a. Cholesterol modulates P-
glycoprotein activity in human peripheral blood mononuc lear cells. Biochem Biophys
Res Commun. 316:705-11.
Troost, J., H. Lindenmaier, W.E. Haefeli, and J. Weiss. 2004b. Modulation of cellular
cholesterol alters P-glycoprotein activity in multidrug-resistant cells. Mol Pharmacol.
66:1332-9.
240
Tropak, M.B., S.P. Reid, M. Guiral, S.G. Withers, and D. Mahuran. 2004. Pharmacological
enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and
Sandhoff Patients. J Biol Chem. 279:13478-87.
Tsai, C.J., Z.E. Sauna, C. Kimchi-Sarfaty, S.V. Ambudkar, M.M. Gottesman, and R. Nussinov.
2008. Synonymous mutations and ribosome stalling can lead to altered folding pathways
and distinct minima. J Mol Biol. 383:281-91.
Tybulewicz, V.L., M.L. Tremblay, M.E. LaMarca, R. Willemsen, B.K. Stubblefield, S.
Winfield, B. Zablocka, E. Sidransky, B.M. Martin, S.P. Huang, and et al. 1992. Animal
model of Gaucher's disease from targeted disruption of the mouse glucocerebrosidase
gene. Nature. 357:407-10.
Ueda, K., D.P. Clark, C.J. Chen, I.B. Roninson, M.M. Gottesman, and I. Pastan. 1987. The
human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation. J
Biol Chem. 262:505-8.
Ueda, K., M.M. Cornwell, M.M. Gottesman, I. Pastan, I.B. Roninson, V. Ling, and J.R.
Riordan. 1986. The mdr1 gene, responsible for multidrug-resistance, codes for P-
glycoprotein. Biochem Biophys Res Commun. 141:956-62.
van den Heuvel-Eibrink, M.M., E.A. Wiemer, M.J. de Boevere, B. van der Holt, P.J. Vossebeld,
R. Pieters, and P. Sonneveld. 2001. MDR1 gene-related clonal selection and P-
glycoprotein function and expression in relapsed or refractory acute myeloid leukemia.
Blood. 97:3605-11.
Van der Bliek, A.M., F. Baas, T. Van der Velde-Koerts, J.L. Biedler, M.B. Meyers, R.F. Ozols,
T.C. Hamilton, H. Joenje, and P. Borst. 1988. Genes amplified and overexpressed in
human multidrug-resistant cell lines. Cancer Res. 48:5927-32.
van der Kar, N.C.J., T. Kooistra, M. Vermeer, W. Lesslauer, L.A.H. Monnens, and V.W.M. van
Hinsbergh. 1995. Tumor necrosis factor a induces endothelial galactosyl transferase
241
activity and verocytotoxin receptors. Role of specific tumor necrosis factor receptors
and protein kinase C. Blood. 85:734-743.
Van Dijk, J., T. Tsuruo, D.M. Segal, R.L. Bolhuis, R. Colognola, R.J. van de Griend, G.J.
Fleuren, and S.O. Warnaar. 1989. Bispecific antibodies reactive with the multidrug-
resistance-related glycoprotein and CD3 induce lysis of multidrug-resistant tumor cells.
Int J Cancer. 44:738-43.
van Helvoort, A., M.L. Giudici, M. Thielemans, and G. van Meer. 1997. Transport of
sphingomyelin to the cell surface is inhibited by brefeldin A and in mitosis, where C6-
NBD-sphingomyelin is translocated across the plasma membrane by a multidrug
transporter activity. J Cell Sci. 110 ( Pt 1):75-83.
van Helvoort, A., A. Smith, H. Sprong, I. Fritzsche, A. Schinkel, P. Borst, and G. van Meer.
1996. MDR1 P-Glycoprotein is a lipid translocase of broad specificity, while MDR3 P-
glycoprotein specifically translocates phosphatidyl choline. Cell. 87:507-517.
van IJzendoorn, S.C., M.M. Zegers, J.W. Kok, and D. Hoekstra. 1997. Segregation of
glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic
route in HepG2 cells. J Cell Biol. 137:347-57.
van Meer, G. 1989. Lipid traffic in animal cells. Annu Rev Cell Biol. 5:247-75.
van Meer, G. 2005. Cellular lipidomics. Embo J. 24:3159-65.
van Meer, G., D. Halter, H. Sprong, P. Somerharju, and M.R. Egmond. 2006. ABC lipid
transporters: extruders, flippases, or flopless activators? FEBS Lett. 580:1171-7.
van Meer, G., and Q. Lisman. 2002. Sphingolipid transport; Rafts and Translocators. J Biol
Chem. 277:25855-8.
van Meer, G., D. Sillence, H. Sprong, N. Kalin, and R. Raggers. 1999. Transport of
(glyco)sphingolipids in and between cellular membranes; multidrug transporters and
lateral domains. Biosci Rep. 19:327-33.
242
van Meer, G., E.H. Stelzer, R.W. Wijnaendts-van-Resandt, and K. Simons. 1987. Sorting of
sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 105:1623-35.
van Veen, H.W., K. Venema, H. Bolhuis, I. Oussenko, J. Kok, B. Poolman, A.J. Driessen, and
W.N. Konings. 1996. Multidrug resistance mediated by a bacterial homolog of the
human multidrug transporter MDR1. Proc Natl Acad Sci U S A. 93:10668-72.
van Weely, S., M. Brandsma, A. Strijland, J.M. Tager, and J.M. Aerts. 1993. Demonstration of
the existence of a second, non- lysosomal glucocerebrosidase that is not deficient in
Gaucher disease. Biochim Biophys Acta. 1181:55-62.
Vankeerberghen, A., W. Lin, M. Jaspers, H. Cuppens, B. Nilius, and J.J. Cassiman. 1999.
Functional characterization of the CFTR R domain using CFTR/MDR1 hybrid and
deletion constructs. Biochemistry. 38:14988-98.
Vassal, G. 2005. Has chemotherapy reached its limits in pediatric cancers? Eur J Cancer.
41:564-75; discussion 576-7.
Veldman, J., K. Klappe, J. Hinrichs, I. Hummel, G. van der Schaaf, H. Sietsma, and J. Kok.
2002. Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due
to uncoupling of glycolipid biosynthesis in the Golgi apparatus. FASEB J. 10:1096.
Veldman, R., H. Sietsma, K. Klappe, D. Hoekstra, and J. Kok. 1999. Inhibition of P-
glycoprotein activity and chemosensitization of multidrug-resistant ovarian carcinoma
2780AD cells by hexanoylglucosyl ceramide. Biochim Biophys Res Comm. 266:492-
496.
Veldman, R.J., A. Mita, O. Cuvillier, V. Garcia, K. Klappe, J.A. Medin, J.D. Campbell, S.
Carpentier, J.W. Kok, and T. Levade. 2003. The absence of functional glucosylceramide
synthase does not sensitize melanoma cells for anticancer drugs. Faseb J. 17:1144-6.
Venkataraman, K., C. Riebeling, J. Bodennec, H. Riezman, J.C. Allegood, M.C. Sullards, A.H.
Merrill, Jr., and A.H. Futerman. 2002. Upstream of growth and differentiation factor 1
243
(uog1), a mammalian homolog of the yeast longevity assurance gene 1 (LAG1),
regulates N- stearoyl-sphinganine (C18-(dihydro)ceramide) synthesis in a fumonisin B1-
independent manner in mammalian cells. J Biol Chem. 277:35642-9.
Vennekens, R., D. Trouet, A. Vankeerberghen, T. Voets, H. Cuppens, J. Eggermont, J.J.
Cassiman, G. Droogmans, and B. Nilius. 1999. Inhibition of volume-regulated anion
channels by expression of the cystic fibrosis transmembrane conductance regulator. J
Physiol. 515 ( Pt 1):75-85.
Vogt, P.K., A.G. Bader, and S. Kang. 2006. PI 3-kinases: hidden potentials revealed. Cell Cycle.
5:946-9.
Volm, M. 1998. Multidrug resistance and its reversal. Anticancer Res. 18:2905-17.
Vunnam, R.R., and N.S. Radin. 1980. Analogs of ceramide that inhibit glucocerebroside
synthetase in mouse brain. Chem Phys Lipids. 26:265-78.
Wacher, V.J., C.Y. Wu, and L.Z. Benet. 1995. Overlapping substrate specificities and tissue
distribution of cytochrome P450 3A and P-glycoprotein: implications for drug delivery
and activity in cancer chemotherapy. Mol Carcinog. 13:129-34.
Wada, R., C.J. Tifft, and R.L. Proia. 2000. Microglial activation precedes acute
neurodegeneration in Sandhoff disease and is suppressed by bone marrow
transplantation. Proc Natl Acad Sci U S A. 97:10954-9.
Waddell, T., A. Cohen, and C.A. Lingwood. 1990. Induction of verotoxin sensitivity in receptor
deficient cell lines using the receptor glycolipid globotriosyl ceramide. Proc. Natl. Acad.
Sci. USA. 87:7898-7901.
Waddell, T., S. Head, M. Petric, A. Cohen, and C.A. Lingwood. 1988. Globotriosyl ceramide is
specifically recognized by the E. coli verocytotoxin 2. Biochem. Biophys. Res. Commun.
152:674-679.
244
Walden, C.M., R. Sandhoff, C.C. Chuang, Y. Yildiz, T.D. Butters, R.A. Dwek, F.M. Platt, and
A.C. van der Spoel. 2007. Accumulation of glucosylceramide in murine testis, caused by
inhibition of beta-glucosidase 2: implications for spermatogenesis. J Biol Chem.
282:32655-64.
Walker, J.E., M. Saraste, M.J. Runswick, and N.J. Gay. 1982. Distantly related sequences in the
alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring
enzymes and a common nucleotide binding fold. Embo J. 1:945-51.
Wandel, C., R.B. Kim, S. Kajiji, P. Guengerich, G.R. Wilkinson, and A.J. Wood. 1999. P-
glycoprotein and cytochrome P-450 3A inhibition: dissociation of inhibitory potencies.
Cancer Res. 59:3944-8.
Wang, E., and A.H. Merrill, Jr. 2000. Ceramide synthase. Methods Enzymol. 311:15-21.
Wang, E., W.P. Norred, C.W. Bacon, R.T. Riley, and A.H. Merrill, Jr. 1991. Inhibition of
sphingolipid biosynthesis by fumonisins. Implications for diseases associated with
Fusarium moniliforme. J Biol Chem. 266:14486-90.
Wang, H., X.P. Chen, and F.Z. Qiu. 2003a. Overcoming multi-drug resistance by anti-MDR1
ribozyme. World J Gastroenterol. 9:1444-9.
Wang, R.B., C.L. Kuo, L.L. Lien, and E.J. Lien. 2003b. Structure-activity relationship: analyses
of p-glycoprotein substrates and inhibitors. J Clin Pharm Ther. 28:203-28.
Wang, T.Y., R. Leventis, and J.R. Silvius. 2000. Fluorescence-based evaluation of the
partitioning of lipids and lipidated peptides into liquid-ordered lipid microdomains: a
model for molecular partitioning into "lipid rafts". Biophys J. 79:919-33.
Wang, Y., T.W. Loo, M.C. Bartlett, and D.M. Clarke. 2007. Modulating the folding of P-
glycoprotein and cystic fibrosis transmembrane conductance regulator truncation
mutants with pharmacological chaperones. Mol Pharmacol. 71:751-8.
245
Ward, A., C.L. Reyes, J. Yu, C.B. Roth, and G. Chang. 2007. Flexibility in the ABC transporter
MsbA: Alternating access with a twist. Proc Natl Acad Sci U S A. 104:19005-10.
Wattenberg, B.W., and D.F. Silbert. 1983. Sterol partitioning among intracellular membranes.
Testing a model for cellular sterol distribution. J Biol Chem. 258:2284-9.
Wenk, J., P.W. Andrews, J. Casper, J.-I. Hata, M.F. Pera, A. von Keitz, I. Damjanov, and B.A.
Fenderson. 1994. Glycolipids of germ cell tumors: extended globo-series glycolipids are
a hallmark of human embryonal carcinoma cells. Int J Cancer. 58:108-115.
Wiels, J., M. Fellous, and T. Tursz. 1981. Monoclonal antibody against a Burkitt- lymphoma-
associated antigen. Proc Nat Acad Sci (Wash.). 78:6485-6488.
Wiels, J., E.H. Holmes, N. Cochran, T. Tursz, and S.-I. Hakomori. 1984. Enzymatic and
organizational difference in expression of a Burkitt lymphoma-associated antigen
(globotriaosylceramide) in Burkitt lymphoma and lymphoblastoid cell lines. J Biol
Chem. 259:14783-14787.
Wiels, J., M. Mangeney, C. Tétaud, and T. Tursz. 1991. Sequential shifts in the three major
glycosphingolipid series are associated with B cell differentiation. Int. Immunol. 3:1289-
1300.
Wiese, M., and I.K. Pajeva. 2001. Structure-activity relationships of multidrug resistance
reversers. Curr Med Chem. 8:685-713.
Wijnholds, J., R. Evers, M.R. van Leusden, C.A. Mol, G.J. Zaman, U. Mayer, J.H. Beijnen, M.
van der Valk, P. Krimpenfort, and P. Borst. 1997. Increased sensitivity to anticancer
drugs and decreased inflammatory response in mice lacking the multidrug resistance-
associated protein. Nat Med. 3:1275-9.
Wilcox, W.R., M. Banikazemi, N. Guffon, S. Waldek, P. Lee, G.E. Linthorst, R.J. Desnick, and
D.P. Germain. 2004. Long-term safety and efficacy of enzyme replacement therapy for
Fabry disease. Am J Hum Genet. 75:65-74.
246
Willingham, M., N.D. Richert, M.M. Cornwell, T. Tsuruo, H. Hamada, M.M. Gottesman, and
I.H. Pastan. 1987. Immunocytochemical localization of P170 at the plasma membrane of
multidrug-resistant human cells. J Histochem Cytochem. 35:1451-1456.
Willingham, M.C., M.M. Cornwell, C.O. Cardarelli, M.M. Gottesman, and I. Pastan. 1986.
Single cell analysis of daunomycin uptake and efflux in multidrug-resistant and -
sensitive KB cells: effects of verapamil and other drugs. Cancer Res. 46:5941-6.
Winchester, B., and G.W. Fleet. 1992. Amino-sugar glycosidase inhibitors: versatile tools for
glycobiologists. Glycobiology. 2:199-210.
Wraith, J.E. 2006. Limitations of enzyme replacement therapy: current and future. J Inherit
Metab Dis. 29:442-7.
Wu, M., and Y.Q. Wei. 2004. Development of respiratory stem cells and progenitor cells. Stem
Cells Dev. 13:607-13.
Yabe, H., M. Yabe, K. Hattori, H. Inoue, M. Matsumoto, S. Hamanoue, A. Hiroi, T. Koike, and
S. Kato. 2005. Secondary G-CSF mobilized blood stem cell transplantation without
preconditioning in a patient with Gaucher disease: Report of a new approach which
resulted in complete reversal of severe skeletal involvement. Tokai J Exp Clin Med.
30:77-82.
Yamaji, T., K. Kumagai, N. Tomishige, and K. Hanada. 2008. Two sphingolipid transfer
proteins, CERT and FAPP2: their roles in sphingolipid metabolism. IUBMB Life.
60:511-8.
Yamashita, T., A. Hashiramoto, M. Haluzik, H. Mizukami, S. Beck, A. Norton, M. Kono, S.
Tsuji, J.L. Daniotti, N. Werth, R. Sandhoff, K. Sandhoff, and R.L. Proia. 2003.
Enhanced insulin sensitivity in mice lacking ganglioside GM3. Proc Natl Acad Sci U S
A. 100:3445-9.
247
Yamashita, T., R. Wada, and R.L. Proia. 2002. Early developmental expression of the gene
encoding glucosylceramide synthase, the enzyme controlling the first committed step of
glycosphingolipid synthesis. Biochim Biophys Acta. 1573:236-40.
Yamashita, T., R. Wada, T. Sasaki, C. Deng, U. Bierfreund, K. Sandhoff, and R.L. Proia. 1999.
A vital role for glycosphingolipid synthesis during development and differentiation.
Proc Natl Acad Sci U S A. 96:9142-7.
Yamashita, T., Y.P. Wu, R. Sandhoff, N. Werth, H. Mizukami, J.M. Ellis, J.L. Dupree, R.
Geyer, K. Sandhoff, and R.L. Proia. 2005. Interruption of ganglioside synthesis produces
central nervous system degeneration and altered axon-glial interactions. Proc Natl Acad
Sci U S A. 102:2725-30.
Yamazaki, M., W.E. Neway, T. Ohe, I. Chen, J.F. Rowe, J.H. Hochman, M. Chiba, and J.H.
Lin. 2001. In vitro substrate identification studies for p-glycoprotein-mediated transport:
species difference and predictability of in vivo results. J Pharmacol Exp Ther. 296:723-
35.
Yang, Q., J. Zhang, S.M. Wang, and J.R. Zhang. 2004. [Expression of glucosylceramide
synthase mRNA in vincristine-resistant KBV200 cell line in association with multidrug
resistance]. Di Yi Jun Yi Da Xue Xue Bao. 24:779-81.
Yildiz, Y., H. Matern, B. Thompson, J.C. Allegood, R.L. Warren, D.M. Ramirez, R.E. Hammer,
F.K. Hamra, S. Matern, and D.W. Russell. 2006. Mutation of beta-glucosidase 2 causes
glycolipid storage disease and impaired male fertility. J Clin Invest. 116:2985-2994.
Yogeeswaran, G., and S. Hakomori. 1975. Cell contact-dependent ganglioside changes in mouse
3T3 gibroblasts and a suppressed sialidase activity on cell contact. Biochemistry.
14:2151-6.
Yoshimitsu, M., T. Sato, K. Tao, J.S. Walia, V.I. Rasaiah, G.T. Sleep, G.J. Murray, A.G.
Poeppl, J. Underwood, L. West, R.O. Brady, and J.A. Medin. 2004. Bioluminescent
248
imaging of a marking transgene and correction of Fabry mice by neonatal injection of
recombinant lentiviral vectors. Proc Natl Acad Sci U S A. 101:16909-14.
Young, J., and I.B. Holland. 1999. ABC transporters: bacterial exporters-revisited five years on.
Biochim Biophys Acta. 1461:177-200.
Yu, R.K., and R.W. Ledeen. 1972. Gangliosides of human, bovine, and rabbit plasma. J Lipid
Res. 13:680-6.
Yuan, H., X. Li, J. Wu, J. Li, X. Qu, W. Xu, and W. Tang. 2008. Strategies to overcome or
circumvent P-glycoprotein mediated multidrug resistance. Curr Med Chem. 15:470-6.
Yunomae, K., H. Arima, F. Hirayama, and K. Uekama. 2003. Involvement of cholesterol in the
inhibitory effect of dimethyl-beta-cyclodextrin on P-glycoprotein and MRP2 function in
Caco-2 cells. FEBS Lett. 536:225-31.
Zaitseva, J., S. Jenewein, T. Jumpertz, I.B. Holland, and L. Schmitt. 2005a. H662 is the linchpin
of ATP hydrolysis in the nucleotide-binding domain of the ABC transporter HlyB. Embo
J. 24:1901-10.
Zaitseva, J., S. Jenewein, C. Oswald, T. Jumpertz, I.B. Holland, and L. Schmitt. 2005b. A
molecular understanding of the catalytic cycle of the nucleotide-binding domain of the
ABC transporter HlyB. Biochem Soc Trans. 33:990-5.
Zhang, F., W. Zhang, L. Liu, C.L. Fisher, D. Hui, S. Childs, K. Dorovini-Zis, and V. Ling.
2000. Characterization of ABCB9, an ATP binding cassette protein associated with
lysosomes. J Biol Chem. 275:23287-94.
Zhang, J.T. 2007. Use of arrays to investigate the contribution of ATP-binding cassette
transporters to drug resistance in cancer chemotherapy and prediction of
chemosensitivity. Cell Res. 17:311-23.
Zhao, H., and G.A. Grabowski. 2002. Gaucher disease: Perspectives on a prototype lysosomal
disease. Cell Mol Life Sci. 59:694-707.
249
Zhao, Y., J. Du, H. Kanazawa, Cen XB, K. Takagi, K. Kitaichi, Y. Tatsumi, K. Takagi, M.
Ohta, and T. Hasegawa. 2002. Shiga-like toxin II modifies brain distribution of a P-
glycoprotein substrate, doxorubicin, and P-glycoprotein expression in mice. Brain Res.
956:246-53.
Zheleznova, E.E., P. Markham, R. Edgar, E. Bibi, A.A. Neyfakh, and R.G. Brennan. 2000. A
structure-based mechanism for drug binding by multidrug transporters. Trends Biochem
Sci. 25:39-43.
Zheleznova, E.E., P.N. Markham, A.A. Neyfakh, and R.G. Brennan. 1999. Structural basis of
multidrug recognition by BmrR, a transcription activator of a multidrug transporter. Cell.
96:353-62.
Zhou, S., J.D. Schuetz, K.D. Bunting, A.M. Colapietro, J. Sampath, J.J. Morris, I. Lagutina,
G.C. Grosveld, M. Osawa, H. Nakauchi, and B.P. Sorrentino. 2001. The ABC
transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular
determinant of the side-population phenotype. Nat Med. 7:1028-34.
Zhou, S.F. 2008. Structure, function and regulation of P-glycoprotein and its clinical relevance
in drug disposition. Xenobiotica. 38:802-32.
Zhu, D., H. Taguchi-Nakamura, M. Goto, T. Odawara, T. Nakamura, H. Yamada, H. Kotaki, W.
Sugiura, A. Iwamoto, and Y. Kitamura. 2004. Influence of single-nucleotide
polymorphisms in the multidrug resistance-1 gene on the cellular export of nelfinavir
and its clinical implication for highly active antiretroviral therapy. Antivir Ther. 9:929-
35.
Zimran, A., and D. Elstein. 2003. Gaucher disease and the clinical experience with substrate
reduction therapy. Philos Trans R Soc Lond B Biol Sci. 358:961-6.
Zoja, J., D. Corna, C. Farina, G. Sacchi, C. Lingwood, M. Doyles, V. Padhye, M. Abbate, and
G. Remuzzi. 1992. Verotoxin glycolipid receptors determine the localization of
250
microangiopathic process in rabbits challenged with verotoxin-1. J. Lab. Clin. Med.
120:229-238.
Zolnerciks, J.K., C. Wooding, and K.J. Linton. 2007. Evidence for a Sav1866-like architecture
for the human multidrug transporter P-glycoprotein. Faseb J. 21:3937-48.
Zschoche, A., W. Furst, G. Schwarzmann, and K. Sanhoff. 1994. Hydrolysis of
lactosylceramide by human galactosylceramidase and GM1-beta-galactosidase in a
detergent-free system and its stimulation by sphingolipid activator proteins, sap-B and
sap-C. Activator proteins stimulate lactosylceramide hydrolysis. Eur J Biochem. 222:83-
90.
