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PhD Thesis
‘APOPTOSIS IN CHRONIC
LYMPHOCYTIC LEUKAEMIA’
Diane King BSc (Hons)
Centre for Mechanisms of Human Toxicityand
Department of Pathology
University of Leicester
UMI Number: U124012
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This thesis is dedicated to Barrie, Jennifer and Angela King
and especially to my husband, James Dodd.
ACKNOWLEDGEMENTS
I would like to thank my supervisors, Professor G.M. Cohen and Dr. J.H. Pringle for their
constructive advice throughout the duration of this study, and Dr. R.M. Hutchinson for his
support and guidance.
Many thanks also to Mr. R. Snowden, Dr. M. MacFarlane, Mr. D. Brown, Dr. J. Shaw, Mrs.
L. Primrose, Mrs. A. Gillies, Mrs. L. Potter and Mrs. T. de Haro for their technical advice and
assistance.
PUBLICATIONS
King D, Cohen GM, Pringle JH, Hutchinson RM: Spontaneous apoptosis in chronic
lymphocytic leukaemia as a possible predictor of response to drug therapy. Br. J. Haematol.,
1997, 97, Suppl. 1; Abstract 112.
King D, Pringle JH, Hutchinson RM, Cohen GM: Processing/Activation of caspases -3, -7
and -8 but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia
cells. Leukemia, 1998,12; 1553 - 1560.
King D, Pringle JH, Hutchinson RM, Cohen GM: Processing/Activation of caspases -3, -7
and -8 but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia
cells. J. Pathol., 1998,186 SS; Abstract 31.
ABSTRACT
B cell chronic lymphocytic leukaemia (B-CLL) is the most common adult leukaemia in the
western world. The disease is characterised by the accumulation of a CD5+ B cell clone. Drug
resistance is a major problem in B-CLL and complete remissions are uncommon. The
lymphoaccumulative nature of B-CLL implies that dysregulation of the apoptotic process
may be responsible for the development and progression of the disease. B-CLL cells were
freshly isolated from patients, and an in vitro apoptosis sensitivity assay was developed using
flow cytometric techniques. Initial studies confirmed the existence of ‘spontaneous apoptosis’
when B-CLL cells were cultured in vitro, and demonstrated a strong correlation between
sensitivity to spontaneous apoptosis and sensitivity to apoptosis induced by the
chemotherapeutic drug chlorambucil in vitro. Immunoblotting of chlorambucil and
prednisolone treated B-CLL cells demonstrated the expression and activation of caspases -3,
-7 and -8 in all samples analysed, whilst activation of caspase-2 was seen in cells from only
one patient. Activation of caspases -3 and -7 was accompanied by the proteolysis of the
DNA repair enzyme, poly (ADP-ribose) polymerase (PARP). Induction of apoptosis and
activation of all the caspases was inhibited by the cell permeable caspase inhibitor,
benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). These results
demonstrated a key role for the activation and processing of caspases in the execution phase
of apoptosis in B-CLL cells. The B cell growth factors interleukin-4 and CD40 were
demonstrated to strongly influence survival of B-CLL cells in in vitro culture, and also to
modulate the response of the cells to chemotherapeutic drugs. Investigations into Fas induced
apoptosis in B-CLL cells demonstrated the expression of the adapter protein, FADD, but no
overexpression of the caspase-8 inhibitory protein, c-FLIP. Additionally, B-CLL cells did not
show rapid assembly of the death inducing signalling complex (DISC) in response to
stimulation of Fas receptor, implying that these cells may preferentially utilise the Bcl-2-
inhibitable Type II (mitochondrial) pathway of apoptosis induction, underlining the important
role that Bcl-2 plays in determining the apoptotic sensitivity of B-CLL cells.
KEYWORDS
Chronic lymphocytic leukaemia; apoptosis; caspases; IL-4; CD40; Fas/CD95
LIST OF ABBREVIATIONS
ATP = Adenosine triphosphate
B-CLL = B cell chronic lymphocytic leukaemia
BSA = bovine serum albumin
CAGE = conventional agarose gel electrophoresis
DISC = death inducing signalling complex
EDTA = ethylenediaminetetraacetic acid
ELISA = enzyme linked immunoabsorbancy assay
FADD = Fas associated death domain protein
FIGE = field inversion gel electrophoresis
FITC = fluorescein isothiocyanate
FLIP = Flice-like inhibitory protein
IL-4 = interleukin-4
ISEL = in situ end labelling
PARP = poly (ADP-ribose) polymerase
PBS = phosphate buffered saline
PI = propidium iodide
PS = phosphatidylserine
RT-PCR = reverse transcriptase PCR
SDS = sodium dodecyl sulphate
TBS = Tris buffered saline
TdT = terminal deoxynucleotide transferase
TNF = Tumour necrosis factor
TRAIL = Tumour necrosis factor-like apoptosis inducing ligand
UP = ultrapure
Z-VAD.fmk = benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone
Apoptosis in Chronic Lymphocytic Leukaemia
CONTENTS
PAGE No1.0 GENERAL INTRODUCTION
Introduction to this thesis 11.0 Chronic Lymphocytic Leukaemia 2
1.1.1 Diagnosis and Prognosis 21.1.2 Clinical staging of CLL 41.1.3 Treatment strategies 5
1.1 Apoptosis 71.2.1 Historical perspectives 71.2.2 Morphological and Biochemical characteristics of
apoptotic cells 71.2.3 The execution phase of apoptosis 81.2.4 Mechanisms of induction of apoptosis 11
1.2.4i Receptor/Ligand signalling pathways 111.2.4ii The mitochondrial pathway of apoptosis
induction - role of the bcl-2 family ofapoptosis regulators 14
1.2.5 Inhibitors of apoptosis 161.2.5i Inhibitors of receptor/ligand induced
apoptosis 161.2.5ii Inhibitor of apoptosis proteins (IAP’s) 17
1.3 Apoptosis and Chronic Lymphocytic Leukaemia 181.3.1 The Bcl-2 family 181.3.2 Caspase expression in CLL 191.3.3 Growth factor dependency of CLL cells 20
1.4 Aims and Objectives 21
2.0 MATERIALS AND METHODS 23
2.0 Drugs, chemicals and stock solutions 242.1 Selection of Patients 242.2 Isolation of B-CLL cells from whole blood 27
2.3.1 Isolation of total lymphocyte fraction 272.3.2 Purification of B lymphocytes 27
2.4 Determination of apoptosis sensitivity 282.5 In situ end labelling (ISEL) 282.6 Annexin V assay for apoptosis 292.7 One-stage DNA fragmentation analysis gels 312.8 Field inversion gel electrophoresis (FIGE) 322.9 Immunoblotting 322.10 Stimulation of B-CLL cells using anti-CD40 monoclonal
antibody and interleukin-4 342.11 Analysis of CD95/Fas receptor expression on B-CLL cells 342.12 Isolation of the death-inducing signalling complex (DISC) 35
Apoptosis in Chronic Lymphocytic Leukaemia
3.0 RESULTS 1 - Development of an in vitro apoptosis sensitivity 36assay for CLL cells
3.1 Introduction 363.2 Spontaneous apoptosis of CLL cells 363.3 Apoptotic CLL cells exhibit limited nucleosomal DNA cleavage,
but evidence of large DNA fragmentation can be observed 393.4 The ISEL assay underestimates the percentage of apoptotic CLL
cells 413.5 In vitro sensitivity to spontaneous apoptosis is a predictor of in
vitro sensitivity to chemotherapeutic drug-induced apoptosis 433.6 As patients undergo chlorambucil therapy, the in vivo level of
apoptosis can decrease, but the sensitivity of the cells to spontaneous and chlorambucil-induced apoptosis in in vitroculture increases 45
3.7 Discussion 48
4.0 RESULTS 2 - Processing/activation of caspases -3 , -7 and -8 , but 53not caspase-2 in the induction of apoptosis in CLL
4.1 Inhibition of spontaneous apoptosis in CLL cells by Z-VAD.fmk 534.2 Activation of caspase-3 and caspase-7 in apoptosis of CLL cells 564.3 Caspase-2 processing does not generally accompany apoptosis of
CLL cells 594.4 Activation of the effector caspases results in the cleavage of PARP 614.5 Activation of caspase-8 during apoptosis of CLL cells 624.6 Z-VAD.fmk inhibits the processing of caspases in CLL cells 644.7 Discussion 66
5.0 RESULTS 3 - Studies on survival factors and the Fas signalling 69pathway in B-CLL
5.1 Introduction 695.2 Purified B-CLL lymphocytes are more sensitive to apoptosis in
vitro culture than unpurified populations of total CLL lymphocytes 695.3 Culture of B-CLL cells with interleukin-4 and CD40 stimulation
results in a reduction in spontaneous apoptosis 725.4 Culture of B-CLL cells with interleukin-4 or CD40 stimulation
increases their resistance to chemotherapeutic drugs 755.5 B-CLL cells are not sensitive to CD95-induced apoptosis 785.6 Upregulation of CD95 receptor on B-CLL cells does not increase
their sensitivity to apoptosis induced by CD95 ligation 805.7 B-CLL cells do not over-express the caspase-8 inhibitory protein,
c-FLIP 855.8 B-CLL cells have all the necessary components to form a
death-inducing signalling complex (DISC) upon CD95 ligation,but do not assemble a DISC in response to CD95 stimulation 87
5.9 Discussion 925.9.1 Survival Factors in B-CLL 925.9.2 Investigations into the Fas signalling pathway in B-CLL 95
Apoptosis in Chronic Lymphocytic Leukaemia
6.0 GENERAL DISCUSSION 100
REFERENCES 105
APPENDIX 119
LIST OF FIGURES AND TABLES
FIGURE1.1 Mechanism of activation of caspase-3 101.2 Pathways leading to cell death 13
2.1 Annexin V/PI dot plot to illustrate positioning of quadrant 31
3.1 CLL cells were isolated from whole blood samples, labelled usingthe ISEL technique and analysed flow cytometrically 38
3.2 CLL lymphocytes were analysed by agarose gel electrophoreticmethods 40
3.4 A comparison of the ISEL and Annexin V labelling techniques 423.5 In vitro sensitivity to spontaneous apoptosis is a predictor of
sensitivity to in vitro chlorambucil-induced apoptosis 443.6 In vivo apoptosis can decrease and sensitivity to spontaneous
apoptosis can increase as drug treatment is administered 47
4.1 Induction of apoptosis in cells from two patients with CLL assessedby phosphatidylserine extemalisation 54
4.2 Induction of apoptosis in a representative B-CLL patient is accompanied by processing of caspase-3 and caspase-7 57
4.3 Activation of caspase-2 only occured in cells from 1 patient 604.4 Cleavage of PARP accompanies apoptosis in CLL cells
Z-VAD.fmk 614.5 Processing of caspase-8 in CLL cells is inhibited by Z-VAD.fmk 634.6 Processing of caspase-3 in CLL cells is inhibited by the caspase
inhibitor Z-VAD.fmk 65
5.1 A Use of CD3+ magnetic beads enriches for CD19+ B cells incultures of CLL lymphocytes
5.1 B Purified B cells are more sensitive to spontaneous apoptosis than cultures of mixed lymphocytes 71
5.2 Interleukin-4 inhibits the induction of spontaneous apoptosis inpurified B-CLL cell cultures 73
5.3 The effect of stimulation of B-CLL cells with interleukin-4 and antibodies to CD40 is case dependent. 74
5.5 Stimulation of B-CLL cells with anti-CD40 and interleukin-4protects against apoptosis induced by chlorambucil. 77
5.6 B-CLL cells are resitant to apoptosis induced by anti-Fasmonoclonal antibody. 79
Apoptosis in Chronic Lymphocytic Leukaemia
5.7 Fas receptor expression is elevated on B-CLL cells following stimulation with antibodies to CD40 and/or interleukin-4.
5.8 Upregulation of Fas receptor on B-CLL cells does not increase their sensitivity to apoptosis induced by Fas stimulation.
5.9 Stimulation of B-CLL cells with CD40 does not confer sensitivity to Fas-induced apoptosis.
5.10 B-CLL cells do not overexpress the Fas signalling pathway inhibitory protein c-FLIP.
5.11 B-CLL cells express the adapter protein, FADD.5.12 Immunoblotting for FADD on immunoprecipitates of Fas
stimulated cells.5.13 Immunoblotting for caspase-8 on lysates of anti-Fas stimulated
cells.
TABLE2.1 List of patients involved in the study
81
83
84
8688
90
91
25/26
4.1 Inhibition of spontaneous apoptosis by Z-VAD.fmk4.2 Clinical information and summary of in vitro apoptosis sensitivity
and incidence of caspase activation
55
58
Chapter 1 Introduction
GENERAL
INTRODUCTION
Chapter 1 Introduction
INTRODUCTION TO THIS THESIS
Described in this thesis is an investigation into the role played by apoptosis in the
etiology and treatment of chronic lymphocytic leukaemia (CLL). This first chapter
serves as an introduction to chronic lymphocytic leukaemia, and as a precis of the
increasingly complex field of apoptosis research. The final section of this chapter
summarises research which has been undertaken to date relating to apoptosis and B-
cell chronic lymphocytic leukaemia (B-CLL), and identifies areas where further
research might be implicated. Subsequent chapters describe the techniques applied
and the results generated during the research project pertaining to this thesis. An
appraisal of the main findings, along with a discussion about future directions for
research in this field, is included at the end.
1.1 CHRONIC LYMPHOCYTIC LEUKAEMIA
1.1.1 Diagnosis and Prognosis
B cell chronic lymphocytic leukaemia (B-CLL) is the most commonly occuring adult
leukaemia in the West and is a result of accumulation of a lymphocyte clone that is
highly resistant to cell death. Since the circulating malignant clone does not undergo
apoptosis, the tumour burden increases in volume and in extent of spread. B-CLL can
therefore be termed a Tymphoaccumulative’ disorder (Dameshek, 1967).
Increasingly B-CLL patients are diagnosed in an asymptomatic phase. This is most
likely attributed to the practice of carrying out blood tests for minor reasons.
Diagnosis is made by the presence of lymphocytosis in peripheral blood and bone
marrow and will include immunophenotypic evaluation to confirm the presence of
characteristic markers. B-CLL lymphocytes express low amounts of surface IgM, or a
combination of slgM and slgD (Rozman & Monserrat, 1995). Other surface markers
commonly expressed on B-CLL lymphocytes include CD20, and CD23, however, the
most common B-CLL marker is CD5 in conjunction with CD 19 (Caligaris-Cappio et
at, 1993).
2
Chapter 1 Introduction
CD5 is a 67 kD glycoprotein which was originally described as a T cell antigen. Only
a small subset of normal B cells found at the edge of the germinal centres of human
lymph nodes are believed to carry CD5. Additionally, a substantial number of foetal B
cells express CD5. In total approximately 5 - 10% of normal B cells express CD5, so
finding a normal counterpart to the B-CLL lymphocyte to use experimentally to
examine the lineage of the B-CLL clone has proved to be difficult.
Morphologically, the B-CLL lymphocyte is not distinct from normal B cells, if
slightly smaller in size. B-CLL cells typically have a high nuclearrcytoplasmic ratio,
and nuclear chromatin often appears clumped. Nucleoli are usually indistinct or not
visible. Conflicting reports exist regarding the stage of maturation of the malignant
cells, but they do appear to be predominantly mature. Any minor variations in shape
or size do not appear to correlate with clinical status or progression.
The volume of neoplastic B lymphocytes increases with time, so even if a patient is
diagnosed in an asymptomatic phase, symptoms will eventually appear. These
typically include lymphadenopathy, splenomegaly and hepatomegaly. The white cell
count, an important monitor of disease progression, will increase, and anaemia and
thrombocytopenia often occur as a result of bone marrow infiltration. Marrow
infiltration can be classified into three types, diffuse, where marrow fat and
interstitium are replaced by extensive lymphocytosis, focal, characterised by distinct,
randomly distributed aggregates of lymphocytes, and interstitial, where the overall
marrow architecture is preserved.
The median survival is approximately nine years, depending on clinical stage at
diagnosis. Good predictors of survival include low clinical stage, low blood
lymphocyte counts, and positive bone marrow histopathological findings (low levels
of lymphocyte infiltration). Positive response to therapy is also a good prognostic
indicator. An indicator of poor prognosis is transformation of B-CLL into large cell
lymphoma (Richters syndrome) (Sawitsky & Rai, 1992).
Cytogenetic abnormalities are also screened for in B-CLL and found in around 50%
of cases (O’Brien et al, 1995). An abnormal karyotype can indicate poor prognosis.
Karyotypic evolution as the disease progresses is rare. The most commonly found
3
Chapter 1 Introduction
cytogenetic abnormality is a deletion at 13ql4. Trisomy of chromosome 12, alone or
with abnormalities of chromosomes 11 and 14 are also found. No ras mutations (chr
12) have been found to date, and translocation of bcl-2 (chr 14) is relatively rare.
However, increased mRNA and protein expression of bcl-2 is found in a majority of
B-CLL cases, although the mechanism by which this is made possible is unclear,
although it has been postulated that hypomethylation of the bcl-2 gene may be
involved (Hanada et al, 1993).
The retinoblastoma (Rb) gene on the long arm of chromosome 13 is rarely deleted or
translocated in B-CLL. When the chromosome 13 breakpoint region was investigated,
a high frequency of deletions was discovered at a locus 530 kb away from the Rb
gene. These deletions are often homozygous, and could indicate the presence of a new
tumour suppressor gene.
1.1.2 Clinical Staging of B-CLL
Two staging systems exist to define disease status, and therefore aid diagnosis, and
choice of treatment strategy. That developed by Rai (Rai et al, 1975) , distinguishes
between five stages of disease :-
Rai staging system
Stage 0 Lymphocytosis in blood and BM.
Stage I + lymph node involvement.
Stage II + organ involvement.
Stage III + anaemia.
Stage IV + thrombocytopenia.
The other commonly used system, is that developed by Binet (Binet et al, 1981),
which classifies patients into three stages of disease depending on the extent of spread
of lymphocytosis (This is the staging system employed in the present study). :-
4
Chapter 1 Introduction
Binet staging system
Stage A Lymphocytosis and < 3 areas of lymphoid
enlargement
Lymphocytosis and > 3 areas of lymphoid
involvement
Lymphocytosis and < lOg/dL Hb,
or < 100 x 109/ L platelets.
Stage B
Stage C
Clinical progression is subsequently assessed by monitoring the peripheral blood
lymphocyte count, platelet count, lymph node appearance and response to treatment.
1.1.3 Treatment Strategies
The alkylating agent chlorambucil has long been the drug of choice in B-CLL. It is
still the most common first-line therapy, and although complete remissions are rare,
chlorambucil does reduce the overall tumour burden in the majority of cases.
In the asymptomatic phase of the disorder it has been demonstrated that treatment
with chemotherapy can actually be detrimental to the patient. For example, patients
with early stage B-CLL treated with chlorambucil have a greater risk of developing
epithelial cancers, and thus have a reduced survival rate when compared with those
patients not receiving early treatment, (Monserrat & Rozman, 1994).
As the disease progresses, and symptoms begin to appear, it is usual to treat with
chlorambucil, at a dose of 0.4 - 0.8 mg/kg every 2-3 weeks. Chlorambucil acts by
alkylating the N7 position of guanine nucleotides producing adducts in the DNA.
Corticosteroids such as prednisolone are often given in conjunction with chlorambucil
to counter immune haemolysis. The dose given is in the range 30 - 60 mg/m2 /day
orally. Prednisolone cytotoxicity is mediated via nuclear receptor interaction.
Combination therapies have been the subject of many clinical trials. These include
COP (cyclophosphamide, vincristine, prednisone), CHOP (as COP but with
doxorubicin), and MOPP (nitrogen mustard, vincristine, procarbazine, prednisone),
5
Chapter 1 Introduction
amongst others. However, results indicate no great improvement on the response rates
for chlorambucil alone (Monserrat & Rozman, 1994).
Recently, much research has centered on the new breed of chemotherapeutic drugs,
the purine analogues, one of which is fludarabine monophosphate (Astrow, 1996).
Fludarabine is transported into the cell by nucleoside transporter proteins. Once inside
the cell, fludarabine is phosphorylated by deoxycytidine kinase to F-ara-adenine
triphosphate (F-ara-ATP). Because of its resistance to deamination by adenosine
deaminase (an enzyme which, incidentally, is naturally depleted in many B-CLL
patients (Sawitsky & Rai, 1992)), F-ara-ATP accumulates in the cell. The result of
this accumulation is suppression of DNA synthesis by inhibition of DNA
polymerase a (O’Brien et al, 1995). Other groups have proposed that incorporation of
F-ara-ATP into replicating DNA is vital to the activity of this drug (Huang &
Plunkett, 1995). The dose given is in the range 25-30mg/m2 for five days, repeated
every four weeks for 4-6 cycles. Since fludarabine is a relatively expensive drug to
administer clinicians are eager to introduce methods of predicting which patients will
respond favourably before treatment commences.
Drug resistance is a major problem in B-CLL and may be linked to an increased
resistance to apoptosis. Several studies have investigated the expression levels of
MDR1 (P-glycoprotein) and glutathione-S-transferases following treatment of B-CLL
patients with chlorambucil. One study showed that MDR1 expression was increased
following treatment (Perri et al, 1989), and GST expression has been shown to be
approximately doubled in a proportion of chlorambucil-treated patients (Schisselbauer
et al, 1990).
6
Chapter 1 Introduction
1.2 APOPTOSIS
1.2.1 Historical Perspectives
Apoptosis, or programmed cell death, was first recognised as being distinct from
necrosis by Kerr and co-workers in the early 1970’s. Electron microscopy was
employed to study some novel histopathological events which had been observed in
lysosomes in hepatic ischemia. These events consisted of the formation of discrete,
rounded bodies, some of which contained chromatin fragments. Electron microscopy
revealed that these bodies contained intact organelles, and that they had arisen by
condensation and disruption of an original hepatocyte. This phenomenon , originally
termed ‘shrinkage necrosis’ (Kerr, 1971), was subsequently observed in many
different tissues. The name ‘apoptosis’ was proposed in 1972 (Kerr et al, 1972) and is
derived from the Greek word meaning ‘falling o ff, as in leaves from trees. Apoptosis
has since been shown to occur throughout many physiological events, from limb
development in the foetus, to cell killing by cytotoxic T lymphocytes.
1.2.2 Morphological and Biochemical Characteristics of Apoptotic Cells
Necrotic cell death is characterised by swelling of the cell and organelles followed by
loss of membrane integrity. When this occurs in tissue, inflammation is inevitable.
Conversely, apoptosis is characterised by compaction of chromatin into crescent
shapes which lie against the nuclear membrane followed by condensation of the
cytoplasm which is not accompanied by swelling of the organelles as occurs in
necrosis. The cell membrane becomes crenellated or ‘blebbed’, and the cell detaches
from its neighbours. Eventually, the cell divides up into multiple membrane bound
‘apoptotic bodies’, some of which contain chromatin. In tissues, these apoptotic
bodies are rapidly cleared, due to the expression of signalling molecules on the cell
surface of the apoptotic cell which are recognised by phagocytic cells. As a result of
the rapid clearance of dead cells from the tissue, inflammation does not occur.
Occuring synchronously with condensation of chromatin is cleavage of double
stranded DNA at nucleosomal intervals resulting in the formation of fragments of
7
Chapter 1 Introduction
DNA in multiples of approximately 200 base pairs (Bortner et al, 1995). An
associated biochemical marker is the production of large fragments of DNA (50-700
kbp), prior to or in the absence of intemucleosomal DNA cleavage. (Oberhammer et
al, 1993). A number of techniques for quantifying apoptosis are based upon
recognition of DNA cleavage. The in situ end labelling (ISEL) and TUNEL methods
can be used on tissue sections or as a flow cytometry method on cell suspensions.
Both techniques use the enzyme terminal deoxynucleotide transferase (TdT) to label
the cleaved DNA fragments with a ‘tail’ of digoxygenin-labelled nuclotides. Anti-
digoxygenin Fab fragments incorporating an colourimetric or fluorescent marker are
used to label the cleaved DNA. Other methods of observing apoptotic cells include
agarose gel electrophoretic methods. The classic ‘ladder’ of nucleosomal fragments
can be visualised using conventional agarose gel electrophoresis (CAGE), and the
formation of the larger DNA fragments can be observed using pulsed field or field
inversion gel electrophoresis (FIGE). Since the early 1990’s techniques have been
developed using other hallmarks of the apoptotic process in order to quantify cell
death, usually flow cytometrically. One of the most widely used techniques is that of
Annexin V / propidium iodide labelling (Koopman et al, 1994). One of the molecules
on the cell surface of an apoptotic cell, which acts as a signalling marker to
phagocytic cells, is phosphatidylserine (PS). PS is a phospholipid usually resident on
the inner leaflet of the cell membrane. During apoptosis PS is flipped onto the outer
surface of the cell. Use of fluorescein isothiocyanate (FITC)-labelled Annexin V, a
protein with high affinity for PS, in conjunction with the red fluorescent dye,
propidium iodide (PI) in a dye exclusion role, results in an extremely efficient flow
cytometric method for analysing apoptosis and for distinguishing apoptotic from
necrotic cells.
1.2.3 The Execution Phase of Apoptosis
Apoptosis can be divided into two phases: an initial condemned phase where cells are
committed to die, without any morphological changes, followed by an execution
phase when the characteristic biochemical and morphological changes of apoptosis
occur, as described above. Apoptosis can occur in response to a variety of stimuli.
Chemotherapeutic drugs, irradiation, and triggering of cell surface ‘death receptors’
8
Chapter 1 Introduction
such as Fas (CD95 / Apo-1) and Tumour necrosis factor receptor (TNF-R1) can all
result in induction of apoptosis. Whatever the nature of the death signal, or the
mechanism by which it is received in the cell, there appears to be a common
‘execution’ pathway, which results in cell death. Much of our present knowledge
regarding this phase of apoptosis comes as a result of studies performed on the
nematode worm Caenorhabditis elegans (Yuan et al, 1993). It is known exactly how
many cells are produced, and how many die during development of this organism, and
the genes which must be mutated in order for the organism to deviate from normal
development. The genes ced-3 and ced-4 were demonstrated to be essential for
developmental cell death in C. elegans since ced-3 or ced-4 null worms have 131
surplus cells at birth.
The mammalian ced 3 homologues are cysteine proteases called ‘caspases’
(previously ICE-like proteases) for cysteine-aspartate proteases. Caspases are
produced as inactive zymogens that require cleavage at the PI position of an aspartate
residue in order to attain their active form. They in turn specifically cleave target
substrates at the PI of an aspartate residue in a defined amino acid sequence. The
caspases are composed of pro-domains followed by two shorter domains, the large
and small subunits. Typically activation involves cleavage at the pro-domain/large
subunit junction, followed by cleavage at the large/small subunit junction and
heterodimerisation of the large and small subunits (Han et al, 1997). A complex
consisting of two heterodimers is thought to make up the active enzyme (figure 1.1).
Caspases can be divided into two groups, initiator caspases and effector caspases.
Initiator caspases preferentially cleave at a (IVL)ExD sequence and typically have
long pro-domains. Initiator caspases such as caspase-8 (FLICE/MACH) (Boldin et al,
1996; Scaffidi et al, 1997) and caspase-2 (Ich-1) (Harvey et al, 1997) are involved in
apoptosis directed through the membrane signalling molecules Fas (CD95/Apo-l) and
TNF-R1. Effector caspases, which include caspase-3 (CPP32) (Nicholson et al, 1995)
and caspase-7 (Mch-3) (Fernandes-Alnemri et al, 1995), cleave at a DEVD target
sequence and are responsible for cleaving substrates which result in cell death. These
targets include poly ADP-ribose polymerase (PARP) (Kaufinann et al, 1993;
Casciola-Rosen et al, 1996), nuclear lamins (Rao et al, 1996) and ICAD, the inhibitor
of the caspase-dependent DNAse (CAD) which is responsible for cleaving DNA into
the characteristic intemucleosomal pattern (Enari et al, 1998).
9
Chapter 1 Introduction
32 kD
PRO 17 kD 12 kD
17 kD12 kD
Caspase-3 zymogen
IStage 1 : Cleavage at 12 kD/17 kD domain junction
PRO 17 kD12 kD
Stage 2 : Cleavage at pro-domain/17 kD domain junction
17 kD12 kD
Stage 3 : Dimerisation o f two active caspase-3 enzymes
17 kD12 kD
Active caspase-312 kD17 kD
Figure 1.1 Mechanism of activation of caspase-3
10
Chapter 1 Introduction
Until recently it was considered that caspase activation was essential for the execution
phase of apoptosis. However, certain instances of apoptotic cell death exist where no
evidence of caspase activation or intemucleosomal DNA cleavage is seen. Caspase-
independent apoptosis in yeast (Green & Reed, 1998), sperm, and
cyclohexamide/staurosporine stimulated chicken erythrocytes (Weil et al, 1998) has
been described, although the molecules involved in this type of apoptosis are yet to be
described.
1.2.4 Mechanisms of Induction of Apoptosis
1.2.4i Receptor-Ligand Signalling Pathways
Triggering of apoptosis can occur via two mechanisms which are not mutually
exclusive. One route for apoptosis initiation is via triggering of the cell surface death
receptors Fas (CD95/Apo-l), TNF-R1 or the TRAIL (tumour necrosis-like apoptosis
initiating ligand) receptors. Binding of Fas ligand to the Fas receptor initiates
trimerisation of the receptor, bring into close contact three intracellular death domains
(DD), a region of homology shared with members of the TNF-family, and some of
the initiator caspases such as caspase-8 (also known as ‘FLICE’). The receptor death
domains are recognised and bound by a death domain containing adapter molecule
called FADD (Chinnaiyan et al, 1996). Other adapter molecules which are involved in
apoptosis signalling through TNF and TRAIL receptors include RAIDD/CRADD
(Duan & Dixit, 1997) and Daxx (Yang et al, 1997). Activation of the receptor/ligand
apoptosis initiating pathways triggers a series of events which result in activation of
the effector caspases leading to cell death. Activation of caspases via Fas signalling
seems to occur by two distinct mechanisms (Scaffidi et al, 1998). Type I is extremely
rapid (within seconds) and requires formation of a ‘death inducing signalling
complex’ (DISC). The DISC consists of caspase-8 which is activated in response to
Fas signalling by binding to the death effector domain (DED) of the adapter molecule
FADD (figure 1.2). Activated caspase-8 then triggers cleavage and activation of
caspase-3 and caspase-7, leading to degradation of the cell. Type II Fas signalling
does not seem to require the formation of a DISC, but appears to be dependent on
mitochondrial activity, in that it can be blocked by Bcl-2 overexpression. Type II
11
Chapter 1 Introduction
cells appear to activate caspase-3 via the formation of an apoptosome consisting of
caspase-9, Apaf-1 and cytochrome c, which is released from mitochondria (figure
1.2). Type II cells show activation of caspase-3 after approximately 60 minutes of Fas
stimulation. Activation of the TNF receptor by TNF binding can promote survival or
apoptosis depending on the adapter and accessory proteins which bind to the
intracellular domain of TNF-R1. TRADD is the primary adapter molecule for TNF
signalling and can recruit FADD and caspase-8 to induce apoptosis (Yuan, 1997).
Alternatively, the adapter molecule RAIDD/CRADD (Duan & Dixit, 1997) can
mediate an NFkB signal in response to binding of the TNF receptor by the kinase RIP
(TNF-receptor interacting protein) (Ahmad et al, 1997; Kelliher et al, 1889). Other
molecules involved in receptor-mediated apoptosis induction include CARDIAK,
which is a RIP-like kinase that can bind and activate caspase-1 via its DED/CARD
domain, and is also involved in triggering of Jun kinase and NFkB (Thome et al,
1999). Daxx is a second adapter molecule in the CD95 system which triggers Jun
kinase, via activation of apoptosis signal-regulating kinase-1 (ASK). FLASH is
another protein involved in receptor-mediated apoptosis. FLASH is a large protein of
approximately 220 kb with homology to C. elegans CED-4, which interacts with
caspase-8 through a DED domain, and appears to be another Fas DISC component
(Imai et al, 1999), although its function is unclear.
The Fas and TNF receptor/ligand partemships are just two of a number of related
pairings which when triggered can cause induction of apoptosis. TRAIL (TNF-related
apoptosis initiating ligand) can bind to a number of receptors. Some of these, such as
DR4 and DR5, when bound by TRAIL, lead to induction of apoptosis. Others, termed
‘decoy receptors’ such as DcRl and DcR2, lack the intracellular death domain
necessary for apoptosis induction. Binding of TRAIL to these receptors, therefore,
does not induce apoptosis (French & Tschopp, 1999 for review). TRAIL has been
shown to induce apoptosis in cells from a range of haematological neoplasms,
including chronic lymphocytic leukaemia (Snell et al, 1997).
12
CELL MEMBRANE
CYTOPLASM
Fas Ligand
Fas receptor
Chemotherapeutic drugs, growth factor withdrawal, mitogenic signals
A FADD
p- | Activated Caspase-8
c-FLIP
Ceram ide
ActivatedCaspase-3
Cytochrome c MITOCHONDRION
Activated caspase-9, dATP, Apaf-1 apoptosome
NUCLEUS
Morphological nuclear events
Figure 1.2 Pathways leading to cell death tCell Death
i
Chapter 1 Introduction
1.2.4ii The Mitochondrial Pathway o f Apoptosis Induction - Role o f the Bcl-2
Family o f Apoptosis Regulators
The mitochondrial pathway of apoptosis induction can be triggered via Fas signalling
as described above, or directly via triggering loss of mitochondrial membrane
potential (Susin et al, 1997). Cytochrome c, released from the mitochondria due to the
opening of a permeability transition pore, triggers the mammalian homologue of the
C. elegans ced-4 gene product, Apaf-1, in the presence of dATP, to activate caspase-9
in a complex which is termed the ‘apoptosome’ (Li et al, 1997). Activated caspase-9
subsequently activates the effector caspases such as -3 and -7. Whether or not the
loss of mitochondrial membrane potential is linked to the release of cytochrome c is
unclear at the present time. One mediator of the mitochondrial apoptotic pathway,
ceramide, is produced by a number of apoptotic initiating agents and accumulates at
the mitochondria, where it can directly bind and release cytochrome c (Garcia-Ruiz et
a l 1997).
Members of the Bcl-2 family exert some of their effects at the mitochondia. Bcl-2 is a
26 kD protein located at 18q21, originally identified during analysis of the t(14; 18)
breakpoint in follicular lymphoma (Tsujimoto et al, 1985). It is homologous to the
apoptosis inhibitory ced-9 gene product in C. elegans. A family of related proteins has
since been discovered, some with anti-apoptotic effects like Bcl-2, and others, such as
Bax, with pro-apoptotic activity (Oltvai et al, 1993), all containing regions of
homology termed the BH domains. Since induction of apoptosis by a wide variety of
stimuli, including anti-cancer drugs, UV irradiation and receptor-ligand signalling
pathways can be blocked by overexpression of Bcl-2 (Cory, 1995), it appears that
Bcl-2 must act at a point in the apoptotic process that is common to many pathways.
Investigations into the mechanism of action of Bcl-2 and related proteins has centered
on their activity in mitochondria during triggering of apoptosis. Early studies on Bcl-2
localised the protein to the mitochondrial outer membrane, particularly at points
where the outer and inner membranes formed a pore. The structure of Bcl-2 and a
related protein, anti-apoptotic B c1-Xl (Boise et al, 1993) resembles that of certain
bacterial toxins which can insert into the mitochondrial membrane and may influence
ion channel activity (Kroemer, 1997). Bcl-2 and Bcl-XL overexpression inhibits the
14
Chapter 1 Introduction
change in mitochondrial membrane potential induced by chemotherapeutic agents
(Decaudin et al, 1997) thus contributing to chemoresistance, and more recent studies
have demonstrated that anti-apoptotic Bcl-2 interacts with pro-apoptotic Bax on the
mitochondrial membrane to prevent activation of downstream caspases, and may
remove Bax to the endoplasmic reticulum to prevent Bax-induced release of
cytochrome c (Bomer et al, 1999). The important role that Bax plays at the
mitochochondria is underlined by the finding that overexpression of Bax can induce
mitochondrial permeability transition (Pastorino et al, 1998). The relevance of
permeability transition to the release of cytochrome c is a matter under much debate,
the two events may be closely related, but reports of cytochrome c release prior to loss
of mitochondrial membrane potential have been made (Reed et al, 1998).
Members of the Bcl-2 family are often involved in amplification or propagation of the
apoptotic signal. In 1998, two pathways for Fas induced apoptosis were reported
(Scaffidi et al, 1998) (as described in section 1.2.4i of this chapter). In Type I
signalling, characterised by the rapid formation of a DISC leading to activation of
significant amounts of caspase-8 and subsequently caspase-3, Bcl-2 is poorly
effective as an inhibitor. In Type II signalling, characterised by activation of small
amounts of caspase-8, Bcl-2 can act as a potent inhibitory factor, indicating that
mitochondria may be involved in this type of apoptosis induction. The link between
activation of caspase-8 and mitochondrial events has been shown to be due to the
activation of the Bcl-2 family member, BID. BID is a 24 kD, BH3 domain containing
protein which has cleavage sites for caspase-8 and granzyme B. Cleavage of BID at
Asp 59 by caspase-8 removes an amino terminus inhibitory domain, to yield a
potently pro-apoptotic protein (Li et al, 1998, Luo et al, 1998). Truncated BID (tBID)
was shown to translocate from the cytoplasm to the mitochondrial and nuclear
membranes where it could induce clustering of mitochondria, loss of mitochondrial
membrane potential and release of cytochrome c, and eventually cell shrinkage and
nuclear condensation (Li et al, 1998). In this way, the Fas induced apoptotic signal
can be amplified by release of mitochondrial factors. A second way in which an
apoptotic signal can be amplified was revealed by Kirsch and co-workers (Kirsch et
al, 1999). Earlier reports had demonstrated that when recombinant anti-apoptotic Bcl-
2 was cleaved at Asp 34 by caspase-3, a 23 kD pro-apoptotic fragment of Bcl-2 was
produced (Cheng et al, 1997). Later studies by the same group identified caspase-3 as
15
Chapter 1 Introduction
the factor required for Bcl-2 cleavage in Fas treated Jurkat cells, and demonstrated the
co-localisation of intact and cleaved Bcl-2 to the mitochondria. There they
demonstrated that cleaved Bcl-2 could promote release of cytochrome c from the
mitochondria in a manner similar to Bax (Kirsch et al, 1999). The authors postulate
that cleavage of Bcl-2 at Asp 34, removing the amino terminal BH4 domain, may
expose the pro-apoptotic BH3 domain, similar to the effect of caspase-8 cleavage of
BID. Thus cleavage of Bcl-2 may act as part of a positive feedback loop, amplifying
the apoptotic signal at a mitochondrial level, by promoting release of cytochrome c
and subsequent activation of caspase-9 and caspase-3.
The release of cytochrome c from mitochondria following an apoptotic stimulus has
been well established. However, it has since been reported that a number of other
factors are released into the cytosol from mitochondria following induction of
apoptosis. These factors include caspase-2 and caspase-9, and a 50 kD ‘apoptosis
inducing factor’ (AIF) (Susin et al, 1999). Bcl-2 may therefore also be involved in
preventing the release of caspase-9 and cytochrome c from mitochondria in the
absence of an apoptotic stimulus, thereby inhibiting apoptosome formation and
activation of downstream caspases such as caspase-3.
1.2.5 Inhibitors of Apoptosis
Since cancer can be viewed as a situation in which the tumour cells are resistant to
cell death, molecules which inhibit apoptosis may play an important role in
development of malignancies. Accordingly, much work has been done to identify and
characterise the expression of molecules such as these.
1.2.5i Inhibitors o f Receptor/Ligand -induced Apoptosis
The identity of several proteins which may act at the proximal stage of apoptosis
induction have been identified. The first of these was c-FLIP (Irmler et al, 1997;
Rasper et al, 1998) (also called Casper, I-FLICE, FLAME-1, CLARP, MRIT and
Usurpin-a), a ‘FLICE-like inhibitory protease’ which can block apoptosis signalling
16
Chapter 1 Introduction
through the CD95/Fas system. c-FLIP has two death effector or caspase-recruitment
domains (DED/CARD’s) at its amino terminus, which enable binding of c-FLIP with
caspase -8 and FADD in the Fas-induced DISC. Interaction of c-FLIP with activated
caspase-8 causes cleavage and activation of c-FLIP and results in the c-FLIP/caspase-
8 interaction becoming more inhibitive. c-FLIP was found to be predominantly
expressed in lymphoid and muscle tissues, and high levels of the protein were
discovered in melanoma cells (Irmler et al, 1997). The extent of expression of c-FLIP
in B-CLL cells has not previously been reported.
The ‘silencer of death domain’ proteins (SODD’s) are a second class of inhibitor
proteins (Jiang et al, 1999). When complexed with the death domain of TNF-R1,
SODD keeps the receptor in a monomeric form. Ligation of TNF-R1 by TNF, causes
SODD to dissociate, allowing formation of the TNF death inducing signalling
complex consisting of the adapter molecules TRADD and TRAF-2 (Yuan, 1997), and
the kinase RIP (Kelliher et al, 1998). A situation could therefore exist where a
dominant negative mutation of a SODD could leave the receptor trapped in a non
functional state. The existence of SODD’s in the TNF receptor-ligand system may
indicate that similar proteins exist in the Fas system, although none have been
described to date.
1.2.5ii Inhibitor o f Apoptosis Proteins (IAPfs)
IAP’s are a family of ‘inhibitor of apoptosis proteins’ (Liston et al, 1996) originally
identified in baculovirus systems, but with mammalian homologues. Mammalian
IAP’s typically contain one or more baculovirus IAP repeat sequence, and have a
carboxy terminus RING finger domain. The IAP’s, which in humans include c-IAP 1,
c-IAP 2, x-IAP and n-IAP, bind to and block the activity of TRAF-2, a protein
involved in TNF receptor-mediated activation of NF-xp.
Survivin, another IAP (Ambrosini et al, 1997) differs from the original IAP’s in that it
contains only one baculovirus IAP repeat, and has no carboxy terminal RING finger.
Survivin is expressed during foetal development, but is not present in terminally
differentiated adult tissue. However, abundant survivin has been detected in several
17
Chapter 1 Introduction
common human cancers, including breast, colon, prostate and lung, in vivo (Kawasaki
et al, 1998). Survivin was also expressed in approximately 50% of high grade non-
Hodgkin’s lymphomas, but not in low grade cases, indicating that this inhibitor may
be important in regulating apoptosis sensitivity in lymphoid malignancies.
1.3 APOPTOSIS AND CHRONIC LYMPHOCYTIC LEUKAEMIA
1.3.1 The Bcl-2 Family
Due to the discovery that Bcl-2 is overexpressed in a majority of B-CLL cases, many
groups have performed investigations in this area. Methods of assessing Bcl-2
expression in B-CLL by different groups range from western blotting of protein
samples, to RT-PCR and flow cytometric techniques. It is frequently reported that
Bcl-2 gene translocations are extremely rare events in B-CLL, and yet mRNA and
protein levels of this factor are elevated in the majority of cases. No satisfactory
explanation exists, as yet, to explain this phenomenon.
Since Bcl-2 is overexpressed in the B-CLL lymphocyte, this may be a possible reason
for accumulation of the malignant B-CLL clone. Several groups have investigated this
possibility. Gottardi et al (1995) confirmed overexpression of Bcl-2 in seven B-CLL
cases by northern blotting and RT-PCR, and postulated that defective apoptosis due to
this overexpression of Bcl-2 may be one reason why B-CLL lymphocytes appear as a
quiescent cell population, accumulating in the Go phase of the cell cycle. In a later
report, the same group analysed twenty-three CLL cases for expression levels of Bcl-
2, Bax, and the two Bcl-X splice variants (Gottardi et al, 1996). They again found
high level expression of Bcl-2 in the majority of cases, and in addition discovered
elevated Bax protein expression in a proportion of cases. Regarding the expression of
B cI-X l and Bcl-Xs, the pattern appears more sketchy, with Bcl-XL more frequently
expressed at a higher level than Bcl-Xs. In all, they found that levels of the apoptosis
inhibitors, Bcl-2 and Bcl-XL, were raised in a significantly higher proportion of cases
than were the apoptosis potentiators, Bax and Bcl-Xs, thus pushing the cells towards
an apoptotic block.
18
Chapter 1 Introduction
A second study examined the expression of Bcl-2 and Bax in relation to in vitro
survival of B-CLL lymphocytes, and clinical progression of the patient. They
concluded that the ratio of Bcl-2 to Bax was an important factor in determining
apoptotic sensitivity in B-CLL (Aguilar-Santelises et al, 1996). This finding was
mirrored by that of Thomas and co-workers (1996) who also looked at the importance
of this ratio in determining apoptotic sensitivity to chemotherapeutic drugs. Of a panel
of eighteen patients, 88% of those with an intermediate to high ratio of Bcl-2 to Bax
were in vitro drug resistant, indicating the importance of these factors in B-CLL.
More recent work has indicated that drug resistance in B-CLL may be due to selection
of subclones which express high levels of Bcl-2 relative to their Bax content (Pepper
etal, 1999).
1.3.2 Caspase expression in B-CLL
The cellular and tissue distribution of different caspases and their substrate specificity
is generally not known, although this is clearly important as exemplified by the
inability of some neuronal but not other cells to undergo apoptosis in caspase-3 7'
mice (Kuida et al, 1996). Multiple species of caspases-3 and -6 appear to be the major
pool of activated caspases in various tumor cell lines induced to undergo apoptosis
(Faliero et al, 1997). In addition to caspases-3 and -6, caspases-2 and -7 are also
activated in human monocytic tumor cells induced to undergo apoptosis by various
stimuli (MacFarlane et al, 1997). Recently it has been shown that peripheral blood
mononuclear cells from B-CLL patients are caspase-3 immunopositive (Krajewski et
al, 1997) and that glucocorticoid-induced apoptosis of B-CLL lymphocytes requires
protease activation and is accompanied by cleavage of PARP and lamin Bi together
with the loss of caspase-3 (Bellosillo et al, 1997; Chandra et al, 1997). The
importance of caspase activation in spontaneous and chemotherapy-induced apoptosis
in leukaemic cells from patients with B-CLL has not previously been reported.
19
Chapter 1 Introduction
1.3.3 Growth Factor Dependency of B-CLL Cells
The characteristic spontaneous apoptosis which occurs when B-CLL cells are cultured
in vitro has been reported previously (Collins et al, 1989). Since removal of B-CLL
cells from their normal environment appears to trigger the cells into apoptosis, it
would seem that the cells are stimulated in vivo by one or more survival factors,
which are not provided by in vitro culture in standard media.
CD40 is a 45-50kD glycoprotein which is expressed on the surface of B cells at all
stages of development, with the exception of terminally differentiated plasma cells.
CD40 is a member of the TNF/NGF family of receptors, which also includes
Fas/CD95. CD40 ligand (CD40L) is a 35kD glycoprotein, expressed on activated T
lymphocytes, follicular and dendritic cells within the germinal centres. Ligation of
CD40 receptor by CD40L requires receptor trimerisation, and activates a pathway that
results in isotype switching and clonal expansion during the B cell maturation
process. CD40 ligation has been demonstrated to rescue isolated germinal centre B
cells from apoptosis and has been reported to rescue B-CLL cells from spontaneous
apoptosis in in vitro culture. One way in which CD40 might increase protection from
apoptosis is by upregulating B cI-X l expression (Ghia et al, 1996). Planken and
collegues (Planken et al, 1996), used the ‘CD40 system’ to induce proliferation of B-
CLL cells in vitro. Using CD40-expressing CD32L cells as a feeder layer, and IL4 as
a media supplement, they demonstrated proliferation of cells from B-CLL patients.
CD40 activation of B-CLL cells has also been used as a method of inducing
upregulation of Fas receptor (Wang et al, 1997).
Interleukin-4 (B cell growth factor) sources include T cells, monocytes and bone
marrow stroma. It is known to stimulate B cell growth and differentiation and antigen
presentation. It has been demonstrated that B-CLL cells express either high or low
affinity receptors for IL4 at levels comparable to normal B cells (Gileece et al, 1993)
Addition of IL4 to cultures of B-CLL cells has been shown to reduce the level of
spontaneous apoptosis and to protect against hydrocortisone-induced apoptosis,
possibly by upregulating Bcl-2 expression (Danescu et al, 1992). Other techniques for
stimulating B-CLL cell growth in in vitro culture include interleukin-2 in combination
with mitogens such as Staphylococcus aureus Cowan I (DeFrance et al, 1991), or
20
Chapter 1 Introduction
pokeweed mitogen (Mainou-Fowler et al, 1995). In both of these studies, IL2 was
shown to have a proliferative effect, whilst IL4 had an ‘anti-apoptotic’ effect. Other
growth factors may be important in B-CLL, some of which will be discussed later in
this thesis.
1.4 Aims and Objectives
The overall purpose of this study was to examine the significance of apoptosis in B
cell chronic lymphocytic leukaemia. The fact that B-CLL cells accumulate due an
inability to undergo cell death (Dameshek, 1967) points to the importance of
understanding the nature of the apoptotic block in B-CLL cells. In addition, since
drug resistance is a major problem in B-CLL, this may also be related to the inability
of the cells to undergo apoptosis. Research into apoptosis, and the causes and
mechanisms of resistance to apoptosis, encompasses a wide range of controlling
factors to be investigated. Regarding B-CLL, much research had been done on the
role of the Bcl-2 family of apoptosis regulating proteins. This work had been done in
response to the finding that B-CLL cells express increased amounts of anti-apoptotic
bcl-2. Work in this area has linked the ratio of Bcl-2:Bax to apoptosis sensitivity
(Thomas et al, 1996; Aguilar-Santelises et al, 1996, Pepper et al, 1996), but only
partially explains the resistance of B-CLL cells to apoptosis induction by
chemotherapeutic drugs.
In order to contribute to research in this field, it was decided to investigate apoptosis
in B-CLL from a variety of different directions. The initial aim of this study was to
successfully apply current methods for analysing apoptosis in cell lines to clinical
samples with a view to developing a method by which cells from B-CLL patients
could be analysed for their sensitivity to apoptosis induced by chemotherapeutic
drugs. To then define what factors could influence the apoptosis sensitivity of B-CLL
cells was the aim of further research in this study. The identification of caspases as the
machinery proteins involved in the execution phase of apoptosis led to a series of
experiments being performed to examine the nature of caspase expression and
activation in B-CLL cells. The objective of this research was to discover whether B-
CLL cells possessed the caspases necessary for apoptosis, and whether these caspases
21
Chapter 1 Introduction
could be activated in response to stimulation of B-CLL cells with chemotherapeutic
drugs. In response to findings produced by this work, the study progressed onto
investigating the Fas receptor/ligand signalling pathway of apoptosis induction in B-
CLL cells. The aim of this research was to analyse the nature of Fas resistance in B-
CLL, by investigating the expression and activation of the various caspases, adapter
molecules and inhibitor proteins involved in Fas-induced apoptosis. In conjunction
with this work, analysis of the dependency of B-CLL cells on stimulation from
survival factors, and the effect that the such stimulation might have on resistance to
chemotherapy was investigated.
In summary, the main objectives of this study were :-
• To develop an assay for analysing apoptosis in freshly isolated B-CLL cells.
• To determine the significance of apoptosis sensitivity in B-CLL and to analyse
apoptosis following treatment of B-CLL cells with chemotherapeutic drugs.
• To investigate the nature of apoptotic resistance in B-CLL, by analysing
expression of proteins involved in the apoptotic pathway.
• To determine whether survival factor dependency plays a significant role in
determining the sensitivity of B-CLL cells to apoptosis.
22
Chapter 2 Materials and Methods
MATF.RIAT.S
AND
METHODS
23
Chapter 2 Materials and Methods
2.0 MATERIALS AND METHODS
2.1 Drugs, chemicals and stock solutions
Laboratory stock solutions of phosphate buffered saline (PBS) (0.15 M NaCl (Fisher
Scientific, Loughborough, UK), 0.01 M Na2HP0 4 (Fisher Scientific), 2.5 mM
NaH2P0 4 2 H2O (Hopkins & Williams, Essex, UK), and Tris-buffered saline (TBS)
(0.05 M Tris (GibcoBRL, Paisley, Scotland), 0.15 M NaCl (Fisher Scientific)) were
used as indicated.
Unless stated otherwise all drugs and chemicals were obtained from Sigma Chemical
Co. (Poole, Dorset, UK). Prednisolone-21-hemisuccinate was stored as a 0.1 M stock
solution in PBS, chlorambucil as a 3 mM solution in ethanol, staurosporine as a 0.2
mM solution in dimethylsulphoxide (DMSO) and fludarabine as a 35 mM solution in
dimethyl formamide (DMF). All drugs were stored at “ 20°C in small aliquots to avoid
repeated freezing and thawing. The recommended maximum storage periods at this
temperature were not exceeded. Dilutions prior to use were made in 1 x PBS, so that
the final concentration of diluent never exceeded 0.1 %.
2.2 Selection of Patients
Local ethical committee approval was obtained prior to this study commencing.
Patients were assessed according to the Binet staging system, and were sourced from
Haematology outpatients clinics. Patients who were undergoing a course of treatment
and those who had completed a course within the previous 4 weeks were excluded
from the study. Table 1 lists the patients from whom blood samples were obtained for
analysis during the study. Of the 44 patients analysed, 55% were male and 45% were
female. The ages of the patients ranged from 40 to 94 years, the average age being
70.1 years. The majority (63%) of patients had received no previous treatment for
their condition at the time of sample collection. Of the patients who had been treated
with chemotherapeutic drugs nine had received chlorambucil alone, four had received
24
Chapter 2 Materials and Methods
chlorambucil and prednisolone in combination, two had received fludarabine alone,
and one had received fludarabine in combination with prednisolone.
Patient no. Sex/Age Binet stage WBC count (x 109/L)
Previoustreatment
11 M/94 A 15.3 none
21 M/85 A 42.0 none
31 M/77 C 3.7 FLD
41 F/73 A 38.2 CHL
51 M/72 A 8.5 PD/FLD
61 F/66 A 51.8 none
71 M/76 C 8.1 PD/CHL
81 F/60 A 68 none
91 F/62 B 35.8 CHL
101 F/84 ND ND none
111 M/67 A 41.7 none
121 M/44 A 15.3 CHL
1 A F/73 A 38.2 CHL
2 A F/80 A 17.1 none
3 A M/74 A 23.1 none
4 A M/72 A 49.7 CHL
5 A M/75 A 20.7 none
6 A F/54 A 29.0 none
7A M/48 A 38.9 none
8 A M/72 A 30.2 none
9 A M/52 A 9.1 FLD
10 A M/91 C 24.5 none
11 A F/74 B 14.2 CHL
12 A F/67 B 17.5 none
13 A M/62 A 34.3 none
25
Chapter 2 Materials and Methods
Patient no. Sex/Age Binet stage WBC count (x 109/L)
Previoustreatment
14 A F/60 A 8.0 none
15 A M/51 A 24.6 none
16 A M/81 A 20.5 CHL
17 A M/71 A 12.7 none
18 A F/84 B 22.7 PD/CHL
19 A M/77 C 7.2 PD/CHL
20 A F/85 A 47.7 none
21 A M/50 B 23.7 none
22 A M/81 C 11.3 PD/CHL
23 A F/75 A ND CHL
24 A F/72 A 10.9 none
25 A F/82 A 66.1 none
26 A M/59 A 22.2 none
27 A F/63 A 29.9 none
28 A F/74 A 39.1 CHL
29 A F/87 A 42.4 none
30 A F/40 A 9.6 none
31 A M/79 A 35.9 none
32 A M/60 A 12.9 none
Table 2.1. List of patients involved in the study (PD = prednisolone, CHL = chlorambucil, FLD = fludarabine). White blood cell counts (WBC) and Binet stage refer to patients’ status at time of sampling. ND indicates not determined.
26
Chapter 2 Materials and Methods
2.3 Purification of B-CLL cells from whole blood
2.3.1 Isolation of whole lymphocyte fraction
Whole blood was collected into K+/ EDTA or Heparin tubes (Sarstedt, Leicester, UK),
and layered over Ficoll (Pharmacia Biotech, Sweden). Following centrifugation for 20
minutes at 400g, the lymphocyte layer was aspirated off, and cells were washed twice
in culture media (as described below), in order to remove any remaining Ficoll.
Freshly isolated lymphocytes were suspended in culture medium (RPMI 1640
(GibcoBRL, Paisley, Scotland), 10% foetal calf serum (FCS) (Sigma), 1%
penicillin/streptomycin (GibcoBRL)) and incubated at 37 °C for 20 minutes in plastic
culture flasks in order to remove adherent cells (macrophages, monocytes). Cells were
resuspended at an average density of 1 x 106 cells /ml. (Cells were counted using a
haemocytometer, and viability was assessed by Trypan Blue exclusion).
2.3.2 Purification of B lymphocytes
Lymphocytes isolated as described above were washed in 1 x PBS/2% FCS, and
resuspended in PBS / FCS at an average density of 10 x 106 cells/ml. Dynabeads M-
450 CD3 (Dynal, Oslo, Norway) were added to achieve a final ratio of dynabeads:— • 7target T cells of at least 4:1, and a minimum concentration of dynabeads of 2 x 10 /
ml. The cells and dynabeads were incubated at 4°C for 30 minutes with rolling action.
The magnetic beads (and bound CD3+ T cells) were removed by placing the tube of
cells into a magnetic particle concentrator (Dynal MPC-1). Unbound cells (CD3* B
cells) were pipetted off, washed and resuspended in media (as previously described) at
an average density of 1 x 106 cells/ml. Purity of the resulting cell population was
determined flow cytometrically using antibodies against the B cell marker CD 19
(CD19-FITC, Dako, High Wycombe, Bucks), and the T cell marker CD3 (CD3-RPE,
Dako) at 5pi per 106 cells. Flow cytometry is the measurement of cells in a flow
system which has been designed to deliver cells in single file past a laser beam.
Fluorescence and scattered light are recorded for each cell and data is depicted
27
Chapter 2 Materials and Methods
graphically in the form of dot-plots and histograms. From these plots, populations of
cells with similar characteristics can be identified and quantified (see figure 5.1, page
71, for an example dot-plot resulting from use of the antibodies described above).
2.4 Determination of Apoptosis Sensitivity
Freshly isolated cells were incubated in six-well plates at 37°C in an atmosphere
containing 5% CO2. Samples of the cells were incubated in the presence or absence of
chlorambucil ( 3 - 1 5 pM), prednisolone (20 - 200 pM), fludarabine (3 pM),
staurosporine (0.2 pM), or anti-Fas monoclonal antibody (CH-11) (0.5 pg/ml)
(Immunotech, Marseilles, France) (Yonehara et al, 1989) as indicated in the text. For
analysis of caspase activation, duplicate samples of untreated and drug-treated cells
were pre-incubated for 30 min with 100 pM Benzyloxycarbonyl-Val-Ala-Asp (OMe)
fluoromethyl ketone (Z-VAD.fmk), a cell permeable caspase inhibitor (Enzyme
Systems Products, Dublin, CA). At specified times samples were analysed for
apoptosis, initially using in situ end labelling (ISEL) and agarose gel techniques for
visualising DNA fragmentation, and later by Annexin V labelling. 1 x 106 cells from
each culture were washed in ice-cold PBS in order to remove media and serum, and
were stored at ”80 °C as cell pellets for immunoblotting.
2.5 In situ end labelling (ISEL)
General Principles
ISEL incorporates digoxygenin-labelled nucleotides to the ends of DNA strand breaks
produced by endonuclease cleavage of DNA during the final stages of apoptosis. The
enzyme utilised is terminal deoxynucleotide transferase (TdT), which adds
nucleotides to the 3' hydroxyl group of a DNA strand, without the need for a template.
The strand breaks are visualised by the addition of an anti-digoxygenin-Fab fragments
with a fluorescein isothiocyanate (FITC) label. The cells are also labelled with
propidium iodide (PI), a red fluorescent DNA staining dye in order to quantify DNA
content. Flow cytometry is used to analyse the resulting cell populations.
28
Chapter 2 Materials and Methods
Method
1 x 106 cells were fixed in 1ml 1% formaldehyde/PBS for 15 minutes on ice, washed
in two changes of ice-cold PBS, and resuspended in 1ml 70% ethanol/PBS for storage
at 4°C until required. For the labelling reaction, the cells were washed first in 1 x
TBS, resuspended in 100 pi of reaction mixture (80 pi UP water, 20 pi 5x TdT buffer,
2 pM digoxygenin-ll-dUTP (Boehringer Mannheim, Germany), and 10 U TdT
(GibcoBRL, Paisley, Scotland)), and incubated for 35 mins at 37°C. To stop the
labelling reaction the samples were removed immediately to ice for 3 mins. The cells
were then washed in staining buffer (1% of 10% Triton X-100, 20% 20x standard
saline citrate (SSC), plus 5% w/v bovine serum albumin (BSA)) and resuspended in
250 pi labelling mixture (250 pi staining buffer and 0.1 pg anti-digoxygenin-FITC
antibody (Boehringer Mannheim)) in which they were incubated for a further 35 mins
at 37°C. Following centrifugation at 400g for 5 minutes the samples were resuspended
in 1ml propidium iodide (PI) (50 pg/ml), and incubated overnight at 4°C. Flow
cytometric analysis was performed the following day on a Becton-Dickinson
FACScan, using Lysys II software. A dot-plot of FL2-Area (DNA content) vs FL1-
Height (FITC-labelled apoptotic cells) was produced for each sample. Regions were
set on FITC-positive and FITC-negative cells for the freshly isolated cell sample in
each case, and remained in place for all further analysis on that patient (see figure 3.1,
page 38, for an example dot-plot which demonstrates the positioning of the quadrant).
Values for the percentage of FITC-positive apoptotic cells in the sample were
generated from these regions. Typically 5000 events were recorded per sample.
2.6 Annexin V assay for apoptosis
General Principles
The Annexin V assay measures a cell surface alteration which occurs during
apoptosis. Phosphatidylserine (PS), a membrane phospholipid, is translocated from
the inner leaflet to the outer leaflet of the cell membrane. The externalised PS assists
recognition of the apoptotic cell by phagocytic cells to aid rapid clearance by
engulfment. Annexin V is a Ca2+ -dependent phospholipid binding protein with high
affinity for PS. When conjugated with a FITC label, this protein can be used as a
29
Chapter 2 Materials and Methods
probe for externalised PS by flow cytometry. The cells can be dual labelled with
propidium iodide, which acts as a dye exclusion assay for necrotic and secondary
necrotic cells. This combination enables a distinction to be made between apoptotic
and secondary necrotic cells. An ideal result using this technique gives three
populations of cells. Viable cells have minimal staining with both fluorochromes
(Pr / FITC ), apoptotic cells are positive for Annexin V-FITC (PI7 FITC+) and
secondary necrotic cells (late stage apoptotic cells) stain highly with both
fluorochromes (PI+ / FITC+).
Method
1 x 106 cells were micro-centrifuged at 400g and then re-suspended in 1ml Annexin V
buffer (10 mM HEPES/NaOH pH 7.4, 150 mM NaCl, 5 mM KC1, 1 mM MgCl2, 1.8
mM CaCh). Annexin V-FITC (Bender MedSystems, Vienna, Austria) was added to a
final concentration of 1.5 pg/ml. After incubation at room temperature for 8 min,
propidium iodide was added (50 pi of 50 pg/ml stock in PBS), and the cells incubated
for a further 2 min at room temperature before being placed on ice prior to flow
cytometric analysis on a Becton-Dickinson FACStar Plus, using Lysys II software. A
quadrant was set on each FL1-H vs FL2-H (Annexin V vs PI) dot plot as illustrated in
figure 2.1. Apoptotic cells were identified as Annexin V+ / PI" cells. Secondary
necrotic (Annexin V+/ PI*) cells were included in the dead cell count as these were
identified as late stage apoptotic cells. Typically 5000 events were recorded per
sample.
30
Chapter 2 Materials and Methods
ANNEXIN V-FITC (3) v s PI (4)
<D’3oHHg.23'3.2
Oh
<N-JOh
&
Secondary necrotic cells A nnexin V+/PI+
Viable cells
r :
■■ ■ ■
■ •
. .. j \ . .
=:• : A poptotic cells l ^ ’iJ l^ ^ ' . - A r m e x m V+/PI-
10“FL1-H (Annexin V-FITC)
Figure 2.1 Annexin V FITC/Propidium iodide dot plot to illustrate positioning of the quadrant used to
quantify cell death in the cell population.
2.7 One-stage DNA fragment analysis gels.
Essentially as described by Sorensen et al (1988), this technique enables visualisation
of oligonucleosomal DNA cleavage without the need to extract DNA using organic
solvents. Briefly, 1 x 106 cells were resuspended in 15 pi of sterile ultrapure (UP)
water. 9 pi loading buffer (0.02% bromophenol blue, 40% glycerol in 1 x TBE) and 6
pi RNase A (50 mg/ml) was added and the samples were incubated at room
temperature for 30 minutes. A 1.8% agarose gel (UltraPure Agarose, GibcoBRL) (in
1 x TBE) was prepared, and a slice cut from the area above the sample wells. A
‘digesting geF of 0.8% agarose (in 1 x TBE, 2 % SDS), supplemented with proteinase
K (50 pl/ml) was poured into the gap above the wells. Once the samples were loaded,
the gel was run in 1 x TBE, and bands were visualised by staining for 30 minutes in
ethidium bromide (50 pg/100 ml) followed by a 60 minutes de-staining in UP water.
31
Chapter 2 Materials and Methods
2.8 Field inversion gel electrophoresis.
Essentially as described by Brown et al (1993) and Anand & Southern (1990). Field
inversion gel electrophoresis (FIGE) is a form of pulsed field gel electrophoresis.
FIGE allows large fragments of DNA up to 700 kb to be resolved on an agarose gel,
by alternating the orientation of the electrical field by 180°, whilst ensuring forward
mobility by increasing the length of the forward pulse over the reverse pulse. In order
to protect the DNA from shear stresses, and to ensure support of the DNA fragments
in an agarose matrix at all times, the cells were embedded in low melting point
agarose plugs (Agarose L, Pharmacia). The plugs were incubated in NDS (1% Lauryl
sarcosine, 0.5 M EDTA, 10 mM Tris) and Pronase (1 mg/ml) (Sigma) for 48 hours at
50 °C, rinsed in NDS followed by three changes of Tris-HCl pH 8.0, before being
loaded on a vertical 1.5 mm thick 1% agarose gel (NA Agarose, Pharmacia). The gel
was run overnight using a pulse controller (PC 750, Hoeffer Scientific Instruments),
before being stained with ethidium bromide and viewed under UV light.
2.9 Immunoblotting
Frozen cell samples were lysed in a 10% SDS sample loading buffer containing
bromophenol blue. Electrophoresis was performed on either a 20 cm x 20 cm Flowgen
apparatus (Flowgen, Ashby de la Zouch, Leics, UK) using 7 % or 13 % resolving gels
with a 4 % stacking gel (Sambrook et al, 1989), or on a 10 cm x 10 cm Novex Mini-
Cell II apparatus (Novex, Germany), using 4 - 12 % pre-cast gradient gels. Samples
were run on SDS-polyacrylamide gels as described before being blotted onto
nitrocellulose membranes (Hybond C-extra, Amersham, Bucks). Membranes were
blocked for 60 min in 5% non-fat dried milk in Tris buffered saline containing 0.1%
Tween 20. The membranes were incubated with the primary antibody for 1 h at room
temperature followed by washing with Tris buffered saline containing 1% Tween-20
and then incubated with horseradish-peroxidase conjugated secondary antibody (rabbit
anti-mouse IgG or goat anti-rabbit IgG, Dako, High Wycombe, Bucks) for a further
hour. Rabbit polyclonal antibodies directed to the 17 kD large subunit of caspase-3
(kindly provided by Dr D Nicholson, Merck-Frosst, Quebec, Canada), which
recognize the proform of caspase-3 and its 17-20 kD large subunit(s), and to the
32
Chapter 2 Materials and Methods
carboxy terminus of caspase-2, which recognize both pro-caspase-2 and the 12 kD
subunits, were used (Santa Cruz Biotechnology, CA). A mouse monoclonal antibody
to poly (ADP-ribose) polymerase (PARP) was used, which recognizes both intact
PARP (116 kD) and a cleavage product of 89 kD (kindly provided by Dr G Poirier,
Laval University, Quebec, Canada). A polyclonal antibody to caspase-7 (kindly
provided by X-M Sun, MRC Toxicology Unit, University of Leicester, Leicester,
UK), which recognizes the pro-caspase-7 and the catalytically active 19 kD large
subunit, was also used as described. A rabbit polyclonal antibody to caspase-8 was
raised against the large subunit of caspase-8 (amino acids 210 - 374) and kindly
supplied by Dr X-M Sun (MRC Toxicology Unit, University of Leicester, Leicester,
UK). The antibody obtained was characterized by ELISA and Western blot analysis,
which verified that it recognized intact procaspase-8 and the 43 kD and 18 kD
subunits. A rabbit polyclonal antibody to c-FLIP (kindly provided by Dr. D
Nicholson, Merck-Frosst) which recognises the intact form of c-F L IP l and the intact
and clipped forms of c-FLIPs (Rasper et al, 1998), and a monoclonal antibody against
FADD which recognises a 26 kD band corresponding to FADD in Jurkat cell lysates
(BD Transduction Labs, Kentucky, USA) were also used. All antibodies were checked
for specificity using lysates prepared from control cell lines. A sample of apoptotic
cells from a cell line was also included on each blot of experimental samples. The cell
lines used were THP-1 (human monocytic) and Jurkat (human T lymphoblastoid)
treated with staurosporine (0.2 pM) for 4 h in order to induce caspase
activation/substrate cleavage. The blots were developed using enhanced
chemiluminescence (ECL, Amersham, Bucks, UK) according to the manufacturer’s
instructions. Blots were exposed to X-OMAT LS film (Kodak, Rochester, NY) for
appropriate time intervals, and were developed using a Kodak X-OMAT film
processor (Kodak, Rochester, NY), in order to visualize caspase activation / substrate
cleavage.
33
Chapter 2 Materials and Methods
2.10 Stimulation of B-CLL cells using anti-CD40 monoclonal antibody and
interleukin-4
Culture flasks were prepared as described by Walker et al (1997), by incubating
overnight with goat-anti-mouse immunoglobulins (Dako, High Wycombe, Bucks) in
TBS at a concentration of 5 pg/ml. 3.4 ml of this solution was used per 25cm3 volume
of flask. Flasks were blocked for lh at room temperature using a solution of TBS/1%
FCS. B-CLL cells were resuspended in culture medium (as previously described) and
dispensed into the prepared flasks. Anti-human CD40 monoclonal antibody (R&D
systems, Abingdon, UK) was added to a final concentration of 1.5 pg/ml. The cells
were cultured overnight at 37°C.
For stimulation with interleukin-4, cells were dispensed into culture flasks, and the
media was supplemented with recombinant human interleukin-4 (R&D Systems) at a
concentration of 10 ng/ml. Cells were then cultured for the time periods indicated in
the text.
2.11 Analysis of CD95/Fas receptor expression on B-CLL cells
1 x 106 cells were resuspended in 100 pi of PBS/1 % FCS. 1 pi of anti-human Fas
monoclonal antibody (CH11, Immunotech, Marseilles) and 5 pi of goat-anti-mouse-
FITC antibody (Dako) were added. As a negative control, 1 x 106 cells were incubated
with 1 pi control (non-human specific) IgM antibody (Dako) and 5 pi goat-anti-
mouse-FITC. The cells were incubated on ice for 30 minutes prior to centrifugation at
6,000 rpm for 2 minutes. The cells were resuspended in 1 ml PBS for analysis by flow
cytometry. Histogram plots of FL1-H (FITC) staining were obtained, the
photomultiplier tube (PMT) voltage settings being determined by the level of staining
in the negative control sample.
34
Chapter 2 Materials and Methods
2.12 Isolation of the death inducing signalling complex (DISC)
Technique courtesy of Dr. M.E. Peter, German Cancer Research Center, Heidelberg,
Germany.
1 x 10 SKW6.4 cells (murine B lymphoblastoid) were resuspended in 5 ml cell
culture media (RPMI 1640, 10% FCS, 1% penicillin/streptomycin, 1% non-essential
amino acids (GibcoBRL)). Anti-Apo-1 (IgG (Bender MedSystems, Vienna, Austria))
was added at 1 pg/ml, and the cells were cultured for 10 minutes at 37°C. Following
stimulation, the cells were washed in ice-cold PBS and resuspended in 1 ml ice-cold
lysis buffer (30 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-X 100, 10%
glycerol, 1 pg/ml each aprotinin, leupeptin and pepstatin A). As negative controls, two
aliquots of unstimulated cells were also lysed. The lysate from one of these samples
was supplemented with 1 pg/ml anti-Apo-1. The samples were vortexed and
incubated on ice for 15 minutes. The samples were then spun at 13,000 rpm at 4°C for
15 minutes. 30 pi of a 50% solution of Protein-A Sepharose CL-4B (Sigma) was
added to each lysate. The samples were then incubated at 4°C with rolling action for 1
h 30 mins. Following this incubation period, the samples were spun at 6,500rpm for 2
minutes, the supernatant removed, and the sepharose washed in 4 changes of lysis
buffer. Sample loading buffer containing 10% SDS, 1% p-mercaptoethanol and
bromophenol blue was then added, the samples were boiled and SDS-PAGE and
immunoblotting was performed as previously described. A sample of whole Jurkat
cells which had been incubated with anti-Fas monoclonal antibody (clone CH11,
Immunotech, Marseille, France) for 4 h was included on the gels as a control for
antibody specificity. The membranes were probed with antibodies against FADD and
caspase-8 (all as previously described). The experiment was repeated using Jurkat
cells and B-CLL lymphocytes, where the initial stimulation period was increased to 60
minutes.
35
Chapter Three Results 1
Chapter 3
Development of an in vitro apoptosis sensitivity assay for CLL cells
3.1 Introduction
For analysing the effects of chemotherapeutic drugs on ex vivo cells, toxicity assays
such as the differential staining cytotoxicity assay (Bosanquet et al, 1993) and the
MTT assay (Campling et al, 1988) have traditionally been used. Apoptosis has been
shown to be the primary form of cell death induced by chemotherapeutic drugs. Many
techniques for observing and quantifying apoptosis have been developed, which
recognise various features of an apoptotic cell. The aim of this study was to develop
an in vitro apoptosis sensitivity assay to measure susceptibility of B-CLL cells to the
chemotherapeutic drugs prednisolone and chlorambucil. Initial experiments in this
project used as a measure of apoptosis the extent of DNA fragmentation in the cells,
using agarose gel electrophoresis to assess nucleosomal DNA cleavage, and the
quantitative technique of in situ end labelling (ISEL), where analysis is performed
flow cytometrically. As the study progressed and the need for an earlier marker of
apoptosis became evident, the Annexin V assay was employed which measures the
extent of extemalisation of phosphatidylserine.
3.2 CLL cells undergo spontaneous apoptosis in in vitro culture - measurement
using the ISEL method
In order to make a valid estimation of the in vivo apoptosis sensitivity of a malignant
cell type in an in vitro system, it must be taken into account that removal of the
malignant cells from their normal cellular environment may alter their innate
susceptibility to induction of apoptosis. This most likely occurs due to the loss of one
or more survival stimuli which are present in vivo, but which are not present in the
cell culture environment. When estimating the sensitivity of ex vivo cells to
chemotherapeutic drugs, this altered sensitivity must be taken into account. The term
‘spontaneous apoptosis’ refers to the apoptosis level induced following culture of the
CLL cells an in vitro culture system, without the addition of any apoptosis-inducing
36
Chapter Three Results 1
stimuli such as chemotherapeutic drugs. The existence of spontaneous apoptosis has
been reported previously (Collins et al, 1989) and was evident from the first few
experiments performed during this study, and will be referred to throughout this
thesis. In order to determine whether cells from different patients were sensitive to
spontaneous apoptosis to varying degrees, samples from nine patients were analysed
as described below.
The total lymphocyte fraction was purified from blood samples obtained from nine
patients with B-CLL. The cells were labelled using the ISEL method to measure DNA
fragmentation and analysed flow cytometrically (figure 3.1 A). In all nine patient
samples, the level of apoptosis in the freshly isolated cells was less than 3% indicating
a low rate of apoptotic cell death in vivo (figure 3.1 B). Lymphocytes purified from
the same B-CLL patients were also cultured for 24 h. Subsequently, an aliquot of
cells (1 x 106 cells) from each culture was labelled using the ISEL method and
analysed flow cytometrically to measure the level of apoptosis in the culture. These
cells analysed following culture in vitro without the addition of anti-cancer drugs
showed a wide variation in spontaneous apoptosis (figure 3.1 B), indicating variable
sensitivity to apoptosis induction between B-CLL cells from individual patients.
37
Chapter Three Results 1
<uX
Pm
Freshly isolated cells
O FL2-Area (6) v s FL1 -Height (3)
Control cells at 24 h
o FL2-Area (6) v s FL1 -Height (3)
3%£97%
"I 1 T 1 1 I—1 1 1 1- 1
/ ' k 93%
90.7%1023 1023
FL2-AreaB
30
_ 25-I LU2 20<0oaa. 10 <* 5
0
□ 24 h
1 I4 J _ □ J J J _
2 1 3 1 4 1 5 1 6 1 7 1 8 1 91 101 11 I 121 Patient Number
Figure 3.1 CLL cells were isolated from whole blood samples, and were labelled using the ISEL
method and analysed flow cytometrically. Samples of the cells were also cultured for 24 h in vitro prior
to being labelled and analysed using ISEL. A- Dot plots showing typical of results obtained from use of
the ISEL method on freshly isolated and cultured CLL cells. On the X-axis, FL2-Area is a measure of
the DNA content of the cells, and on the Y-axis, FL1-Height is a measure of green (FITC) flourescence.
Cells expressing high green fluorescence are apoptotic. B- Analysis of freshly isolated cells revealed a
low ex vivo rate of apoptosis (0 h). After 24 h culture in vitro, the level of spontaneous apoptosis varied
widely between patient samples.
38
Chapter Three Results 1
3.3 Apoptotic CLL cells exhibit limited intermicleosomal DNA cleavage, but
evidence of large DNA fragmentation can be observed
In situ end labelling measures the extent of DNA fragmentation in apoptotic cells. In
order to visualise the extent of this DNA fragmentation in apoptotic CLL cells,
agarose gel DNA fragmentation analysis was performed on freshly isolated CLL cells,
and those which had been cultured in the presence of chemotherapeutic drugs.
Following the culture of CLL lymphocytes for 24 h alone or in the presence of
prednisolone (0.1 - 10 mM), chlorambucil (15 pM), fludarabine (3 pM) or etoposide
(2 pM), samples of cells were analysed by agarose gel electrophoresis for evidence of
intemucleosomal DNA fragmentation. In all of the patient samples, after 24 h of
culture, there was no clear evidence of intemucleosomal DNA cleavage (figure 3.2
A). Samples from five of the patients were analysed using field inversion gel
electrophoresis (FIGE). This analysis revealed the presence of large fragments of
DNA of 300 kb and 50 kb produced following induction of apoptosis by
chemotherapeutic dmgs (figure 3.2 B).
From the agarose gel electrophoresis analysis it appears that within 24 h of removal
from the in vivo environment, with or without treatment with chemotherapeutic dmgs,
CLL cells do not exhibit significant amounts of nucleosomal DNA cleavage, but do
undergo cleavage of the DNA into large (50 - 700 kb) fragments. This finding raised
significant questions as to the accuracy of using DNA fragmentation as a measure of
apoptosis in CLL cells. It was decided to employ an alternative flow cytometric
method, the Annexin V assay, which measures the extemalisation of
phosphatidylserine, a common feature of apoptotic cells (Koopman et al, 1994)
39
Chapter Three Results 1
4 h 24 h( \ ( 'i
M M 0 10 1 0.1 0 10 1 0.1 mM Pd
B1 2 3 4 5 6 7 8 9 10 11
300 k B > 5 0 k B >
< 700 kB
Figure 3.2 B-CLL lymphocytes were analysed by agarose gel electrophoretic methods in order to
determine the extent of DNA fragmentation occuring during apoptosis in these cells. A. Cells were
analysed for nucleosomal DNA cleavage after 4h and 24h culture alone or with prednisolone (Pd). B.
Cells were analysed for formation of large (>50kB) DNA fragments in response to culture alone (lanes
2,3,c,d) or with prednisolone (10 mM, lanes 4 and 5; 1 mM, lanes 6 and 7; 0.1 mM, lanes 8 and 9) or
chlorambucil (15 pM, lanes e and f) or etoposide (2 pM, lanes 10 and 11). (Lanes 1 and a are lambda
phage genome concatamers, and lane b is S. cerevisiae chromosome marker).
40
Chapter Three Results 1
3.4 The ISEL assay underestimates the percentage of apoptotic CLL cells
In situ end labelling measures the extent of apoptotic DNA fragmentation in a
population of cells. Previous results had demonstrated that CLL cells undergoing
apoptosis exhibited only a limited amount of intemucleosomal DNA fragmentation
(section 3.3), and so an alternative method of assessing apoptosis was chosen, the
Annexin V assay. In the hope that results obtained using ISEL would relate to results
gained using the Annexin V assay, comparative experiments were performed to
establish the relationship between results from the two techniques
CLL cells from six patients were cultured for 24 h alone (control) or in the presence of
prednisolone (20 pM) or chlorambucil (7 pM) prior to being analysed using both the
Annexin V and ISEL labelling techniques. The results obtained from use of the ISEL
method were plotted against the results obtained from using the Annexin V assay
(figure 3.4). Regression analysis confirmed that the two sets of data had a linear
relationship which was highly significant (P < 0.0001) at the 99.5% confidence level.
The equation for the regression line was calculated (y = 2.0lx), the coefficient of 2.01
indicating that the Annexin V values are double those recorded using the ISEL
method. As a result of this finding, it was deemed unsuitable to continue using DNA
fragmentation as a marker for apoptosis in CLL cells, and the Annexin V assay was
used in all subsequent analysis in this thesis.
41
Chapter Three Results 1
y = 2.0185x80
ao< 50
c 30
10 ^
20 25 30 350 5 10 15 40ISEL % Apoptosis
Figure 3.4 A comparison of the ISEL and Annexin V labelling techniques
demonstrates that measuring DNA fragmentation using ISEL underestimates the
number of apoptotic CLL cells in a population by approximately 50 %. Cells were
isolated from six CLL patients. At 0 h and after 24 h of culture alone or in the
presence of 20 pM prednisolone or 7 pM chlorambucil, duplicate samples were
labelled using ISEL and the Annexin V assay. The flow cytometry results obtained
using each technique were plotted against each other. Regression analysis was
performed to check for a linear relationship, and the regression line was plotted. The
equation for the line is stated on the graph.
42
Chapter Three Results 1
3.5 In vitro sensitivity to spontaneous apoptosis is a predictor of in vitro
sensitivity to chlorambucil-induced apoptosis
Previous results had demonstrated the variability between patient samples in
spontaneous apoptosis sensitivity. To determine whether this sensitivity was reflected
in the response of the cells to chemotherapeutic drug-induced apoptosis, CLL cells
from 30 patients were tested for chlorambucil sensitivity. For each case, duplicate
cultures were set up. One of these cultures was left untreated (control), and the
chlorambucil was added to the second culture (7 pM). The cells were incubated at
37°C for 24 h. Apoptosis sensitivity in the two cultures was assessed flow
cytometrically using the Annexin V assay to measure externalised phosphatidylserine.
Control cultures were scored as sensitive or resistant to spontaneous apoptosis.
(Sensitive samples were given the value 1, and resistant values were scored 0). The
value chosen to delineate between the two groups was the median value of 20.75 %
spontaneous apoptosis. The chlorambucil-treated samples were then split into two
groups based on the sensitivity to spontaneous apoptosis of their corresponding
control cultures and the results were plotted (figure 3.5). A one-tailed T-test
(assuming unequal variances) was performed in order to determine if the means of the
two groups were significantly different. A significant P-value of < 0.0001 confirmed
that the patients which were scored as sensitive to spontaneous apoptosis were most
likely to be highly sensitive to chlorambucil-induced apoptosis. This implies that the
relationship between sensitivity to spontaneous apoptosis and sensitivity to in vitro
drug-induced apoptosis is significant, a finding which would greatly simplify any
predictive testing technique based upon apoptosis sensitivity.
43
Chapter Three Results 1
(0
CO
80
70
60
50
■g 40■T
l ^E| 206̂
10
t
I
♦♦♦♦♦♦
0 1
Sensitivity to spontaneous apoptosis
Figure 3.5 Cells from thirty CLL patients were cultured for 24 h alone (control) in
order to determine sensitivity to spontaneous apoptosis, or in the presence of 7 pM
chlorambucil. After 24 h, flow cytometric analysis of the cells was performed using
the Annexin V labelling technique to measure externalised phoshatidylserine. The
control cultures were scored as sensitive (1) (n = 15) or resistant (0) (n = 15) to
spontaneous apoptosis. The value obtained for chlorambucil-induced apoptosis for
each sample was plotted against the spontaneous apoptosis score in each case.
44
Chapter Three Results 1
3.6 As patients undergo chlorambucil therapy, the in vivo level of apoptosis can
decrease, but the sensitivity of the cells to spontaneous and chlorambucil-induced
apoptosis in in vitro culture increases
In order to attempt to determine how closely the in vitro system of apoptosis induction
by chemotherapeutic drugs was related to the in vivo reaction of CLL cells to drug
therapy, a small in vivo study was performed. Blood samples were taken from two
CLL patients (8 A and 12 1) immediately prior to them beginning a 14-day course of
chlorambucil. This was to be the first course of treatment for patient 8 A, but patient
12 I had received chlorambucil therapy previously. In addition to the pre-treatment
blood sample, further samples were taken from the same patients at day 7 of the
course of treatment, and at day 30, two weeks post-treatment. On each sample day, the
in vivo level of apoptosis was analysed by labelling the freshly isolated lymphocytes
using the Annexin V technique. Flow cytometric analysis was performed immediately.
This method attempts to give as accurate a measure of in vivo apoptosis rate as is
possible, the time lapse from phlebotomy to flow cytometric analysis being less than
60 minutes. This analysis revealed that the in vivo apoptosis rate for patient 8 A
remained relatively stable, whereas the in vivo apoptosis level for patient 12 I
decreased as the course of treatment was administered (figure 3.6).
A proportion of cells from each isolate were resuspended in culture medium and
cultured for 24 h. Assessment of the level of spontaneous apoptosis induced in the
cultures was made usisng the Annexin V assay. This analysis revealed that the in vitro
apoptosis sensitivity of the ex vivo CLL cells was elevated during administration of
chemotherapy. In both cases, the level of spontaneous apoptosis increased during and
after the course of treatment when compared with the level of spontaneous apoptosis
obtained pre-treatment (figure 3.6).
In order to further compare the in vivo and in vitro responses of CLL cells to
chlorambucil, the remaining cells from each sample were cultured in vitro in the
presence of 7 pM chlorambucil. After 24 h culture, the sensitivity of these cells to
chlorambucil-induced apoptosis was assessed flow cytometrically using the Annexin
V technique. The cells from patient 12 I showed a decrease in sensitivity to
45
Chapter Three Results 1
chlorambucil as treatment commenced, which increased again by the 30 day sample
point (figure 3.6). However, the sensitivity of the cells did not recover to the levels
observed prior to the treatment beginning, possibly indicating the loss of a particularly
chlorambucil-sensitive clone. Since this patient had in the past had one course of
chlorambucil therapy, this result may indicate some degree of resistance of the
remaining clone to apoptosis induction by chlorambucil. Additionally, the level of
apoptosis induced by chlorambucil at the 30 day sample point was not significantly
higher than the level of spontaneous apoptosis induced at the same timepoint. When
compared with the greater difference in the cells’ sensitivity to spontaneous and
chlorambucil-induced apoptosis at the pre-treatment sample point, this may indicate
that the cells are becoming increasingly resistant to chlorambucil as a result of the
therapy.
Cells from patient 8 A showed a steady increase in sensitivity to chlorambucil as the
course of treatment progressed, mirroring the increase in sensitivity to spontaneous
apoptosis. Of interest is the observation that the post-treatment level of chlorambucil-
induced apoptosis in this patient was 37% higher than the pre-treatment level (figure
3.6), which implies that a number of chlorambucil-sensitive CLL cells remain in
circulation following the end of the course of treatment.
46
Chapter Three Results 1
C/5cooQ.OQ.<
100
80
o 60
40
20
Patient 12 I
Re-treatment (Day 0) Day 7
Sample Day
□ Freshly isolated cells ■ Control□ 7 uM Chlorambucil
Fbst-treatment (Day 30)
100
Patient 8 A
<0'(02Q.OQ.<
80
60
40
20
Re-treatment (Day 0) Day 7
Sample DayFbst-treatment (Day 30)
Figure 3.6 In vivo apoptosis can decrease and sensitivity to spontaneous apoptosis
can increase as drug treatment is administered. Blood samples were taken from
patients 121 and 8 A prior to them starting a two week course of chlorambucil (day 0).
Blood samples were also taken at day 7 of their courses of treatment, and at day 30
(post-treatment). At each sample point, freshly isolated lymphocytes were labelled and
analysed using the Annexin V technique. Other cells from the same isolate were
cultured for 24 h in culture medium alone (control) or with 7 pM chlorambucil before
being analysed using the Annexin V assay. For flow cytometry histograms of freshly
isolated and control cells at each of the sample points for both patients, please refer to
the Appendix, figure A1.
47
Chapter Three Results 1
3.7 Discussion
The initial aim of this reseach project was to investigate the relationship between
chemotherapy and apoptosis in chronic lymphocytic leukaemia (CLL). CLL is often
diagnosed in an asymptomatic phase, and patients can live with the disease for several
years before in some cases the tumour burden and its accompanying effects on the
body mean that chemotherapeutic treatment is necessary. The treatment of choice has
been the alkylating agent, chlorambucil with or without the glucocorticoid,
prednisolone. More recently, the purine analogues, of which fludarabine is one, have
shown increasing promise and a higher rate of remissions when used as first line
therapy, when compared with the remission rate obtained using chlorambucil.
However, drug resistance is a major problem in CLL, and second or third treatments
with chlorambucil do not usually produce high remission rates. Since the advent of
chemotherapy, it has been a goal for many researchers to develop a means of
analysing the toxicity or killing efficacy of the drugs on the target cells in order to
reduce the incidence of unsuccessful treatment (Hanson et al, 1991; Bosanquet, 1993).
In addition the effect of the drugs on other cells in the body needs to monitored, a
process which plays a crucial role in the development and testing of new drug
therapies. Toxicity tests have been in existence for many years, and have been used
extensively for purposes such as those outlined above. Examples include the MTT
assay which assesses the amount of insoluble formazan produced by living cells
(Campling et al, 1988) and the differential staining cytotoxicity assay (Bosanquet et
al, 1983). In the 1970’s apoptosis was identified as a mode of cell death distinct from
necrosis (Kerr, 1972). Since then, investigations into the effects of chemotherapy on
mammalian cells have revealed that apoptosis is the primary method by which cells
die as a result of exposure to these drugs.
This study began with the development of a method to analyse apoptosis sensitivity of
lymphocytes sourced from CLL patients attending outpatient clinics, some untreated,
and some who were receiving chemotherapy. At the time that this study commenced
there were numerous techniques available for analysing or quantifying apoptotic cells
(Carbonari et al, 1995), several of which were based upon the characteristic
48
Chapter Three Results 1
intemucleosomal DNA cleavage that occurs in an apoptotic cell. Qualitative agarose
gel electrophoresis techniques and flow cytometric methods, such as in situ end
labelling, appeared to be the ideal techniques to apply to CLL samples. In particular,
in situ end labelling (ISEL) (Wolfe et al, 1996) was chosen over some newer flow
cytometric labelling techniques (such as the Hoerscht 33342 ‘high blue’, method
(Brown et al, 1996)), because of the inclusion of a fixation step in the procedure. This
enabled the samples to be stored until a sufficient number were available to be
analysed, and also decreased the risks asscociated with handling unfixed clinical
specimens. The data obtained from the flow cytometric analysis of the CLL cells were
supplemented using agarose gel electrophoretic methods to enable visualisation of
intemucleosomal and larger DNA fragmentation (Figure 3.2 A and B).
Initial results using ISEL analysis revealed a low level of in vivo apoptosis in the cells
from every patient analysed, demonstrating that the CLL lymphocytes are not
undergoing cell death in the patient to any significant degree (Figure 3.1 B). It is
possible that the in vivo level could be higher than this, but that apoptotic cells are
being cleared effectively from the blood, and so cannot be measured as such.
However, if these figures do accurately represent levels of in vivo apoptosis, then this
supports the lymphoaccumulative model of CLL (Dameshek, 1967), in which CLL
lymphocytes remain for a long period in circulation due to reduced levels of cell
death.
ISEL analysis also confirmed the existence of spontaneous apoptosis, a feature of
cultured ex vivo CLL cells which had been observed previously (Collins et al, 1989).
From a common low level of in vivo apoptosis, CLL cells cultured for 24 h prior to
being labelled using the ISEL method and analysed flow cytometrically varied
dramatically in their sensitivity to spontaneous apoptosis (Figure 3.1 B). This implies
that differences may exist between the CLL lymphocytes of the spontaneous apoptosis
sensitive and resistant patients. Differences in the expression levels of apoptosis-
controlling factors could be one reason for this variability. The relative levels of anti-
apoptotic Bcl-2 and pro-apoptotic Bax in these cells may be instrumental in
determining their apoptosis sensitivity, a theory which has been investigated by a
number of research groups (McConkey et al, 1996; Gottardi et al, 1996; Pepper et al,
49
Chapter Three Results 1
1996; Thomas et al, 1996). A high Bcl-2:Bax ratio has been shown to correlate with
resistance to chlorambucil in vitro (Thomas et al, 1996), and Bax upregulation
appears to be required for chemotherapeutic drug-induced apoptosis to take place,
whilst a high expression level of Bcl-2 confers survival (Pepper et al, 1999). It is also
likely that CLL cells differ in their sensitivity to growth factors in their surrounding
environment, a subject which will be discussed in later sections of this thesis.
Populations of cells which depend heavily on growth factor stimulation (such as less
mature and less well differentiated CLL clones) are likely to be more susceptible to
cell death upon growth factor withdrawal (such as occurs in in vitro culture).
Use of gel electrophoretic methods to confirm that apoptosis was indeed the
prevailing mode of cell death in CLL cells exposed to chemotherapeutic drugs led to
the observation that, following 24 h of in vitro culture, there was little evidence of the
intemucleosomal cleavage characteristic of apoptosis (Figure 3.2 A). However, large
DNA fragmentation analysis demonstrated that the cells were in the early stages of
DNA fragmentation confirming that apoptosis was occuring (Figures 3.2 B and 3.3).
This phenomenon of little or no intemucleosomal cleavage in apoptotic CLL cells had
been reported previously (Huang & Plunkett, 1995), but the presence of
intemucleosomal DNA fragments may be dependent on the sampling point chosen
following apoptosis induction. Later time points may pick up DNA cleavage more
effectively than earlier time points. However, since the ISEL technique is based upon
labelling the fragmented DNA with digoxygenin-labelled nucleotides in order to
visualise the apoptotic cells, it was of some concern that the cells in question were not
undergoing intemucleosomal DNA fragmentation within the timescale of the assay.
In addition, since the study was moving towards more in-depth analysis of apoptosis
in CLL cells, it became more pmdent to use an earlier marker than DNA
fragmentation in order to quantify apoptosis in these cells.
Annexin V has a high affinity for the membrane phospholipid, phosphatidylserine.
During apoptosis, phosphatidylserine (PS) is flipped from the inner leaflet of the cell
membrane to the outer surface, where it serves as a recognition marker to phogocytic
cells (Koopman et al, 1994), and where it can also be bound by fluorescently labelled
recombinant Annexin V and used as a marker for flow cytometric analysis of
50
Chapter Three Results 1
apoptosis (Martin et al, 1995). In order to introduce this new technique into the study,
a small series of comparitive experiments were performed. Cells from six CLL
patients were cultured alone or for 24 h in the presence of chemotherapeutic drugs
prior to being labelled and analysed using both the ISEL and Annexin V techniques.
When the results obtained with each technique were plotted against the other, it was
discovered that the proportion of apoptotic cells in the CLL cell population was being
underestimated by ISEL by as much as 50%, confirming the observation that DNA
fragmentation was not taking place in these cells to any great degree (Figure 3.4). All
further analysis of apoptosis levels in this study was made using the Annexin V assay.
Large variation had been observed in the sensitivity of CLL cells from different
patients to spontaneous apoptosis. To determine if this sensitivity to spontaneous
apoptosis was mirrored in the response of the cells to chlorambucil-induced apoptosis,
cells from thirty patients were cultured alone or in the presence of chlorambucil for 24
h, prior to assessment of apoptosis using the Annexin V assay. When the results were
compared, it was discovered that cells which were sensitive to spontaneous apoptosis
were also more likely to be sensitive to apoptosis induced by chlorambucil (p <
0.0001). This implies that there is a relationship between sensitivity to spontaneous
apoptosis and sensitivity to in vitro drug-induced apoptosis, a finding which could
greatly simplify predictive testing techniques based upon apoptosis sensitivity.
In order to assess how closely this in vitro analysis of CLL cells response to
chemotherapeutic agents was related to the in vivo response to chemotherapy, and to
analyse how the apoptosis sensitivity of the cells altered during a course of treatment,
a small in vivo study was performed. Cells were taken from two CLL patients prior to,
during and after they had received a 2-week course of chlorambucil. As the treatment
commenced the in vivo level of apoptosis in one of the patients decreased, whilst the
level in the other patient remained stable. More interesting was the effect on the in
vitro spontaneous apoptosis sensitivity of the cells. As the patients’ course of
treatment progressed, the CLL cells became increasingly sensitive to spontaneous
apoptosis, possibly indicating that the treatment was having the desired effect, in that
the cells were being triggered into apoptosis, removal to the in vitro situation serving
only to hasten their eventual death. The fact that the cells were more sensitive to
51
Chapter Three Results 1
spontaneous apoptosis two weeks after the end of the course of treatment than they
were prior to the treatment commencing may have some bearing on treatment
decisions for future patients. However, a much larger group of patients would need to
be analysed using this method before this observation could be confirmed.
Samples of cells taken from the treated patients were also cultured for 24 h in the
presence of 7 pM chlorambucil. Analysis of the chlorambucil sensitivity of these cells
using the Annexin V assay revealed differences between the two patients studied.
Patient 12 I had previously undergone treatment with chlorambucil. The cells from
this patient decreased in their sensitivity to in vitro chlorambucil-induced apoptosis as
the course of therapy progressed. Two weeks post-treatment, the cells had recovered
some sensitivity to chlorambucil-induced apoptosis, but not to the level seen at the
pre-treatment sample point. This could signify the loss of a chlorambucil-sensitive
clone of cells and may indicate some degree of resistance of the remaining clone to
apoptosis induction by chlorambucil. Since the patient had been treated with this drug
in the past, it may be conceivable that some degree of chlorambucil-resistance had
developed. In support of this theory, increases in p-glycoprotein (Perri et al, 1989) and
upregulation of glutathione-s-transferase mRNA expression (Schisselbauer et al,
1990) have been demonstrated in some cases of chlorambucil-resistant CLL.
The cells from patient 8 A behaved in a slightly different manner. For this patient, this
was the first course of treatment with chlorambucil or any other chemotherapy. The
cells from this patient showed a steady increase in sensitivity to apoptosis induction in
vitro by chlorambucil as the course of treatment progressed. Of some interest is the
observation that the post-treatment level of chlorambucil-induced apoptosis in this
patient was higher than the pre-treatment level. This may imply that a significant
number of chlorambucil-sensitive CLL cells remain in circulation following the end
of the course of treatment, a finding which also may have relevance to the way in
which CLL patients are treated therapeutically. Again, a larger group of previously
treated and untreated patients would need to be analysed before this observation could
be confirmed.
52
Chapter Four Results 2
Chapter 4 - Processing/Activation of caspases -3 , -7, and -8 , but not caspase-2 in the induction of apoptosis in B-chronic lymphocytic leukaemia cells
4.1 Introduction
The importance of caspase activation in spontaneous and chemotherapy-induced
apoptosis in leukaemic cells from patients with CLL had not previously been
described. It had been shown that peripheral blood mononuclear cells from CLL
patients were caspase-3 immunopositive (Krajewski et al, 1997) and that
glucocorticoid-induced apoptosis of CLL lymphocytes requires protease activation
and is accompanied by cleavage of PARP and lamin Bi, together with loss of caspase-
3 (Bellosillo et al, 1997; Chandra et al, 1997). The aim of the work described in this
chapter, was to further define the role of caspases in induction of spontaneous and
drug-induced apoptosis in CLL cells.
4.2 Inhibition of spontaneous apoptosis in CLL cells by Z-VAD.fmk
Apoptosis was assessed in CLL cells using Annexin V to measure extemalisation of
phosphatidylserine (figure 4.1). Freshly isolated cells, prior to culture, showed a very
low level of spontaneous apoptosis, as measured by Annexin V (figure 4.1 A). During
culture, the cells underwent spontaneous apoptosis (figure 4.IB), which was increased
in the presence of chemotherapeutic agents, such as chlorambucil (figure 4.1C). A
variable amount of spontaneous apoptosis was observed (Tables 4.1 and 4.2) in
agreement with other studies (Collins et al, 1989). The measurement of
phosphatidylserine exposure allowed a clear quantification of the percentage of
apoptotic cells. In order to assess the role of caspases in the execution phase of
apoptosis in B-CLL cells, Z-VAD.fmk, a cell permeable caspase inhibitor, was used
which inhibits apoptosis in many different model systems (MacFarlane et al, 1997,
Zhu et al, 1995, Feamhead et al, 1995). Z-VAD.fmk (100 jliM ) inhibited spontaneous
apoptosis in all but one of the patient samples examined (Table 4.1) supporting the
involvement of caspases in the spontaneous apoptosis of CLL cells. In order to obtain
more definitive evidence for the involvement of caspases, the processing/activation of
the effector caspases was examined as well as the proteolysis of PARP, which has
been used as a marker of apoptosis ( Kaufmann et al, 1993).
53
Chapter Four Results 2
54.5%
93.6%
CO66 .2%
M2
33.9%
4
M1 -----145% M2 -----1
55% 16A
Annexin V - FITC
10* 1 0 1 102 1 0 J 10*
►
Figure 4.1 Induction of apoptosis in cells from two patients with CLL assessed by
phosphatidylserine extemalisation. A - Freshly isolated CLL cells, from patients 15 A
and 16 A, were examined for phosphatidylserine exposure by binding of Annexin V as
described in Chapter 2, section 2.6. The percentages of cells with low and high
Annexin V binding representing normal (marker 1) and apoptotic (marker 2) cells are
shown. CLL cells were also cultured for 20 h either B. alone to measure spontaneous
apoptosis or C. in the presence of chlorambucil (7.5 pM).
54
Chapter Four Results 2
Patient No % Spontaneous apoptosis
- Z-VAD.fmk + Z-VAD.fmk
2 A 37 18
3A 17 8
4A 14 5
11A 21 11
12A 18 11
19A 7 7
Table 4.1 Inhibition of spontaneous apoptosis by Z-VAD.fmk
Freshly isolated CLL cells from patients were cultured for 24 h at 37°C either alone or
in the presence of Z-VAD.fmk (100 pM) as indicated. Apoptosis was assessed by
measuring extemalisation of phosphatidylserine using Annexin V staining.
55
Chapter Four Results 2
4.3 Activation of caspase-3 and caspase-7 in apoptosis of CLL cells.
The time course of induction of apoptosis was examined in four previously untreated
Binet Stage A cases of B-CLL. In each patient, a time dependent induction of
spontaneous apoptosis was observed (figure 4.2 A). Cells from these patients exposed
to chlorambucil, showed a concentration dependent increase in the induction of
apoptosis compared to control cells (figure 4.2 A). These cells were also analysed by
immunoblotting for activation of caspases -3 and -7. Freshly isolated untreated CLL
cells contained primarily the proform of caspase-3 (figure 4.2 B, lane 1). Processing of
caspase-3 at Asp 175 between the large and small subunits yields a 20 kD subunit,
which is further processed at Asp 9 and Asp 28 to yield 19 kD and 17 kD large
subunits (Fernandez-Alnemri et al, 1996). A time dependent processing of caspase-3
to its catalytically active large subunit(s), 17-20 kD, was observed (figure 4.2 B, lanes
2-5), which was increased in the presence of chlorambucil (figure 4.2 B, lanes 6-9).
Activation of caspase-3 was observed in both spontaneous and drug-induced apoptosis
in all 9 samples examined to date (Table 4.2).
Caspase-7 was also present in freshly isolated CLL cells as a ~35 kD proform without
any detectable large 19 kD subunit (figure 4.2 C, lane 1). Processing of caspase-7
initially occurs at Asp 198 between the large and small subunits, followed by cleavage
at Asp 23 to yield the 19 kD large subunit (MacFarlane et al, 1997). A time dependent
processing of caspase-7 to its 19 kD subunit was observed in spontaneous apoptosis,
which was also increased as a result of exposure to chlorambucil (figure 4.2 C).
Caspase-7 was activated in all 10 samples of spontaneous and drug-induced apoptosis
examined (Table 4.2). Thus induction of apoptosis in CLL cells was accompanied by
the processing of both the effector caspases-3 and -7 to their catalytically active large
subunits.
56
Chapter Four Results 2
(/>o+•>a.o£ 20
0 4 8 12 16 20 24
Time (h)
B Control 7 pM Chir
Time (h) 0N /"
10 20 2 10 20
32 kD ►
LS ►
Control 7 pM Chir "n r - \
Time (h) 0 2 6 10 20 2 6 10 2035 kD
19 kD ►•xO l '
Figure 4.2 Induction o f apoptosis in cells from a representative CLL patient is accompanied by
processing of caspase-3 and caspase-7. (A) Freshly isolated CLL cells were incubated either alone ( ♦ -
♦ ) or in the presence o f chlorambucil (3 pM) ( ■ - ■ ) or (7.5 pM) (A -A ) and apoptosis assessed at the
indicated times by extemalisation o f phosphatidylserine as shown in Fig 4.1. (B) Processing of
caspase-3 in CLL cells undergoing apoptosis. CLL cells from patient 15 A were cultured for the
indicated times either alone (Con) or in the presence of chlorambucil (Chi, 7.5 pM) and examined by
western blot analysis for caspase-3 as described in Chapter 2, section 2.9. The arrows denote either the
32 kD proform o f caspase-3 or the catalytically active large subunits (LS) of approximately 17 - 20 kD.
(C) Processing of caspase-7 in CLL cells undergoing apoptosis. CLL cells from patient 15 A were
cultured for the indicated times either alone (Con) or in the presence of chlorambucil (Chi, 7.5 pM) and
examined by western blot analysis for caspase-7. The arrows denote either the 35 kD proform of
caspase-7 or the large subunit o f 19 kD.
57
Chapter Four Results 2
PatientNo
Sex/Age
Binetstage
Previoustherapy
WBC
x 109/1% Apoptosis
Spon Pd Chi -2Caspase -3 -7 -8
PARP
2A M/74 A - 23.1 36 55 57 + + + ND ND3A M/72 A Chi 49.7 17 19 21 - + + ND ND8A M/72 A Chi 30.2 14 71 36 ND + ND ND ND10A M/91 A - 24.5 7 22 18 - + + ND ND11A F/74 B Chi 14.2 20 24 19 - + + ND ND12A F/67 B - 17.5 18 27 20 - + + + +13A M/62 A - 34.3 13 27 22 - + + ND ND14A F/60 A - 8.0 22 ND 42 ND + + ND +15A M/51 A - 24.6 27 ND 48 ND + + + +16A M/81 A Chi 20.5 42 ND 66 ND ND ND “f* +17A M/71 A - 12.7 7 19 25 - ND ND + ND
18A F/84 B Chi, Pd 22.7 41 58 67 - ND + + ND
19A M/77 C Chi, Pd 7.2 29 42 67 - ND + + ND
20A F/85 A - 47.7 20 44 15 ND ND ND + ND
Table 4.2 . Clinical information and summary of in vitro apoptosis sensitivity and
incidence of caspase activation. Peripheral blood lymphocytes from patients
diagnosed with B-CLL were cultured in vitro for 20 h either alone or in the presence
of prednisolone (Pd) and chlorambucil (Chi). Both spontaneous (spon) and drug-
induced apoptosis were then determined by Annexin V binding. Activation of
caspases -2, -3, -7 and -8 as well as proteolysis of PARP was determined by western
blot analysis. Expression of the pro-forms of the caspases was found in all cases
examined. A *+’ indicates activation of the pro-enzyme to an active subunit was
observed following in vitro culture with and without addition of the drugs, whereas a
indicates no such activation was observed. (ND indicates not determined).
58
Chapter Four Results 2
4.4 Caspase-2 processing does not generally accompany apoptosis of CLL cells.
Several recent studies have shown that caspase-2 (ICH-1, Nedd2) is processed in
some but not all cells during the induction of apoptosis (MacFarlane et al, 1997, Li et
al, 1997, Harvey et al, 1997). The prodomain of caspase-2 binds to the adaptor
molecule RAIDD/CRADD which also binds the receptor-interacting protein RIP and
may thus regulate apoptosis (Duan & Dixit, 1997, Ahmad et al, 1997). It is not known
which if any intracellular substrates are cleaved by caspase-2 in apoptosis. In order to
investigate the possible importance of caspase-2 in apoptosis of CLL cells, an
antibody was used which recognizes both the proform and the small p i2 subunit of
caspase-2. In agreement with previous studies (MacFarlane et al, 1997), induction of
apoptosis in the human monocytic tumor cell line, THP. 1, resulted in the activation of
caspase-2 to its ~ 12 kD small subunit (figure 4.3, lane 1) and was included as a
positive control for the processing of caspase-2. In cells undergoing spontaneous and
drug-induced apoptosis, processing of procaspase-2 was seen in only 1/9 samples
examined to date (Table 4.2). The results from this case are shown in more detail
(figure 4.3). Untreated freshly isolated CLL cells from this patient contained primarily
the ~ 48 kD proform of caspase-2 with no detectable small 12 kD subunit (figure 4.3,
lane 2). These freshly isolated cells also contained an immunologically reactive
protein of ~ 33 kD, which may represent an early processed form of caspase-2 (figure
4.3, lane 2). In cells undergoing spontaneous apoptosis, little or no cleavage of
procaspase-2 to its 12 kD small subunit was observed (figure 4.3, lane 3). Induction of
apoptosis with prednisolone resulted in the processing of procaspase-2 to its 12 kD
subunit (figure 4.3, lane 4), which was more marked with chlorambucil (figure 4.3,
lane 5). Thus although processing of caspase-2 was observed in cells from this patient,
it did not appear to be a general feature accompanying either spontaneous or drug-
induced apoptosis of CLL cells.
59
Chapter Four Results 2
+ ve Oh Con Pd
+ Z - VAD.fink
Chi Con Pd Chi
48 kD^*
33 kD^*
12 kD
Figure 4.3 Activation of caspase-2 only occurred in cells from one patient. Freshly
isolated CLL cells, from patient 2A, were cultured for 20 h either alone (Con) or with
prednisolone (Pd, 200 pM) or chlorambucil (Chi, 15 pM) in the absence or presence
of the caspase inhibitor Z-VAD.fmk (100 pM) as indicated. Freshly isolated cells (0
h) were included as a control. The cells were examined by western blot analysis for
caspase-2 as described in Chapter 2, section 2.9. The arrows indicate the proform of
caspase-2 and its 12 kD small subunit. Of the 9 patients examined, this was the only
one to show activation of procaspase-2. Human monocytic THP.l cells treated with
etoposide (25 pM) for 4 h were included as a positive control (+ve) as caspase-2 is
processed in these cells to its 12 kD small subunit.
60
Chapter Four Results 2
4.5 Activation of the effector caspases result in the cleavage of PARP.
As processing of caspases-3 and -7 was observed, CLL cells were also examined for
the cleavage of PARP, a substrate for both these caspases (Nicholson et al, 1995,
Fernandez-Alnemri, 1995). Induction of spontaneous apoptosis was accompanied by a
time dependent cleavage of PARP to a characteristic 89 kD signature fragment (figure
4.4 - Patient 14A, lanes 2-5; Patient 17A, lanes 1-4). The induction of apoptosis and
the cleavage of PARP were both induced following exposure of the CLL cells to
chlorambucil (figure 4.4 - Patient 14A, lanes 6-9; Patient 16A, lanes 5-12), further
supporting the involvement of caspases in the execution phase of apoptosis in CLL
cells.
Patient 14 A
rTime (h) 0
Control 7 pM Chia r
10 20 2 10 20
116kD >
89 k D >
Patient 16 A
Time (h)
Control 3 pM Chi 7 pM Chi1 r
6 10 20 2> r
10 20 2 10 20
116kD>"
8 9 k D >
Figure 4.4 Cleavage o f PARP accompanies apoptosis in CLL cells. Freshly isolated CLL cells, from patients 14 A and 16 A, were cultured for the indicated times either alone (Con) or with chlorambucil (Chi, 3 pM or 7.5 pM) and then analysed by western blot analysis for intact PARP (116 kD) or its cleaved product (89 kD).
61
Chapter Four Results 2
4.6 Activation of caspase-8 during apoptosis of CLL cells.
Although caspase-8 has been implicated as one of the main “activator” caspases in
receptor-mediated apoptosis (Boldin et al, 1996, Muzio et al, 1996) , its role if any in
drug-induced apoptosis is not clear. In order to examine its possible involvement in
apoptosis of CLL cells, a caspase-8 antibody was utilised. In freshly isolated CLL
cells from 7/7 patients, the antibody recognised two protein(s) of -55 kD (figure 4.6),
most probably corresponding to two different isoforms of caspase-8, MACHal and
MACHa2 (Boldin et al, 1996, Scaffidi et al, 1997). In addition the freshly isolated
cells also contained two immunoreactive proteins of - 43 kD (figure 4.5 A and B, lane
1), which probably resulted from loss of the small 12 kD subunit following cleavage
at Asp 374 (Medema et al, 1997). The cells also contained two immunoreactive
proteins of -28 kD, which may have arisen following removal of the two death
effector domains following cleavage at Asp 216. Culture of the CLL cells resulted in a
time dependent processing of the -55 kD protein(s) to fragments of -43, -28 and a
small amount of an 18 kD fragment (figure 4.5 A, lanes 2-5). Formation of the 18 kD
large subunit most probably occurs following further cleavage of the -43 kD
fragments at Asp 216. Formation of all these fragments was slightly increased
following treatment of CLL cells with chlorambucil (figure 4.5 A, lanes 6-9). The
time course of cleavage of caspase-8 appeared similar to that of caspase-3 and
caspase-7. Some variation in the processing of caspase-8 was noted in the samples
analysed to date. For example in patient 17A, caspase-8 was activated to fragments of
-43 and -28 kD with little or no 18 kD being formed (figure 4.5 B). Thus
interindividual variation was observed in the activation of caspase-8. Further
investigation indicated that this interindividual variation in caspase-8 activation was
apparent in a larger number of cases. Particularly striking in all samples was the
apparently high levels of the proform of caspase-8 and the relatively small amounts of
the proform which were processed during culture.
62
Chapter Four Results 2
Time (h) 0 2
5 5 k D > 4 3 k D >2 8 k D >
1 8 k D >
Con Chi
6 10 20 2 6 10 20
B + Z-VAD.fmk ( ̂
0 h Con Chi Pd Con Chi Pd
5 5 k D >4 3 k D >
2 8 k D > _______ ________
III1 8 k D >
+ ve
Figure 4.5 Processing of caspase-8 in CLL cells is inhibited by Z-VAD.fmk. (A)
CLL cells from patient 15 A were cultured for the indicated times either alone (Con)
or in the presence of chlorambucil (Chi, 7.5 pM). (B) CLL cells from patient 17 A
were cultured for 20 h either alone (Con) or in the presence of prednisolone (Pd, 20
pM) or chlorambucil (Chi, 7.5 pM) in the absence or presence of Z-VAD.fmk (100
pM) as indicated. Cells from both patients were analyzed for activation of caspase-8
by immunoblotting. Freshly isolated cells (0 h) were included as a control. In both
cases THP.l cells induced to undergo apoptosis by exposure to staurosporine (0.5
pM) were included as a positive control (+ ve) for the processing of caspase-8.
63
Chapter Four Results 2
4.7 Z-VAD.fmk inhibits the processing of caspases in CLL cells.
In order to determine at what stage of the apoptotic process, Z-VAD.fmk was
inhibiting apoptosis (Table 4.1), its ability to inhibit the processing of different
caspases was examined. Freshly isolated CLL cells from patient 2A contained
primarily the proform of caspase-3 (figure 4.6, lane 2). Spontaneous apoptosis was
accompanied by the processing of caspase-3 to its catalytically active large subunit(s)
(figure 4.6, lane 3), which was increased following treatment with both prednisolone
and chlorambucil (figure 4.6, lanes 4 and 5 respectively), commensurate with their
ability to induce apoptosis. In both spontaneous and drug-induced apoptosis, Z-
VAD.fmk almost completely inhibited the processing of procaspase-3 to its
catalytically active large subunits (figure 4.6, lanes 6-8). Z-VAD.fmk also inhibited
the drug-induced processing of caspase-2 observed in patient 2A (figure 4.3, lanes 7
and 8). In patient 17A, Z-VAD.fmk inhibited the activation/processing of caspase-8 to
a p43 fragment (figure 4.5 B, lanes 5-7). Thus Z-VAD.fmk acts to block drug-induced
apoptosis of CLL cells by blocking the activation/processing of caspases.
64
Chapter Four Results 2
+ Z - V A D .fm k „ A __________ .r ^
+ ve Oh Con Pd Chi Con Pd Chi
w (mm ■ •|£ 1 1 ^ ^ ‘m m m m m m m m m - -.-■■■ ■■■
LS
Figure 4.6. Processing of caspase-3 in CLL cells is inhibited by the caspase inhibitor,
Z-VAD.fmk. CLL cells from patient 8 I were cultured for 20 h either alone (Con) or in
the presence of prednisolone (Pd, 200 pM) or chlorambucil (Chi, 15 pM) either in the
absence or presence of Z-VAD.fmk (100 pM) as indicated. Freshly isolated cells (0 h)
and THP.l cells treated with etoposide to induce apoptosis were included as controls.
The cells were analyzed for activation of caspase-3 by immunoblotting. The arrows
denote either the 32 kD proform of caspase-3 or the catalytically active large subunits
(LS) of approximately 17-20 kD.
65
Chapter Four Results 2
4.8 Discussion
In this study it has been demonstrated that induction of apoptosis of CLL cells leads to
the selective induction of some but not all caspases. The processing/activation of at
least three caspases (caspases-3, -7 and -8) has been shown during the execution phase
of apoptosis of CLL lymphocytes whereas caspase-2 does not generally appear to be
activated. Recent studies have implicated a role for the release of mitochondrial
cytochrome c in the activation of procaspase-3 provided Apaf-3, now identified as
caspase-9, and dATP are present (Zou et al, 1997; Li et al, 1997). In all the patients
examined, activation of the effector caspases, -3 and -7, were observed (Table 4.2 and
figures 4.2 and 4.6) suggesting that these caspases may be responsible for the cleavage
of PARP found in CLL cells in this and other studies (Bellosillo et al, 1997; Chandra
et al, 1997). Further support for this hypothesis was provided by the observations that
Z-VAD.fmk, a cell permeable caspase inhibitor, inhibited the activation/processing of
all the caspases studied, as well as the cleavage of PARP and both spontaneous and
drug-induced apoptosis of CLL cells (Table 4.1 and figures 4.2, 4.3, 4.5 and 4.6).
Thus these effector caspases also play a central role in the execution phase of
apoptosis in CLL cells as they do in other cell systems (Cohen, 1997; Nicholson &
Thomberry, 1997).
In 8/9 patients, caspase-2 was not activated (Table 4.2). In only one case was
activation observed (figure 4.3). At the time of sampling this patient was Binet stage
A, but in the last year has progressed to stage C. Activation of caspase-2 during the
execution phase of apoptosis has been observed in different tumor cell lines
(MacFarlane et al, 1997; Li et al, 1997; Harvey et al, 1997). The precise mechanism
by which procaspase-2 is activated in cells is not known. It may involve recruitment
through an adapter such as RAIDD/CRADD (Duan & Dixit, 1997; Ahmad et al,
1997) or based on activities of recombinant enzymes, it has been proposed that
caspase-3 activates pro-caspase-2 (Li et al, 1997; Harvey et al, 1996). No activation of
procaspase-2 was observed even in the presence of activated caspase-3 and caspase-7
demonstrating that neither caspase-3 nor -7 activates procaspase-2 in CLL cells. The
data also demonstrates that activation of caspase-2 is not required for the induction of
apoptosis in CLL cells.
66
Chapter Four Results 2
The results on the activation/processing of caspase-8 are particularly intriguing.
Triggering of the Fas/CD95 receptor leads to the recruitment and activation of
caspase-8, which may then act as the apical “initiator” caspase responsible for the
activation of other caspases and the execution of apoptosis (Boldin et al, 1996; Muzio
et al, 1996). Whilst caspase-8 is activated early in Fas/CD95-induced apoptosis, little
is known as to its role if any in drug-induced apoptosis. Of particular interest were the
high levels of procaspase-8 present in 7/7 patients (Table 4.2). Procaspase-8 was
present as two main forms of —55 and 53 kD, which correspond to caspase-8/a
(MACHal) and caspase-8/b (MACHa2) (Scaffidi et al, 1997). At the end of the
culture, despite the induction of significant apoptosis (25 - 65 %), most of the caspase-
8 was still present as the proform (figure 4.5), in contrast to Fas/CD95-induced
apoptosis when all the caspase-8 is rapidly processed (Scaffidi et al, 1997). In addition
in 2/4 cases, caspase-8 was activated concurrently with caspases -3 and -7 after 6 to 10
h of in vitro culture. These results would seem to preclude an initiator role for
caspase-8 in drug-induced apoptosis of CLL cells, and suggest that it may be activated
non-specifically by other caspases.
Some cytotoxic agents, such as doxorubicin, induce apoptosis in human leukaemic
cells lines and neuroblastoma cells via the Fas/CD95 receptor/ligand system, a
hypothesis supported by the finding that these cells also display cross-resistance
between the cytotoxic agent and Fas/CD95-induced apoptosis (Freisen et al, 1996;
Fulda et al, 1997; Freisen et al, 1997). However, other studies have suggested that
chemotherapy-induced apoptosis is not dependent on Fas/CD95 receptor/ligand
interaction (Eischen et al, 1997; Gamen et al, 1997). While the reasons for these
discrepancies are not clear, they may be related to the rate of induction of apoptosis.
For example, in those systems where a positive relationship between chemotherapy
and involvement of the Fas/CD95 receptor/ligand system has been implicated (Freisen
et al, 1996; Fulda et al, 1997; Freisen et al, 1997), apoptosis is induced over a long
period of time (24 - 48 h) so allowing the synthesis of new proteins. The possible
involvement of the Fas/CD95 receptor/ligand system in CLL cells remains to be
determined.
67
Chapter Four Results 2
Interestingly CLL cells have been reported to have undetectable or very low levels of
CD95 expression (Mapara et al, 1993; Moller et al, 1993; Wang et al, 1997). This
expression can be increased following in vitro activation with interleukin-2,
Staphlyococcus aureus 1 or CD40 although in some cases this may lead to
proliferation (Marpara et al, 1993; Wang et al, 1997). Thus CLL cells express high
levels of caspase-8 in conjunction with very low levels of CD95 receptor. These
results highlight the possibility of developing new therapies for CLL based on the
upregulation of CD95- or other death receptors, which would synergize with the high
levels of caspase-8 in CLL cells. Freshly isolated B-CLL cells possess caspase-2, -3, -
7 and -8. Some of these caspases can be activated to cleave protein substrates such as
PARP. These results demonstrate that B-CLL cells possess the complete apoptotic
machinery required to execute the apoptotic programme. Thus the dysregulation of
apoptosis in CLL cells does not appear to be due either to a deletion of pro-caspases or
to point mutations leading to their inactivation. Rather, this study suggests that the
molecular basis of dysregulated apoptosis in vivo may reside in the signalling leading
to the activation of caspases, or the presence of inhibitory proteins at the apoptosis
induction stage.
68
Chapter 5 Results 3
Chapter 5 - Studies on survival factors and the Fas signalling
pathway in B-CLL
5.1 Introduction
Spontaneous apoptosis occurs when B-CLL cells are removed from their normal
environment and placed into in vitro culture (Collins et al, 1989 and section 3.2 of
this thesis). This implies that one or more survival factors needed by the B-CLL cells
are not being provided by the in vitro culture conditions. All previous analysis in this
study had been performed on populations of mixed lymphocytes, both B and T cells.
The T cell fraction can be assumed to be small due to the high proportion of tumour B
cells, but still the presence of T lymphocytes may be reducing the accuracy of the
studies. For experiments investigating the effect of survival factors in chronic
lymphocytic leukaemia it was considered important to be studying a purer population
of what will now be referred to as B-CLL cells. Accordingly, a T cell depletion step
was incorporated into the lymphocyte isolation procedure. Further work in this chapter
describes an investigation into the finding that B-CLL cells have large amounts of
caspase-8 which is not cleaved to any great extent during induction of apoptosis (see
Chapter 4, section 4.5). Since caspase-8 is known to play a major role in the Fas
signalling pathway, the response of B-CLL cells to stimulation of this pathway was
investigated.
5.2 Purified CLL B lymphocytes are more sensitive to apoptosis in in vitro
culture than unpurified populations of total CLL lymphocytes
To further increase specificity when analysing apoptosis sensitivity of CLL cells, it
was decided to purify the B lymphocyte fraction (B-CLL) from the total lymphocytes
obtained from the patients. By incubating a proportion of the isolated lymphocytes
with CD3+ dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s
instructions, depletion of T cells was performed. A determination of the purity of the
pre- and post-depletion fractions was obtained flow cytometrically using antibodies to
CD3 and CD 19 (see Chapter 2, section 2.3.2) (Figure 5.1 A).
69
Chapter 5 Results 3
Blood samples were obtained from six patients and the lymphocyte fraction was
isolated as described in chapter 2. A proportion of these cells were cultured for 24 h
alone or in the presence of chlorambucil (7 pM) or staurosporine (0.2 pM). The
remaining cells from each isolation were further purified by T cell depletion prior to
being cultured for 24 h alone or in the presence of chlorambucil (7 pM) or
staurosporine (0.2 pM). After the 24 h culture period, cells from all of the cultures
were labelled using the Annexin V technique and analysed flow cytometrically. In all
six cases, the level of spontaneous apoptosis in the purified B cell cultures was
slightly increased over that in the total lymphocyte cultures (Figure 5.1 B), which does
indicate that the B-CLL cells may be losing one or more survival stimuli as a result of
the T cell depletion step.
70
Chapter 5 Results 3
WPhIcnQCJ
Total lymphocyte populationo FL1 -Height (3) v s FL2-Height (4)
11.6% -T cell
• - 8 .
. • •83.4%B cells
™l---..........10° 101
Post-T cell depletion
10"
1.3% -T cell
*92%B cells
10° W io2 10J
CD19-FITC
B70
50
WB 602 CLoQ.<w 40 o£ 30 2 § 20
£ 10
\ J
15 A 21A 23A 24A 25APatient number
28A
m B cells ■ Total cells
Figure 5.1 (A) Use of CD3+ magnetic beads enriches for CD 19+ B cells in cultures
of CLL lymphocytes. (B) Purified B cells are more sensitive to spontaneous apoptosis
than cultures of mixed lymphocytes. Lymphocytes were isolated from CLL blood
samples and were split into duplicate cultures. T cells were depleted from one fraction
using CD3+ magnetic beads. Both fractions of cells were cultured for 24 h prior to
being labelled using the Annexin V technique to analyse the level of apoptosis in the
samples.
71
Chapter 5 Results 3
5.3 Culture of B-CLL cells with Interleukin-4 and CD40 stimulation results in a
reduction in spontaneous apoptosis
In order to investigate the requirement of B-CLL cells for stimulation by survival
factors, two B cell growth factors, interleukin-4 and CD40, were incorporated into the
study. Both of these factors had previously been identified as B cell growth stimulants
in normal and malignant cells (Nakanishi et al, 1996; Crawford et al, 1993).
Interleukin-4 has been reported to have anti-apoptotic effects when applied to in vitro
cultures of B cells (Panayiotidis et al, 1993) and acts as an apoptosis inhibitory agent
by maintaining the Bcl-2 expression level in B-CLL cells (Danescu et al, 1992). CD40
stimulation promotes survival by activation of NFkB (Rothe et al, 1995) and protein
tyrosine phosphorylation (Laytragoon-Lewin et al, 1998).
Freshly isolated, purified B-CLL cells from 10 patients were cultured alone or in the
presence of recombinant human interleukin-4 (10 ng/ml). In all instances, the addition
of interleukin-4 reduced the level of spontaneous apoptosis after 24 h of culture
(figure 5.2 A), and increased viability of IL-4-treated cells was maintained to 48 h
(figure 5.2 B). The effect of interleukin-4 on inhibition of spontaneous apoptosis
varied between patient samples, but a reduction in spontaneous apoptosis level was
seen in all of the samples. Addition of a monoclonal antibody directed against CD40
also reduced the level of spontaneous apoptosis in the majority of cases, however, in
the cells from patient 27A, addition of anti-CD40 monoclonal antibody actually
increased the level of spontaneous apoptosis (figure 5.3). Overall, the level of
protection afforded by CD40 stimulation against spontaneous apoptosis was less
dramatic than the effect provided by interleukin-4 stimulation. In order to investigate
the combined effect of these two survival stimuli on B-CLL cell survival, samples of
cells from eight patients were cultured with a combination of interleukin-4 (10 ng/ml)
and anti-CD40 monoclonal antibody (1.5 pg/ml). The level of apoptosis in the
cultures was assessed after 24 h using the Annexin V assay. In cases 23A, 27A, 29A
and 31 A, anti-CD40 increased the protection provided by interleukin-4, in the
remaining 4 cases, anti-CD40 abrogated the protective effect of interleukin-4 (figure
5.3). This trend may indicate a variable dependency on survival factors between
patients.
72
Chapter 5 Results 3
A
15A 23A 27A 28A 29A 30A 31A 32A
Patient Number
a Control B 10ng/ml IL4
B
100
= 60 B .2> /in
20
0 4 ------Oh 24 h 48 h
Time (h)
ControlIL4
Figure 5.2 Interleukin-4 inhibits the induction of spontaneous apoptosis in purified B-
CLL cell cultures. (A) Freshly purified cells were cultured for 24 h alone (control) or
in the presence of 10 ng/ml IL-4. Apoptosis was assessed using the Annexin V assay
to measure externalised phosphatidylserine. (B) B-CLL cells were cultured alone or
in the presence of IL-4 (10 ng/ml) for 48 h. Samples (1 x 106 cells) were taken from
each culture at 24 and 48 h following culture setup, viability was assessed by
propidium iodide exclusion. Results plotted are the mean of six experiments. Standard
error bars are also depicted.
73
Chapter 5 Results 3
70
60
50_co
23A 24A 27A 28A 29A 30A 31A 32A
Patient Number
□ Control ■ IL4H anti-CD40 m anti-CD40 + IL4
Figure 5.3 The effect of stimulation of B-CLL cells with interleukin-4 and antibodies
to CD40 is case dependent. B-CLL cells were cultured for 24 h alone or in the
presence of IL-4 (10 ng/ml), anti-CD40 monoclonal antibody (1.5 pg/ml) or both
stimuli. Following the culture period, 1 x 106 cells from each culture were analysed
for apoptosis level using the Annexin V assay.
74
Chapter 5 Results 3
5.3 Culture of B-CLL cells with Interleukin-4 or CD40 stimulation increases
their resistance to chemotherapeutic drugs
In order to determine the level of protection afforded by interleukin-4 against
apoptosis induced by chemotherapeutic drugs, B-CLL cells from patients 28A and 29
A were pre-incubated for 24 h alone (control) or in the presence of interleukin-4 (10
ng/ml). After 24 h pre-incubation, chlorambucil (7 pM) or staurosporine (0.2 pM)
were added and the cells were cultured for a further 24 h. The protective effect of
interleukin-4 against drug-induced apoptosis was then analysed by assessing the
extent of apoptosis in these cultures compared to apoptosis levels in cultures of cells
incubated for 24 h with chlorambucil or staurosporine alone. In both cases the
addition of interleukin-4 to the cells reduced the level of spontaneous apoptosis. The
level of apoptosis induced by chlorambucil and staurosporine was also reduced in the
cells pre-incubated with interleukin-4, compared to the levels in cultures after 24 h
incubation with the drugs alone. In cells from patient 28 A, the protection from drug-
induced apoptosis was no more than 7%. However, in cells from patient 29A, the
level of apoptosis in the cultures pre-incubated with interleukin-4 were reduced by as
much as 22% (figure 5.4). This finding, although concerned with a limited sample of
B-CLL patients, again indicates that B-CLL cells from different patients vary in their
level of dependence on survival factors, and also demonstrates the important role that
interleukin-4 may play in maintaining survival of the malignant lymphocyte clone and
protecting the cells from chemotherapeutic drug-induced apoptosis in vivo.
In order to determine the protection from drug-induced apoptosis afforded by a
combination of CD40 and interleukin-4 stimulation, B-CLL cells from three patients
were cultured for 24 h in the presence of anti-CD40 mAb (1.5 pg/ml) and IL-4 (10
ng/ml). Following this pre-incubation period, chlorambucil (7 pM) was added to the
cultures. The cells were incubated for a further 24 h, prior to assessment of apoptosis
levels in the cultures by Annexin V labelling. Control cultures were also analysed,
where the cells had been incubated for the full 48 h in the presence of IL-4 and anti-
CD40, without the addition of chlorambucil.
75
Chapter 5 Results 3
Spontaneous apoptosis was inhibited in all the three patient samples analysed, and
chlorambucil-induced apoptosis was also inhibited in the samples which had been pre-
incubated with IL-4 and anti-CD40 (Figure 5.5 A). The average protective effect of
IL-4 + anti-CD40 against spontaneous apoptosis was 21.2%, and the average
protection against chlorambucil-induced apoptosis was 26.8% (Figure 5.5 B).
However, there was wide variation between the patient samples as to the effectiveness
of IL-4 and CD40 stimulation. Cells from patients 21A and 27A were highly receptive
to the protective effects of IL-4 and CD40 against chlorambucil-induced apoptosis,
whereas the cells from patient 24A were not protected to such a great extent (Figure
5.5 A). The extent of protection against chlorambucil-induced apoptosis afforded by
the two stimuli was not always to the same degree as the protective effect against
spontaneous apoptosis. Cells from patient 21A were protected to a similar degree
from spontaneous and chlorambucil-induced apoptosis, cells from patient 24A were
protected to a greater degree from spontaneous apoptosis than from chlorambucil-
induced apoptosis, and, interestingly, cells from patient 27A were protected to a
greater extent against chlorambucil-induced apoptosis than against spontaneous
apoptosis (figure 5.5 A). This again underlines the wide variability in survival factor
dependency between individual B-CLL cases, a dependency which may also have
implications in determining the response of different patients to chemotherapy.
76
Chapter 5 Results 3
A
Patient No. Control IL-4 + CD40 Chi IL-4 + CD40
+ Chi
21 A 39 42 57.9 16.4
24 A 62 43.5 84.7 79.8
27 A 14.1 3.4 72.7 38.7
B
90
Control CD40+IL4 CHL CD40+IL4+CHL
Figure 5.5 Stimulation of B-CLL cells with anti-CD40 and interleukin-4 protects against apoptosis
induced by chlorambucil. B-CLL cells from three patients were cultured for 24 h alone (Control), or in
the presence of chlorambucil (7 pM) (CHL), prior to assessment of apoptosis using the Annexin V
assay. Identical cultures of cells from the same patients were pre-incubated for 24 h in the presence of
anti-CD40 monoclonal antibody (1.5 pg/ml) and interleukin-4 (10 ng/ml) (CD40 + IL-4) prior to
chlorambucil (7 pM) being added (CD40 + IL-4 + CHL) and the cells being cultured for a further 24 h.
A. Table of results obtained from each patient analysed in this way. All numerical values are percentage
apoptosis. B. Plotted mean results from the three patients. Standard error bars are also depicted.
77
Chapter 5 Results 3
5.4 B-CLL cells are not sensitive to Fas-induced apoptosis
High levels of the initator caspase, caspase-8 had previously been observed in B-CLL
cells (see chapter 4, section 4.5). Caspase-8 cleavage in response to induction of
apoptosis by chlorambucil was also observed (figure 4.5 A, lanes 7-9 and 4.5 B, lane
4). The role of caspase-8 activation in drug-induced apoptosis is unclear, but in
proportion to the amount of caspase-8 pro-form present in these cells, there appeared
to be little activation taking place as a result of treating the cells with
chemotherapeutic drugs (figure 4.5). Since caspase-8 is known to play a major role in
the Fas signalling pathway, the response of B-CLL cells to stimulation of this pathway
using an anti-Fas (IgM) monoclonal antibody was investigated.
B-CLL cells isolated from eight patients were cultured alone or in the presence of
anti-Fas monoclonal antibody (clone CH11, 0.5 pg/ml) for 24 h. Following the
incubation period, 1 x 106 cells from each culture were labelled using the Annexin V
assay, and analysed flow cytometrically (figure 5.6). Fas stimulation induced
apoptosis above the level of spontaneous apoptosis in only two instances (patients
22A and 29A). In the other six cases addition of anti-Fas monoclonal antibody
inhibited the induction of spontaneous apoptosis. This finding mirrors that of
Laytragoon-Lewin and co-workers (1998) who also determined that B-CLL cells were
generally not sensitive to Fas stimulation. Further studies were initiated in order to
determine the nature of Fas resistance in B-CLL cells.
78
Chapter 5 Results 3
</)8Q.O*
22A 23A 24A 25A 26A 27A 28A 29A
Patient Number
■ Control 24 h■ Fas 24 h
Figure 5.6 B-CLL cells are resistant to apoptosis induced by anti-Fas monoclonal
antibody. B-CLL cells were cultured for 24 h alone or in the presence of anti-Fas mAb
(0.5 pg/ml). Analysis of apoptosis levels in the cultures was made using the Annexin
V assay.
79
Chapter 5 Results 3
5.5 Upregulation of Fas receptor on B-CLL cells does not increase sensitivity to
apoptosis induced by Fas ligation.
Previous reports have confirmed the low density of expression of the Fas receptor on
B-CLL cells (Mainou-Fowler et al, 1995). Freshly isolated B-CLL cells were labelled
with anti-Fas monoclonal antibody and a FITC-conjugated secondary antibody, as
described in Chapter 2, and analysed flow cytometrically for the level of expression of
Fas receptor. Previous reports had demonstrated that Fas receptor levels on B-CLL
cells could be upregulated by culturing the cells with either CD40 or interleukin-2 and
Staphylococcus aureus Cowan I (Wang et al, 1997). To induce upregulated expression
of Fas receptor, B-CLL cells were cultured for 24 h in the presence of anti-CD40
monoclonal antibody (1.5 pg/ml) alone or in combination with interleukin-4 (10
ng/ml). Since culturing B-CLL cells for 24 h (with or without CD40 stimulation)
results in induction of apoptosis (see figure 5.3), interleukin-4 was added to the
cultures in order to inhibit apoptosis to a level from which analysis of Fas-induced
apoptosis could be made following on from the initial 24 h culture to upregulate Fas
receptor expression. The level of Fas receptor on the freshly isolated cells was
compared with the level of Fas receptor on the CD40 +/- IL-4 -stimulated CLL cells
(figure 5.7 A). For all five patient samples, the level of expression of Fas receptor
increased following stimulation of the cells with CD40, compared to the level of Fas
receptor on the freshly isolated cells (figure 5.7 B). The addition of interleukin-4 did
not appear to alter the effectiveness of CD40-induced Fas receptor upregulation
(figure 5.7 B).
80
Chapter 5 Results 3
A.
Control IgM (MFI = 2.3)
/ Freshly isolated B-CLL cells (MFI = 8.2)
B-CLL cells + anti-CD40 mAb (24 h) ^(M FI = 27.2)
Log green fluorescence (FITC)
B.
21A 23A 24A 27A 29A 30A 31A 32A Patient Number
■ Fresh cells @ CD40 24h■ CD40 + IL4 24h
Figure 5.7 Fas receptor expression is elevated on B-CLL cells following stimulation with antibodies to
CD40 and/or interleukin-4. A. B-CLL cells (from patient 30A) show increased Fas receptor expression
following 24h incubation with anti-CD40 mAb (0.5 fig/ml) when compared with the level of expression
on the freshly isolated cells. B. Culture of B-CLL cells with anti-CD40 alone or in combination with
interleukin-4 results in upregulation of Fas receptor expression. Freshly isolated B-CLL cells were
labelled with anti-Fas monoclonal antibody (as described in Chapter 2, section 2.11) and analysed flow
cytometrically for the amount of expression of the cell surface receptor, Fas. Subsequently, cells from
the same patients were cultured for 24 h in the presence of anti-CD40 monoclonal antibody alone or
with interleukin-4 (10 ng/ml) added, before analysis of Fas receptor expression was made again.
81
Chapter 5 Results 3
To determine if the increased expression of Fas receptor on the B-CLL cells would
confer increased sensitivity to Fas-induced apoptosis, B-CLL cells from three patients
which had been cultured with anti-CD40 (1.5 pg/ml) and IL-4 (10 ng/ml) for a period
of 24 h to enable upregulation of Fas receptor expression as described above, were
exposed to an anti-Fas monoclonal antibody (clone CH-11, 0.5 pg/ml). Following a
further 24 h culture period, the level of apoptosis in the cultures was analysed using
the Annexin V assay. From the three cases analysed using this method, the level of
apoptosis induced by anti-Fas was not increased over that in the control (anti-CD40 +
IL-4 alone) cultures (figure 5.8). To check that the addition of interleukin-4 to the
cultures was not inducing Fas resistance, cells from patient 27A were cultured for 24 h
with anti-CD40 alone prior to addition of anti-Fas and a further 24 h culture period. At
48 h, the level of apoptosis in the CD40 alone culture was 59.7%, and the level of
apoptosis in the culture pre-incubated with anti-CD40 and with anti-Fas added for the
second 24 h, was 57.1%. This analysis demonstrated that CD40 stimulation alone,
whilst effectively upregulating Fas receptor, was not conferring Fas sensitivity on the
B-CLL cells. It also demonstrated that the addition of interleukin-4 to the cultures for
the first 24 h was not contributing to this resistance to Fas-induced apoptosis.
82
Chapter 5 Results 3
91©ao
70Patient 21 A60
50
40
30
20
10
00 12 24 36 48
Time (h)
Patient 27 A60
•23 5 0912 40 ag . 30
20
10
00 12 3624 48
Time (h)
70 T60 .. Patient 29 A
Time (h)
Control
CD95
A — CD40 + IL4
CD40+ IL4 + Fas
Figure 5.8 Upregulation of Fas receptor on B-CLL cells does not increase their
sensitivity to apoptosis induced by Fas stimulation. B-CLL cells from three patients
were cultured for 24 h in the presence of anti-CD40 monoclonal antibody (1.5 fig/ml)
and IL-4 (10 ng/ml). Subsequently, anti-Fas antibody was added (CH11, 0.5 pg/ml).
The cells were cultured for a further 24 h, before the extent of phosphatidylserine
extemalisation in the cultures was assessed using the Annexin V assay. Results were
compared against samples of cells stimulated for the 48 h period with anti-CD40 + IL-
4 alone, control cells which had received no stimulation, and cells which had received
stimulation from anti-Fas antibody for 48 h.
83
Chapter 5 Results 3
80 n70 -
co 60 ■ ® 50
O 40 -Q_< 30 -
20 -
120 24
Time (h)
• — Control ■•—Fas tIc—CD40 ■ m - CD40 + Fas
Figure 5.9 Stimulation of B-CLL cells with CD40 does not confer sensitivity to Fas-
induced apoptosis. Cells from patient 27A were cultured for 24 h in the presence of
anti-CD40 monoclonal antibody (1.5 pg/ml). Fas receptor upregulation was checked
flow cytometrically (see figure 5.7B), anti-Fas monoclonal antibody (clone CHI 1, 0.5
pg/ml) was added and the cells were cultured for a further 24 h (CD40 + Fas).
Samples of cells from the same patient were cultured for 48 h alone (Control), with
Fas stimulation alone (Fas) or with CD40 stimulation alone (CD40). The apoptosis
level at 48 h in each of the cultures was assessed using the Annexin V assay to
measure externalised phosphatidylserine.
84
Chapter 5 Results 3
5.7 B-CLL cells do not overexpress the caspase-8 inhibitory protein, c-FLIP
Several proteins have been identified which can inhibit Fas-induced apoptosis by
blocking the interaction of caspase-8 with the death effector domain (DED) of the
adapter molecule FADD. One such protein is c-FLIP. c-FLIP is present in mammalian
cells as two isoforms, c-FL IP l (-55 kD) and c-FLIPs (—33 kD) (Irmler et al, 1997;
Rasper et al, 1998). High levels of c-FLIP have been detected in some melanoma
tumours, and c-FLIP is predominantly expressed in lymphoid and muscle tissue
(Irmler et al, 1997), indicating that this protein may play a role in regulating apoptosis
of lymphoid malignancies.
The following experiments were performed in order to determine the relative
expression of c-FLIP in B-CLL cells, compared to the expression levels of caspase-8,
and to examine any alterations in expression as the cells were exposed to
chemotherapeutic drugs and Fas stimulation. B-CLL cells from patients 21 A, 31A and
32A which had been cultured alone or in the presence of chlorambucil (7 pM),
interleukin-4 (10 ng/ml) or anti-CD40 (1.5 pg/ml) were labelled using the Annexin V
method, and analysed flow cytometrically to record the level of apoptosis in the
cultures (figure 5.9 A). Samples of cells from these cultures were examined using
immunoblotting for the presence of the caspase-8 inhibitory protein c-FLIP (figure 5.9
B). Blots were subsequently stripped, and re-probed with anti-caspase-8 polyclonal
antibody (figure 5.9 C), allowing a comparison of the relative levels of c-FLIP and
caspase-8 to be made. The immunoblot results demonstrate that B-CLL cells do
express c-FLIP and caspase-8, but the level of expression of c-FLIP does not appear to
be increased over that of caspase-8. This implies that c-FLIP overexpression is not
responsible for the apoptotic block in Fas-induced apoptosis in B-CLL cells. Upon
examination of the immunoblot results, it was noted that in one case (21 A)
chlorambucil had induced clipping of the short form of c-FLIP (figure 5.9 B, lane 3),
which occured concurrently with activation of caspase-8 (figure 5.9 C, lane 3). The
positive control lane for this experiment was THP-1 monocytic cells treated for 4 h
with anti-Fas monoclonal antibody. In the positive control lane, c-FLIP is also
cleaved. This phenomenon may represent a caspase dependent cleavage of c-FLIP.
85
APatient 21A Patient 31A Patient 32A
t
55 k D -^ '« * 4 3 k D -> »
28 k D -> *
18kD->- —
Figure 5.7 B-CLL cells do not overexpress the caspase-8 inhibitory protein c-FLIP. Freshly isolated B-CLL cells (1) from patients 21 A, 31A and 32 A were cultured in vitro alone (2) or in the presence of chlorambucil (7 J»M) (3), interleukin-4 (10 ng/ml) (4) or anti-CD40 monoclonal antibody (1.5 jpg/ml) (5). (A) The level of apoptosis in the cultures was assessed after 24 h using the Annexin V assay. (B) Cells from the cultures were analysed by immunoblotting for the presence of c-FLIP. (C) The blots were stripped and re-probed with an antibody against caspase-8.
Chapter 5 Results 3
5.6 CLL cells have all the necessary components to form a death inducing
signalling complex (DISC) upon Fas ligation but do not assemble a DISC upon
Fas stimulation
The formation of a death-inducing signalling complex (DISC) in response to Fas
ligation on Jurkat cells was reported in 1997 (Medema et al). The DISC consists of
the intracellular ‘death domains’ (DD) of the trimerised Fas receptors, the adapter
molecule FADD, which binds to the Fas receptor DD through its own death domain.
FADD also consists of a region called a ‘death effector domain’ (DED) which recruits
caspase-8 to the DISC. Cells which respond to Fas stimulation by assembling a DISC
can be split into two categories. Type I cells assemble the DISC in a matter of
seconds, whilst type II cells take 15-30 minutes to form a DISC and activate
downstream caspases such as caspase-3 (Scaffidi et al, 1998). In order to investigate
the possible reasons for the resistance of B-CLL cells to Fas-mediated apoptosis,
immunoblotting for FADD was performed on freshly isolated and cultured B-CLL
cells (figure 5.10), the presence of caspase-8 already having been confirmed in these
cells (figures 4.5 and 5.9). The expression level of FADD appeared to increase
slightly after the culture period of 24 h, but no significant alterations in expression of
FADD were seen as a result of culturing the cells with chlorambucil, anti-Fas or anti-
CD40 monoclonal antibodies or interleukin-4 (figure 5.10). In the cells from patient
22 A, there is evidence of a slight decrease in FADD expression following treatment
of the cells with staurosporine (figure 5.10 A, lane 4), which corresponded with
induction of a high level of apoptosis and activation of caspase-8 (figure 5.10 B, lane
4). In samples from patient 22 A, FADD appeared as a single band at approximately
26 kD. In all other patient samples, FADD appeared as a doublet band (Figure 5.10 C
and D), which may correspond to different FADD isoforms, or serine phosphorylation
states (Xerri et al, 1999).
SKW6.4 (murine B cells, Type I) and Jurkat (human T cells, Type II) cell lines were
used as positive controls for DISC formation and isolation. Using the method of Peter
and co-workers (1998) as described in chapter 2, section 2.12, SKW6.4 and Jurkat
cells were stimulated with anti-Fas monoclonal antibody (anti-Apo-1, IgG) for 15
minutes.
87
Chapter 5 Results 3
24 h B ? 4 h
f ^ Oh Con Chi STS FasO h C o n C hi STS Fas
55/53 * ■ * mm mmF A D D > — — - A - — 4 3 k D > mm~ 26 kD 28 kD >■
18 kD >
7.3 13.9 11.8 67.3 27.4 % A po
24 hThp-1+ STS
O h rCon C hi IL 4 C D 40 |
FADD > 9 ~ 26 kD
DPatient Number
Single or double band corresponding to FADD
15 A21 A22 A28 A29 A31 A32 A
DoubleDoubleSingle
DoubleDoubleDoubleDouble
Figure 5.10 B-CLL cells express the adapter protein, FADD. (A) Freshly isolated B-CLL cells from
patient 22 A, and those cultured for 24 h alone (Con) or with chlorambucil (Chi, 7 pM), staurosporine
(STS, 0.2 pM), or anti-Fas monoclonal antibody (Fas, 0.5 pg/ml) as indicated, were run on a 4-12%
gradient polyacrylamide gel, blotted onto nitrocellulose filter and probed with a monoclonal antibody
directed against the DISC adapter molecule, FADD. In patient 22 A, FADD appears as a single band at
approximately 26 kD. (B) Samples of cells from the same patient were run on a gel, blotted and probed
with a polyclonal antibody to caspase-8, as described previously. The percentage apoptosis level in the
cultures is indicated below the blot. (C) Cells from patient 32 A, were cultured alone (Con), or with
chlorambucil (Chi, 7 pM), interleukin-4 (IL4, 10 ng/ml) or anti-CD40 monoclonal antibody (CD40, 1.5
pg/ml), prior to being run on a 12 % polyacrylamide gel, blotted onto nitrocellulose and probed with a
monoclonal antibody directed against FADD. In this patient, FADD appears as a doublet band. (D)
Table showing FADD expression in all 7 cases analysed. FADD appears as a doublet band in the
majority of cases.
88
Chapter 5 Results 3
Samples o f stimulated and unstimulated (control) cells were lysed, and the same
concentration of anti-Fas antibody used for stimulation was added to the control
samples as a negative control. The cells were incubated with Protein A-Sepharose to
immunoprecipitate any complexes which may have formed as a result of stimulating
the Fas receptor with an IgG anti-Apo-1 monoclonal antibody. Immunoblotting of
immunoprecipitates of anti-Fas stimulated cells showed a band corresponding to
FADD (~26 kD), in the lane containing stimulated SKW6.4 cells, which was absent in
the lane containing stimulated Jurkat cells (figure 5.12A, lanes 1 & 3). This
demonstrated that FADD had been recruited to a DISC within 15 minutes in SKW6.4
cells, but not in Jurkat cells. The negative control lanes (lysate supplemented samples,
figure 5.12A, lanes 2 & 4) showed higher levels of FADD than was evident in the
lanes containing stimulated cells. This was most likely due to inefficient cell lysis
leaving functional, membrane bound Fas receptor in the lysate which could be
stimulated to form DISCs. FADD molecules which are also freely available in the
lysate appear to bind more effectively in this situation.
In order to investigate whether or not Fas-stimulated B-CLL cells were capable of
forming a DISC, similar experiments on B-CLL cells were performed which included
SKW6.4 cells as a positive control. B-CLL cells from patients 31A and 32A were
cultured for 24 h in the presence of anti-CD40 in order to upregulate Fas receptor
expression, which was checked flow cytometrically. The cells were subsequently
treated with anti-Fas monoclonal antibody (anti-Apo-1, IgG) for 60 minutes.
Following lysis of the cells the anti-Fas bound complex was immunoprecipitated
using Protein A-Sepharose, subsequently the DISC complex was immunoblotted for
FADD and caspase-8.
SKW6.4 cells assembled FADD to the DISC after only 15 minutes (figure 5.12A),
however, after stimulation with anti-Fas monoclonal antibody for 60 minutes, B-CLL
cells did not show FADD binding to a DISC (figure 5.12B). The blots were stripped
and re-probed with anti-caspase-8 polyclonal antibody, however, due to the presence
of a strong band on the blots corresponding to protein A (~42 kD), the presence of
caspase-8 proform and activated fragments (55 and 43 kD) could not be established
(figure 5.13).
89
Chapter 5 Results 3
c lCL3C/3
S 3 B• ^ C/3 \ g■+T* IX rr>
o.D.3coSC3
£C /3
&i4C /D
c313
c3•i3
(26 kD)
B
C3-j£
a.a.3
S '• - P
C/3
&C3 CO
c CO3 V ©
£ £ £>4 *C /3 C /3 C /3
JVCQ
I3
JuCQ
< IgGI g G >
E .gVP to3 -<1-3 M5£ £*C /1 c / 3
S
13 t ohJ_lu o
CQ CQ
< - FADD (26 kD) - > « * » * *
Patient 32 A Patient 31 A
Figure 5.12. Immunoblotting for FADD on immunoprecipitates of Fas-stimulated cells. (A) SKW6.4
and Jurkat cells were stimulated for 15 minutes with anti-Fas monoclonal antibody (anti-Apo-1, IgG).
Samples of stimulated and unstimulated cells were lysed, and anti-Apo-1 was added to the unstimulated
lysates as a control. The protein complexes formed by Fas stimulation were immunoprecipitated using
Protein-A-Sepharose. The lysates were run on a 4-12% gradient SDS-polyacrylamide gel, and
transferred onto a nitrocellulose filter. The filter was probed with a monoclonal antibody directed
against FADD. (B). B-CLL cells from patients 31A and 32A were stimulated for 60 minutes with anti-
Fas (anti-Apo-1, IgG). Lysates from stimulated and unstimulated cells, along with samples of SKW6.4
cells treated as above, were run on 4-12% polyacrylamide gels, transferred onto nitrocellulose filters
and probed with anti-FADD monoclonal antibody.
IgG
FADD
90
Chapter 5 Results 3
a.a.
55 kD ^ " 43 k D -> »
*
FADD (26 k D )> -. 18 k D >
ts*3i—> JUo
3C/5 • Eg M d on'■&a
c3C/5
C/5on
C3
3 VO J£ £ _ )
VJVu i t
C/5 C/5 C/5 0Q 0Q
Figure 5.13 Immunoblotting for caspase-8 on lysates of anti-Fas stimulated cells.
Cells from patient 32A were stimulated with anti-Apo-1 for 60 minutes, lysed and
incubated with Protein-A-Sepharose for 90 minutes. Samples of stimulated and
unstimulated B-CLL cells were run on a 4 -12 % polyacrylamide gel along with
samples of stimulated (15 minutes), unstimulated and lysate supplemented samples of
SKW6.4 cells. The proteins were transferred onto a nitrocellulose filter, which was
probed with a polyclonal antibody to caspase-8 (as described previously).
91
Chapter 5 Results 3
5.7 Discussion
5.7.1 Survival factors in B-CLL
In order to increase specificity when analysing apoptosis in CLL, it was decided to
purify B-CLL cells from the total lymphocyte fraction, as had been used in previous
experiments. B-CLL patients have a high proportion of tumour B cells in their
peripheral blood compared with the ratio of T to B cells in normal blood. The patients
sampled in this study had white blood cell counts ranging from 3.7 to 68 x 109/L, the
average white cell count being 28 x 109/L. In cases such as these the T cell fraction
can be assumed to be minimal, but nevertheless still present. For studies into the
effect of growth factors of B-CLL cells, the presence of T cells may provide a
contaminating source of survival stimuli and so removal of this fraction was deemed
necessary. Accordingly, a T cell depletion step was incorporated into the B-CLL cell
purification procedure. In order to assess the degree to which T cell-mediated stimuli
could influence B-CLL cell survival, a series of comparitive experiments were
performed. In all six of the cases analysed, the level of spontaneous apoptosis was
elevated in the purer B-CLL culture compared to the culture containing B and T cells
(Figure 5.1 B). The difference in sensitivity was significant in only one case, patient
24A, where the B-CLL cells were 21.9% more sensitive to spontaneous apoptosis than
cells in the unpurified culture. In the remaining cases the difference was slight (4.6%
average), but still demonstrates the effect that T cells may have in stimulating survival
of B-CLL cells.
The effects on cell growth and apoptosis of growth factor additions to cultures of CLL
cells has been investigated by a number of groups, (DeFrance et al, 1991, Mainou-
Fowler et al, 1995) but information regarding variable requirements between cases is
limited. In order to investigate the requirement of B-CLL cells for stimulation by
survival factors, the effects of two B cell growth factors, interleukin-4 and CD40,
were studied. Both of these factors had previously been identified as B cell growth
stimulants in normal and malignant cells (Nakanishi et al, 1996; Crawford et al,
1993).
92
Chapter 5 Results 3
Interleukin-4 had previously been reported to have anti-apoptotic effects when applied
to in vitro cultures o f B cells (Panayiotidis et al, 1993) and so the ability of
interleukin-4 to inhibit spontaneous apoptosis of B-CLL cells was investigated.
Interleukin-4 inhibited spontaneous apoptosis in all of the patient samples analysed,
and could significantly improve the survival of the cells (Figure 5.2A and B). The
extent of inhibition of spontaneous apoptosis ranged from 5% to 53.8%, indicating
that B-CLL cells vary in their dependency on interleukin-4 for protection against
apoptosis induction. In order to investigate this further, it would have been interesting
to monitor the Bcl-2 expression levels in these cells before and after culture with
interleukin-4, since this is one of the ways in which IL-4 might protect cells from
apoptosis (Danescu et al, 1992).
An inter-patient variability in sensitivity to interleukin-4 stimulation has been
described above. It was of interest, therefore, to discover if IL-4 sensitive B-CLL cells
could be equally as sensitive to a second survival stimulus, and to determine whether
an additional survival advantage could be provided by stimulation of the cells with a
combination of growth factors. A second B cell survival factor is CD40. CD40
stimulation promotes survival by activation of the anti-apoptotic transcription factor,
NFkP (Rothe et al, 1995) and protein tyrosine phosphorylation (Laytragoon-Lewin et
al, 1998). In this study, samples of B-CLL cells from eight patients were cultured in
the presence of interleukin-4 and a monoclonal antibody to CD40. This culture system
for delivery of the CD40 stimulation was as described by Dive et al, 1998. This
analysis revealed that B-CLL cells were less sensitive to CD40 stimulation than to
interleukin-4 stimulation (Figure 5.3). In fact, in one of the patient samples analysed,
addition of anti-CD40 monoclonal antibody induced apoptosis above the level in the
control culture, a phenomenon which had been reported previously (Wang et al,
1997). Stimulation of B-CLL cells with a combination of interleukin-4 and CD40
stimulation revealed further inter-patient variability. In 50% of the samples, the
combination of survival factors reduced the level of apoptosis below that resulting
from stimulation of the cells with interleukin-4 alone. In the remaining 50% of
samples, the combination of stimuli was less effective at inhibiting spontaneous
apoptosis than interleukin-4 alone.
93
Chapter 5 Results 3
Since B-CLL cells appear to vary in their sensitivity to inhibition of spontaneous
apoptosis by growth factors, it would be interesting to discover if these stimuli could
also promote resistance of the cells to apoptosis induced by chemotherapeutic drugs.
The level of apoptosis in drug-treated cultures pre-incubated with IL-4 was less than
the level of apoptosis in the cultures incubated with the drugs alone (Figure 5.4).
Samples of cells from two patients were analysed for the protective effect of
interleukin-4 against drug-induced apoptosis, and the cells from the two patients
differed in the degree of protection afforded by interleukin-4. A combination of
interleukin-4 and CD40 stimulation also inhibited induction of apoptosis by
chlorambucil (Figure 5.5). Again, the response differed between cells from the three
patient samples analysed, the percentage reduction in apoptosis level in the survival
factor pre-incubated samples, compared to the cultures incubated with the drugs alone,
ranging from 4.9% to 41.5%. Because this reduction in apoptotic rate might be due to
inhibition of spontaneous apoptosis, and not due to inhibition of drug-induced
apoptosis, samples from the same patients were cultured alone and with the survival
stimuli alone as controls. Results from one patient (27A) showed that IL-4 and CD40
stimulation had inhibited drug-induced apoptosis to a much greater extent than
spontaneous apoptosis, indicating that the survival factors were affecting the cells’
response to chemotherapeutic drugs. Of the remaining two cases, one showed the
reverse pattern, indicating that in this patient’s cells the survival stimuli were only
effective in reducing spontaneous apoptosis, and in the third case, spontaneous and
drug-induced apoptosis were inhibited to roughly the same extent.
These findings demonstrate a wide inter-patient variability in dependence of the B-
CLL cells on stimulation from survival stimuli. Spontaneous apoptosis and
chemotherapeutic drug-induced apoptosis can be inhibited by culturing B-CLL cells
with interleukin-4 and/or CD40 stimulation. This raises the question of how relevant
these factors are in determining apoptosis sensitivity in vivo. B-CLL patients have
already been shown to have elevated levels of CD40 ligand in their serum (Younes et
al, 1998), and this, in conjunction with a normal level of expression of CD40
receptors on the CLL cells (Laytragoon-Lewin et al, 1998), could be one mechanism
in which B-CLL cells escape apoptotic cell death. B-CLL cells have also been shown
to express normal levels of the interleukin-4 receptor (Gileece et al, 1993), and so the
94
Chapter 5 Results 3
levels of interleukm-4 in the peripheral blood of B-CLL patients could also be of great
importance. In relation to this, it has been shown that T cells derived from B-CLL
patients have increased levels of cytoplasmic IL-4 compared to normal control T cells
(Mu et al, 1997), although whether or not this IL-4 was secreted was not determined.
Further studies could be performed, possibly using ELISA-based assays, which would
determine whether or not this IL-4 is secreted by the B-CLL T cells, and whether or
not B-CLL patients have normal or elevated levels of serum interleukin-4.
5.7.2 Investigations into the Fas signalling pathway in B-CLL cells
Previous experiments in this study had demonstrated that B-CLL cells have caspase-8
proform present in abundance. However, caspase-8 does not seem to be cleaved to any
great extent during drug-induced apoptosis (Figure 4.5). Caspase-8 is the apical
caspase in the signalling pathway induced following stimulation of the Fas receptor.
B-CLL cells are known to express reduced levels of Fas receptor, and this, in
conjunction with high levels of inactive caspase-8 may be one mechanism whereby B-
CLL cells are resistant to apoptosis. Investigations into the Fas signalling pathway in
B-CLL cells began with a series of experiments to determine whether the cells could
be triggered into apoptosis via Fas signalling. In only 2/8 patients did Fas stimulation
induce apoptosis above the level of spontaneous apoptosis. In the remaining cases, Fas
stimulation inhibited spontaneous apoptosis. One reason for the resistance of B-CLL
cells to Fas-induced apoptosis may be the low level of expression of Fas receptor on
the cells’ surface (Mainou-Fowler et al, 1995; Wang et al, 1997). There are a number
of ways in which Fas receptor expression can be upregulated on B cells. Tonsillar B
cells and Burkitt’s Lymphoma cells can be induced to upregulate Fas receptor
expression by CD40 stimulation (Garrone et al, 1995; Scattner et al, 1995), and B-
CLL cells have been shown to upregulate Fas receptor upon stimulation with a
combination of interleukin-2 and a mitogenic stimulus such as Staphylococcus Aureus
Cowan I (Mapara et al, 1993). In all of these studies the cells became sensitive to Fas
stimulation following upregulation of receptor levels. In this study, B-CLL cells were
cultured for 24 h with a monoclonal antibody to CD40 in order to upregulate Fas
receptor expression. Comparison of the level of Fas receptor on freshly isolated B-
CLL cells with the levels on CD40 stimulated cells demonstrated that CD40
95
Chapter 5 Results 3
stimulation was indeed inducing increased expression of Fas receptor on these cells
(Figure 5.6).
The aim of upregulating Fas receptor levels was to analyse the functionality of the Fas
signalling pathway in B-CLL cells. Following 24 h pre-incubation with CD40
stimulation, the cells were cultured for a further 24 h with anti-Fas monoclonal
antibody in order to assess Fas sensitivity. However, by the time the 48 h time point
was reached, the level of spontaneous apoptosis in the control cultures was very high,
as was the level of apoptosis in the CD40-stimulated samples. This meant that
examination of the Fas signalling pathway would be hampered by a high baseline
level of apoptosis. In order to promote survival of the B-CLL cells over the first 24 h
culture period, interleukin-4 was added to the cultures. The combination of CD40 and
interleukin-4 stimulation also resulted in upregulation of Fas receptor, but successfully
inhibited the induction of high levels of spontaneous apoptosis making analysis of
Fas-induced apoptosis possible.
To determine whether or not the upregulated Fas receptor was functional, cells which
had been cultured with CD40 and interleukin-4 for 24 h were stimulated with anti-Fas
monoclonal antibody for a further 24 h. After this time, apoptosis levels in the cultures
were measured using the Annexin V assay. None of the three patient samples analysed
in this manner were sensitised to Fas-induced apoptosis following CD40 plus
interleukin-4 stimulation (Figure 5.8). Interleukin-4 had previously been shown to
inhibit Fas-induced apoptosis in B-CLL cells (Mainou-Fowler et al, 1995). To
determine whether the addition of interleukin-4 to the cultures for the first 24 h was
causing resistance to Fas-induced apoptosis, cells from one patient were cultured with
CD40 alone prior to fas stimulation. However, no increased sensitivity to Fas-induced
apoptosis was seen (Figure 5.9).
Reports that CD40-upregulated Fas receptor can be functional have been published.
Stimulation of murine B cells with anti-CD40 results in upregulation o f Fas receptor,
and renders the cells sensitive to Fas-induced apoptosis (Nakanishi et al, 1996). When
human tonsillar B cells are stimulated with anti-CD40 to upregulate Fas receptor, they
too become sensitive to Fas-induced apoptosis (Garrone et al, 1995), as do Burkitt’s
96
Chapter 5 Results 3
lymphoma cells (Scattner et al, 1995). However, the situation in B-CLL cells appears
to differ somewhat. Wang and co-workers (1997) reported that, regardless of the
stimulus used to upregulate Fas receptor on B-CLL cells (including CD40 stimulation
or culture with interleukin-2 and pokeweed mitogen), the cells did not become
sensitive to Fas-induced apoptosis. This result conflicts with that of Marpara and co
workers (1995) who demonstrated increased sensitivity to Fas-induced apoptosis in B-
CLL cells pre-incubated with interleukin-2 and S. aureus Cowan I. The sample group
of patients analysed in this study was very small, but this study has demonstrated that
upregulation of Fas receptor by CD40 stimulation does not increase sensitivity to Fas-
induced apoptosis in B-CLL cells.
To further define the nature of Fas resistance in B-CLL cells, components of the
signalling pathway upstream of caspase-8 activation were investigated.
Immunoblotting for proteins involved in the formation of the death inducing
signalling complex (DISC) was performed. This analysis confirmed that B-CLL cells
contain the adapter molecule, FADD (Chinnaiyan et al, 1996), which is required for
caspase-8 activation in response to stimulation of the Fas receptor (Boldin et al,
1996). Since B-CLL cells had now been shown to express both FADD and caspase-8,
and since upregulation of the Fas receptor had been confirmed, it was decided to
analyse expression of a known inhibitor of Fas signalling. c-FLIP (FLICE-like
inhibitory protein) is an inhibitor of the Fas receptor/ligand system, and acts at the
level of FADD/caspase-8 binding. Interaction of c-FLIP with activated caspase-8
causes cleavage and activation of c-FLIP, which results in the c-FLIP/caspase-8
interaction becoming more inhibitive. Since c-FLIP is predominantly expressed in
lymphoid and muscle tissue (Irmler et al, 1997), this indicates that this protein may
play a role in regulating apoptosis of lymphoid malignancies. To determine whether
B-CLL cells were overexpressing c-FLIP, in comparison with the level of caspase-8,
immunoblotting using a polyclonal antibody to c-FLIP (Rasper et al, 1998) was
performed on B-CLL cells which had been stimulated with chlormabucil or anti-Fas
monoclonal antibody. The blots were re-probed with anti-caspase-8 polyclonal
antibody, so that the relative expression of each protein could be compared. This
analysis demonstrated that, while B-CLL cells do express c-FLIP, the level of
expression does not appear to be elevated above that of caspase-8 (Figure 5.9), which
97
Chapter 5 Results 3
means that c-FLIP overexpression is unlikely to be responsible for the block in
apoptosis signalling through Fas in B-CLL cells.
Fas signalling occurs by two distinct mechanisms (Scaffidi et al, 1998). Type I Fas
signalling is extremely rapid, occuring in under 10 minutes, and requires the
formation of a death inducing signalling complex (DISC). Type II Fas signalling is
slower, and requires amplification of the apoptotic signal through release of
cytochrome c from the mitochondria, which activates the apoptosome, a complex
consisting of caspase-9 and Apaf-1. Since B-CLL cells do not undergo apoptosis in
response to Fas signalling, regardless of the level of expression of Fas receptor on the
cells’ surface, it was postulated that the B-CLL cells may be unable to form a DISC to
transmit the apoptotic stimulus into the cell. Accordingly, analysis of the ability of B-
CLL cells to form DISC’S was carried out. SKW 6.4 murine T cells were used as
positive controls for Type I DISC formation, and Jurkat T cells were used as controls
for the Type II Fas response. As expected, SKW 6.4 cells showed binding of FADD to
a Fas-induced DISC after only 10 minutes of stimulation (Figure 5.13 A). B-CLL and
Jurkat cells, however, did not show any evidence of DISC formation even after 60
minutes of Fas stimulation (Figure 5.13 B), confirming that B-CLL cells cannot be
classified as Type I cells. Whilst this evidence does not fully reveal the nature of Fas
resistance in B-CLL cells, it does throw up some interesting possibilities. The Type II
signalling pathway, unlike the Type I pathway, can be inhibited by Bcl-2 (Scaffidi et
al, 1998), most probably at the level of amplification of the apoptotic signal at the
mitochondria leading to apoptosome activation. Several studies have correlated
apoptotic resistance in B-CLL with increased expression of Bcl-2 (Thomas et al,
1996; Aguilar-Santelises et al, 1996; Pepper et al, 1996). This evidence in conjunction
with the discovery that B-CLL cells are most likely to act in a Type II manner in
response to Fas stimulation, may be one reason for apoptotic resistance in B-CLL
cells. In addition, other groups have reported that some cytotoxic drugs, such as
doxorubicin, can induce apoptosis via the Fas signalling pathway (Freisen et al, 1997;
Fulda et al, 1997). If this pathway is inhibited in B-CLL by the overexpression of bcl-
2, this may account for some of the drug resistance evident in B-CLL patients. This
present study was limited in the number of patient samples analysed, and further
investigations could monitor expression levels of Bcl-2 in conjunction with analysis
98
Chapter 5 Results 3
into apoptosome formation, possibly by monitoring activation of caspase-9, to
determine whether B-CLL cells respond to Fas signalling or stimulation with
cytotoxic drugs in a Type II manner.
Chapter Six General Discussion
Chapter Six - General Discussion and Suggestions for Future Work
This thesis has described an investigation into the significance of apoptosis in B cell
chronic lymphocytic leukaemia. The lymphoaccumulative nature of B-CLL implies
that dysregulation of the apoptotic process may be responsible for the development
and progression of the disease. Additionally, apoptosis is known to result from
treatment of malignant cells with chemotherapeutic drugs. The fact that drug
resistance is a major problem in CLL, indicates that there may be a problem in
apoptosis induction in B-CLL cells.
In order to investigate this relationship, preliminary work was performed in order to
assess the application of techniques for analysing apoptosis to specimens of freshly
isolated CLL cells. Use of flow cytometric techniques, supplemented with agarose gel
electrophoretic methods allowed quantification of the percentage of apoptotic cells in
any given sample of CLL cells, although the labelling techniques used altered as the
study progressed and new techniques became available.
Initial studies demonstrated a low in vivo level of apoptosis in B-CLL patients, and
confirmed the existence of ‘spontaneous apoptosis’ when freshly isolated B-CLL cells
were cultured in vitro. Variations in the level of spontaneous apoptosis between cases
indicated that B-CLL cells differed from patient to patient in their dependence on
external survival stimuli, which were not provided by the standard in vitro culture
environment. Preliminary findings in this study also showed that the sensitivity of B-
CLL cells to drug-induced apoptosis was closely related to the sensitivity of the cells
to spontaneous apoptosis. Taken together, these findings would appear to implicate
the degree of survival factor dependency of the malignant cells as an important factor
in determining the response of patients to chemotherapy, thus making this an
important area for future research. Candidates for survival stimuli in B-CLL include
anti-apoptotic members of the Bcl-2 family, type II cytokines (e.g. interleukin-4), and
members of the TNF/NGF receptor/ligand superfamily, in particular CD40 and its
ligand. The role of two of these factors (CD40 and interleukin-4) was investigated in
this thesis, where it was shown that both factors could inhibit some degree of
spontaneous or drug-induced apoptosis (see chapter 5). In relation to this, a
100
Chapter Six General Discussion
combination of CD40 stimulation and overexpression of Bcl-2 has been shown to
increase the clonogenic survival of chlorambucil-treated B-CLL cells, and as such
may contribute towards the acquisition of drug resistance (Walker et al, 1997), a
major problem in advanced B-CLL cases.
In this study, the effects of only two B cell survival factors have been investigated.
Other factors may be important in promoting survival of B cells. Enhanced survival
of B-CLL cells when cultured in direct contact with bone marrow stromal cells has
been reported, the effect mediated by p i and p2 integrins and linked to maintainence
of Bcl-2 levels (Lagneaux et al, 1998). The authors of this report postulate that this
contact derived survival stimulus could play a role in accumulation of B-CLL cells in
the bone marrow. These findings and the results contained within this thesis
demonstrate the extent to which B-CLL cells depend on extracellular stimuli for
enhanced survival and escape from apoptosis. As discussed above, this may also be a
crucial factor in determining the response of B-CLL cells to chemotherapy. Further
investigations in this area should incorporate a wider selection of B cell survival
factors and use a larger group of patient samples in order to better determine the
significance of the findings. The relative levels of the factors in the peripheral blood
and bone marrow of B-CLL patients, or the availability of such factors accessible
through contact with other cell types could also be investigated. In addition, samples
of B-CLL cells derived from bone marrow rather than peripheral blood may have an
altered dependence on growth factor stimulation for survival. One way in which this
may occur, the survival stimulus being derived from contact with bone marrow
stromal cells, is mentioned above (Lagneaux et al, 1998). Analysis of bone marrow
derived B-CLL cells could provide insights into the nature of more advanced or drug
resistant B-CLL.
Other investigations performed during this study focused on components of the
apoptotic machinery. Proteases belonging to the caspase family are the machinery
enzymes involved in the degradation of an apoptotic cell, and are related to the ced-3
death gene of the nematode worm, C. elegans. They reside in the cell as inactive
zymogens which require cleavage at specific aspartate residues in order to attain their
active form. Caspase-3 and caspase-7 are two of the main ‘effector’ caspases,
101
Chapter Six General Discussion
responsible for initiating the terminal events of the apoptotic cascade such as DNA
degradation. Other caspases, including caspase-2, caspase-8 and caspase-9 have been
termed ‘initiator’ caspases due to their involvement in upstream events such as
Fas/CD95 receptor mediated apoptosis and initiation of apoptosis via mitochondria.
The important role that these proteases play is underlined by the finding that caspase-
3 null mice develop brain tumours. Also, it has recently been reported that in human
lymphoma the cellular location of caspase-3 can be an important prognostic indicator
(Donoghue et al, 1999). Work described in this thesis has demonstrated the
expression of caspase-2, caspase-3, caspase-7 and caspase-8 in B-CLL cells, all of
which, with the exception of caspase-2, are activated as a result of induction of
apoptosis by the chemotherapeutic drugs, chlorambucil and prednisolone. It has been
postulated that the inability of B-CLL cells to undergo apoptosis may have been due
to the absence of one or more caspases, however, this would not appear to be the case.
Caspase-8 pro-form was shown to be present in abundance, but was cleaved in only
small amounts in response to chlorambucil treatment. Since this caspase is primarily
involved in apoptosis mediated induced by the Fas/CD95 receptor, the ability of B-
CLL cells to undergo apoptosis in response to Fas stimulation was subsequently
investigated. This included an investigation into the expression of Fas inhibitory
proteins, and an examination of the ability of Fas-stimulated B-CLL cells to form a
‘death inducing signalling complex’ (DISC). In this study, a combination of CD40
and interleukin-4 was used to upregulate expression of the Fas receptor on B-CLL
cells. Despite this fact, it appears that the choice of interleukin-4 may not have been
prudent. A recent report has been published which describes a system for upregulating
Fas receptor using interleukins -2, -7 and -12 in combination with lipopolysaccharide
(LPS) (Williams et al, 1999). Fas receptor upregulated in this system was functional
in that the Fas bearing cells could be lysed by effector cells expressing Fas ligand.
The addition of interleukin-4 to the combination of stimulatory cytokines resulted in
delayed upregulation of Fas receptor, which was not functional in that apoptosis could
not be triggered by fas ligand. In the study presented in this thesis, although Fas
receptor was upregulated by CD40 and IL-4 stimulation, no increase in sensitivity to
Fas-induced apoptosis was evident, which may be due to the effects of interleukin-4.
However, in addition to the effects of interleukin-4, there may be a second factor
involved in determining Fas-mediated apoptosis sensitivity. The report described
102
Chapter Six General Discussion
above (Williams et al, 1999) used Fas ligand-expressing hybridoma cells as effectors,
whereas other reports, including this present study, used a monoclonal IgM anti-Fas
antibody to stimulate the Fas apoptosis pathway. It appears that this may be
significant. Analysis of the Fas sensitivity of cells carrying mutant Fas receptor
constructs and chimeric Fas/TNF constructs, demonstrated a difference in sensitivity
to fas ligand and anti-Fas antibody (Thilenius et al, 1997). The authors postulate that
the altered sensitivity observed in their system could be due to the different binding
characteristics of the antibody when compared with the ligand. It appears that the
large IgM molecule may cluster as many as ten Fas receptor molecules in a back-to-
back formation, rather than the cluster formed when Fas ligand binds to its receptor
(two hydrophobic faces together, ligand on the inside, receptor clustered on the
outside) (Hahne et al, 1995). Whether this could directly influence the downstream
signalling of Fas is not clear, but it could be one reason for the differences seen, and
as such should be considered in the planning and execution of any future research into
Fas-induced apoptosis.
From the analysis performed in this study on the apical events of Fas-induced
apoptosis in B-CLL cells, it appears that there is no evidence for overexpression of
the caspase-8 inhibitory protein, c-FLIP, although it is plausible that other, yet to be
identified proteins, may exert their effects at this point. One possibility is the
existence of a SODD-like ‘silencer’ protein, as is found complexed with the TNF
receptor (Jiang et al, 1999). In relation to the ability of B-CLL cells to form a ‘death
inducing signalling complex’, necessary for apoptosis induced via the Fas receptor, it
appears that, whilst the cells contain the death domain containing adapter molecule
FADD, along with significant amounts of caspase-8, a DISC is not readily assembled
upon stimulation of the cells with antibodies against the Fas receptor. It may be the
case therefore, that apoptosis in B-CLL cells could be mediated mainly via the
mitochondria. Since the mitochondrial pathway of apoptosis can be inhibited by anti-
apoptotic members of the Bcl-2 family, the fact that Bcl-2 is known to be
overexpressed in many instances of B-CLL could be significant. This may also have
implications in the development of drug resistance in B-CLL, since many
chemotherapeutic agents have been shown to induce apoptosis via the mitochondrial
pathway. Further research could be performed in order to characterise the pathway of
organelle involvement and caspase activation induced during apoptosis of B-CLL
103
Chapter Six General Discussion
cells. Should further analysis be undertaken, it should include monitoring of
expression levels of the Bcl-2 family in conjunction with analysis of loss of
mitochondrial membrane potential, release of cytochrome c, and activation of the
apoptosome caspase, caspase-9.
Overall, this study has confirmed that chronic lymphocytic leukaemia is a disease
linked closely with the process of apoptosis. Arguably the most significant findings
described here are the discovery that B-CLL cells possess the necessary caspases and
adapter molecules to execute the apoptotic process, and that the aberration which
contributes to the longevity of B-CLL cells most probably lies in the control or
upstream signalling events of apoptosis. The importance that members of the Bcl-2
family play in this disease has again been highlighted, and this, in conjunction with
the findings related to B-CLL cell dependency on extracellular survival factors, may
hold the key to understanding the nature of the malignant B-CLL cell.
104
References
References
Aguilar-Santelises M, Rottenberg ME, Lewin N, Mellstedt H, and Jondal M: Bcl-2, Bax
and p53 expression in B-CLL in relation to in vitro survival and clinical
progression. International Journal o f Cancer, 1996, 69; 1 -6 .
Ahmad M, Srinivasula SM, Wang L, Talanian RV, Litwack G Femandes-Alnemri T,
Alnemri ES: CRADD, a novel human apoptotic adaptor molecule for caspase-2,
and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Research,
1997, 57; 615-619.
Ambrosini G, Adida C, Altieri DC: A novel anti-apoptosis gene, survivin, expressed in
cancer and lymphoma. Nature Medicine, 1997, 3; 917-921.
Anand R, Southern EM: in Gel Electrophoresis of nucleic acids: A practical approach,
1990, Ed. Rickwood & Flames, 2nd Ed., IRL Press, Oxford. pplOl-123.
Astrow AB: Fludarabine in chronic leukemia - commentary. Lancet, 1996, 347; 1420-
1421.
Bellosillo B, Dalmau M, Colomer D, Gil J: Involvement of CED-3/ICE proteases in
the apoptosis of B-chronic lymphocytic leukemia cells. Blood, 1997, 89; 3378 -
3384.
Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, Vaugier G,
Colona P, Oberling F, Thomas M, Tchemia G, Jacquillat C, Boivin P, Lesty C,
Duault M, Monconduit M, Belabbes S, Gremy F: A new prognostic classification
of chronic lymphocytic leukemia derived from a multivariate survival analysis.
Cancer, 1981, 48; 198-206.
Boise LH, Gonzalez-Garcia M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X,
Nunez G, Thompson CB: bcl-x, a bcl-2-related gene that functions as a dominant
regulator of apoptotic cell death. Cell, 1993, 74; 597 - 608.
Boldin MP, Goncharov TM, Goltsev YV, Wallach D: Involvement of MACH, a novel
MORT1/FADD interacting protease, in Fas/Apo-1 and TNF receptor-induced cell
death. Cell, 1996, 85; 803 - 815.
Bomer C, Monney L, Olivier R, Rosse T, Hacki J, Conus S: Life and death in a
medieval atmosphere. Cell Death and Differentiation 1999, 6; 201-206.
Bortner CD, Oldenburg NBE, Cidlowski JA: The role of DNA fragmentation in
apoptosis. Trends in Cell Biology., 1995, 5; 21 - 26.
105
References
Bosanquet AG, Bird MC, Price WJP, Gilby ED: An assessment of a short-term tumour
chemosensitivity assay in chronic lymphocytic leukemia. British Journal o f
Cancer, 1983, 47; 781 - 789.
Bosanquet AG: In vitro drug sensitivity testing for the individual patient: an ideal
adjunct to current methods of treatment choice. Cinical Oncology, 1993, 5; 195 -
197.
Brown DG, Sun X-M, Cohen GM: Dexamethasone-induced apoptosis involves
cleavage of DNA to large fragments prior to intemucleosomal fragmentation.
Journal o f Biological Chemistry, 1993, 268; 3037-3039.
Calligaris-Cappio F., Gottardi D., Alfarano A., Stacchini A., Gregoretti M.G., Ghia P.,
Bertero M.T., Novarino A., Bergui L: The nature of the B lymphocyte in B-
Chronic Lymphocytic Leukemia. Blood Cells, 1993,19; 601-613.
Campling BG, Pym J, Galbraith PR, Cole SP: Use of the MTT assay for rapid
determination of chemosensitivity of human leukemic blast cells. Leukemia
Research , 1988,12; 823 - 831.
Carbonari M, Cibati M, Fiorilli M: Measurement of apoptotic cells in peripheral blood.
Cytometry, 1995, 22; 161 - 167.
Casciola-Rosen L, Nicholson DW, Chong T, Rowan KR, Thomberry NA, Miller DK,
Rosen A: Apopain/CPP32 cleaves proteins that are essential for cellular repair: A
fundamental principle of apoptotic cell death. Journal o f Experimental Medicine,
1996,183; 1957- 1964.
Chandra J, Gilbreath J, Freireich EJ, Kliche K-O, Andreeff M, Keating M, McConkey
DJ: Protease activation is required for glucocorticoid-induced apoptosis in chronic
lymphocytic leukemic lymphocytes. Blood, 1997, 9; 3673 - 3681.
Cheng EHY, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, Hardwick JM:
Conversion of Bcl-2 to a Bax-like death effector by caspases. Science, 1997, 278;
1966-1968.
Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM: FADD, a novel death domain-
containing protein, interacts with the death domain of Fas and initiates apoptosis.
Cell, 1995, 85; 505 - 512.
Chinnaiyan AM, Tepper CG, Seldin MF, O’Rourke K, Kischkel FC, Hellbardt S,
Krammer PH, Peter ME, Dixit VM: FADD/MORT1 is a common mediator of
CD95 (Fas/Apo-1) and tumour necrosis factor receptor-induced apoptosis. Journal
o f Biological Chemistry, 1996, 271; 4961 - 4965.
106
References
Cohen GM: Caspases: the executioners of apoptosis. Biochemical Journal, 1997, 326; 1
-16.
Collins RJ, Verschuer LA, Harmon BV, Prentice RL, Pope JH, Kerr JFR: Spontaneous
programmed cell death (apoptosis) of B-chronic lymphocytic leukemia cells
following their culture in vitro. British Journal o f Haematology , 1989, 71; 343 -
350.
Cory S: Regulation of lymphocyte survival by the Bcl-2 gene family. 1995, Annual
Review o f Immunology 13; 513-543.
Crawford D, Catovsky D: In vitro activation of leukaemic B cells by interleukin-4
and antibodies to CD40. Immunology, 1993, 80; 40-44.
Dameshek W: Chronic lymphocytic leukemia - an accumulative disease of
immunologically incompetent lymphocytes. Blood, 1967, 29; 566 - 584.
Danescu M, Rubio-Trujillo M, Biron G, Bron D, Delespesse G, Sarfati M:
Interleukin-4 protects chronic lymphocytic leukemia B cells from death by
apoptosis and upregulates bcl-2 expression. Journal o f Experimental Medicine,
1992,176; 1319-1326.
Decaudin D, Geley S, Hirsch T, Castedo M, Marchetti P, Macho A, Kofler R, Kroemer
G: Bcl-2 and Bcl-XL antagonise the mitochondrial dysfunction preceding nuclear
apoptosis induced by chemotherapeutic agents. Cancer Research, 1997, 57; 62 -
67.
DeFrance T, Fluckiger A-C, Rossi J-F, Rousset F, Bancherou J: In vitro activation of B-
CLL cells. Leukaemia & Lymphoma, 1991, 5; 13-19.
Donoghue S, Baden HS, Lauder I, Sobolewski S, Pringle JH: Immunohistochemical
localization of caspase-3 correlates with clinical outcome in B-cell diffuse large
cell lymphoma Cancer Research, 1999, 59; .5386-5391.
Duan H, Dixit VM: RAIDD is a new ‘death’ adaptor molecule. Nature, 1997, 385; 86 -
89.
Eamshaw WC: Apoptosis: lessons from in vitro systems. Trends in Cell Biology, 1995,
5; 217-220.
Eischen CM, Kottke TJ, Martins LM, Basi GS, Tung JS, Eamshaw WC, Leibson PJ,
Kaufmann SH: Comparison of apoptosis in wild-type and Fas-resistant cells:
chemotherapy-induced apoptosis is not dependent on Fas/Fas ligand interactions.
Blood 1997, 90; 935-943.
107
References
Enari M, Sakahira H, Yokoyama H, Okawa K, Iwamatsu A, Nagata S: A caspase-
activated DNase that degrades DNA during apoptosis and its inhibitor ICAD.
Nature, 1998, 391; 43-50.
Faleiro L, Kobayashi R, Feamhead H, Lazebnik Y: Multiple species of CPP32 and
Mch2 are the major active caspases present in apoptotic cells. EMBO Journal,
1997,16; 2271-2281.
Feamhead HO, Dinsdale D, Cohen GM: An interleukin-1 P-converting enzyme-like
protease is a common mediator of apoptosis in thymocytes. FEBS Letters, 1995,
375; 283-288.
Femandes-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L, Tomaselli KJ,
Wang L, Yu Z, Croce CM, Salveson G, Eamshaw WC, Litwack G, Alnemri ES:
Mch-3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer
Research, 1995, 55; 6045 - 6052.
Femandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F,
Fritz C, Trapani JA, Tomaselli KJ, Litwack G, Alnemri ES: In vitro activation of
CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing
two FADD-like domains. Proceedings o f the National Academy o f Science USA ,
1996, 93; 7464 - 7469.
Fraser A, Evan G: A license to kill. Cell, 1996, 85; 781 - 784.
Friesen C, Herr I, Krammer PH, Debatin K-M: Involvement of the CD95 (Apo-l/Fas)
receptor/ligand system in drug-induced apoptosis. Nature Medicine. 1996, 2; 574 -
577.
Freisen C, Fulda S, Debatin K-M: Deficient activation of the CD95 (APO-1/Fas) system
in drug resistant cells. Leukaemia 1997,11; 1833-1841.
French LE & Tschopp J: The TRAIL to selective tumour death. Nature Medicine, 1999,
5; 146-147.
Fulda S, Sieverts H, Friesen C, Herr I, Debatin K-M: The CD95 (APO-1/Fas) system
mediates drug-induced apoptosis in neuroblastoma cells. Cancer Research 1997,
57; 3823-3829.
Gamen S, Anel A, Lasierra P, Alava MA, Martinez-Lorenzo MK, Pineiro A, Naval J:
Doxorubicin-induced apoptosis in human T-cell leukaemia is mediated by
caspase-3 activation in a Fas-independent way. FEBS Letters 1997, 417; 360-
364.
108
References
Garcia-Ruiz C, Colell A, Mari M, Morales A, Femandez-Checa JC: Direct effect of
ceramide on the mitochondrial electron transport chain leads to generation of
reactive oxygen species. Role of mitochondrial glutathione. Journal o f Biological
Chemistry, 1997, 272; 11369 - 11377.
Garrone P, Neidhardt E-M, Garcia E, Gailbert L, van Kooten C, Banchereau J: Fas
ligation induces apoptosis of CD40-activated human B lymphocytes. Journal o f
Experimental Medicine 1995,182; 1265-1273.
Ghia P, Boussiotis VA, Manie S, Cardaso AA, Gribben JG, Freedman AS, Nadler LM:
Activation of follicular lymphoma through CD40 upregulation of Bcl-XL
thereby promoting survival. Blood 1996, 88, Suppl. 1; 2672
Gottardi D, Alfarano A, De Leo AM, Stacchini L, Bergui F, Caligaris-Cappio F:
Defective apoptosis due to Bcl-2 overexpression may explain why B-CLL cells
accumulate in GO. Current Topics in Microbiology and Immunology 1995,194;
307-312.
Gottardi D, Alfarano A, De Leo AM, Stacchini A, Rigo A, Veneri D, Zanotti R, Pizzolo
G, Caligaris-Cappio F: In leukemic CD5+ B cells the expression of the Bcl-2 gene
family is shifted toward protection from apoptosis. British Journal o f Haematology,
1996, 94; 612-618.
Green DR & Reed JC: Mitochondria and apoptosis. Science, 1998, 281; 1309-1312.
Hahne M, Peitsch MC, Irmler M, Schroter M, Lowin B, Bron C, Renno R, French L:
Characterisation of the non-functional Fas ligand of GLD mice. International
Immunology, 1995, 7; 1381- 1386.
Han Z, Hendrickson EA, Bremner TA, Wyche JH: A sequential two-step mechanism
for the production of the mature p 17;p 12 form of caspase-3 in vitro. Journal o f
Biological Chemistry, 1997, 272; 13432-13436.
Hanada M, Delia D, Aiello A, Stadtmauer E, Reed JC: bcl-2 gene hypomethylation and
high level expression in B-cell chronic lymphocytic leukaemia. Blood 1993, 82;
1820-1828.
Hanson JA, Bentley P, Bean EA, Nute SR, Moore JL: In vitro chemosensitivity testing
in chronic lymphocytic leukemia patients. Leukemia Research, 1991,15; 565 - 569.
Harvey NL, Trapani JA, Femandes-Alnemri T, Litwack G, Alnemri ES, Kumar S:
Processing of the Nedd2 precursor by ICE-like proteases and granzyme B. Genes
to Cells, 1996,1; 673 - 685.
109
References
Harvey NL, Butt AJ, Kumar S: Functional activation of Nedd2/ICH-1 (Caspase-2) is an
early process in apoptosis. Journal o f Biological Chemistry, 1997, 20; 13134 —
13139.
Huang P, Plunkett W: Fludarabine and gemcitabine induced apoptosis: incorporation of
analogues into DNA is a crucial event. Cancer Chemotherapy Pharmacology,
1995, 36; 181-188.
Imai Y, Kimura T, Murakami A, Yajima N, Sakamaki K, Yonehara S: The CED-4-
homologous protein FLASH is involved in Fas-mediated activation of caspase-8
during apoptosis. Nature, 1999, 398; 777 - 785.
Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer J-L,
Schroter M, Bums K, Mattmann C, Rimoldi D, French LE, Tschopp J: Inhibition of
death receptor signals by cellular FLIP. Nature, 1997, 388; 190- 195.
Jiang YP, Woronicz JD, Liu W, Goeddel DV: Prevention of constitutive TNF receptor 1
signaling by silencer of death domains. Science 1999, 283; 543-546.
Kaufmann SH, Desnoyers S, Ottaviano Y, Davidson NE, Poirier GG: Specific
proteolytic cleavage of poly(ADP-ribose) polymerase: An early marker of
chemotherapy-induced apoptosis. Cancer Research, 1993, 53; 3976 - 3985.
Kawasaki H, Altieri DC, Lu C-D, Toyoda M, Tenjo T, Tanigawa N: Inhibition of
apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer
Research, 1998, 58; 5071 - 5074.
Kelliher MA, Grimm S, Ishida Y, Kuo F, Stanger BZ, Leder P: The death domain
kinase RIP mediates the TNF-induced NFkB signal. Immunity, 1998, 8; 297 - 303.
Kerr JFR: Shrinkage necrosis : A distinct mode of cellular death. Journal o f Pathology,
1971,105;13-15.
Kerr JFR, Wyllie AH, Currie AR: Apoptosis : A basic biological phenomenon with
wide ranging implications in tissue kinetics. British Journal o f Cancer, 1972, 26;
239-243.
Kirsch DG, Doseff A, Chau BN, Lim D-S, de Souza-Pinto NC, Hansford R, Kastan
MB, Lazebnik YA, Hardwick JM: Caspase-3 dependent cleavage of Bc-2
promotes release of cytochrome-c. Journal o f Biological Chemistry 1999, 274;
21155-21161.
110
References
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, Peter ME:
Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins from a death-
inducing signaling complex (DISC) with the receptor. EMBO Journal, 1995, 14;
5579 - 5588.
Koopman G, Reutelingsperger CPM, Kuijten GAM, Keehnen RMJ, Pals ST, van Oers
MHJ: Annexin V for flow cytometric detection of phosphatidylserine expression
on B cells undergoing apoptosis. Blood, 1994, 84; 1415 - 1420.
Krajewski S, Gascoyne RD, Zapata JM, Krajewska M, Kitada S, Chhanabhai M,
Horsman D, Berean K, Piro LD, Fugier-Vivier I, Liu Y-J, Wang H-G, Reed JC:
Immunolocalization of the ICE/CED-3-family protease, CPP32 (caspase-3) in non-
Hodgkin’s lymphomas, chronic lymphocytic leukemias, and reactive lymph nodes.
Blood, 1997, 89; 3817-3825.
Kroemer G: The proto-oncogene bcl-2 and its role in regulating apoptosis. Nature
Medicine, 1997, 3; 614-640.
Kuida K, Zheng TS, Na S, Kuan C-Y, Yang D, Karasuyama H, Rakic P, Flavell RA:
Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.
Nature, 1996, 384; 368 - 372.
Lagneaux L, Delforge A, Bron D, De Bruyn C, Stryckmans P: Chronic lymphocytic
leukaemic B cells but not normal B cells are rescued from apoptosis by contact
with normal bone marrow stromal cells. Blood 1998, 91; 2387-2396.
Laytragoon-Lewin N, Duhony E, Bai X-F, Mellstedt H. (1998) Downregulation of
CD95 receptor and defect CD40-mediated signal transduction in B-chronic
lymphocytic leukemia cells. European Journal o f Haematology, 1998, 61; 266-
271.
Lazebnik YA, Kaufinann SH, Desnoyers S, Poirier GG, Eamshaw WC: Cleavage of
poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature,
1994, 371; 346 - 347.
Li H, Bergeron L, Cryns V, Pasternack MS, Zhu H, Shi L, Greenberg A, Yuan J:
Activation of caspase-2 in apoptosis. Journal o f Biological Chemistry, 1997, 272;
21010-21017.
Li H, Zhu H, Xu CJ, Yuan J: Cleavage of BID by caspase-8 mediates the mitochondrial
damage in the Fas pathway of apoptosis. Cell, 1998, 94; 491 - 501 .
I l l
References
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X:
Cytochrome c and dATP-dependent formation of Apaf-l/caspase-9 complex
initiates an apoptotic protease cascade. Cell, 1997, 91; 479 - 489.
Liston P, Roy N, Tamui K, Lefebvre C, Baird S, Cherton-Horvat G, Farahani R,
McLean M, Ikeda J-E, MacKenzie A, Korneluk RG: Suppression of apoptosis in
mammalian cells by NAIP and a related family of LAP genes. Nature, 1996, 379;
349-353.
Luo X, Budihardjo I, Zou H, Slaughter C, Wang X: Bid, a bcl-2 interacting protein,
mediates cytochrome c release from mitochondria in response to activation of cell
surface death receptors. Cell, 1998, 94; 481 -501.
MacFarlane M, Cain K, Sun X-M, Alnemri ES, Cohen GM: Processing/activation of at
least four interleukin-lp converting enzyme-like proteases occurs during the
execution phase of apoptosis in human monocytic tumor cells. Journal o f Cell
Biology 1997,137; 469 - 479.
Mainou-Fowler T, Craig VA, Copplestone AJ, Hamon MD, Prentice AG. Effect
of anti-APO-1 on spontaneous apoptosis of B cells in chronic lymphocytic
leukaemia: the role of bcl-2 and Interleukin-4. Leukemia and Lymphoma, 1995,19;
301-308.
Mapara MY, Bargou R, Zugck C, Dohner H, Ustaoglu F, Jonker RR, Krammer PH,
Dorken B: APO-1 mediated apoptosis or proliferation in human chronic B
lymphocytic leukemia: correlation with bcl-2 oncogene expression. European
Journal o f Immunology, 1993, 23; 702 - 708.
Martin SJ, Reutelingsperger CPM, McGahon AJ, Rader JA, van Schie RCAA, LaFace
DM, Green DR: Early redistribution of plasma membrane phosphatidylserine is a
general feature of apoptosis regardless of the initiating stimulus: Inhibition by
overexpression of Bcl-2 and Abl. Journal o f Experimental Medicine, 1995, 182;
1545 - 1556.
McConkey DJ, Chandra J, Wright S, Plunkett W, McDonnell TJ, Reed JC, Keating M:
Apoptosis sensitivity in chronic lymphocytic leukemia is determined by
endogenous endonuclease content and relative expression of Bcl-2 and Bax.
Journal o f Immunology, 1996,156; 2624 - 2630.
112
References
Medema JP, Scaffidi C, Kischkel FC, Schevchenko A, Mann M, Krammer PH, Peter
ME: FLICE is activated by association with the CD95 death-including signaling
complex (DISC). EMBO Journal, 1997,16; 2794 - 2804.
Moller P, Henne C, Leithauser F, Eichelmann A, Schmidt A, Briiderlein S, Dhein J,
Krammer PH: Coregulation of the APO-1 antigen with intercellular adhesion
molecule-1 (CD54) in tonsillar B cells and coordinate expression in follicular
center B cells and in follicle center and mediastinal B-cell lymphomas. Blood,
1993, 81; 2067 - 2075.
Monserrat E., Rozman C: Current approaches to the treatment and management of
Chronic Lymphocytic Leukemia. Drugs, 1994, 47 (Suppl 6); 1-9.
Moriishi K, Huang DCS, Cory S, Adams JM: Bcl-2 members do not inhibit apoptosis
by binding the caspase activator Apaf-1. Proceedings o f the National Academy o f
Science USA, 1999, 96; 9683 - 9688.
Mu X, Kay NE, Gosland MP, Jennings CD: Analysis of blood T-cell cytokine
expression in B-chronic lymphocytic leukaemia: evidence for increased levels of
cytoplasmic IL-4 in resting and activated CD8 cells. British Journal o f
Haematology, 1997, 96; 733-735.
Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C,
Bretz JD, Zhang M, Gentz R, Mann M, Krammer PH, Peter ME, Dixit VM:
FLICE, a novel FADD-homologous ICE /CED-3-like protease, is recruited to the
CD95 (Fas/APO-1) death-inducing signalling complex. Cell, 1996, 85; 817 - 827.
Nakanishi K, Matsui K, Kashiwamura S, Nishioka Y, Nomura J, Nishimura Y,
Sakaguchi N, Yonehara S, Higashino K, Shinka S,: IL-4 and anti-CD40 protect
against Fas-mediated apoptosis and induce B cell growth and differentiation.
International Immunology, 1996, 8; 791-798.
Nicholson DW, Ali A, Thomberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau
Y, Griffin PR, Labelle M, Lazebnik YA, Munday NA, Raju SM, Smulson ME,
Yamin T-T, Yu VL, Miller DK: Identification of the ICE/CED-3 protease
necessary for mammalian apoptosis. Nature, 1995, 376; 37 - 43.
Nicholson DW, Thonberry NA: Caspases: killer proteases. Trends in Biochemical
Science 1997, 22; 299 - 306.
O’Brien S., del Giglio A., Keating M: Advances in the biology and treatment of B-cell
chronic lymphocytic leukemia. Blood, 1995, 85; 307-318.
113
References
Oberhammer F, Wilson JW, Dive C, Morris ID, Hickman JA, Wakeling AE, Walker
PR, Sikorska M: Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or
50 kb fragments prior to or in the absence of intemucleosomal fragmentation.
EMBO Journal 1993,12; 3679-3684.
Oltvai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 heterodimerises in vivo with a conserved
homologue, Bax, that accelerates programmed cell death. Cell, 1993, 74; 609 - 619.
Panayiotidis P, Ganeshaguru K, Jabbar SAB, Hoffbrand AV: Interleukin-4
inhibits apoptotic cell death and loss of the bcl-2 protein in B-chronic lymphocytic
leukaemia cells in vitro. British Journal o f Haematology, 1993, 85; 439-445.
Pastorino JG, Chen S-T, Tafani M, Snyder JW, Farber JL: The overexpression of Bax
produces cell death upon induction of the mitochondrial permeability transition.
Journal o f Biological Chemistry, 1998, 273; 7770 - 7775.
Pepper C, Bentley P, Hoy T: Regulation of clinical chemoresistance by bcl-2 and bax
oncoproteins in B-cell chronic lymphocytic leukemia., 1996, British Journal o f
Haematology 95; 513 - 517.
Pepper C, Thomas A, Hoy T, Bentley P: Chlorambucil resistance in B-cell chronic
lymphocytic leukemia is mediated through failed Bax induction and selection of
high Bcl-2-expressing subclones. British Journal o f Haematology, 1999,104; 581
-588.
Perri R.T, Louie SW, Espar WG: Expression of the multi-drug resistance (MDR) gene
MDR1 in chronic lymphocytic leukemia (CLL) B cells. Blood , 1989, 74 (Suppl.
1); 198a.
Planken EV, Dijkstra NH, Willemze R, Kluin-Nelemans JC: Proliferation of b cell
malignancies in all stages of differentiation upon stimulation in the ‘CD40 system’.
Leukaemia, 1996,10; 488-493.
Rao L, Perez D, White E: Lamin proteolysis facilitates nuclear events during apoptosis.
Journal o f Cell Biology, 1996, 135; 1441-1455.
Rasper DM, Vaillancourt JP, Hadano S, Houtzager VM, Seiden I, Keen SLC, Tawa P,
Xanthoudakis S, Nasir J, Martindale D, Koop BF, Peterson EP, Thomberry NA,
Huang J, MacPherson DP, Black SC, Homung F, Lenardo MJ, Hayden MR, Roy S,
Nicholson DW: Cell death attenuation by ‘Usurpin’, a mammalian DED-caspase
homologue that precludes caspase-8 recruitment and activation by the CD95
(Fas/APO-1) receptor complex. Cell Death and Differentiation, 1998, 5; 271-288.
114
References
Reed JC, Jurgensmeier JM, Matsuyama S: Bcl-2 family proteins and mitochondria.
Biochemica et Biophysica Acta 1998,1366; 127-137
Robertson LE, Plunkett W, McConnell K, Keating MJ, McDonnell TJ: Bcl-2 expression
in chronic lymphocytic leukaemia and its correlation with the induction of
apoptosis and clinical outcome. Leukemia, 1996,10; 456 - 459.
Rothe M, Sarma V, Dixit VW, Goeddel DV: TRAF2-mediated activation of NF-kB by
the TNF receptor-2 and CD40. Science 1995, 269; 1424-1427
Rozman C, Montserrat E: Chronic lymphocytic leukemia. New England Journal o f
Medicine, 1995, 333; 1052 - 1057.
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 2nd
Edition, Cold Spring Harbor Laboratory Press, Plainview, NY, 1989.
Sawitsky A., Rai K.R: 1992, ‘The Chronic Lymphoid Malignancies’, Chapter 20 in
‘Leukemia’, Ed. Whittaker J.A., Blackwell Scientific Publications.
Scaffidi C, Medema JP, Krammer PH, Peter ME: FLICE is predominantly expressed as
two functionally active isoforms, caspase-8/a and caspase-8/b. Journal o f
Biological Chemistry, 1997, 272; 26953 - 26958.
Scaffidi C, Fulda S, Srinivasan A, Freisen C, Li F, Tomaselli KJ, Debatin K-M,
Krammer PH, Peter ME: Two CD95 (Apo-1/Fas) signalling pathways. EMBO
Journal, 1998,17; 1675 - 1687.
Schattner EJ, Elkon KB, Yoo D-H, Tumang J, Krammer PH, Crow MK, Freidman SM:
CD40 ligation induces Apo-1/Fas expression on human B lymphocytes and
facilitates apoptosis through the Apo-1/Fas pathway. Journal o f Experimental
Medicine 1995,182; 1557-1563.
Schisselbauer JC, Silber R, Papadopoulos E: Characterisation of glutathione S-
transferase expression in lymphocytes from CLL patients. Cancer Research , 1990,
50; 3562-3568.
Schulze-Osthoff K, Bauer MKA, Vogt M, Los M: Role of ICE-related and other
proteases in Fas-mediated apoptosis. Cell Death and Differentiation, 1996, 3; 177 —
184.
Sen S, D’Incalci M: Apoptosis: Biochemical events and relevance to cancer
chemotherapy. FEBS Letters, 1992, 307; 122 - 127.
115
References
Snell V, Clodi K, Zhao S, Goodwin R, Thomas EK, Morris SW, Kadin ME, Cabanillas
F, Andreef M, Younes A: Activity of TNF-related apoptosis -inducing ligand
(TRAIL) in haematological malignancies. British Journal o f Haematology 1997,
99; 618-624.
Sorenson CM, Eastman A: Mechanism of cis-diamminedichloroplatinum(II)-
induced cytotoxicity - role of G2 arrest and DNA double strand breaks. Cancer
Research 1988, 48; 4484-4488
Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G, Alnemri ES: Molecular
ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-
inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases.
Proceedings o f the National Academy o f Science USA, 1996, 93; 14486 - 14491.
Susin SA, Zamzami N, Castedo M, Daugas E, Wang H-G, Geley S, Fassy F, Reed JC,
Kroemer G: The central executioner of apoptosis: multiple connections between
protease activation and mitochondria in Fas/Apo-1/ CD95- and ceremide-induced
apoptosis. Journal o f Experimental Medicine, 1997,1; 25 - 37.
Susin SA, Lorenzo HK, Zamzami N, Marzo I, Brenner C, Larochette N, Prevost M-C,
Alzari PM, Kroemer G: Mitochondrial release of caspase-2 and -9 during the
apoptotic process. Journal o f Experimental Medicine, 1999,189; 381-393.
Ihilenius AR, Braun K, Russell JH: Agonist antibody and Fas ligand mediate different
sensitivity to death in the signalling pathways of Fas and cytoplasmic mutants.
European Journal o f Immunology, 1997, 27; 1108-1114.
Thomas A, El Rouby S, Reed JC, Krajewski S, Silber R, Potmesil M, Newcomb EW:
Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship
between p53 gene mutation and Bcl-2/Bax proteins in drug resistance. Oncogene,
1996,12; 1055 - 1062.
Thome M, Hofmann K, Bums R, Martinon F, Bodmer JL, Mattmann C, Tschopp J:
Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.
Current Biology 1998, 8; 885-888.
Thompson CB: Apoptosis in the pathogenesis and treatment of disease. Science
1995, 267; 1456 - 1462.
Tsujimoto Y, Cossman J, Jaffe E, Croce CM: Involvement of the bcl-2 gene in human
follicular lymphoma. Science, 1985, 228; 1440-1443
116
References
Walker A, Taylor ST, Hickman JA, Dive C: Germinal centre-derived signals act with
bcl-2 to decrease apoptosis and increase clonogenicity of drug-treated human B
lymphoma cells. Cancer Research, 1997, 57; 1939-1945.
Wang D, Freeman GJ, Levine H, Ritz J, Robertson MJ: Role of the CD40 and CD95
(APO-l/Fas) antigens in the apoptosis of human B-cell malignancies. British
Journal o f Haematology 1997, 97; 409 - 417.
Weil M, Jacobson MD, Raff MC: Are caspases involved in the death of cells with a
transcriptionally inactive nucleus? Sperm and chicken erythrocytes. Journal o f Cell
Science, 1998, 111; 2707-2715.
Williams JF, Petrus MJ, Wright JA, Husebekk A, Fellowes V, Read EJ, Gress RE,
Fowler DH: Fas-mediated lysis of chronic lymphocytic leukaemia cells: role of
type I versus type II cytokines and autologous FasL-expressing T cells. British
Journal o f Haematology, 1999,107; 99-105.
Wolfe JT, Pringle JH, Cohen GM: Assays for the measurement of DNA fragmentation
in apoptosis. ‘Techniques in apoptosis: a user’s guide’ 1996
Xerri L, Devilard E, Bouabdallah, R, Stppa A-M, Hassoun J, Birg F: FADD expression
and caspase activation in B-cell lymphomas resistant to Fas-mediated apoptosis.
British Journal o f Haematology 1999,106; 652-661.
Yang X, Khosravi-Far R, Chang HY, Baltimore D: Daxx, a novel Fas-binding protein
that activates JNK and apoptosis. Cell 1997, 89; 1067 - 1076.
Yonehara S, Ishii A, Yonehara M: A cell-killing monoclonal antibody (anti-Fas) to a
cell surface receptor antigen co-downregulated with the receptor of tumour necrosis
factor. Journal o f Experimental Medicine 1989,169; 1747 - 1756.
Younes A, Snell V, Consoli U, Clodi K, Zhao S, Palmer JL, Thomas EK, Armitage RJ,
Andreef M: Elevated levels of biologically active soluble CD40 ligand in the serum
of patients with chronic lymphocytic leukaemia. British Journal o f Haematology
1998,100; 135-141.
Yuan J, Shamam S, Ledoux S, Ellis HM, Horvitz HR: The C. elegans cell death gene
ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting
enzyme. Cell, 1993, 75; 641-652.
Yuan J: Transducing signals of life and death. Current Opinion in Cell Biology 1997, 9;
247-251.
117
References
Zhu H, Feamhead HO, Cohen GM: An ICE-like protease is a common mediator of
apoptosis induced by diverse stimuli in human monocytic THP.l cells. FEBS
Letters, 1995, 374; 303 - 308.
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X: Apaf-1, a human protein homologous
to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-
3. Cell 1997, 90; 405 - 413.
118
Appendix
Appendix
Patient 121
Pm -treatm ent, fM shly isolated cells
Ml*33*.
M2
J 1 10* FL1 -Height
T natm en t day 7, fieshly isolated cells Post-treatment, freshly isolated cells
Ml92.3 *A
in®
M2
‘ 7.5%
1 '0 * 1 0 1 1 0 * 1 0 1 1 0 *
FL1-Height
CO
M2
UJ
FL1 -Height
2Pie-treatm ent, Control 24 h
Ml77.6%h- M2
in ® i n • V n t V r
22.4%
10* 1 0 ’ 10* 1 0 J 10 * FL1-Height
Treatm ent day 7, Control 24 h
Ml
M2
1 0 ' 10FL1-Height
M159.9%
i- M240.1%
10* 1 0 1 10* 1 0 J 10* FL1 -Heigiift
Patient 8 A
Pre-treatment, freshly isolated cells
S w l| 93%* 1 . M2| I '----- T l %
O JW hy-s- .... i .iwm ■. ....10* 10’ 102 1 0 J 10*
FL1-Height
Day 7, freshly isolated cells M1
V)
CD>UJ
. 93.7% M2
5.9%
1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height
Post-treatment, freshly isolated cells M1
93.2%
1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height
Pre-treatment, Control 24 h M1co 1
v>■ECD>UJ
63.6%M2
34.6%
10* 10 ’ 102 10* 10*
FL1-Height
Day 7, Control 24 h M1
■ECD>
59.5%i--- M2
40.3%
1 0 * 1 0 ’ 102 1 0 J 10* FL1 -Height
Post-treatment, Control 24 h M1
«■ECD>UJ
51.2%I--- M2
48.6%
1 0 * 1 0 ’ 102 1 0 J 10* FL1-Height
Figure A l. Flow cytometry histograms for in vivo analysis, chapter 3, section 3.6. Cells were taken from patients 121 and 8A prior to, during and post treatment. The cells were analysed using the Annexin V assay at 0 h and 24 h of in vitro culture. The histograms shown are plots of Annexin V-FITC (X-axis) against number of cells (Y- axis). The two peaks are live cells (Annexin V negative) and apoptotic cells (Annexin V positive). Markers were set on the pre-treatment, 0 h sample for each case, and remained in place for all further analysis.
119
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