IDElYTIFICATION OF NKLAMRE, THE EWMAN HOMOLOGUE TO THE MAPKKDK RELATED PROTEIN KINASE NKIATRE, AND ITS

LOSS IN LEUKEMIC BLASTS WITH THE Sq- SYNDROME

Michael Midmer

A thesis submitted in conformity with the requirements for the Degree of Master of Science, Graduate Department of Medical Biophysics, in the

University of Toronto

O Copyright by Michael Midmer 1999

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Identification of NKIAMRE, the Human Homologue to the MAPWCDK Related Protein Kinase NKIATRE, and its

Loss in Leukemic Blasts with the 5q- Syndrome.

Michael Midmer Master of Science, 1999

Department of Medical Biophysics University of Toronto

ABSTRACT

Myelodyspiastic syndromes W S ) are a group of pre-mdignant hematologic

di sorders characterized b y anemia, abnormai maturation of the granulocytic lineage, and

the potential to transfom into malignant acute myelogenous leukemia (AML). Loss of

al1 or part of the long arm of chromosome 5 (5q- syndrome) has been associated with

these myeloid disorders. To identie lost turnor suppressor genes in MDS and AML,

attempts have been made to define the minimdly deleted region on chromosome 5q3 1.

We have identified human NKIAMRE, a novel cyclin dependent kinase-related

rnolecule closely relatesi to the rat serindthreonine kinase NKIATRE. NKLAMRE also

shares features of the mitogen activated protein kinase family including the Thr-X-Tyr

motif in subdomain VIII. It also contains the NKIAMRE domain, for which it is named,

which is a potential binding site for a cyclin.

Human NKIAMRE localizes to 5q3 1.1, centromeric to the IL-9 locus and

telomeric to IRF- 1. NKIAMRE was deleted at both aileles in 9 of L8 leukemic samples

with 5q3 1 abnormalities studied by fluorescence in situ hybndization. This study implies

that loss of NKZAMRE is associated with the deveIopment or progression of myeloid

disorders. Furthemore, NKIATRE is not expresseci in any ce11 lines exarnined

suggesting that it may be detrimentai to ceIl proliferation.

ACKNOWLEDGEMENTS

This study could not have been accomplished without the help fiom the following

people. Primarily, I woufd like to t h d xny supervisory cornmittee, Dr. Brent Zanke, Dr.

Jeremy Squire, and Dr. Robert Rottapei. In addition, 1 would like to îhank the Zanke iab

including Jason Goncalves and Ben Beheshti for their guidance and wisdom. In

particular, Barbara Iahte my gossip partner and relationship counselor, Susan Moore for

putting up with rny singing and dancing, and Matthew M c h e s for keeping me in

perspective. Finally, and most importantiy, my family for th& continueci support and

joining me on this roller coaster ride.

TABLE OF CONTENTS

Abstract

Acknowledgements

Table of Contents

List of Tables

List of Figures

List of Abbreviations

List of Symbols

Chapter 1 : Introduction

Introduction importance of MDS The Hemopoietic System Clinical Features of MDS 5q- Syndrome Smallest Commonly Deleted Region Candidate Genes Mapping to 5q3 1

Tumor Suppressor Genes History The Re~obiastoma Gene and Gene Product

Signal Transduction Conserved Kinase Features Mitogen Activated Protein Kinase Signaling The ERK Subgroup of MAPKs The JNK Subgroup of MAPKs The p38 Subgroup of MAPKs Functional Separation of MAPK Pathways

The Eukaryotic Cell Cycle Cyclin Dependent Kinases C yclins Cyclin-CDK Complexes CDK Activation by Phosphorylation CDK inhibition by Phosphorylation CDK Inhibition by inhibitory Subunits

ii

iii

iv

vii

-. - vlll

ix

xii

1 .5 Cdc2-related Protein Kinases 43

Chapter 2: The Molecular Cloning of NKIAMRE, the Human Homologue to the MAPK/CDK-related Pro tein Kinase NKIATRE.

Introduction

Materials and Methods Molecular Clonin& PCR, and Sequencing Northern Blot Analysis Ce11 culture and Transfection Tet-On Ce11 Lines

R e d t s Isolation of the Human cDNA Sequence Andy sis Northern Blot Analysis Tet-On Cell Lines

Discussion

Chapter 3: nie Chromosomal Localization of NKIAMRE to 5q3 1.1 and its Loss in Leukernic Blasts with the 5q- Syndrome.

Introduction

Materials and Methods Patients Southern Blo t Analysis Isolation of Human Genomic NKIAMRE Fluorescent Chromosomal in sim Hybridization Statistical Analysis Northern Blot Analysis Differential Hemopoietic Gene Expression Analysis

Results Isolation and LocaIization of Genomic NKIAMRE Southern Analysis on Patients with 5q- Syndrome The NKIAMRE Locus is Deleted in Myeloid Leukemia Northern Blot Analysis Differential Hemopoietic Gene Expression Andysis

3 -4 Discussion

3.5 Future Work

3.6 Appeudix: S tatisticai Analysis

Chapter 4: References

LIST OF TABLES

Table la: FAB Classification

Table Ib: Survival and Leukemic Transformation

Table 2: Sequencing Primers

Table 3: Smdy Patients

Table 4: NKlAMRE Deletion

Table 5: Differentiation of HL60, U937, and K562 Ce11 Lines

Table 6: DNA Samples on Hemopoietic Gene Expression Blot

vii

LIST OF FIGURES

Figure 1 :

Figure 2:

Figure 3:

Figwe 4:

Figure 5:

Figure 6:

Figure 7a:

Figure 7b:

Figure 8:

Figure 9:

Figure 10:

Figure 1 1:

Figure 12:

Figure 13:

Figure 14:

Figure 15:

Figure 16:

Figure 17:

Figure 1 8 :

Figure 19:

Figure 20:

The Hemopoietic Cascade

Schematic representation of 5q3 1433

MAPK Signaling

Eukaryotic Celi Cycle

Tet-On System

Sequence of NWAMRE

Sequence analysis of NKIAMRE

Sequence cornparison of NKIAMRE

Sequence alignment of MUAMRE

Expression of NKIAMRE in PC 12, H9C2, and L6 Ce11 Lines

NKIATRE Tet-On Cell Lines

Schematic of Fluorescent in situ Hybridization

Southern Blot of human BAC DNA for NKIATRE

Localization of NKIAMRE to 5q3 1

Southexn Blot of human genomic DNA for NKIAMRE

FISH on interphase nuclei

Expression of MUAMRE in hurnan bone marrow and leukemic ce11 lines

Expression of NKIAMRE in human leukernic ce11 lines following differentiation

Hernopoietic Gene Expression Blot for N K W

Hemopoietic Gene Expression Blot for p-actin

Gene Mapping of NKLAMRE

LIST OF ABBREWATIONS

ADP A.ML ATP ATF BAC CAK CDC CD1 CDK cDNA CKI CMIML CSFlR d m DEPC DIG DMSO DNA m Dox D-rr EDTA EGF EGFR EGR ERK FAB FBS FGF FITC FISH GO G1 G2 GABRAl GDP GMCSF Grb2 GRL GTP HA HOG HCl IL nzF INK J-NK J-NKK KIP

adenosine diphosphate acute myelogenous leukemia adenosine triphosphate activating tianscnption factor bacterial artificial chromosome CDK activating kinase ceIl division cycle cyciin-dependent kinase inhibitor cyclin-dependent kinase complimentary DNA cyclin-dependent kinase Ùihiïbitor chronic myelomonocytic leukemia teceptor for macrophage-colony stimulating factor deoxycytidine triphosphate diethyl pyrocarbnate digoxigenin dimethyl sulfoxide deoxyrr'bonucleic acid deoxyribonucleoside triphosphates deoxycyciine dithiothreito 1 ethylenediaminetetraacetic acid epidermal growth factor epidermal growth factor receptor early growth response extracellw signal-regulated kinase French, American, and British group fetal bovine senun fibroblast growth factor fluorescein isothiocyanate fluorescent in situ hybridization quiescent state first gap phase second gap phase gamma-aminobutyric acid A receptor alpha guanosine diphosphate granulocyte-macrophage colony stimulating factor growth factor receptor binding protein 2 glucocorticoid receptor guanosine triphosphate hernagglutinin high osmolarity glycerol response hydro~hloric acid interleukin interferon regdatory factor inhibitor of CDK c-Jun NI&-terminal kinase c-Jun NI&-terminal kinase kinase kinase inhibitor protein

KR LB LOH LPS M phase MAP MAPK MAPKK MAPKKK MAT MBP M D S MEK MEKK MKK MLK MPF mRNA NLS NTP PAC PAGE PCR pfu P U 4 PUR0 RA RAEB RAEB-t RARS RB RIF- 1 RNA RSK RT-PCR rtTA rTetR SAPK SDS SEK SH2 SH3 SOS S phase SSC SSCP TAK1 TCF- 1 TDY TE TGF

kinase dead version luria broth loss of heterozygosity lipopolysaccharide mitosis microtubule-associateci protein mitogen-activated protein kinase mitogen-activated protein kinase kinase mitogen-activated protein kinase kinase kinase menage a trois myelin basic protein myelodysplastic syndrome MAPKERK kinase MEK kinase MAP kinase kinase mixed lineage kinase maturation promoting factor rnessenger RNA nuclear localization signal ribonucleotide triphosphates P I artificial chromosome polyaaylamide gel electrophoresis polymerase chah reaction plaque fonning units phorbol- 12-myristate- 13-acetate puromycin refi-actory anemia refiactoxy anemia with excess blasts rehctoxy anemia with excess blasts in transformation refhctory anemia with ringed sideroblasts retinoblastorna protein radiation-induced fibrosarcoma n'bonucleic acid ribosomal protein S6 b e reverse transcriptase PCR reverse tet trânsactivator reverse tet repressor stress activated protein kinase sodium dodecyl sulphate SAPK/ERK kinase src homology 2 domain src homology 3 domain son of sevenless DNA synthesis phase sodium chloriddsodium citrate single strand conformation polyrnorphism TGF-beta-activated kinase- l T ce11 factor -nine-aspartate-tyrosine tris-EDTA transfonning growth factor

TNF-a TRE UV WT YAC

tumor necrosis factor-a tet responsive element ultraviolet wild type yeast artificial chromosome

LIST OF SYMBOLS

base pair degree celcius centimet er counts per minute kilo base kilodafton molar milligram millilitre millimeter dlimolar mole -''gram revolutions per minute lulits microcurie microgram microliter micromolar micromole

CHAPTER 1

INTRODUCTION

1.1 INTRODUCTION

1.1.1 Importance of MDS

A group of pre-rnalignant hematologic disorders, characterized by anemia

and abnormal myeloid diffêrentiation, have been recognized for over half a century (Lee

et al., 1993). These disorders are most fommonly refmed to as myelodysplastic

syndromes (MDS). Factors known to be associated with MDS are age and cytotoxic

therapy, especially alkylating agents and radiation therapy (Lee et al., 1993). Patients

with poor prognosis MDS can develop malignant acute myelogenous leukemia (AML),

which is rapidly fatai.

Patients are classified according to guidelines developed by a co-operative

French, American, and British @AB) group, based on blood and bone marrow

morphology. Patients are usually elderly women with a mean age at presentation of 67

years and occasionally suffer weight loss and fever or infections, bniising, and abnormal

bleeding (Lee et al., 1993). Most patients with MDS do not require treatment. In fact,

many patients never have symptoms related to MDS and eventually die of unrelated

causes (Lee et ai., 1993).

MDS is associated with chromosomal disorders of the hematopoietic progenitor.

Most commonly, an interstitial deletion involving the long arm of chromosome 5 (Se) is

obswved (Van den Berghe, 1974). MDS associated with a deletion in chromosome 5 is

most commonly refmed to as the Sq- syndrome. There is a variable patteni of

chromosomal loss among a predominant cluster* of hematopoietic growth regulating

genes within chromosome 5q, which may explain the diverse clinical presentation of this

syndrome. Many different groups have reported smali conseneci deletions of 5q3 1.1 and

are attempting to fùrther define the minimally deleted region.

1.1.2 The Hernopoietic System

The hierarchical organization of the hemopoietic system originates fiom a rare

subpopulation of multipotential hemopoietic stem cells (HSCs) present in the bone

marrow (TiU & McCulloch, 1980). The HSC has the ability to regenerate itself through

the process of self-renewal, thereby maintainhg mature blood ce11 production (Keller and

Snodgrass, 1990). The HSC differentiates into intmediate pluri-, tri- or bipotential

precursors which eventuaily become terminally-diflerentiated cells (Figure 1). These

mature cells encompass the myeloid lineage (erythrocytes, platelets, eosinophils,

neutrophils, macrophage) and the Iymphoid lineage (T cells and B cells).

Hernopoietic disorders such as MDS are characterized b y inappropriate

hemopoietic maturation leading to changes in the proportion of myeloid ce11 precursors.

Norrnally, myeloblasts represent less than 5% of the bone marrow. In contrast,

myeloblasts make up as much as 30% of the bone marrow in MDS patients (Lee et al.,

1993). in some cases, ringed sideroblasts or abnormal erythroblasts are observed. These

cells are identified by a brown ring around the nucleus attributed to an iron stain which

becomes incorporatecl in the rnitochondria.

The FAB classification of MDS consists of five clinifal syndromes: (1) rehctory

anemia (RA), (2) rehctory anemia with ~ g e d sideroblasts (RARS), (3) refractory

mernia with excess blasts (RAEB), (4) refiactory anemia with excess blasts in

transfomation (RAEB-t), and (5) chronic myelomonocytic leukemia (CMML) (Lee et

ai., 1993).

The criteria proposed by the FAB group as well as characteristic cytogenetic

abnorrnalities, such as 5q-, define the stage of the disorder (Tablela). Patients with bone

marrow myeloblasts representing less than 5% of hematopoietic cells are classified as RA

(Lee et al., 1993). RARS is classified as a patient with RA having ringed sideroblasts

representing 15% or more of erythroid precursors. Patients presenting with 5-20% of

bone marrow cells as myeloblasts are classified as RAEB, and those with 20 to 30%

m yeloblasts are classified as transformed RAEB (RAEB-t). Finally, monocytosis in

excess of 1 X 1 0 ' ~ is sufficient to classify a MDS as CMML, regardless of the other

morphologie features (Lee et al., 1993).

Evolution from MDS to acute myelogenous leukemia (AML) occurs infrequently

and varies with FAB category. Analysis of data fkom several studies indicates that

evolution to leukemia occurs in 19% of patients (Sam and Sanz, 1992). Leukemic

transformation ranges f?om 5% in RARS to 48% in RAEB-t with a median survival of 19

months (Sam and Sam, 1992) (Table lb). Patients presenting with RA and/or RARS are

considered to have a good prognosis and RAEB and/or RAEB-t a poorer one, while

TABLE la. Criteria for FAB classification of Myelodysplastic Syndromes (Lee et al.,

RARS CMML RAEB RAEB-t

---Borie Marrow-

Monocytes Blasts (%) (5 ( 5 >20 5-20

>2û-30 andor Auer rods

Ringed sideroblasts. The Ringed sideroblasts are expressed as a percentage of totai bone marrow cells as recommended by the FAB. More commonly, they are expressed as percent of total erythroid precursors. RA, rehctory anemia; RARS, RA with ringed sideroblasts; CMML, chronic myelomonocytic leukemia; RAEB, RA with excess blasts; RAEB-t, RAEB in transformation.

Ringed Sid* (96) c l 5 >15 - - -

TABLE 1 b. Survival and Leukemic Transformation Accordhg to FAB Subtype (Lee et al., 1993)

FAB Subtype (%) 1 Median Survivd in 1 % Leukemic

RARS (18) RAEB (28) RAEB-t (12) CMML (17) AI1 patients

RA (25) Transformation (Range)

1 1 (0-20) Months (Range)

3 7 ( 1 9-64)

RA, refiactory anemia; RARS, RA with ringed sideroblasts; CMML, chronic myelomonocytic leukemia; RAEB, RA with excess blasts; RAEB-t, RAEB in transformation.

CMML is intennediate both with respect to the likelihood of development of AML and to

survival (Lee et al., 1993).

Loss of ail or part of the long a m of chromosome 5 has been associated with a

reIatively benign clinical course characterized by rehctory anemia with excess blasts

(RAEB), modest leukopenia, macrocytic anemia, hypolobular rnegakaryocytes and a low

risk of transformation to acute leukemia (Tefferi et al., 1994; Mahmood et al., 1979).

The 5q- syndrome has been descnlbed in all FAB classes of myelodysplôstic syndromes

(MDS), in de novo acute myeloid 1eukeLnia (AML) and in leukemias and MDS secondary

to cytotoxic therapy (Lewis et al., 1995). Patients presenting with the de novo AML or

transfonning to AML, ofien display additional karyotypic abnomalities, suggesting that

important genetic modulators of hematopoietic growth and differentiation may be lost as

early events in al1 of these clinicai conditions (L,ewis, 1995).

1.1.4 5q- Syndrome

An abnormality of chromosome 5 in myeloid disorders was first documented by

Rowley et al. (1981), who observeci a loss of the entire chromosome or a deletion of the

long arm in c e k fkom 23 of 26 MDS patients. Other investigators, such as Le Beau et al.

(1986a), confïrmed this observation by demonstrating a chromosome 5 abnormality in

120 of 129 consecutive patients with MDS. Other abnomalities associated with the 5q-

syndrome include -7, +8, 1 lq-, 12p, and 20q- (Lee et al., 1993). Cytogenetic

abnomalities may therefore occur as a single abnormal chromosome or a more complex

karyotype, consisting of two or more chromosome defects, resulting in a poorer

prognosis. The occurrence and fiequency of these abnomalities varies considerably,

however, a region of consistent loss between bands 5q23-31 has been observed in aU

cases (Le Beau, l986a).

The high fiequency of loss of genetic material associated with chromosome 5

suggests that the loss of one or severai genes, in this region, may result in MDS and

subsequently AML. The genes encoding several growth factors and growth factor

receptors have been localized to this region. These include the interféron regdatory

factor ( m l ) , the gene for interleulàn 9, granulocyte-macrophage colony stimulahg

factor (GMCSF), and early growth response-1 gene (EGR1) (Wasmuth et al., 1989).

Atternpts have been made to identiQ these and other genes to define the muiimally

deleted region through a systernatic cornparison of a series of karyotypes derived fiom

myeloid disorders.

1.1.5 Smdest Commoniy Deleted Region

A region of consistent loss at band 5q3 1.1 has been observed in al1 cases of

patients with preleukemic myelodysplasia and acute myelogenous leukemia. Shown in

Figure 2 is a schematic representation of 5q3 1q33 illustrating the different studies which

have attempted to define the critical region. Several groups have identified a 2.4 Mb

region with proximal and distal ends consisting of the gene for interleukin 9, and the zinc

finger transcription factor, EGRl (Fairman et al., 1995; Le et al., 1993). In addition,

polymorphic loci have been used which provide markers that define the minirnaily

deleted region. Polymorphic loci are variations in DNA sequence that create or destroy

restriction-enzyme recognition sites. DSS89 is a two-allele polymorphism that destroys a

Sac1 restriction endonuclease site in the normal human population. Fairman et al. (1995)

dernonstrated that the polymorphic locus DSS89, flanked centromeric by IL9 and

telomeric by EGR-1, resides within the critical region. Many MDS patients are

characterized by loss of one DSS89 allele. The patient's genotype is heterozygous

defined by one wild-type D5S89 allele and loss of the other. Nagarajan et al. (1994b),

however, demonstrate loss of heterozygozity &OH) for D5S89 sequence in four patients

with the 5q deletion. That is the patient's genotype is characterized by loss of both

D5S89 alIeIes. Loss of both DSS89 alleles suggests that this region contains a gene that

may be involved in the progression of this disease.

More recently, Homgan et al. (L996), defineci a critically deleteci region of

approximately 1 Mb bounded by markers IL4 and the polymorphic locus DSS4 14. The

study fùrther defïned this region by examuiing the LOH of several intemal polymorphic

loci in patients with AML and MDS. PCR prirners were generated based on a panel of

pol ymorphic markers within the IL-9 to D5S4 1 4 intmal. PCR amplification

demonstrated LOH in seven of 29 sarnples collected fiom a series of AML and MDS

cases.

This region has been further refined, focussing on a 1 -0-1 -5 Mb segment, found

between polymorphic loci D5S479 and D5SS00 (Zhao et al., 1997b). This region was

defined by generating a physical map using fluoresence in siîu hybridization (FISH) of

P 1 (P AC), bacterial (BAC), and yeast arti ficial chromosomes. These arti ficial

chromosomes allow cloning of LOOkb (PACBAC) or lûûûkb (YAC) inserts which can

be used to generate a contig of the investigated region. The contig represents a series of

overlapping clones that define the physical map between two known markers. To refine

the commonly deleted segment of 5q3 1 in myeloid leukemias, FISH of PAC clones

within the contig was performed on metaphase cells fiom 28 patients with loss of 5q. in

this group, hemizygous deletions were detected, consistent with cytogenetic records,

however, no homozygous deletions were detected, in addition, two known genes, the

DNA-binding zinc finger protein EGRl and the phosphatase CDC25C have been placed

in this interval. The role of EGRl and CDC25C was examuied by single-strand

conformation polymorphisrn reveahg no mutations in myeloid cells with a 5q- deletion.

These studies have been helpfùl in de£ïning the critical region associated with the 5q-

syndrome in order to localize genes responsible for this disorder.

1 .l.6 Candidate Genes Mapping to Sq31

The majority of gens localized to 5q3 1 encode growth factors and growth factor

receptors. These include interleukui-3,4,5, and 9 and granulocyte-macrophage colony

stimulahg factor (GMCSF) (Huebner et al, 1985; Le Beau et al, 1986% 1987b, 1989~).

Others include the early growth response 1 protein @GR-1), a T ceil specific

transcription factor (TCF-7), endothelid ce11 growth factor (FGFA), and the

glucocorticoid receptor (GRL) (Wasmuth et al, 1989). With the exception of GMCSF,

each of the genes listed were found to be deleted in bone marrow aspirates fkom several

AML patients (Le Beau et al., 1987b).

Recently Zhao et al. (1998a) identifiai and mapped the gene encoding human

CDC23, a subunit of the anaphase-promoting cornplex. The gene for CDC23 is located

in the 1 to 1 . S M segment of Sq3 1, flanked by D5S479 and D5S500, which is proposed

to be the smallest commonly deleted region. The role of CDC23 in the 5q- syndrome was

evaluated by examining myeloid leukania celis for mutations. Arnong 14 patients with

MDS/AML examined, no mutations were detected suggesting that CDC23 is not

responsible for the pathogenesis of myeloid leukemias.

Another group, Zavadil et al. (1997), localized the gene encoding the

transcriptional transactivator SmadS to 5q3 1.1. This group demonstrated its hemizygous

loss in the human leukemia ce11 line, HL60, but were unable to demonstrate that the

Srnad5 gene was mutated in the remaining allele. A physical map of the 5q3 1.1 critical

region was generated which aliowed Zavadil to localize Srnad5 between markers D5S393

and DSS399, w i t b the minimally deleted region (Zavadil et al., 1997). FISH was

performed, using a pool of cosmid clones derived from a YAC spanning the gene

encoding SmadS, revealing loss of one SmadS allele. RT-PCR of the remaining SmadS

allele revealed that the leukemic phenotype in HL60 is not caused by Srnad5 mutations

(Zavadil, 1 997).

Finally, Willman et al. (1993) localized the gene encoding the interferon

regulatory factor- 1 (IRF- 1) to 5q3 1.1 revealing its consistent deletion of both alleles in 6

of 1 1 patients samples exarnined, with individual proportions as hi& as 20%. The [RF-1

gene is approxirnately 200 kb telomeric to IL-5 and 100 kb centromeric to CDC25C

which is outside the smailest commonly deleted region. iRF-1 demonstrates the

instability associateci with the 5q region may result in deletion or rearrangement of the

residual allele. Thus deletions or rearrangernents, rather than point mutations, may be

more fiequent at this genetic locus in human leukemia and MDS (Willman et al., 1993).

1.2 Tumor Suppressor Genes

Attempts at understanding the inherited predisposition to cancer have focused

attention on tumor suppressor genes. Turnor suppressor genes encode proteins involveci

in regulating ce11 growth and cd1 death. In human cancer, tumor suppressor genes o h

become inactivated, contributing to tumor development. Initiai experiments in somatic

ce11 genetics (Harris et al., 1969) demonstrated that one or more gene products could

suppress or inhiiit the growth of cells in a turnor. For example, a normal ce11 in culture

fiised witb a ce11 able to produce tumors resulted in a hybrid that grew well in culture but

no longer produced tumors in animals (Levine, 1992). Normal tumor suppressor genes in

the donor ce11 somehow repressed the unregulated growth potential of the -or.

Furthermore, in 197 1, Knudson demonstrated the role of inherited alterations in the

ongins of the childhood eye tumor, retinoblastoma. in most cases, retinoblastoma &ses

sporadically (1:20,000), however, in one third of the cases, the disease is heritable.

Tumors arise because of two separate mutations, at the RB locus, kading Knudson

( 1 97 1 ) to postulate his 'Ywo-hit" theory. The retinoblastoma susceptibility gene, RB,

defined the tumor suppressor gene that in the normal state inhibited tumor growth.

Loss of heterozygosity (LOH) results in ce11 proliferation in an uncontrolled manner

giving rise to a retinal tumor. Bilateral tumors, for example, result fiom the inheritence

of a single mutant allele fiom one parent followed by mutation in the remaïning allele

during fetal life or shortly after birth. The instability of ce11 regulation associated with

cancer demonstrates the need to identify tumor suppressor genes and understand how

tumor development occurs when they become inactivated. Defining the retinoblastoma

gene, for example, has aiiowed greater understanding of its role in retinal tumorigenesis

as well as in ce11 cycle control.

1.2.2 The Retinoblastoma Gene aad Gene Product

The RB gene, shown to encompass 2ûûkb of human chromosome 13, was isolated

by molecular cloning in 1986 (Fnend et al., 1986). Cytological studies revealed deletion

of chromosomal material surrounding and including the q 14 band of chromosome 13 in

sorne retinoblastomas (Yunïs and Rarnsey, 1978). interstitial deletions in this

chromosomal region providecl M e r evidence that RB was the gene critical for

retinoblastorna tumorigenesis. Furthermore, the majority of inactive, tumor associated

RE3 alleles had lost fûnction through subtle alterations, specifically point mutations.

Functional testing of the cloned gene revealed that loss of growth controi in

retinoblastoma cells and other tumor ce11 types occurs because of loss of RB gene

function. In fact, reintroduction of wild type RB through the use of retrovirus

transducing vectors results in inhibition of tumor ce11 growth and a profound loss of

tumorigenicity in vivo (Huang et al-, 1988; Bookstein et al., 1990). Furthermore,

phosphorylation of the RB protein disrupts the inhibitory complexes formed between the

RB protein and members of the E2F family of transcription factors resulting in

progression to the DNA synthesis phase of the ce11 cycle (Weinberg, 1995).

Mmy tumors, in addition to retinoblastoma, have demonstrateci consistent loss of

selected chromosomes or specific portions of chromosomes (deletions). in human

tumors, for example, deletions in chromosomes 1 lp and 13q are commonly found in

breast cancers and deletions of 3p, 13q, and 17p in lung cancers (Levine, 1992). These

observations have directed attention to the long arm of chromosome 5 as a likely tumor

suppressor locus which may be lost early in the development of maiignant hematopoiesis,

since accmuiation of additional karyotypic a b n o d i t i e s characterizes diseases of

higher grade (Tefferi et al., 1994). In patients with a 5q deletion, progression fiom MDS

to acute leukemia could also resdt fkom mutation or structural rearrangernent of the

single remaining tumor suppressor locus, analogous to the molecular pathogenesis of

retinoblastoma (Tefferi et al., 1994).

1 3 Signal Transduction

Extracellular stimuli are interpreted by multicellular organisms through intracellular

signaling pathways triggering cellular processes such as ce11 division, differentiation or

celf death. The stimulus is translated by a number of regulatory molecules leading to a

range of nuclea. and cytoplasmic events. Signal transduction cascades o h contai. a

series of kinase proteins which communicate through phosphorylation events. Kinases

catalyse the transfer of the terminal phosphate group of ATP to tyrosine, tfireonine, or

serine residues on their target substrates. Kinases, therefore, serve to mediate the

response of eukaryotic cells to extemai stimuli.

13.1 Conserved Kinase Features

Protein kinases are classified by catalytic domains which define enzymatic

activiw. The catalytic domain consists of a range of 250 to 300 amino acid residues

encompassing regions of high conservation interspersed with regioas o f low

conservation. An overall similarity exists in the catalytic domain of 65 protein kinases

studied (Hanks et al., 1988). Protein kinases consist of 12 major conserved subdomaim

sequentially numbered fiam subdomain I to subdomain XI, including two suMomaios

VIA and VI%. The nonconserved domains are thought to f o m looping regions allowing

essentially conserved domains to form the active site (Hanks et al., 1988). Intersperseci

within the conserveci regions are twelve invariant or nearly invariant amino acids which

play important roles in catal ysis. Notable residues include the consensus Gly-X-Gly-X-

X-Gly motif in subdomain I which is found in many nucleotide binding proteins in

addition to protein kinases (Wierenga and Hol, 1983). This motif provides an ATP-

binding pocket, forming an elbow around the nucleotide, with the first glycine contacting

the ribose ~g and the second glycine contacting the terminal phosphate (Hanks et al.,

1988).

Protein kinases have been classified into two subgroups based on substrate

specificity. The two broad classes include the s&ne/thrmnine-specific and the tyrosine-

specific protein kinases. Both classes share the conserved catalytic domain however

several stretches of amino acids among the subgroups are unique and can be used to

predict the kinase substrate specificity. Strong indicators of kinase specificity can be

found within two motifs in subdomain M and subdomaui W. The consensus sequence

DLKPEN, in nibdomain VI, predicts serine/threonine specific kinase activity, whereas

DLRAAN or DLAARN indicates tyrosine specificity. In subdomain VIII, the consensus

sequence PIVK/RWT/M predicts tyrosine specific kinase activity and GT/SXXY/FX for

se~e/threonine specificity.

13.2 Mitogen Activated Protein Kinase Signaling

The response to environmental stress is mediated by several adaptive signaling

pathways. Signaling pathways transmit messages received at the ce11 surface to the

nucleus which result in cytoplasmic and nuclear events. Key to this communication is

the rnitogen-activated protein kinase (MAPK) family of enzymes. The MAPK family

consists of severai isoforms each activated following different stimuli. Physiological

processes are o h regulated by signaling through different MAPK signal transduction

cascades. In the yeast Saccharomyces cerevisiae, four pathways have been described

which regulate ceIl processes such as sporuiation, ceil wall integrïty, mating, and osmotic

stability. In mammals, five distinct pathways have been described some of which

regulate processes such as osmotic stability, the response to stress, and differentiation.

A common backbone of three kinase molecules defines the MAPK signal

transduction pathway (Figure 3). Activation of a MAPKKK results in activation of a

MAPKK which finally activates a MAPK. The MAPKKKs are serindthreonine specific

which target the MAPKKs, a unique group of dual specificity kinases activahg MAPKs

through phosphorylation on threonine and tyrosine- The serindthreonine specific

MAPKs have a characteristic tripeptide motif Thr-X-Tyr present within the Liz loop

between the protein kinase subdomains VI1 and VlTI (Zhang et al., 1994).

Although multiple MAPK homologùes exist, many studies have focused on the

ERK (BouIton et al., 199 l), SAPK (Derijard et al., 1994; Kynakis et ai., 1994; Sluss et

aI., 1994; Kallimki et al., 1994), and p38 subgroups (Han et al., 1994; Lee et al., 1994;

Rouse et al., 1 994). The ERIC subgroup is activated by growth factors, while the SAPK

and p38 subgroups are activated by cytokines and environmental stress (Davis, 1994)

(Figure 3).

As mentioned these subgroups are activated by dual phosphorylation of the

tripeptide motif, however, each protein has a different X amino acid which characterizes

the substrate specificity.

13.3 The ERK Subgroup of MAPKs

In marnrnalian cells, the extracellular-signal-regulated kinase (ERK) farnily of

MAPK enzymes is involved in the regulation of différentiation and ce11 growth. The

ERK family is composed of p44 ERK and p42 ERK (aiso refmed to as ribosomal S6

kinase (RSK) kinase, MAP-2 kinase, MBP kinase, and MAPK) and was initially

Growth Factor

MEK

ERK ( T W

Stress

SEK

O

High Osmolarity, Stress

TAKl MLK3

Figure 3: The ERK, SAPK, and p38 signaling pathways. Each molecule represents a MAPK homologue which becomes activated following several different environmental stimuli. Growth factors lead to the activation of the ERK pathway while the SAPK pathway is activated by stress and p38 pathway by stress and high osmolarity.

identified by SturgiU and colleagues (1991). ERKs are proline-directeci protein kinases

that phosphorylate Serm-Pro motifs based on their ability to activate RSK, a kinase

thought to be involveci in niosomal protein S6 phosphorylation (Chung et al., 1991).

Activation of ERKs occurs by the dual specificity kinase MEK which recognizes and

phosphorylates the tripeptide phosphorylation motif Thr-Glu-Tyr. The MEK kinase in

response to growth stimulation, for example, becomes activated following

phosphorylation by the kinase Raf (a MAPKKK). Upstream regdation of the ERIC

signaling pathway is controlled by receptor tyrosine kinases which bemme activated

during meiotic maturation and upon exposure to various stimuli which include epidennal

growth factor (EGF), insulin, nerve growth factor, platelet derived growth factor, insulin-

like growth factor, and phorbol esters (Sanghera et al., 1990; Ahn et al., 1990; Hoshi et

al., 1988; Gomez et al., 1990; Ray and Sturgill, 1987).

Activation of ERK by EGF, for exampIe, occurs by the following mechanism. Some

receptor tyrosine kinases can dimerize upon EGF bindiag ieading to auto-

phosphorylation of the receptor cytoplasmic domains. The phospho-tyrosine residues of

the activated tyrosine receptor serve as dockîng sites for many molecules including the

adapter protein, Grb2. Grb2 is composed of an SH2 domain, which interacts with the

receptor phospho-tyrosine residues, and two SH3 domains which interact with the

proline-rich binding sites on the guanine-nucleotide exchange factor SOS (Lowenstein et

al., 1992; Egen et al., 1993). Relocation of SOS fiom the cytoplasm to the plasma

membrane allows for the interaction with the membrane-bound Ras protein. SOS

catalyses the exchange of GDP to GTP which results in the activation of Ras (Buday and

Downward, 1993) Activated Ras then binds to the N-terminal domain of the MAPKKK

enzyme Raf (Vojtek et al., 1993;Wame et al., 1993; Zhang et al., 1993). MEKl/MEK2

can then bind to the C-terminal catdytic domain of Raf and is phosphorylated by the Raf

serinelthreonuie kinase activity. Upon activation, MEKlMEK.2 phosphorylates the

threonine and tyrosine residues within the tripeptide motif of ERK(Nakie1ny et ai., 1992).

Consequently, active MAP kinase phosphorylates severai substrates including

transcription factors , kinases and other cytosolic proteins.

13.4 The JNK Subgroup of MAPKs

The JNK protein kinases (SAPKs), isolated fiom cyclohexamide-treated rats, are

described as protein bases with the ability to phosphorylate the transcription factor Jun.

Activation of cellular Jun (c-Jun) occurs through phosphorylation at Ser63 and Ser73 in

the amino-terminal domain (Hibi et al., 1993). Like the ERG, the c-Jun kinases are

proline-directed and activated upon phosphorylation in the tripeptide consensus motif

Thr-Pro-Tyr. Although they are 4045% identical to ERKs 1 /2 they are distinct fiom the

ERKs in their substrate specificity. Furthexmore, while Erks are activated by mitogens or

phorbal esters, JNK activation is associated with the response to stress factors such as UV

irradiation, cycloheximide, heat shock, sodium arsenite, and TNF-a (Derijard et ai.,

1994; Kyriakis et al., 1994; Pombo et al., 1994).

SAPK is activated by dual phosphorylation of Thr and Tyr by the protein kinase

SAPK/ERK kinase- 1, SEK 1 (also refmed to as MKK4 and JNKK). The specificity of

SEKl was detennined by assessing the activation of SAPK and ERKI under a range of

different environmental factors. SEKl was initially identified based on its ability to

activate both SAPK and ERIC However, SEKl drarnatically activates SAPKs in

response to stress but does not activate ERK after mitogenic stimulation (Sanchez, 1994)-

Activation of SEKl occurs through the serine-threonine specific kinase MEKK1.

MEKKl was originally characterized as a protein kinase acting on MEK (Yan et al.,

1994). Overexpression of MEKKl does result in phosphorylation and activation of

MEK, however, at physiologic levels, activation of the SAPK pathway through

coincident phosphorylation of SEKl is observeci (Yan et al., 1994).

13.5 The p38 Subgroup of MAPKs

HOGl and PBS2 are two yeast genes required for restoring the osmotic gradient

across the ce11 membrane in response to increased extemal osmolarity (Brewster et al,

1993). HOGl is a member of the MAPK family which is activated by PBS2, a member

of the MAPKK family. Yeast strains lacking these genes, identified by screening

O smoregulation-de fective mutants, are unable to g o w on high-osrnolarity medium. The

HOGl protein contains the signature 300 amino acid catalytic kinase subdomain and

retains the tripeptide motif, Thr-Gly-Tyr. The kinase is most similar to the other MAPK

members: FUS3 and KSS1, requred for the mating pheromone response and ERKI,

required for the growth response.

The mammalian homologue of HOGl, p38, was first identified as a protein kinase

that is phosphorylated on tyrosine in response to endotoxin nom Gram-negative bacteria

(lipopolysaccharide), hyperosmolar medium, interieukin-1, and heat shock (Han et al.,

1994; Rouse et al., 1994). Both JNKl and p38 can substitute for stress-activated HOGL

in S. cerevisiue suggesting that the p38 may be related to the INK signal transduction

pathway (Han et al-, 1994).

13.6 Funetionai Sepmation of MAPK Pathways

The recognition that MAPK pathways share a common backbone

raises questions regarding the specificity of these pathways. In vivo studies suggest that

the signal transduction pathways are independent. in S. cerevisiae, three MAPK

pathways exist that fùnction in rnating, cell-wall biosynthesis, and osmoregulation.

When one of these pathways is rendered non-fùnctional the other two pathways remain

active. 'ïhat is, ceus lacking HOGl are defective in sensing osmotic stress but are d l 1

capable of mating (Brewster et al., 1993).

in contrast, SEK1 has been s h o w to phosphorylate p38 in vin0 and in transfected

COS cells, indicating that SAPK and p38 may both be regulated through this kinase (Lin

et al., 1995). A more recent study, however, demonstrateci that SEKL associates in vivo

with SAPK but not p38 (Zanke et al., 1996). Similarly, the expression of a dominant-

negative version of SEK (SEK-AL) iabibits the activation of SAPK but not p38 after

hyperosrnolar stress in RIF- 1 cells (Zanke et al., 1996). These two observations suggests

that although both pathways respond to similar agonists, SAPK and p38 are activated

independently implying a different physiological role for each kinase.

1.4 The Edcaryotic CeU Cycle

The process of cell division results in equal segregation of one copy of each

duplicated chromosome into each of two daughter cells. Regulation of ce11 division is

critical to the normal development of a multicellular organism. Loss of control, due to

the inability to arrest the ce11 cycle, apoptosis a d o r differentiation, is the basis for the

development of cancer. The study of the cell cycle has lead to an understanding of the

rnechanisrn responsible for keepiag the ce11 cycle in order. The ce11 cycle is typically

divided into four stages. The ce11 begins chromosomal replication during the S

(synthesis) phase which results in cell division d e r the M (mitosis) phase with each

phase being separateci by gaps of varying length called G1 and G2 (Grana and Reddy,

1995). A mal1 number of protein kinases regulate the transition between these phases,

termed 'ccheckpoints", to ensure the proper tirning and coordination of ce11 cycle events

(Figure 4). The kinases are heterodimeric proteins consisting of the regdatory subunit or

cyclin and the catalytic subunit or cyclin-dependent kinase (CDK) (Morgan, 1995).

1.4.1 Cyclin Dependent Kinases (CDKs)

Initial studies in the budding yeast, S. cerevisiae, and the distantly related fission,

yeast S. pombe, have been instrumental in isolating the molecules responsible for ce11

cycle progression. The cdc2 gene of S. pombe and the homologous CDC28 gene in S.

cerevisiae, were discovered by isolathg mutants that were blocked at specific steps of the

cell cycle or that extil'bit altered reguiation of the ce11 cycle (Lee and Nurse, 1987). The

defect was based on temperature sensitivity preventing mutant yeast from growing on

selective medium. Mutants were identified based on morphology as yeast progressed

throughout the ce11 cycle. For example, mutant S. cerevisiae cells with a cell cycle defect

arrest in the budding process at the non-permissive temperature. These cells were

therefore called cdc (cell division cycle) mutants- The wild-~pe alleles were isolated by

rescuing mutant cells, at the non-permissive temperature, with a libfary prepared fkom

wild-type cells (Lee and Nurse, 1987). The wild-type allele complements the mutant cell

and allows colony growth.

A mammalian homologue of cdc2 was identified based on its ability to rescue a

temperature sensitive S. pombe strain (Lee and Nurse, 1987). In mamrnalian cells, CDC2

controls the GuM transition and the cdc2 homologues, CDK2 and CDK4, control the

G 1/S transition (Morgan, 1995). CDKs are closely related in size (35-40kD) and share

greater than 40% sequence identity. The typical CDK contains a 300 amino acid

catalytic core which is regulated by phosphorylation and dephosphorylation (Gao and

Zelenka, 1997). Substrate binding is blocked by an extended loop texmed the T loop

which must be phosphorylated at a position corresponding to Thr 16 1 of yeast cdc2. In

addition, negative reguiation occurs by phosphorylation at Thrl4 and Tyr1 5 of yeast cdc2

by the activity of cyclin-dependent kinase inhi'bitors. The final condition required for the

ce11 to proceed into S phase or mitosis is the availability of a group of molecules texmed,

cyclins.

1.4.2 Cychs

Although CDKs are constitutively expressed through the ce11 cycle their activity

is dependent on the transient expression of cyclins. Cyclins are defined by a 100 amino

acid, cyclin box, which is responsible for CDK binding and activation (Kobayashi et al.,

1992; Lees and Harlow, 1993). Cyclins are fwther dehed by the stage at which they are

expressed. For exarnple, cyclins D and E are observed at the G 1 /S transition while cyclin

A and B are generally observed at the GUM transition. Although synthesis is relatively

constant throughout the ce11 cycle, oscillation occurs because of marked periods of

degradation during mitosis. For example, in the earl y Xenopus embryo, oscillations in

the mitotic cyclin, cyctin B, occurs by degradation involving the ubiquitin-dependent

proteolytic machinery. Cyclin B, like many other cyclins, contains an N-terminal cyclin

destruction box which provides a recognition site for a protein that directs poly-

ubiquitination. Poly-ubiquitination involves the covalent attachment of ubiquitin to

residues conferred by PEST amino acid sequences that are carboxy-terminal to the cyclin

box (Glotzer et al., 1991).

1 . 4 Cych-CDK Complexes

The transition £rom GUS is marked by the association of a D-type cyclin and

either CDK4 or CDK6 dependhg on the cell type (Matsushime et al., 199 1 ; Meyerson

and Harlow, 1994). Cyclin levels are fairly constant throughout the ce11 cycIe with a

modest accumulation observed in late G1. Cyclin D levels are absent in quiescent ceUs

and their levels are dependent on the constant supply of growth factors. Cyclin D is

required for passage through the restriction point as fibroblasts injected with anti-cyclin

D 1 antibodies are unable to progress fiom G 1 to S phase (Baldin et al., 1993).

The subsequent activation of CDK2 is associated with the expression of cyclin E.

Both cyclin D and E are rate-lïrniting steps to the GUS transition as ectopic expression of

either cyclin can aaxlerate the Gl phase and can complement Gl cyclin mutants fiom

yeast (Sherr, 1994; Heichman and Roberts, 1994). Like cyclin D, micro-injection of an&

cyclin E or anti-CDK2 anti idies arrest cells in GI, indicating that both are required for

progression to S phase (Pagano et al., 1993).

DNA replication is dependent upon the synthesis of cyclin A as microinjection of

anti-cyclin A antiiodies r e d t s in inhibition of DNA synthesis (Labbe et al., 1989).

Furthermore, cyclin A and CDK2 have been shown to localize to the nucleus which

indicates that this protein cornplex is involved in the onset of DNA replication (Cardoso

et al., 1993). Finally entry into mitosis is controlled by the Maturation Promoting Factor

(MPF), which is composed of cdc2 and cyclin B (reviewed Grana and Reddy, 1995).

1.4.4 CDK Activation by Phosphory latïon

As mentioned, CDK activation is regulated by the binding of a cyclin as well as

phosphorylation at a consewed threonine residue (Thr 16 1 in hurnan CDC2, Tbr 160 in

CDK2). The crystal structure of CDK2 shows that it rernains in an inactive state because

the substrate binding site is blocked by the T loop (De Bondt et al., 1993). The threonine

residue lies within the T loop and must be phosphorylated potentially enhancing CDK

binding to its cyciin partner (Ducommum et al., 1 99 1).

Phosphorylation of this critical residue is performed by the enzyme, CDK

activating kinase ( C M ) . CAK is a muiti-subunit enzyme consisting of the CDK-related

protein kinase tenned CDK7, a cyclin binding partner termed cyclin H, and an assembly

factor termed MAT1 Fisher and Morgan, 1994; Matsuoka et al., 1994; Makela et al.,

1994). CDK7, Like its substrates, is potentidy regulated by phosphorylation on Thri70

(human CDK7) as mutation of this residue greatly decreases kinase activity. Both

Starfish and Xenoplcs CDK7 can substitute for the mammalian CDK7/cyclinH/MATI

complex by phosphorylaîing CDC2 suggesting that a single CAK may be responsible for

activating al1 of the major CDKs (Fisher and Morgan, 1994).

The oscillation of Thr 1 6O/ 1 6 1 phosphorylation parallels the levels of the cyclins

during the ce11 cycle. Changes in phosphorylation are probably not due to changes in

CAK activity, but the ability of cyclin binding to stimulate phosphorylation, suggesting

that CAK activity is not a rate-limiting step during normal cell proliferation (Matsuoka et

al., 1994).

1 -4.5 CDK Inhibition by Phosphorylation

Activation of CDKs by phosphorylation of Thr16OI 16 1 and binding to cyclin

would suggest that inhibition could occur through phosphorylation or cyclin degradation.

CDK-cyclin complexes can be inhibited by phosphorylation at two sites near the amino

terminus (Thrl4 and Tyr1 5 in human CDC2 and CDK) (reviewed by Morgan, 1995).

Although the mechanism of inhiiition is unknown, these residues are located in the Gly-

X-Gly-X-X-Gly ATP binding motif Phosphorylation at these residues may affect ATP

binding or prevent ATP fkom being catalysed.

Phosphorylation at Thrl4 and Tyr15 provides a mechanism of keeping CDKs

inactive until the appropnate time in the ce11 cycle. For example, CDC2 phosphorylation

at these residues parallels the rise in cyclin B levels that occurs as cells approach mitosis

(Krek and Nigg, 1 99 I ;Solomon et al., t 990). The CDC2-cyclinB cornplex, therefore,

remains inactive until dephosphorylation at nir14 and Tyr15 occurs at the end of G2.

In S. pombe, the Weel kinase phosphorylates Tyr1 5 after cyclin binding.

Although Weel is capable of phosphorylating peptide substrates on both threonine and

tyrosine residues it phosphorylates CDC2 in vitro at Tyr15 but not Thrl4, supporting the

existence of a separate T M 4 kinase (Parker and Piwnica-Worms, 1992).

Both Thrl4 and Tyr15 are dephosphorylated by a dual-specificity phosphatase

called CDC25. The activity of CDC25 peaks during mitosis resulting fiom increased N-

terminal phosphorylation. Recent studies suggest that CDC2 may be responsible for this

phosphorylation forming the basis for a positive-feedback loop (Hofnnan et al., 1993;

Inimi, 1993).

1.4.6 CDK Inhibition by Iahibitory Subunits (CKIs)

A further mechanism of CDK-cyclin inhibition focuses on a diverse family of

proteins, termed cyclin-dependent kinase inhibitors (CKIs). Among the family members

identified, the mamrnalian CKIs fa11 into two classes. Class 1 consists of the iNK family

members: pl6 MK4a , pl s ~ ~ ~ ~ , p18MKk, and 1 9 ~ " while class 2 consists of the KLP

family members: p2 1 CIP 1 /WAF 1 , p27m1, and p57uP2 (review by Morgan, 1995).

Inhibition occurs by CKI binding to Thr 16011 6 1 phosphorylated CDKkyclin

complexes resulting in inhibition of kinase activity. CKI's are transcriptionally

regulated. For exampie, p21 transcription is induced by p53, following DNA damage, or

senescence. The p21 protein, binds and inhibits a variety of cyclin-CDK complexes

including cyclinD-CDK4 and cyclùiElA-CDK2 (Harper et ai., 1993).

Ce11 cycle status is o h associated with the levels of CKis. In murine Balbk-

3T3 fibroblasts depriveci of semm mitogens, p27 accumulates resulting in GL ce11 cycle

arrest (Coats et al., 1996). Ce11 cycle arrest of Balbk-3T3 cells was comelated with

down-regulation of the cyclinE-CDK2 and cyc1in.A-CDK2 protein kinases- The length of

G1 also correlates with the amount of p27 expressed in proliferating cells. Increased

amounts of p27 prevent CDK activation resulting in ce11 cycle arrest, whereas, decreased

p27 expression results in premature CDK activation and a shortening of the G1 (Coats et

ai., 1996).

1.5 Cdc2-related Protein Kinases

Among the CDK molecules previously described, a group of cdc2-related

molecules exist with greater than 50% amino acid identity with p34CdE2. Seven novel

genes have been described (Meyerson et al., 1992). Although none of these kinases have

been shown to associate with a cydin partner, the ernerging group has been named after

the putative cyclin binding motif, PSTAIRE. The novel group consists of PITSLRE,

PCTAIRE, PITAIRE, PITALRE, PISSLRE, KKIAMRE, and KEUALRE (reviewed by

Grana and Reddy, 1995).

The presence of a large group of cdc2-related kinases suggests that their

expression or function may be limitecl to certain cell types correspondhg with restricted

patterns of mRNA expression. in fact, Northem blot analysis of RNA corn various

human sources connmis that expression varies across different ceil lines, with each gene

showing a distinct pattern. One such pattem that may refiect a fimetional difference was

seen by cornparhg the expression levels of the cdk2 and PSSALRE transcripts

(Meyerson et al., 1992). Both genes are abundantly expressed in various ce11 lines but

show divergent patterns of expression in primary tissues. For exarnple, cdk2 is expressed

in rapidly dividing tissues such as the placenta, however, PSSALRE expression is limited

to differentiated tissues such as the brain, which contains few dividing cells.

The most recentiy identified cdc2-related kinase is the epidexmal growth factor-

stirnuiated p56 KKZAMRE (Taglienti et al., 1996). KKIAMRE was cloned by screening

a human cDNA library with a PCR product amplified using degenerate oligonucleotide

primas based on known MAPK sequences. Sequence analysis revealed that it not only

shared homology to cdc2 but also to the MAPK kinase group. The 56 kD protein also

shares 50% amino acid identity to the p42 KKIALRE, intially identified by Meyerson et

al. (1 992). Both KKIAMRE and KKIALRE are expressed in differentiated tissues such

as the kidney, brain and lung as well as testis @56 KKIAMRE) and ovary @42

KKIALRE). They both also contain the conserved tripeptide phosphorylation motif Thr-

Asp-Tyr located in subdomain W. interestingly, the tripeptide motif differs from the

other MAPK members, (ERKIR, INK, and p38) suggesting the emergence of a novel

signahg pathway. Furthermore, both KKIAMRE and KKIALRE protein kinase activity

can be stimulated by epidemal growth factor, however, in contrast with the MAPKs,

phosphory lation within the tri peptide motif is not necessary for activation.

1.6 Summary

Protein phosphorylation is central to signal transduction and to cell cycle

response. The cdc2-related kinases represent an interesting hybrid of MAPK and CDK

members which may have important roles in growth factor induced ce11 cycle change.

This group of molecules will corne under greater understanding and their roles in biology

will be better understood- Furthermore, the possible roles of dc2-related molecules as

tumor suppressor genes will be important in understanding their relevance to disease.

There are several examples of kinase molecules that also fdl into the class of

tumor suppressor genes. For example, a recent publication demonstrated the deletion and

mutation of SEKl in human pancreatic, lung, breast, testicle, and colorectal cancer ce11

lines. This study suggests that SEKl, an important SAPK activator, may play an

additional role in -or suppression (Teng et al., 1997). Furthennore, some of the

PITSLRE genes have been altered in human neuroblastoma tumors, suggesting that they

may be tumor suppressors (Lahti et al., 1995).

Chapter 2 of this thesis describes the molecular cloning of MUAMRE, the human

homologue to the MAPWCDK related protein kinase NKIATRE. In addition, Chapter 3

describes the chromosomal localization of NKIAMRE to 5q3 1.1 and its loss in leukemic

blasts with the 5q- syndrome. These observations suggest that NK[AMRE may be

another example of a kinase that serves as a tirmor suppressor gene. Future experiments

dedicated to understanding the role NKLAMRE plays in the context of the ce11 will be

crucial to proving its candidacy.

The Molecular Clonhg of NKIAMRE, the Human Eomologue to the MAPWCDK-related Protein Kinase NKIATRE

Parts of this chapter have been accepted in: "Identification of MUAMRE, the human homologue to the MAP WC DK-related protein kinase NKIATRE, and its loss in leukemic blasts with the 5q- Syndrome." M. Midmer, R. Haq, J.A. Squire, B. Zanke Cancer Research

Parts of this chapter have been submitted in: "NUATRE is a novel multi-isofom kinase related to both MAPKs and cyclin-dependent kinases." Haq, R., Randall, S., Midmer, M., Zanke, B.W. Oncogene

2.1 INTRODUCTION

Eukaryotic cells use a variety of conserveci intracellular signaling paîhways in

order to respond to the extemal environment leading to outcornes such as ce11 division,

differentiation, or death. Among these pathways, the mitogen-activated protein kinase

(MAPK) family plays a key role in regulating growth and differentiation, osmotic

stability, and the response to stress stimuli. MAPK signaling is important in regulating

transcription as well as controlling ce11 cycle progression by regulating, directly or

indirectl y, the activi ties of the cyclinaependent kinases (CDK) and their regul atory

subuni ts, the cyclins.

In a previous report, a novel serine/threonine kinase was àescribed, based on its

homology to both the MAPKs and CDKs (Haq et al., 1 999). Its similarity to the MAPKs

resides in its TDY motif in subdomain VI11 and its activation by epidennal growth factor

(EGF). Sirnilar to the CDKs are the potential regdatory amino acids (S14 and Y15)

which conform to the ATP binding motif (GXGXXG). It also contains the NKIATRE

domain, for which it is named, which is a potentid binding site for a cyclin. The gene is

expressed exclusiveIy in difkentiated tissues and may have a role in negatively

regulating ce11 growth and differentiation.

NKIATRE was cloned by screening both a rat jejunum and brain cDNA library

with a PCR product amplifid h m MAPK degenerate primers. Three independent

clones were obtained. Two brain clones, each named NKIATRE a, contain the entire

open reading h e encoding a protein of 505 amino acids, with a predicted molecdar

weight of 57kD. The jejunum clone, named NKIATRE p, is himcated with a premahire

stop codon after amino acid 457, with a predicted molecular weight of 52kD.

With the goal of isolating the human NKIATRE homologue, a human fetal heart

cDNA library was screened with a human expressed sequence tag (EST) which had

sequence similarity to the rat NKIATRE gene. The human library isolate contained the

expected EST sequence and had 79% amino acid identity to the rat NKIATRE f3 isoforrn.

The hurnan clone was fiirther compareci to the rat NKIATRE to reveal that the core

catalytic domain with the various structural motifs is conserveci.

2.2 MATERIALS AND METHODS

2.2.1 Molecular cloning, PCR, and sequencing

To clone the NKJATRE hwnan homologue, one unspliced 394 base pair

sequence, Genbank accession R.21498, having 89% nucleotide identity over a 113bp

exonic region was used to screen a human fetal heart cDNA library. The sequence

encoded an expressed sequence tag (EST), which represents a cDNA amplified fiom

human mRNA based on poly-A priming. There were several ESTs in the database that

had sequence identity to NKIATRE. R21498 was chosen as it contained the largest

cloned fiagrnent which would be needed to generate a probe. The EST was obtained

fiom Genome Systems and was sequenced using vector-specific primers to c o n h its

identity. A 426bp Notl-HindIII hgment digested from the Lafinid BA cloning vector

was used as a probe.

For Iïbrary screening, plating cultures were prepared by inocdating LB broth,

supplemented with 0.2% maltose and lûmM MgS04 , with a single colony of the

bacterial sfraui Y 1090. Cultures were grown ovemight at 3 7 ' ~ and spun down for 10

minutes at 2000 rpm and resuspended in lOmM MgS04 at an ODW of 0.5. To 6 0 0 ~ 1 of

the cells, 5 x 10' p h of the library were added and the solution was gently mixed and

incubated at 37% for 15 minutes. To each of 15, 1 Sûtnm, NZY plates, the phage and

bacteria mixture added to 8ml of NZY top agar was poured. The plates were incubated at

3 7 ' ~ until the bacteria cells were completely lysed (approximately 12 hours).

The phage DNA fiom the lyseci bacteria cells was tramferreci to duplicate

Hybond-N membranes (Amersham, Amersham UK) for 2 minutes. After transfer, the

filters were denatured for 2 minutes in a solution of 1.5M NaCl and 0.5M NaOH, and

neutralized for 5 minutes in 1 .SM NaCl and 0.5M Tris-HCL @Hg). The filters were then

bnefly rinsed in 2X SSC and ailowed to dry at room temperature. DNA was UV

crosslinked to the filters using the UV Stratalinker (Stratagene, La Joua CA, USA) and

the agar plates were stored at 4 ' ~ .

For screening, the filfers were pre-hybridized in a solution of 50% formamide, 5X

Denhardt's reagent, 6X sodium saline citrate (SSC), 0.5% sodium dodecyl sulfate (SDS),

and 50 pg/ml denatured, hgmented salmon sperm DNA for three hours at 4 2 ' ~ .

Hybridization was carried out overnight at 4 2 ' ~ in a solution of 50% fomamide, 1X

Denhardt's reagent, 6X sodium saline citrate (SSC), 0.5% sodium dodecyl sulfate (SDS),

10% dextran sulphate, and 10 pg/ml denaîured, fiagmented salmon spemi DNA.

The probe was made fiom a 426bp Norl-HindID hgment digested fiom the

human EST in the Lannid BA cloning vector. 30ng of the digested insert was

radiolabelled with 50 pCi of [ U - ~ ~ P I ~ C T P using the Multiprime DNA labelluig system

(Amersham, Amershm UK) and unincorporated label was removed by gel filtration

through lcc of Sephadex G-50.

Following hybridization, the filters were washed twice at 6 5 ' ~ in lx SSC and

0.1% SDS for thkty minutes and once at 6 5 ' ~ in O. LX SSC and 0.1% SDS for thirîy

minutes. Filters were exposed to film ovemight at - 7 0 ' ~ with an intensifjring screen.

The hybndizing plaques were purified by secondary and tertiary screpns, as desmbed

above. Excision of the cDNA inserts, fiom the purified phage, was perfionned by usuig

QIAGEN's Lambda phage DNA extraction kit The insert was PCR amplifie. and

cloned into the pCR2.1 vector (Invitrogen). in brief, PCR was performed in a 1 ûûul

reaction consisting of 50pmol of h g 1 1 fornard and reverse primers (Table 2), 4mM

Mg& 0.4m.M dNTP, 1X GibcoBRL PCR buffer, and 0.2U Taq DNA Polymerase

(GibcoBRL). Before the addition of Taq DNA polymerase each reaction was heated to

94OC for Sminutes. For 30 cycles the reaction mixtures were denatured at 94°C for 2min.,

allowed to anneal at SO°C for Lmin., and extended at 72OC for lmin30s, and finally the

reaction was extended at 72°C for 7min.

The 100%~ product was sequenced at the York University Core Molecular

Biology Facility (Toronto) using PCR based methodology. Sequencing primers used

included the Forward Ml3 Primer and the Reverse Ml3 Primer (Perkin Elmer,

Branchburg NJ, USA)(Table 2). in addition, two primers, 8 Fonvard and 8 Reverse,

were constnicted to detexmine intemal sequence not obtained through the use of the

vector-specific primers (Table 2).

TABLE 2: Sequencing Pnmers. The nucleotide sequences of the primers used for PCR and sequencing.

PRIMER NAME SEQUENCE 5'03'

Forward Ml 3/LacZ

I

H594 ATG GTC GAC CAC CTT CAT ACT CTT 1

GCC AGG GTI' TTC CCA GTC ACG A

Reverse M 1 3ILacZ

hgt 1 1 Forward

hgt 1 1 Reverse

HS9 1

H592

H593

GAG CGG ATA CAA TIT CAC ACA GG

GGT GGC GAC GAC TCC TGG AGC CCG

TTG ACA CCA GAC CAA CTG GTA ATG

ATG GTC GAC ATG GAG ATG TAT GAA ACC

ATG GTC GAC ATG GAG TAA ATC CAA ATC

ATG GTC GAC TTC TlT CAT CTT TCC T

8 Forward GAT CTC CTG GGC TTT T

1

8 Reverse GGC AGA TAT AGT TCA T

2.2.2 Northern BIot Analysis

Total cellular RNA was prepared using the RNeasy Mini Kit (QIAGEN). in some

cases, lOpg of poly(A)+RNA was prepared ushg the Oligotex mRNA Midi Kit

(QIAGEN). Ten pg of either total or poly(A)+RNA diluted in 1X RNA loading buffer

(20mM MOPS, 5mM sodium citrate, 1mM EDTA, 0.25M fomaldehyde) fiom various

ce11 Iines was used to generate the blots. RNA was fiactionated for three hours by

electrophoresis on a 1.2% fomaldehyde agarose gel. Prior to fhctionation the RNA was

heated at 6 5 ' ~ for 5 minutes. RNA was transferred to a nylon membrane by capillary

transfer (Sambrook et al., 1989). The Northern blots were probed with either the 1 S kb

BamH 1 fragment of the rat cDNA, the 426bp Not I-HindIII human EST or the 1005bp

library human NKIATRE purifieci clone. The probes were radiolabeled as described in

section 2.2.1. To control for the toading and quality of RNA in each lane, a rat p-actin

probe was used. Prehybndization, hybridization and wash conditions were carried out as

described in section 2.2.1. Filters were exposed ovemight ont0 a Phosphohager Screen

and visualized by a Phosphohager (Molecula. Dynamics) using the imageQuant

software (Molecdar D ynamics).

2.2.3 CeM culture and transfection

HeLa ceiis, maintaineci in Ddbecco's Modified Eagle's Medium (DMEM,

GibcoBRL) containing 1 0% fetal bovine senmi (FBS) (GibcoBRL), were transiently

transfected with rat pCDNA3-NKIATRE-HA and vector alone. Prior to transfection,

80% confluent cells were exposed to 150 pl vaccinia virus directing the expression of Ti

RNA polyrnerase (Blakely et al., 1991), in 2.5d media without sediOOmm plate for

one h o u with rocking. To media (41x11) without serum, liposomes (60~1) and rat

pcDNA3-NKIATRE-HA (10 pg) or vector alone were incubated for 30 minutes (Felgner

et al., 1987). After one hour the 4ml mixture. containhg the T7 polymerase-driven

expression plasmids, was added to the lOOmm plates for four hours without rockuig.

After four hours, the plates were washed and lOml media with 10% serum was added and

the plates were incubated for 20 hours at 37'~. After 20 hours, the media was aspirated,

the plates were rinsed with Sm1 1 X PBS and trypsinized for 5 minutes at 37'~. Senun

containing media was added to neutralize the trypsin and the cells were centrifùged for 5

minutes at 1 SOûrpm. The pellet was then us& to extract RNA as describeci in 2.2.2.

2.23 Tet-On CeU Lines

To generate a stable ce11 line the Tet-On Gene Expression Systern was used (Figure

5). The Tet-On system is based on a "reverse" Tet repressor (rTetR) which binds the

TRE in the presence of deoxycycline @ox) (Hillen&Berens, 1994; Gossen, 1995).

Transcription 'Jl VI

m m

Integrated copy of pUHD 1 0.3 response plasmid v

Figure 5: Schematic of gene regulation in the Tet-On System. The reverse tet-responsive transcriptional activator (rtTA) is a fusion of the "reverse" Tet repressor (rTetR) to the VP16 activation domain of herpes simplex virus. The rtTA binds the tet responsive element (TRE) and activates transcription in the presence of Dox.

wtien rTetR is fûsed to the VP16 activation domai. a "reverse" tTA @TA) is created

that activates transcription in the presence of Dox. The negatively charged VP 16 domain

fbnctions as a transcriptional activation domain in mammalian cells (Triezenberg et al.,

1988). The rtTA is encoded by the pTet-On regdatory plamid which had been

previously stably transfected into MH3T3 172 -1- PURO cells. The Tet-On System

utilizes the plasxnid vector pUHD 10.3 which contains the promoter and operator elements

required to control gene expression by altering Dox concentration. Both NKIATRE

wildtype (WT), and a kinase dead mutant NKLATRE KR were cloned into pUHD 10.3.

The kinase dead mutant was generated by mutating a lysine residue (K-22) to arginine

(R-22). This lysine residue, as part of the ATP binding domain, is essential for cataiytic

activity (Tien et al., 1990) Both constnicts were epitope tagged with HA

(hemaglutinin).

The constmcts were transiently transfected into NIH3T3 172 -1- PURO cells in

order to confim expression. In brief, 5 pg of WT, KR, or empty vector was transfected

using Fugene 6 Transfection Reagent (Boehringer Mannheim) into subconfluent cells

grown in 60mm dishes. After three days, the ceus were lysed in hypotonie lysis buffer

(25mM Tris pH 7.5, ImM EDTA, 0.2mM EGTA, 1mM DTT, 1X protease inhibitors, 1X

aproteninfleupeptin, 1X PMSF) for 20 minutes. After scraping, passing the lysate

through a 22 G needle, and centrifbgation at 10,000 rpm for 10 minutes, the supernatant

was added to anti-HA antibody and rocked at 4Oc for one hour. The lysate was then

transfmed to 50 pl of protein A beads (Pharmacia Biotech) and rocked at 4 ' ~ for one

hou. The beads were then washed three times in Id lysis buffer, added to 30 pl protein

loading dye (50mM Tris-HCL pH6.8, lOOmM DTT, 2%SDS, 0.1% BPB, 10% glycerol),

and boiled for 5 minutes,

The smples were fiactionated by electmphoresis on an 8% SDS-Polyacrylamide

Gel (8% acrylamide mix, 0.1% SDS, 0.375 M Tris (pH 8.8), 0.1% ammonium

persulphate, and 0.06% TEMED) for two hours at 100mA. The gel was transferred to an

Immobilon-P membrane (Millipore) for 1 hour at 1.2 m ~ l a d . The membrane was

blocked in 5% no fat milk dissolved in 0.1% lgepal (Sigma)/lX PBS for I hour at m m

temperature.

A 111 0 0 diiution of anti-HA mouse ascites in lûrnl of 0.1% Igepal (Sigma)/lX

PBS was added to the membrane and vigorously shaken for 45 minutes at room

temperature. The membrane was then washed three times for 5 minutes each with 0.1 %

Igepal (Sigrna)/lX PBS. A 1/2000 dilution of anti-mouse HRP (horseradish peroxidase)

conjugated anti'body was added to the membrane and vigorously shaken for 30 minutes at

room temperature. The membrane was washed again as describeci above. The membrane

was then incubated with lm1 each of Solution 1 and 2 ECL Western blotting detection

reagent for one minute and the blot was exposed on a filter for 5 minutes.

To generate stable ce11 lines the constructs were transfeçted into NIH3T3 172 -/-

PUR0 cells. In brief, 10 pg of WT, KR, or vector alone as well as lug of pCDNA3 was

transfected into IOOmm dishes through the use of Fugene 6 Transfection Reagent

(Boehringer Mannheim). After three days, the cells were split (1120 dilution) each into

10, lOOmm dishes and exposed to selection media (Spg/ul puromycin, Img/mI G418,

10% tet-fi-ee fetal bovine senim). Media was changed every four days until ai i the cells

on the control plates died. At this point, surty colonies were selected and tested for

expression of NKIATRE WT or KR. CeUs were plated in 1- dishes at 50%

confluency and induced for 48 hours with 2 pg/ml of deoxycycline. Harvesting,

immuno-precipitation, and western analysis was performed as describeci.

23 RESULTS

23.1 Isolation of the human cDNA

S e w d expressed sequence tags (ESTs), which have sequence similarity to the rat

NKLATRE gene, have been found within the Genbank data base

~ttp://www.ncbi.nlm.nih,gov/).

To isolate the human NKIATRE homologue, a 426bp NotI-HindIII probe f?om a human

Expressed Sequence Tag (EST), noted to be 89% identical to the previously isolated rat

NKIATRE sequence (Genbank accession # R21498) was used to probe a human fetal

heart cDNA library. The longest library isolate contained 1OOSbp of open reading frame,

including the human EST sequence, and had remarkable similarity to the rat NKIATRE P

isofonn, with 79% amino acid identity overall. Comparison of this human library isolate

to rat NKlATRE demonstrateci that it lacked 358 bp of coding sequence immediately

following the initiation codon. A further data base search revealed that the human partial

genomic sequence of NKIATRE had been deposited in Genbank for which we could

identim the first three exons contained in a Pl artificial chromosome, PAC H59

(Lawrence Livermore National Laboratory, Genbank accession number AC005354).

Cornparison of the human library isolate to PAC H59 revealed the distai 1 8 1 bp of the

third coding exon was identical to the 5' terminus of our human cDNA library isolate,

confirming that the cDNA was derived h m a transcript of the genomic sequence. Using

the genornic sequence we were able to deduce the complete human coding sequence,

which predicts a 455 amino acid serine threonine kinase 84% identical to the rat gene

overall (Figure 6).

23.2 Sequence Analysis

Cornparison of the human version of NKIATRE to the rat revealed that it has

feaîures of both the MAP kinases and the cyclinaependent kinases (CDKs). The human

clone contains the same core catalytic domain with various structural motifs conserved.

Furthemore it contains ail twelve of the invariant or nearly invariant residues that are

characteristic of the kinase cataiytic domain; Glyll and Gly13 within subdomain 1

(GXGXXGXV motif), Lys33 in subdomain II, Glu50 in subdomain III, Aspl25 and

Asnl30 in subdomain VIB, Asp 143 and Gly145 in subdomain VII @FG motif), Glu 170

in subdomain VLU (APE motif), Aspl83 and Gly188 in subdomain IX, and Arg274 in

subdomain XI (Haq et al., 1999) (Figure 7a & 7b). Similar to NKIATRE, the human

clone contains the two short motifs DMSEN in subdornain VI (residues 125-130) and

ATRWYR in subdomain ViII (residues 162-167) which are strong predictors of

serine/threonine kinase activity (Hanks et al., 1988) (Figure 7a & 7b).

Like the MAP kinases, potentiai regdatory phosphorylation sites at threonine 158

and tyrosine 16 1 in kinase domain VI11 are conserved (Ioannou et al., 1994). While

SAPK, ERK, and p38 contain the three regulatory amino acid motif TPY, TEY, and TGY

respectively, the human and rat version mainain the TDY motif. Like the CDKs, the

human clone maintains threonine 158 which corresponds to a criticai site of activating

M E M Y E T L G K V G E G S Y G T ATGGAGATGT ATGAAACCCT TGGAAAAGTG GGAGAGGGAA GTTACGGAAC

V M K C K H K N T G Q I V A K AGTCATGAAA TGTAAACATA AGAATACTGG GCAGATAGTG GCCATTAAGA I F Y E R P E Q S V N K I A M R E TATTTTATGA GAGACCAGAA CAATCTGTCA ACAAAATTGC GATGAGAGAA I K F L K Q F H H E N L V N L I E

ATAAAGTTTC TAAAGCAP-TT TCATCACGAA AACCTGGTCA ATCTGATTGA V F R Q K K K I H L V F E F I D

AGTTTTTAGA CAGAAAAAGA AAATTCATTT GGTATTTGAA TTTATTGACC H T V L D E L Q H Y C H G L E S K ACACAGTATT AGATGAGTTA CAACATTATT GTCATGGACT AG&-GAGTPPG R L R K Y L F Q I L R A I D Y L H

CGACTTAGAA AATACCTCTT CCAGATCCTT CGAGCAATTG ACTATCTTCA S N N I I H R D I K P E N I L V

CAGTAATAAT ATCATTCATC GAGATATAAA ACCTGAGMT ATTTTAGTAT S Q S G I T K L C D F G F A R T L CCCAGTCAGG AATTACTAAG CTCTGTGATT TTGGTTTTGC ACGAACACTA A A P G D I Y T D Y V A T R W Y R GCAGCTCCTG GGGACATTTA TACGGACTAT GTGGCCACAC GCTGGTATAG A P E L V L K D T S Y G K P V D

AGCTCCCGAA TTAGTATTAA AAGATACTTC TTATGGA- CCTGTGGATA I W A L G C M I I E M A T G N P Y TCTGGGCTTT GGGCTGTATG ATCATTGAGA TGGCCACTGG AAATCCCTAT L P S S S D L D L L H K I V L K V

CTTCCTAGTA GTTCTGATTT GGATTTACTC CATAAAATTG TTTTGAAAGT G N L S P H L Q N I F S K S P I

GGGCILATTTG TCACCTCACT TGCAGAATAT CTTTTCCAAG AGCCCCATTT F A G V V L P Q V Q H P K N E s R K TTGCTGGGGT AGTTCTTCCT CAAGTTCMC ACCCCAAAAA TGCAAGAAAA K Y P K L N G L L A D I V H A C L

AAATATCCAA AGCTTMTGG ATTGTTGGCA GATATAGTTC ATGCTTGTTT Q I D P A D R I S S S D L L H H

ACAAATTGAT CCTGCTGACA GGATATCATC TAGTGATCTT TTGCATCATG E Y F T R D G F I E K F M P E L K AGTATTTTAC TAGAGATGGA TTTATTGAAA AATTCATGCC AGPACTGm A K L L Q E A K V N S L I K P K E GCTPMTTAC TGCAGGMGC W G T C A A T TCATTAATAA AGCCAAAAGA

S S K E N E L , R K D E R K T V Y GAGTTCTAAA GAAAATGAAC TCAGGAAAGA TGAAAGAAAA ACAGTTTATA T N T L L S S S V L G E E I E K E CCAATACACT GCTAAGTAGT TCAGTTTTGG GAGAGGAAAT AGAAAAAGAG K K P K E I K V R V I K V K G G R

AAAAAGCCCA AGGAGATCAA AGTCAGAGTT ATTAAAGTCA AAGGAGGAAG G D 1 S E P K K K E Y E G G L G

AGGAGATATC TCAGAACCAA AAAAGAAAGA GTATGAAGGT GGACTTGGTC Q Q D A N E N V H P M S P D T K L AACAGGATGC AAATGAAAAT GTTCATCCTA TGTCTCCAGA TACAAAACTT V T I E P P N P I N P S T N C N G GTAACCATTG PACCACCAAA CCCTATCAAT CCCAGCACTA ACTGTAATGG

L K E N P H C G G S V T M P P I 1251 CTTGAAAGAA AATCCACATT GCGGAGGTTC TGTAACPATG CCACCCATCA

N L T N S N L M A A N L S S N L F 1301 ATCTAACTAA CAGTAATTTG ATGGCTGCPA ATCTCAGTTC AAATCTCTTT

H P S V R 1351 CACCCCAGTG TGAGGTGAGC TGTAACAGAG AAGAAACCTA AATAATACPA 1401 CATTCCTGTA TAATGGTATT TCAAAGAATC GTGTTCATAG TGTCTGTATG 1451 TAAACTGAAC TTGPIIGAAAA TATATTGAAA T T M G C T G T ATAATGGGCC 1501 AAAAAAAAAA Am

Figure 6: The nucleotide and deduced amino acid sequence of NKIAMRE. Protein kinase consensus residues are highlighted. The NKIAMRE motif and two sites of potential regulation, the SY duplex characteristic of the cyclin-dependent kinases and the TDY W base-like regulatory region, are shown in bold face type. The sequence correspondhg to EST R2 1498 is shown underlined.

Figure 7a: Sequence analysis of NKIAMRE. NKIAMRE contains several regulatory sites of phosphorylation including a potential cyclin binding motif, for which it is named.

Figure 7b: Sequence cornparison between rat NKIATRE and human NKIAMRE. Major differences lie in the postulated cylin-binding motif [NKIA(T/M)RE] including the C-terminal truncation of PEST and nuclear localization sequence (NLS).

phosphorylation by the CDKs. Furthennore, the human clone maintains serine 14 and

tyrosine 15 which may correspond to CDK negative regdation sites.

The putative cyclin binding domain, NKIATRE, for which the kinase was named

is substituted with NKIAMRE. In keeping with the naming convention of CDK-related

molecules, this human gene has been named NKIAMRE. NKIAMRE has 84% arnino

acid identity to NKIATRE, and 41% and 42% identity to the closest matches identified

by Blast search @ttp://www.ncbi.nlm.nihnihgov/), the cdc-2 related kinases KKIAMRE and

KKLALRE respectively (Figure 8).

As mentioned, the complete rat bfain (NKIATRE a) and rat jejunal (NKLATRE

p) open-reading h e s enmde putative protein kinases, with predicted rnolecular

weights of 57kDa and 52kDa respectively (Haq et al., 1999). NKIAMRE is a 455 amino

acid protein with a predicted rnolecular weight of 52 kDa- NIUAMRE sequence

terminates at precisely the same location as NKIATRE P redting in the tnincation of

two potentially important molecular features (Figure 7b). Firstly, putative nuclear

localization motifs are present in NKIATRE a between amino acids 245-252

(f KNARKK) and 473477 (RTKKRR) (Haq et al., 1999). A fùndamental hexapeptide

motif containhg four arginines and lysines, flanked b y hydrophobie residues, has been

proposed as a "core nuclear localization signal (NLS)" (Boulikas, 1993). Secondly, a

putative PEST sequence is present in NKIATRE a between arnino acids 480 and 493

(RQEDTGPTQVQTEK) (Haq et al., 1999). PEST sequence, named after the individual

amino acids, is associated with ubiquitin-dependent degradation of nuclear proteins

(Rechsteiner et al., 1996; Rogers et al., 1986).

101 111 121 13 1 141 NKIAXRE KRLRKTLIQI w U a , Y L 8 S 1 MSXHRûXKPB HILVSQSOIT KLCDIGFART HRIATaE KRLRKYLIQI LEU- MISERDIKPI NILVSQSOIT KLCDFGIART KKIAXRE QWQKYLFQI INGIGFCHSH NIIHRDIKPE NILVSQSGVV KLWFGFART KKIALRS HLVnSITWQT LQAVNFCHKH NCIHRDVKPE NILITKHSVI KLCDFGFARL

151 161 171 181 191 NïCIAMRE LAAPODSTTD IVATRWPIUP BLVLXDTSYO rPVDIWALGC MIIEMATGNP NICIATRB LAAPûDVltTO YVATRUTRAP ELVLICDTTYO IPVDIWALGC MIIEMATGNe lCKLAWZLB LAAPGEWTD YVATRWYRAP ELLVGDVKYG KAVDVWAIGC LVTEMFMGEP KKIALRE LTGPSDYYTD YVATRWYRSP ELLVGDTQYG PPVDVWAfGC VFAELLSGVP

201 211 221 231 241 NKXAMRB YLPSSSDLDL LHKIVLKVGN LSPHLQNTFS KSPIFAGVVL PQVQHPKNAR NXIATRE YLPSSSDLDL LHKIVLKVGN LTPHLHNIFS KSPIFAGVVL PQVQHPKNAR KXIABfRE LE'PGDSDIDQ LYHIMMCLGN LIPRüQELFN KNPVFAGVRL PEIKEREPLE KKIALRB LWPGKSDVDQ LYLIRICTLGD LIPRHQQVFS TNQYFSGVKI PDPEDMEPLE

2 S l 261 271 281 291 NKIAXRE KKYPKLNGLL ADfVHACtQI DPADRISSSD UHHEYFTRD GFIEKFMPEL NXIATRE KKYPKLNGLL ADIVHACLQI DPAERISSTD LLHHDYFTRD GFIEKFIPEL KXIMZRE RRYPKLS~VV rnLAmccuu DPDKRPPCAB LLHHDFFQMD GFAERFSQEL KKIALRE LKFPNISYPA LGLLKGCLFiM DPTBRfrTCEQ LLHHPYE'ENI REIEDLAKEH

301 311 321 331 341 HKIAHRB KAKLLQEAKV NSLIKPKESS KENELRKüER W S SSV-L-GEEI NKIATRB RAKLLQEAKV NSFIKPKENE' RENGPVRDEK KPVFTNPLLY GNPTLYGKEV KRfAnaE QLKVQKDARN VSLSKKSQNR XKKKEKDDSL VEERKTLWQ DTNADPKIKD ICKiALRE DKPTRRlltRK SRKHBCFTET SKLQYLPQLT GSSUPALDN KKYYCDTKKL

351 361 371 381 391 NKIUIRE EXEKKPKEIK VRVIKVKGGR GDISBPKKKE YEGGLGQQDA NENVHPMSPD NKUTRB DRDKRAKELK VFLVIKAKGGK G D V P D m SEGEEIRQQGT AEDTHPTSLD KRIAHRE YKLFKIKGSK IDGEKAEKGN RASNASCLEID SRTSEINRIVP STSLKDCSNV KltIALELB NYRFPNI

401 411 421 431 441 NKXlUEB TKLVTIEPPNP INPSTNCNG LKENPHCGGS VTMPPïNLTM -SS NKIATRS RKPSVSELTNP VHPSANSDT VKEDPHSGGC MIMPPINLTS SNLLAANPSS KKïAXRB SVDHTRNPSVA IPPLTHNLS AVAPSINSGn GTBTXPIQGY RVDEK!I!KKCS

451 461 471 4 8 1 491 NKIAMRB NLFHPSVR NKIA- NLSHPNSR KKïAXRB XPFVKPNRHSP SGrYNINVT TLVSGPPLSD DSGADLPQME HQH

Figure 8: Sequence alignment of NKIAMRE (hurnan) compareci to the NKIATRE B isofom (rat), and the closest related kinases KKIAMRE (human), and KKlALRE (human). Residues varying from NKIAMRE are highlighted. NKIATRE is 84% identicai to NKIAMRE while KKLAMRE and KKMLRE show 41 and 42 percent identity respectively.

233 Northern BIot Anaiysis

To determine the expression of NKIATRE in rat ce11 lines, a Northem blot of

poly(A)+mRNA ti-om H9C2 cardiac myoblasts, L6 skeletal myoblasts, and the adrenal

pheochromocytoma ce11 PC12 was probed with NKIATRE a isofonn cDNA (Figure 9).

These ce11 lines were chosen as they were derived fiom tissue having strongest

expression of NKIATRE. Although expression was detected in control transiently

tmnsfected HeLa cells, no hybnduing signai was detected in any of the cell lines

examineci. Equal loading and quality of poly(A)+ mRNA was determineci by probing

with rat P-actin. Absence of NKIATRE expression was aiso observed in HeLa, COS-7,

HL60, U937, DP 16, A43 1, NIH-3T3, EC 18, K562, HEK 293, MDA 468 and MDA 469

ceUs using the same probe. This suggests that NKIATRE expression may be resrricted to

non-proliferating a d o r differentiated ce11 types.

2-3-4 Tet-On Cell Lines

The lack of expression observed in the ce11 lines descriied above prompted us to

Produce a stable NKIATRE expressing ce1 line. The NIH3T3 172 -/- PUR0 ce11 line

was transiently transfected with wild type NKIATRE-pUHD10.3, kinase dead

NKIATRE-pUHD 10.3 and vector alone to test for expression using these constnicts. The

cells were stimulateci with or without deoxycycline for 48 hours prior to harvesting.

Figure 10 illustrates that both mnstructs were being expressed. The constructs were

subsequently used to generate

Figure 9: (A) Expression of NKIATRE in PC 1 2, H9C2, and L6 ce11 lines was examined by Northern blot analysis of poly(A) mRNA with rat NKIATRE as a probe. Hela cells transfected (tf) with NKIATRE-pCDNA3 or vector alone were used as a control. (B) The same blot probed for B-actin.

68

Figure 10: NKIATRE Tet-On ce11 lines. (1) Protein expression analysis of transient transfected NM3T3 172-1- PURO ce11 lines with WT-NKIATRE-pUHD, KR-NKIATRE-pUHD, and vector alone after +/- deoxycline (Dox) stimulation for 48 hrs. (2) Stable protein expression of Wr NKI ATRE-pUHD or vector alone in NIH3T3 172-/- PURO ce11 line after +/- Dox stimulation for 48 hours. (3) Stable protein expression of KR-NKIATRE-pUHD or vector alone in NIH3T3 172-1- PURO ce11 line after +/- Dox stimulation for 48 hours. 69

stable ce11 lines expressing the wild type or kinase dead versions. After G418 selection

two ce11 lines were obtained fiom the screening of approximately sixty clones. Cells

stably expressing NKIATRE wild-type (clone 2) and kinase dead KR (clone 14) were

examineci for expression foliowing stimulation with deoxycycline for 48 hours (Figure

1 O).

2.4 DISCUSSION

In an attempt to understand more about the MAPWCDK related kinase

NKIATRE, we cloned the human version, which has been named NKIAMRE-

NKIAMRE most closely resembles the rat jejunum isoform, NKIATRE B, a 455 amino

acid protein with predicted molecular weight of 52 kDa. Like NKLATRE, the human

version maintains al l residues characteristic of a kinase cataiytic domain (Figure 7a &

7b). Furthermore, NKIAMRE maintains sequences DIKPEN in kinase subdomain VI

(residues 125-130) and ATRWYR in kinase subdomain VI11 (residues 162-167), which

are strong predictors of serine/threonine kinase activity (Hanks et al., 1988) (Figure 7a &

7b). Like the jejunum isoform, NKLAMRE, lacks both a putative nuclear locaiization

signal and PEST sequence suggesting that these versions may have differences in nuclear

localization, and modes of degradation. Finally, compared to NKIATRE, the human

version shows 95% amino acid identity in the core catalytic domain however identity is

drastically reduced to 68% as the molecule becornes more C-terminal. This suggests that

homology among species is maùitained only in areas of fùnctional importance.

The first three exons of human genomic NKLAMRE have been deposited in

Genbank and are found in a Pl-artificial chromosome, H59. Compared to NKIATRE,

the first three exons show 98% identity to the rat cDNA sequence at the amino acid level.

We dso identified several human expressed sequence tags (ESTs) which represent cDNA

clones derived fiom NKIAMRE. The ESTs show an inconsistent pattern of expression

suggesting alternative splicing. To identie the human homologue the EST with the

highest sequence similarity to rat NKIATRE was used in order to screen the human fetal

heart cDNA library. It was more advantageous to use the human EST, as opposed to rat

NKIATRE, as the library could be screened at a much higher stringency which

eiiminated fdse positives. Severai of the isolated clones wae the same PCR amplifieci

size, suggesting that only one version of human NKIATRE was represented in the

library. The 5' terminus of the isolated clone was identical to the distal 181 bp of the

third exon of H59, which confirmed that the cDNA and genornic sequence represented

the sarne gene.

The tissue distriiution of NKIATRE, indicates that expression is restncted to

differentiated or non-prdiferative tissues. The strongest expression is seen in brain,

muscle and h a r t (Haq et al., 1999). In ce11 lines; H9C2, PC 12, and L6, representative of

these primary tissues, the expression of NKlATRE was not shown by Northern analysis

(Figure 9). Furthennore, NKIATRE expression was not observeci in untransfected HeLa,

COS-7, HL60, U937, DP 16, A43 1, NIH-3T3, IEC 18, HEK 293, MDA 468 and MDA

469 cells. The absence of expression suggests that NKIATRE may play a role in the

suppression of growth or the induction of differentiation.

In order to test this hypothesis, a stable cell h e with regulated expression of wild

type (WT) or kinase dead (KR) NKIATRE was coastructed. The Tet-On gene expression

system was chosen which allows for regdated expression of the gene of interest after

induction with deoxycycline- Many experimental systems have been developed in an

attempt to regulate gene expression. Most systems have several disadvantages in that

they lack quantitative control, have pleiotropic effects and are prone to "leakiness"

(Gossen et al., 1995). The Tet-On expression system was chosen as the maximal levels

of expression are quite high and compare favorably with the maximal levels obtainable

f?om strong, constitutive mammalian promoters such as CMV (Yh et ai., 1996).

Furthmore, unlike other inducible mammaiian expression systems, the Tet-On systern is

highly specific and can be finely regulated so interpretation of results is not complicated

by pleiotropic effects. This ce11 line will be instrumental in hture experiments to

understanding NKIATRE' s possible role in negative ce11 growth regdation and

di fferentiation.

The Chromosomal Locaiization of NKIAMRE to 5q3l.l and its Loss in Leukemic Blasts with the 5q- Syndrome

Parts of this chapter have been accepted in: "Identification of MUAMRE, the human homologue to the MAPWCDK-related protein kinase NKIATRE, and its loss in leukemic blasts with the 5q- Syndrome." M. Midmer, R. Haq, J.A. Squire, B. Zanke Cancer Research

3.1 INTRODUCTION

Loss of al l or part of the long ami of human chromosome 5 (5q- syndrome) is

commonl y seen in human myelod ysplastic disorders (MDS) and leukemia (Boultwood et

al., 1994a). Patients with such deletions as the sole karyotypic abnomality often suffer

from a wide range of rnyeloid disorders, including rehctory anemia (RA), refractory

anernia with excess blasts (RAEB), rehctory anemia in transformation W B T ) ,

m y elodysplastic syndrome (MDS), and acute myelogenous Leukemia (AML), (Lee et al .,

1993). Neoplastic transformation occurs by deletion in the multipotential hemopoietic

stem ce11 resulting in cells failing to mature beyond the blast stage, leading to progressive

accumulation of myelob1ast.s in the bone marrow (Lee et al., 1993).

To identiQ lost tumor suppressor genes in MDS and acute leukenGa, atternpts

have been made to define the minimally deleted region on chromosome 5q through the

cornparison of malignant myeloid karyotypes. Among the gens Iocalized to the region

include several growth factors and growth factor receptors, including the interleukin

genes (TL3, L4, ILS, IL9), the receptor for macrophage-colony sîimulating factor

(CSF 1 R), interferon regdatory factor (IRF 1 ), garnma-aminobuîyric acid A receptor alpha

1 (GABRA 1 ), and early growth response 1 (EGR- 1 ). (Wasmuth et al., 1 99 1).

With the goal of further characterizhg NKIAMRE, we performed Fluorescence in

situ Hybndization (FISH) on normal human metaphase chromosomes. Here, we report

the localization of NKIAMRE to 5q3 1.1, an active site of deletion in patients with 5q-

syndrome. We have perfomed the molecular examination of MUAMRE in a group

patients with the Sq- syndrome and have show loss of both copies of NICIAMRE in 9

out of the 18 patients. Furthemore, the expression of NKIAMRE in human bone marrow

and leukemic celi lines was examineci-

3.2 M A T E W S AND METEODS

3.2.1 Patients

The Princess Margaret Hospital treats annually over one hundred patients with

acute leukemia Since 1 990, patient material, including cxyogenically presewed

leukemic blasts and genomic DNA, has been archived. Two cases were randomly

selected in which deletion of 5q31 on one chromosome was documented and used for

Southern blot analysis. In addition, eighteen cases were randomly selected in which

deletion of 5q31 had been identifiai during routine cytogenetic analysis. Aiiquots of

methanol-acetic acid fixeci cells derived fiom the original cytogenetic preparations were

stored a 4 ' ~ prior to use for FISH studies. For ail patient samples good quality

interphase nuclei were present and for some samples a lirnited number of metaphase cells

of adequate quality were present. For each patient the clinical records were abstracted

(Table 3).

3.2.2 Southern Blot Analysis

For Southern blot analysis, 10 pg of normal or patient human genomic DNA was

digested ovenight at 3 7 ' ~ with HindIII. Each of these digests were hctionated

ovemight by electrophoresis on a 1 .O% agarose gel. Following electrophoresis, the gel

was incubate. in 2 volumes of 0.25M HC1 for 30 minutes at room temperature. The gel

was then rinsed and incubated in 1 SM NaCVO.5M NaOH denaturation solution as before

for 20 minutes. Finally, the gel was rinsed and incubated in 1 S M NaCVOSM Tris-HCI

(pH7.0) neutralization solution as before for 20 minutes. The genomic DNA was

transfmed to a nylon membrane by capillary transfer (Sambrook et al., 1989). The blot

was probed with the 1OOSbp human Iïbrary NKIAMRE clone. The probe was

radiolabelleci as desmbed in section 2.2.1. To control for the loading and quality of the

DNA in each lane, a rat B-actin probe was use& Prehybridization, hybridization and

wash conditions were carried out as descriied in section 2.2.1. FiIters were exposed

overnight ont0 a Phosphoimager Screen and visualized by a Phosphohnager (Molecular

Dynamics) using the ImageQuant software (Molecular Dynamics).

3.2.3 Isolation of human genomic NKIAMRE

To facilitate subsequent chromosome hybridization studies, rat NKIATRE cDNA

was hybridized to a commercial array of human bacterial artificiai chromosomes

(generated by Dr. Pieter de Song, Roswell Park Cancer Institute, Buffalo NY:

http:/hacpac.med.buffalo.edu(hdhf.htm). One identified clone, BAC 2 15P23, was

partially sequenced using the NKIAMRE-specific primers H59 1-4 (Table 2) confimiing

the presence of NKIAMRE coding sequence. The product was sequenced at the York

University Core Molecular Biology Facility (Toronto) using PCR based methodology.

3.2.4 Fluorescent Chromosornai in situ Hybriàization (FISB)

Methanol k e d leukemic blasts were dropped ont0 coverslips, washed with a 3: 1

mixture of methanoVacetic acid, and ailowed to evaporate to encourage chromosome

spreading, Slides were dehydrated by consecutive treatment in 75%, 90%, and 100%

ethano1 for 5 minutes respectively in prepmtion for hybndization.

Coincident hybridization was performed using the NKIAMRE locus-specific

BAC 215P23 and with a hybridization mntrol probe, BAC 42H21

fittp :/hacpac.rned.buffalo.edu/) (Icindly provided by Dr. Barbara Beatty, Toronto) which

had been mapped to human chromosome 5p15 by the CGAT FISH Mapping Resource

Center (Ontario Cancer Lnstitute). BAC 2 15P23 was labeled with biotin using a BioNick

labeling system (GibwBRL), while BAC 42H21 was labeled with digoxigenin (DE)

using a DIG-NICK Translation Mix (Boehringer Mannheim). The labeled probes as weil

as salrnon spenn DNA was fiactionated by electrophoresis on a 1.0% ethidium bromide

agarose gel and size was estirnateci to detexmine efficiency of labeling. The ideal

fiagrnent size is between 100-500 bps in length. Both probes were precipitated in the

presence of salmon spenn DNA (Strategene). For each pg of labeled probe, 50 pg of

salmon spemi DNA was used as a carrier. The precipitated DNA was resuspended in

water at a final concentration of 10 ng/pl. An aliquot of 250 ng of each probe was then

m e r precipitated with 4 pg of human Cot- 1 DNA (GibcoBRL). The precipitated DNA

was hal ly resuspended in L 5 pl of 37'~ preheated Hybrisol VI1 (Oncor).

The probes and slides were denatured in the presence of formamide. The probes

were denaîured for 5 minutes in a 7 5 ' ~ water bath and then incubate in a 3 7 ' ~ dry oven

for 2 hours. The slides were denatured in a 7 0 ' ~ water bath in a coplin jar contaking

preheated 70% deionized formamide and 2X SSC for 2 minutes. A critical h a 1

temperature of ~O'C was obtained by assuming a 1 OC drop in temperature for each slide

added. If the final temperature is greater than 7 0 ' ~ DAPI (G) banding is changed to C

banding. If the final temperature is less than 7 0 ' ~ denaturation is incomplete. The slides

were immediately transferred to consecutive ice cold coplùl jars containing 70%, 90%,

and 100% ethanol for 5 minutes respectively. After the slides were dried the probes

(dissolved in Hybrisol) were mounted ont0 the slides with 24 X 4Omm wver slips ,

sealed with rubber cernent, placed in a damp container, and incubated at 3 7 ' ~ in a dry

oven for 16 hours.

A f k incubation the cover slips were removed and the slides were washed three

times consecutively with 70% formamide12X SSC and 2X SSC for 5 minutes at 4 5 ' ~ .

The slides were then mounted with 40 pl of blocking reagent (2X SSC, 1 -5% bovine

s e m m albumin, 0.75% Tween 20,O. 15M NaCI, 0.1 M TrisHC1) and incubated in a damp

container for 20 minutes in a dry oven at 37 '~ . After incubation, slides were mounted

with anti-DIG (Boehnnger Mannheim) and FITC-avidin (Oncor) antibodies and

incubated as above. After incubation, cover slips were removed and slides were washed

three times consecutively for 5 minutes at 4 5 ' ~ in phosphate buffered detergent (Oncor).

Slides were re-mounted with DIG-anti-mouse (Boehringer Mannheim) and anti-avidin

(Oncor) antibodies and incubated followed by washing as above. Slides were re-mounted

with Rhodamine-anti-DIG (Boehringer Mannheim) and FITC-avidin (Oncor) antibodies

and incubated followed by washing as above. Finally, cells were stained with DAPl

(Oncor) to visualize G-banding under a fluorescent microscope. Fifty cells were counted

on each slide by two independent reviewers, blinded to sample identity.

3-2.5 S tatisticd Gnaîysis

Deletion of the NKIAMRE locus was evaiuated by cornparhg the proportion of

marrow cells having loss of either one or two BAC 215P23 signals in leukemic and in

normal control samples. Each of these cornparisons was done assuming two independent

proportions wi th qua1 variance (Pagano et al., 1 992). S tatistical significance was

detennined by comparing the test-statistic to the normal distribution and using a 0.05 cut-

off (Appendix). This means that the probability that observeci differences in proportions

occmed due to chance alone is l e s than or equal to 5%.

3.2.6 Northern Blot Analysis

Northem Blot analysis was perfomed as describeci in 2.2.2. The 1005 bp library

human NKIAMRE clone was used as a probe and radiolabelled as described in 2.2-1.

Transfection and ceii culture was performed as described in 2.2.3. which established a

positive control for hybridization. Ten pg of poly(A)+RNA was prepared from human

bone marrow, HL60, U937, and pcDNA3-NKIATRE-HA transfected HeLa cell lines. In

another experiment, ten pg of total RNA was extracted fiom HL60, U937, and K562 cells

that had been induced to differentiate. Differentiation was induced by the addition of

1 -25% dimethyl sulfoxide (DMSO) and 100 n g h l phorbol- 12-myrïstate- 13-acetate

(PMA) for 0, 12,24, and 48 hours. DiEerentiation was assessed by plate adherence and

change in morphology (Table 5).

3.2.7 Dinerentiai Hemopoietic Gene Expression Analysis

To determine the expression of NKIAMRE in human hemopoietic cells a slot blot

was probed with the 1005 bp human Ii'brary NKlAMRE clone. The slot blot was kindly

provided by Dr. Norman Iswve, Toronto. The blot is an array of PCR ampiified cDNA

fiom individual hemopoietic precursor ceIIs. The precursors cells were cultured in

various cytokines to induce diffentiation into daughter cells that are committed to

differing limages- Termuially maturing erythroid cells, megakaryocytes, neutropbils,

and macrophages were isolated fiom single-lineage colonies grown fiom marrow-ce11

precursors in methyl cellulose containing IL- 1, IL-3, and Epo (Brady et al., 1995). The

celk were lysed and cDNA with an average length of 400 bases was generated with

reverse transcriptase and a oligo (dT)Z4 primer. A homopolymeric 3' (dA) tail was

generated with terminal deoxynucleotidyl tramferase and the resulting cDNA mixture

was amplified by PCR using a 3' (dT)24-contaïning 60-base primer (Brady and Iscove,

1993). The dot biot was dso probed with B-actin as a control for hybndization. Both

probes were radiolabelleci as described in section 2.2.1.

3.3 RESULTS

33.1 Isolation and locaiization of genomic NKIAMRE

As mentioned in 2.3.1, a data base search identifid three exons within a genomic

PAC sequence having 88% nucleotide and 94% predicted amino acid identity to the 554

bp following the rat NKLATRE initiation codon @AC H59, Lawrence Livennore

National Laboratory, Genbank accession number AC005354). Prior to the recognition of

NKIAMRE within the genomic PAC H59 sequence, we identified hybndization of rat

NKIATRE to the hurnan BAC 2 15P23 containing a 174 kb genomic insert. Southern

hybridization of the complete NKIATRE a sequence to EcoRl-digested gene hgments

produced three hybridizing bands of size 1.2 kb, 3.7 kb and 7.6 kb (Figure 12). The

southem blot was kindly provided by Dr, Steve Sherer, Toronto. In order to confïxm the

presence of NKIAMRE genomic sequence, the clone was partially sequenced which

demonstrateci secpence identity to NKIAMRE cDNA between bp; 30- t 64, 540-604, 882-

955, 1034-1 120, and to PAC H59 between bp 5748-6207. The chromosomal localization

of NKIAMRE was detennined by FISH of labeled BAC 215P23 DNA to a normal human

chromosome metaphase spread. Coïncident chromosomal G-banding confirmed the

localization of NKIAMRE to chromosome 5q3 1 (Figure 13). This localization is

consistent with the reference location of PAC HS9

( h t t p : / / w w w - h g c . l b l . g o v / b i o l o g y / m a p p ~ which maps telomeric to the

TCF-1 gene and centromeric to the IL-9 gene. This site is approximately 1 Mb

centromeric to the myeloid leukernia locus defmed by Le Beau et al. (1994) and 1.8 Mb

telomeric to RF-1 locus, also implicated in acute leukemia (Willman et al., 1993)

prompting us to evaluate NKIAMRE's deletion in primary acute leukemia cells.

3.3.2 Southern Analysis on patients with Sq- syndrome

To examine NKIAMRE's possible deletion in acute leukernia, the 1005 bp

hgrnent of the human heart cDNA clone was used to screen human genomic DNA

digested with the HindIII restriction endonuclease (Figure 14). Genomic DNA was

obtained fiom two nonnal control patients and two patients with 5q- syndrome. Results

Figure 12: Southem hybridization of NKIATRE a sequence to EcoR1 -digested gene fragments contained in bacterial artificial chromosomes (BACS). BAC2 15P23 was used for FISH analysis.

Figure 13: Locdization of NKIAMRE to 5q3 1 by Fluorescent in situ Hybridizattion. Metaphase chromosome shows the green NKlAMRE signal localinng to band 5q31 (left). DAPI staining of the same chromosome (middle) and schematic representation of chromosome 5 (right) are shown .

Figure 14: (A) Southem hybridization of NKIAMRE sequence to HindIII-digested human genornic DNA. (B) The same blot probed for P-actin.

of Southem blotting revealed that three bands hybridized to EcoRL digested normal

human genomic DNA with approximate sues of 10.0 kb, 9.0 kb, and 2.5 kb. In both

patients, however, the 2.5 kb band was absent and in the patient with a 5q13q3 1 deletion

the intensity of the 10.0 kb and 9.0 kb bands was decreased by almost 90%. In order to

control for quality and loading of genomic DNA the Southern blot was probed with P-

actin. This would suggest that a deletion of NKIAMRE may occur in these two patients.

The limitation with Southern analysis is that the genomic DNA is obtained h m a

heterogeneous mixture of cells and is therefore not a tnie representation of oniy leukemic

clones. Consequently, the more sensitive method of FISH was used which enabled

precise detection of deletion in individuai cells.

3.33 The NKI[GMRI., locus is deleted in acute leukemia

Deletions involving chromosome 5q are the most commoniy observed genetic

abnormalities in acute leukemia, occurring in 50% of cases presenting after pior

chemotherapy and in 15% of cases arising de novo (18-20). Since NKIAMRE locaiizes

to this cornmonly deleted region, its mono ailelic loss could simply reflect its proximity

to other tnily relevant loci. However, loss of both NKIAMRE loci, would have a greater

significance, suggesting that MUAMRE, or a closely luiked gene, is important for the

generation or maintenance of the pathologie -te.

Eighteen methanol-acetic acid fixecl samples of primary leukemic bone marrow

were selected on the basis of an identified chromosomal aberration of band 5q31 by

conventional G-banded analysis of metaphase spreads. To evaluate potential bi-allelic

loss of NKIAMRE within these samples, slides derived fiom cryopreserved interphase

nuclei were hybridized to biotin-conjugated BAC 215P23 and detected with FITC-

conjugated avidin antiï.ody. This collection containeci one case of rehctory anemia with

excess blasts (RAEB), three cases of acute lymphocytic leukemia (ALL) of the L2

subtype, and 14 cases of acute myelogenous Ieukemia (AML). Of the 14 AML cases, 8

had a prior clinical history or marrow morphologie evidence of myelodyspiasia.

In al1 experiments hybridization efficiencies were monitored within and between

individual samples, by simultaneously scoring hybridization signals fkm the BAC

42H2 1, a singIe copy genomic probe mapping to chromosome band 5p 15. 42H2 1 was

labeled with digoxigenin and detected by rhodamine conjugated to anti-digoxigenin

antibody, producing red fluorescence, distinct fiom the green fluorescence of BAC

2 15P23 hybridization (Figure 15). Complete loss of chromosome 5 would result in loss of

both green and red signais and this was not present in any of our samples, so that analysis

was restricted to cells having two 5p15 (probe 42H21) hybridizing signais. Since bone

marrow usually contains normal stroma1 cells in addition to the malignant clone, cells

having 2 hybridizing signais within the 5q31 locus were cowidered to be normal

contaminating marrow cells and were excluded fkom the detection analysis.

Hybridization efficiencies of >90% were observed for both probes when FISH

was performed on nuclei fiom diploid control samples, indicating each probe had

comparable detection and sensitivities. Similady, the 5p 12 control probe (4282 1 )

exhibite. two signals in >90% of nuclei when hybndized to leukemic nuclei. Table 4

shows the percentage of nuclei in which a homozygous, hemizygous, and no deletion was

apparent. As expected, most leukemic ceils had at l e s t one deleted NKIAMRE locus

(1 3/18). In 5 samples, the proportion of cells having loss at this locus did not differ from

Figure 15: Fluorescent in situ Hybndizaîion performed on marrow of patient 8 with del(5q3 1) kaqotype (see Table 3). ALI ceiis have 2 copies o f the red Sp15 telomeric control. Ceils with two green signals are remalning normal IMITOW

cells. Maliguant celis have one green signal or absence of hybridization if the remahhg NKIAMRE allele bas been lost.

TABLE 3

P2 69 M M . 45JCY,de1(5)(q22q33),-7

P3 60 M L2 46,XY,de1(5)(q3 l),t(7; 12Mq36;q 13)[4]/46XYI 131

P4 73 M M6 50,XY,add(3)(q12),de1(5)(q 1 12q33),de1(7)(q3 13q34),+8,+9,+ 10,- 1 1, add(14)(pI2),+15,+22[4]

P5 14 M U 43,XY,t(3;3)(q2 i;q26)&1(5)(q3 l),der(5)t(5; 12m l5;q 131,-7,- 1 1,- 12, add(W(q 1 1)PI

P6 18 M RAEB 50,XY,del(S)(q3 1),+6,de1(6)(qn)+ 16,+16,+2 11W46XY131

Study Patients. Eighteen patients were selected for study based on cytogenetic evidence for 5q3 1 abnormalities. FAB pathologie classification is shown. The complete karyotype of al1 metaphases are listed. If more than one karyotype was identified within individual samples the numbers of cells of each type is shown in square brackets.

TABLE 4

NKIAMRE fluorescence in situ chrornosomal hybridization in leukemic samples and nomal conîrols. The proportion of leukemic cells in each sample haWig zero, one or two NKIAMRE signals were compareci to normal bone marrow controls analyzed concurrently. Values statistically differing fiom controls are shown in bold faced type.

Pl P2 P3 P4 C l

TwoNKIAMRE Signais (%)

46 8 22 24 82

OneNKIAMRE Signal (94)

30 72 16 26 14

ZeroNKIAMRE Signrls (54)

24 20 62 50 4

controls, niggesting either inaccuracies in the karyotypic analysis of these cases, or

significant admixture of n o d manow hematopoieiic or stromal cells which prevented

the detection of statistical significant deletion. Of thirteen samples having BAC 2 1 SP23

loss, nine displayed statistically significant bi-dlelic NKIATRE deletion. Withüi these

individual samples, the percentage of cells having bi-allelic deletion was quite variable,

ranging fiom 34-70% (Table 4)-

The clinical records of patients were reviewed to dettxmine if NKIAMRE

deletion was associateci with a distinct clinical profile. Within this small sample size,

patients with bi-ailelic NKIAMRE deletion were not distinguished by age at diagnosis,

gender, F AB leukemic subtype, previous chemotherap y, antecedent myelodysplasia, or

survival afier diagnosis (Table 3).

3.3.4 Northern Blot Analysis

To determine the expression of NKIAMRE in huma. cells and ce11 lines, a

Northern blot of poly(A)tmRNA fkom normal bone marrow, the promyelocytic leukemia

ce11 line HL60 , and the monocytic leukemic ce11 line U937 was probed with the 1005 bp

fragment fiom the 1005 bp fi-agment of the human heart cDNA clone (Figure 16). These

celI lines were chosen to demonstrate expression of NKIAMRE in human bune marrow

or derived ce11 lines in order to conclude that it is a likely candidate gene involved in

dysmyelopoiesis. Although expression was detected in control transientiy transfected

HeLa cells, no hybridizing signal was detected in any of the ce11 lines examined. Equal

loading and quality of poly(A)+mRNA was detemiinai by probing with rat B-actin.

Figure 16: (A) Expression of NKIAMRE in human bone marrow, and leukemic ce11 lines: HL60 and U937 was examined by Northern blot analysis of poly(A) mRNA with the human clone as a probe. Hela cells transfected (tf) with NKIATRE-pCDNA3 or vector alone were used as a control. (B) The same blot probed for P -actin.

92

This result somewhat conflicts with our hypothesis that NKIAMRE is a gene

reponsible for a myeloid disease. The expression may however be induced following a

specific stimulus or expression may be transient in cases such as differentiation. This has

been observed previously (2.3.3) by the lack of expression in cell lines representative of

di fferentiated primary tissues. To determine expression foiiowing differentiation, a

Northem blot of total RNA fiom HL60, U937, and the chronic myelogenous leukemia

cell line K-562 was probed with the 1005 bp bgment of the hman heart cDNA done.

The cells were stimulated with either PMA or DMSO for 0, 12,24, and 48 hours, prior to

harvesting. Diffentiation was determined by ce11 adherence and change in morphology

(Table 5). in the absence of PMA or DMSO the FIL60 cells grow in suspension and are

predominantly typical promyelocytes with large well rounded morphology (Collins et al.,

1978). The addition of PMA or DMSO causes monocytic d i f f ia t ion as demonstrated

by plastic adherence and characteristic features of monocytes (Yang and Shaio, 1994).

At 48 hours p s t induction 95% of the cells had adhered to the tissue culture plastic.

Similady, the addition of PMA or DMSO to U937 and K562 cells induces cellular

differentiation into monocytes as reflected by growth inhibition and increased plastic

adherence (Vrana et ai., 1998). Although expression was detected in control transiently

transfected HeLa cells, no hybndizing signal was detected in any of the ce11 lines

examined (Figure 17). Equal loading and quality of RNA was determined by ethidium

bromide stainhg.

- . - PMA PMA PMA ii&

(A) $& 0122448 0122448 0122448

$$ IIU93711K562' 4-4kb-

2.3 kb- 2.0kb-

0.56kb-

%Fi. PMSO DMSO DMSO L L.

Figure 17: (A) Expression of NKIAMRE in human leukemic ce11 lines (HL60, U937, and K562) was examined by Northern blot analysis of total RNA with NKIAMRE as a probe. Differentiation was induced following PMA and DMSO stimulation for 0, 12,24, and 48 hours. Hela cells transfected (tf) with NKIATRE-pCDNA3 or vector alone were used as a control. (B) Ethidium bromide stain of total RNA illustrating the 28s and 18s ribosomal RNA subunits.

TABLE 5 Differentiation of HL60, U937, and K562 based on percent adherence to tissue culture plastic.

' Time O hrs.

Time O hrs. 12hrs.

HL6O-PMA 0%

BL60-DMSO 0%

K562-PMA 0% 40%

K562-DMSO 0% 5%

Figure 18: Slot blot containhg DNA fkom samples shown in table below. Probed with human EST revealing band in mature megakaryocyte slot.

Table 6: Table denotes DNA samples on slot blot. G (granulocytes), Eo (eosinophil), N (neutrophil), E (erythroid), Ma (macrophage), Mu (multipotential), Mg (megakaryocyte), M (mature), S (small), L (large), P (EST control plasmid).

96

Figure 19: Slot blot containing DNA samples from Table 6 . Probed with p-actin.

33.5 DWerentiai Hemopoietic Gene Expression Anaiysis

The expression of NKIAMRE was W e r examined by probing a slot blot which

contained a set of cDNA samples fkom individual hemopoietic precursor ceils mapped to

a variety of positions in the hemopoietic hierarchy (Table 6). The cDNAs were made by

amplification of poly(A)+RNA transcripts nom a variety of hemopoietic precursors. The

ampIification is representative of the extreme 3' untranslateci ends of the original

tmmcripts. The human EST was chosen as it was rmiplified in the same way and would

consequently be the most representative of the 3' untrmlated region of NKIAMRE. The

blot was probed with the human EST (R21498) revealing expression in mature

megakaryocytes (Figure 18). The probe also hybridized to the 30 ng fraction of the

human EST that was added to the blot to control for hybridization effieciency. The blot

was also probed with p-actin demonstrating expression in ail hemopoietic ceIl types

(Figure 19).

3.4 DISCUSSION

Considerable progress has been made in understanding the role of tumor suppressor

genes in human malignancies. The major emphasis of the molecular analysis of the

chromosomaI abnonnalities has fociissed on recwruig translocations resulting in an

active oncogene by combining two unrelated genes Further, molecuiar analysis has

focussed on the loss of genetic material, resulting fiom chromosomal loss or deletion,

amibuting to a gene dosage effect or the unmasking of a recessive allele on a stmcturally

normai homologue (Klein, 1988). Acute leukemia and myelodysplastic syndromes are

associated with deletion or Ioss of chromosome Sq, refmed to as the 5q- syndrome, The

Tel -c fms

Figure 20: Gene mapping of NKiAMRE. Schematic representation of 5q3 1 illustrates NKIAMRE' localization with respect to other known genes and markers. NKLAMRE localizes centromeric to the IL-9 locus and telomeric to IRF-1.

5q- syndrome, first descn'bed by Van den Berghe et al- (1974) is characterized by

rehctory anemia, morphological abnomalities of megakaryocytes, and an interstitial

deletion of the long arm of chromosome 5 as the sole karyotyic abnormality (Boultwood

et al., 1994a; Van den Berghe and Michaux, 1997). Consistent loss or deletion of the

long arm of chromosome 5 suggests a tumor suppressor locus, which may be lost early in

the development of malignant hematopoiesis, since accumulation of additional karyotypic

abnormdities characterize diseases of higher grade (Tefferi et al., 1994).

Many groups have been attempting to delineate the critical deleted region in the

5q- syndrome. The majority of genes assigned to the long a m of chromosome 5 encode

growth factors and growth factor receptors. These genes include interferon regdatory

factor (IRFI), the gene for interleukin 9, granulocyte-macrophage çolony stimulating

factor (GMCSF), and early growth response-1 gene (EGRl), and fibroblast growth factor

acidic (FGF 1) (Wasmuth et al.,1989). Wilhan et aI. (1993), demonstrated the consistent

deletion of IRFI in 13 cases of leukemia or myeIodysplasia with aberration of Sq3 1.

IRF-1 defined a critical region teIomeric to IL-5 and centromeric to GM-CSF (Figure

20). Another study, Zhao et al. (1 997a), defineci a 1 - 1.5 Mb critical region, flanked by

the anonymous markers D5S479 and D5S500, which also excluded the possibility of

EGRl and CDC25C as candidate tumor suppressor genes. Homgan et al. (1996)

identified a 1Mb region that overlaps with that of Zhao et al. (199%) between IL9 and

DSS414, through the use of PCR-based allelotyping. A study focussing on

poIyrnorphisms of the anonymous genomic marker, D5S89, identified LOH within this

region in 5 patients with Sq deletion (Nagarajan et al., 1994). DSS89 sequence,

encompased within a 300 kb YAC clone, maps telomeric to the interleukin 9 gene, but

centromeric to the EGR-1 gene, thus excluding the involvement of the loci for iL3, IL4,

IL5 and GM-CSF which are found centromeric to this site- In contrast, a study of 2

patients with rnyelodysplasia and uncommonly small 5q deletions utilized pulse field gel

electrophoresis to map 5q deletion breakpoints to a non-overlapping site telomeric to the

EGR-1 locus (Boultwood et al., 1994a &1994b). These observations suggest that

phenotypically uniform myeloproliferative disorders may have a complex and variable

polygenic etiology.

The partial human genomic sequence of NKIAMRE bas been depositeù in

Genbank and is containesi within the 28 Kb PAC H59 derived fiom a 20 Mb YAC contig

localizing to 5q31 (http://www-hp;c.lb1.gov/biology/mapping. html#Chrom5),

NKIAMRE lies immediately telomeric to the TCFl locus, and is found approximately 2

Mb centromeric to the IL-9 gene. WhiIe MUAMRE lies centromeric to the minimally

deleted regions of Zhao et al. (1997), Boultwood et al- (1994a), and Nagarajan et al.

(1994), it lies approximately 2.5 Mb telomeric to the IRF-1 locus, a site implicated in

myeloid dyscrasias as described above.

The localization of NKIAMRE was confirmeci by Fluorescent in situ

Hybridization (FISH) on metaphase chromosomes. The probe was obtained by screening

a commercial array of human genomic bacterial artificid chromosomes with NKLATRE

a. Southern hybridization of the complete NKIATRE a sequence to EcoR1-digested

gene fragments produced 3 hybridizing bands of size 1.2 kb, 3.7 kb, and 7.6 kb (Figure

14). The isolated BAC, BAC 215P23, was partial sequenced revealing identity to both

the NKIAMRE cDNA, and to PAC H59, coofirming the presence of NKIAMRE genomic

sequence. The probes were biotin labeled and the chromosome locaiization was

visualized by the interaction with FITC-avidin antibody, generating a green signal

localized to 5q3 1.1 by fluorescent microscopy (Figure 12)- Coincident chromosomal G-

banding by DAPI staining confimied the localization of NKIAMRE to chromosome

5q3 1. This chromosome localization corresponds to the reference location of PAC H59

which maps telomeric to the TCF-I gene and centromeric to the IL-9 gene.

NKLAMREYs localization to human chromosome 5q suggests that it may be

involvecl in the etiology of the 5q- syndrome. To date, a tumor suppressor gene

responsible for the syndrome has not been identified. NKIAMRE's deletion in patients

with dysmyelopoiesis would strongly support its role in the association with the disease.

To address NKIAMRE's relevance to acute leukemia, the genomic DNA fiom two

patients, with a previously identified abnormality in the long arm of one chromosome 5,

was used in Southern analysis (Figure 13). Hybridization of the complete NKIAMRE

sequence to two normal patients produced three bands of size 10.0 kb, 9.0 kb, and 2.5 kb.

In the patient with a 5q31 deletion, the 2.5 kb band was absent. Furthermore, in the

patient with a 5q13q31 deletion, the 2.5 kb band was absent and the 10.0 kb and 9.0 kb

band intensity was decreased by almost 90%. This resuIt gave the first evidence to

suggest that NKIAMRE may be deleted in patients with 5q- syndrome. The problem

with this technique is that the genomic DNA is derived fiom a heterogeneous population

of cells comprising both normal and malignant clones. Consequently, it is difficult to

access the extent of mono or bi-allelic NKIAMRE deletion in these patients. Since

NKIAMRE locaiizes to this commonly deleted region, its mono allelic loss could simpiy

reflect its proximity to other tnily relevant loci. Loss of both NKIAMRE loci, however,

wodd have greater significance, suggesting that NKIAMRE, or a closely linked gene, is

important for the generation or maintenance of the pathologic state.

The technique of fluorescent in si& hybridization was chosen as metaphase

chromosomes and interphase nuclei can accurately be assesseci for mono or bi-allelic

deletion of MUAMRE. Eighteen patients were randomly selected which demonstrated a

previous deletion within the long arm of chromosome 5 detennined by G-banding during

routine cytogenetic andysis. Dual colour FISH was performed to evaluate for deletion of

the remaining locus (Figure 15). The probes consistai of the genomic BAC 2 15P23 and

an intemal control BAC 42Hî 1, which recognizes a marker locus on choromosome 5p f 5.

Fifty cells were scored on the basis of two copies of 5p15 and the data was then

statistically analysed for deletion of NKIAMRE. The 5p 15 probe served as a marker for

hybridization efficiency and allowed us to confïrm that bi-allelic deletion was associated

with the 5q3 1.1 locus and not to entire loss of chromosome 5. We observed statistically

significant NKlAMRE loss of heterozygosity in half of the leukemic cell populations.

Statistical significance was determined by wmparing the proportion of marrow cells

having loss of either one or two BAC 215P23 signals in leukemic and in normal control

samples (Appendix). The proportion of cells having bi-allelic loss varied fiom 34% to

70% within individual leukemic samples (Table 4). A similar observation has been made

with respect to loss of the IRF-1 domain in kaxyotipically similar group of patients

(Willman et al., 1993). In this study, leukemic cells rnissing both iRF-1 hybridization

domains were seen in 6 of 1 1 samples tested, with individual proportions as high as 20%.

Thae subpopulations of cells may be emerging leukemic clones, selected for absence of

a dose-sensitive tumor suppressor locus. Although the evolution of leukemic

subpopulations with increased karyotypic complexity signals a poor clinical prognosis,

clinical outçome was not statistically worse for patients lacking the NKIAMRE locus in a

significant proportion of their malignant cells (Estey et al., 1995; Horiike et al., 1988)-

NKIAMRE's candidacy as a potential tumor suppressor gene wodd be reinforcd

if the gene was expressed in hematopoietic ceils. Expression in human boue marrow,

U937, and HL60 cells, however, was not observed (Figure 16) The expression of

MUAMRE may be associated with cells undergoing diffefentiation or =est. Expression

of NKIAMRE, however, was not observed following differentiation of U937, K562, and

HL60 cells (Figure 17). To date, expression has only been observed in mature

megakaryocytes (Figure 18). MDS is characterized by the presence of atypical

micromegakaryocytes de fined by cytoplasmic vacuolation, variable nuclear/cytoplarnic,

and varible cytoplasmic granulation (Ohshima et al., 1995; Rabellino et al., 1984). An

immunohistochemical and morphological analysis dernonstrated in 3 1 of 40 patients,

with different subtypes of MDS, the numbers of megakaryocytes were increased with a

large proportion being of smaller forms (Thiele et al., 199 1 )- NKIAMRE expression in

normal megakaryocytes suggests that its deletion in patients with MDS may be associated

with the disproportionate increase in megakaryocyte precursors and abnormal

morphology of micromegakaryocytes.

The molecular analysis of the loss of genetic material in the long arm of

chromosome 5 suggests the presence of a tumor suppressor locus. Deletion of a turnor

suppressor gene is centrai to the development of cancer and may be responsible for the

progression f?om MDS to acute leukemia Patients with a previously identified deletion

at 5q31 may s u e disease of higher grade if the remaining tumor suppressor allele is

deleted or mutated. NKLAMRE bi-allelic deletion in 9 of 18 patients with 5q- syndrome

suggests that it may be a tumor suppressor gene involved in the disease or confineci to

region where a tumor suppressor gene exists. However, NKIAMRE's isolated expression

in mature megakaryocytes suggests that it may have lunited if any involvement with the

progression and pathogenesis of acute myelogenous leukemia.

3.5 FUTURE WORK

The possible role that NKIAMRE plays in the progression and pathogenesis of

MDS needs to be fùrther exarnined. In this study, most leukemic cells had at least one

deleted NKIAMRE locus (Wl8) . The remaining allele may also be inactivated by point

mutations which can not be detected by FISH analysis. Consequently, single-stranded

conformation polymorphic analysis (SSCP) can be used to detect subtle nucleotide

changes. This technique is based on the property that single-stranded DNA molecules of

the sarne length assume different conformations depending on their nucleotide sequence.

Single-stranded DNA molecules that differ by oniy one base can be detected by

electrophoresis on nondenaturing polyacrylamide gels.

NKIATRE's preferential expression in differentiated tissues and mature

megakarycytes suggests it rnay have limited involvement in leukemia Although, its

expression may be h e l y regulated and not interpretable based on the techniques used in

this study. NKIATRE's expression can be examineci by immunohistochemisûy on bone

mmow smears. This technique offers a sensitive approach to determining expression.

The physiological significance of NKIAMRE to myelodysplasia can be assessed

by targeted mutation in a mouse. Absence of the gene rnay Iead to a murine form of

MDS or acute leukemia. Furthemore, examining the development of mice Iacking

NKIAMRE rnay provide M e r insight into the role NKiAMRE plays in differentiation

and ce11 proliferation.

It will also be necessary to understand how NKIATRE is regulated. NKIATRE

has been s h m to be activated by EGF, but not by stress stimuli such as sorbitol, UV

light and PMA (Haq et al., 1999). Mitogen activated protein kinases are reguiated by

phosphorylation of the Thr-X-Tyr activation motif. However, a mutant form of

NIUATRE in which the TDY putative activation domain has been mutated to AEF also

retains enzymatic activity. This suggests that regdation rnay occur by phosphorylation at

other residues. These sites can be determineci by western analysis using anti-phospho

tyrosine antibodies.

NKIATRE has been named based on the presence of a putative cyclin-binding

domain. NKIATRE activity rnay occur by modulation by cyclins or cyclin-like

molecules. Cyclin 1, for example, is expressed excIusively in differentiated tissues of the

brain and skeletal muscle. NKIATRE activity rnay require cyclin binding and this

interaction çan be assessed by yeast two hybnd analysis. Furthermore, the yeast two

hybnd system offers the possibility to screen other modulating molecules that rnay be

important to fünction.

The differential expression of NKIATRE in pst-mitotic tissues and its absence in

cultured ce1 lines suggests that it rnay have a role in negatively regulating ce11 growth.

The Tet-On ce11 lines generateù will be instrumental in understanding NKIATRE's

physiological d e . Determining the effects of stable NKIATRE expression on ce11

growth, viability, and differentiation can be assessed with this ce11 line.

Clearly, there are many possible experiments that r w a h to elucidate the role of

NKIAMRE in either myelodysplasia, differentiation, and ce11 growth. Whiie NKIAMRE

loss may contribute to the transformed phenotype of our study population, loss of closely

linked genes may contribute, or be solely responsible. The role of NKlAMRE in a

broader leukemic population awaits dennition through more exteasive FISH studies of

the region, together with mutational analysis of NKIAMRE. While NKLATRE has been

shown to be activated in response to mitogenic stimulation its mode of activation remains

to be understood. It will be important to identify upstream activators as well as

downstream targets. Finally, the lack of expression observed in ce11 lines suggests that

NKIATRE is important in regulating ceil growth, Future experiments with stable ce11

lines or mice lacking NKIATRE will be important in understanding its physiological role.

APPENDIX: Statistical Analysis: Cornparison of proporrim: Calculation performed to detexmine statisticai significance of marrow cells having loss of one or two BAC 215P23 signals in leukemic sampla. This statistic test compares two populations to determine if they statistical differ from each other. In our case, one population was the patient cells and the other the normal cells. If the differeme in standard enor (S.E.) between these two populations is greater than 1.96 (significant at 5%) than the populations significantly diffa fiom each other.

P 1 = # ceiis with zero/one signais X 100 P2= # cells with two sipals X 100 Total # cells counted Total # cells counted

(cl P l -P2 vs 1.96 (significant at 5%) S.E. (P 1-P2)

Example calcuiation: Patient # 5

In this case 50 patient cells were counted. Of the 50 cells; 27 had zero green BAC 215P23 signals, 14 had one green BAC 215P23 signais, and 9 had two green BAC 2 15P23 signals. Al1 of the cells scored had two red BAC 42H2 1 signals. Fi* normal cells were counted of which 92 had two green BAC 215P23 signals, 6 had one green BAC 2 15P23 signals, and 2 had zero green BAC 2 15P23 signais. Al1 of the cells scored had two red BAC 42H21 signals.

Calculation: Determine statistical significance for ceils with zero green BAC2iSP23 signals.

( A ) S.E.(Pl)= 54(100-54 r-77 = 49.70 + 7.67 = 7.57

( C ) 54-4 = 6.61 > 1.96 7.57

S.E. (P2) =/F

Since 6.6 1 is greater than 1 -96 this result is statisticaily signi fiant.

CEAPTER 4

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