CHARACTEREATION OF C-MYC AS A TRANSCRIPTIONAL REPRESSOR Wilson Marhin 1998, Doctor of Philosophy Graduate Department of Molecular and Medical Genetics University of Toronto, Canada

ABSTRACT

The c-myc proto-oncogene encodes a nuclear phosphoprotein, whose expression

has been tightly linked to cellular transformation in vivo and in vitro. The strong

tumorigenic potential of Myc is reflected in its biological activities. Myc can promote cell

cycle progression, inhibit growth arrest in the form of quiescence and senescence, and

abrogate cellular differentiation in a diverse array of cell types. In essence, the net effect of

Myc is to enhance cell growth by promoting cell proliferation and inhibiting growth arrest.

Myc is believed to mediate these diverse biological activities by acting as a transcription

factor, inducing or repressing the expression of specific subsets of genes. Analysis of the

function of the Myc-induced genes that have been identified, reveals that some of these

genes are required for progression through the cell cycle, particularly the transition from

G1 to S phase. This observation suggests that Myc may enhance cell growth through the

induction of genes which are essential to cell cycle progression. In comparison, less is

known about the importance or mechanism of Myc repression activities, due to the small

number of genes that have been reported to be repressed by Myc. Identification of the

subset of genes which are repressed by Myc will help us to understand the mechanism of

Myc-mediated gene repression, and how the repression of genes by Myc can promote cell

proliferation. The crux of my research has been to identify and characterize novel gene

targets for Myc repression, with the intention of revealing novel Myc-dependent pathways

to drive cells out of growth arrest and promote cell proliferation. We have focused

primarily on three genes: the cyclin Dl gene; the Platelet-derived growth factor P receptor

@d@-pr) gene; and the growth arrest gene, gadd45; analyzing their expression in response

to Myc-activation. In contrast to previous resuits obtained in selected immortalized cell

lines, we clearly demonstrate that in primary mouse embryonic fibroblasts, cyclin Dl

expression is not repressed by Myc. Indeed, loss of cyclin Dl expression is only evident

in cells which exhibit Myc-activation and lack retinoblastoma tumour suppressor protein

expression. Our data hrther suggest that the decrease in cyclin Dl expression is an indirect

effect of cellular transformation rather than a direct effect of Myc. In addition, we have

identified and characterized the repression of gadd45 and thepdgf-pr genes by Myc.

Analysis of these Myc-mediated repression mechanisms revealed that unlike Myc-mediated

transactivation, Myc-mediated repression is comparatively more complicated and can be

mediated by multiple pathways. The precise nature of these repression mechanisms is

currently under study. The identification of these genes as targets for Myc repression has

revealed that Myc repression pathways serve an important role in the ability of Myc to

rebwlate the cellularresponse to changes in the extracellular environment, to drive cells out

of growth arrest, and aid in tumour progression. Thus, it would appear that we are at the

dawn of a new era of Myc research. Deciphering the significance of Myc repression will

help us to understand how both Myc transactivation and Myc repression cooperate together

to regulate normal cell proliferation, through the induction of cell cycle progression and the

inhibition of growth arrest.

ACKNOWLEDGMENTS

As I sit here thinking about my acknowledgments, I realize that this is one of the

most difficult things that I have ever had to write. A number of individuals have helped

and guided me to this moment. How do you adequately thank everyone for their support

and friendship over the years? My appreciation and gratitude to these individuals cannot be

solely expressed in mere words, but I will try. I will start with thanking the one person

who has been and still is the biggest influence in my life, Dr. Linda J. Z. Penn. I am not

exaggerating when I say that she has helped me become who I am today. I reminisce about

the old days when I started my research career, Linda was in the lab helping me to do my

first Northern. That was typical of Linda, she was always available for help and

discussion. She was also very understanding, allowing me the freedom to pursue

additional experiments during my training, even though they were completely unrelated to

my main project. I have benefited greatly ftom her guidance, and so I would like to

express my gratitude to her for her support, and more importantly her friendship over the

years.

The next person on my list is Mr. Gerald Yates, my grade 10 science teacher. He

made science more than just another high school subject. In my distant past, when I was

unsure about my future, he steered me onto the path of science. As such, I owe my life in

research to him. I thank him for his mentorship, and stimulating my interest in science. I

also thank the members of my committee, Drs. Eleanor Fish and Andrew Bognar, whose

knowledge and advice supported me through my training. I would also like to thank the

members of the lab, past and present, for their friendship during my studies. The days just

fly past with guys like that in the lab. Thanks to Linda Facchini, it is upon her shoulders

that I stand. The other members of the triangle of knowledgeable sarcasm, Patrick

"Captain Quebecois" Dion and Justin "Kahuna" Lear. Gihane for being my sympatico.

The current gang Mary, Cynthia, Sara, Gina and Jim for making me laugh. Mary for the

caring, supportive person that she is. Cynthia and Sara for teaching me more than I ever

wanted to know about life. Jim for being just Jim. I reserve special thanks to my parents

and my siblings for their continual love, and support. I acknowledge the financial support

of the Ontario Graduate Scholarship Program and the University of Toronto.

Hey .... it is true, once you write that first sentence the rest is easy.

ATTRIBUTION OF DATA

I am responsible for all of the work described in this thesis with the following

exceptions. The in vitro kinase assays in Chapter 2 were performed by a collaborator, Dr.

Yong-J. Hei. The somatic cell hybrids Rat-1 x NIH3T3v3 and Rat-1 v-myc x NIH3T3v3

v-myc cells in Chapter 3 were generated by Dr. Linda M. Facchini. The H015.19 gfp and

H015.19 gfpmyc cells in Chapters 3 and 4 were generated by Sara K. Oster.

TABLE OF CONTENTS

Page

ABSTRACT

ACKNOWLEDGMENTS

AlTRIBUTION OF DATA

TABLE OF CONTENTS

LIST OF FIGURES

LIST OF ABBREVIATIONS

CHAPTER 1 INTRODUCTION

The molecular basis of cancer.

1.1.1 Normal cell signalling cascades in growth control.

1.1.2 The cell cycle.

1.1.3 Cancer: genetic aberration of growth control genes.

Myc activation and cancer.

The c-myc proto-oncogene.

1.3.1 The myc family of genes.

1.3.2 Structure of the myc gene.

1.3.3 Regulation of Myc expression.

1.3.4 Structure of the Myc protein.

1.3.5 Post-translational modification of the Myc protein.

The biological activities of Myc.

1.4.1 Myc in normal cell proliferation.

1.4.2 Myc: inhibitor of differentiation.

1.4.3 Myc and apoptosis.

vii

ii

iv

vi

vii

xiii

xvi

1.4.4 Myc and cdkIs.

1.4.5 Myc transformation in vitro and in vivo.

1.4.6 Myc cooperativity in transformation.

1.5 Mechanism of action.

1.5.1 Myc interacting proteins.

1.5.2 Transcriptional activation.

1.5.3 Transcriptional repression.

1.6 The emerging significance of Myc repression.

1.6.1 Cotrelation of repression with transformation.

1.6.2 Activation and repression co-operate to promote

cell proliferation.

1.7 Objectives.

CHAPTER 2 LOSS OF RB AND MYC ACTIVATION CO-OPERATE TO

SUPPRESS CYCLIN Dl AND CONTRIBUTE TO

TRANSFORMATION

2.1 Abshct

2.2 Introduction

2.3 Materials and Methods

2.3.1 Cell culture.

2.3.2 Genomic DNA extraction and mouse embryonic

fibroblast genotyping.

2.3.3 Retroviral infection.

2.3.4 RNase protection.

2.3.5 Immunoblotting and immunoprecipitation.

2.3.6 In vitro kinase assay.

2.4 Results

2.4.1 Primary mouse embryonic fibroblasts lacking pRb

protein expres normal levels of cyclin Dl protein.

2.4.2 Constitutive Myc expression suppresses cyclin Dl

protein in pRb-deficient but not wild-type MEFs.

2.4.3 The suppression of cyclin Dl protein in MEFs can

occur by regulatory mechanisms controlling either

RNA or protein expression.

2.4.4 pRb-deficient MEFs exhibit a delay in growth

compared to wildtype MEFs.

2.5 Discussion

2.6 Acknowledgments

CHAPTER 3 MYC IS AN ESSENTIAL NEGATIVE REGULATOR OF

PLATELET DERIVED GROWTH FACTOR BETA

RECEPTOR EXPRESSION

3.1 Abstract

3.2 Introduction

3.3 Materials and Methods

3.3.1 Cell culture, and somatic cell hybridizations.

3.3.2 Rekoviral vectors.

3.3.3 Retroviral infection.

3.3.4 RNase protection.

3.3.5 Northern blot analysis.

3.3.6 PDGF BB binding studies.

3.3.7 Luciferase assays.

3.4 Results

PDGF P receptor mRNA expression is suppressed

following serum or PDGF BB stimulation of Rat-1

cells.

3.4.2 PDGF P receptor RNA is down-regulated in Myc

-activated non-transfomed cells.

3.4.3 The suppression of PDGFP receptor mRNA levels

following Myc-activation occurs with rapid kinetics.

3.4.4 Myc-induced repression of celi surface PDGF P receptors.

3.4.5 The mechanism of PDGF P receptor suppression

differs from Myc autosuppression.

3.4.6 Myc represses transcription from thepdgf-Pr promoter.

3.4.7 Characterization of the mousepdgf-jr promoter.

3.4.8 Myc is required for the suppression inpdgf-fir mRNA

levels following mitogen stimulation.

3.5 Discussion

3.6 Acknowledgments

CHAPTER 4 MYC REPRESSES THE GROWTH ARREST GENE GADD45

4.1 Abstract

4.2 Introduction

4.3 Materials and Methods

4.3.1 Cell 4ture.

4.3.2 Retroviral infection.

4.3.3 RNase protection and luciferase assays.

4.3.4 Immunoblotting and irnmunoprecipitation.

4.4 Results

Sequential induction of c-myc and suppression of

gadd45 mRNA levels following serum stimulation

of quiescent Rat-1 fibroblasts.

Ectopic Myc expression in primary cell culhxes and

immortalized cell l i e s results in a suppression of

endogenous gadd45 mRNA levels.

4.4.3 The suppression ofgadd45 in response to Myc

-activation occurs with rapid kinetics.

4.4.4 The suppression ofgadd45 in response to Myc does

not require wildtype p53 activity.

4.4.5 Myc and p53 co-regulategadd45 transcription.

4.4.6 Myc regulates transcription fiom the gadd45 promoter.

4.4.7 Deletion analysis of the hamstergadd45 promoter.

4.4.8 Myc is an essential negative regulator ofgadd45

expression.

4.5 Discussion

4.6 Acknowledgments

CHAPTER 5 FUTURE DIRECTIONS

Summary of research.

Analysis of the relationship between Myc, pRB, and cyclin Dl.

Characterization of the repression of PDGF-PR expression by Myc.

5.3.1 Biological role of PDGF-PR suppression by Myc

following ligand stimulation.

5.3.2 Analysis of the mechanism ofpdgf-pr mRNA

suppression by Myc.

Characterization of the mechanism of gadd45 repression by Myc.

xi

5.4.1 13iological role ofgadd45 suppression by Myc 179

following rnitogen stimulation.

5.4.2 Myc abrogates the stress-induced upregulation in 181

gadd45 expression.

5.4.3 Analysis of the mechanism ofgadd45 mRNA 182

suppression by Myc.

5.5 Models of Myc repression. 183

5.6 Does Myc transactivation or Myc repression lead to cell transformation? 186

5.7 Perspectives 188

CHAPTER 6 REFERENCES 190

LIST OF FIGURES

Page

Chanter 1

Figure 1.1 Simplified schematic of a mitogenic signal transduction

pathway.

1.2 The cell cycle.

1.3 C-myc gene organization.

1.4 Myc mRNA and protein expression, in non-transformed and

transformed cells, in response to external stimuli.

1.5 Functional and structural features of the human cMyc protein.

1.6 Myc-induced Apoptosis.

1.7 Possible mechanisms for the role of Myc in driving cell cycle

progression, based on the induction of the indicated target

genes by Myc.

Chaoter 2

Figure 2.1 Determination of RB-1 gene status and pRb protein expression

in primary mouse embryonic fibroblast (MEF) cultures derived

from 3 litters of embryos produced *om the mating of RB-1

heterozygotes.

2.2 MEFs lacking pRb protein express cyclin Dl protein and

cyclin Dl associated kinase activity at levels comparable to

their wildtype littermates.

2.3 Cyclin Dl protein expression is induced in quiescent wildtype

and pRb-deficient MEFs in response to serum-stimulation.

xiii

2.4 Constitutive ectopic Myc expression in primary MEFs lacking 77

pRb protein suppresses cyclin Dl protein expression and activity.

2.5 Cyclin Dl expression in Myc-activated, pRb-deficient MEFs is 80

suppressed either at Ihe RNA or protein level.

2.6 Proliferation profiles of wildtype (MEF 4) and pRb-deficient 82

MEFs (MEF I), infected with control retrovirus (CONTROL)

or with retrovirus canying a v-myc gene (MYC).

2.7 pRb-deficient MEFs constitutively expressing ectopic Myc

protein exhibit an increased proliferative capacity.

Chapter 3

Figure 3.1 Expression of c-myc andpdgf-pr mRNAs in quiescent Rat-1

cells stimulated with serum or PDGF BB.

3.2 Suppression of endogenous c-nryc andpdgf-Dr mRNA levels

in rodent cells constitutively expressing exogenous Myc.

3.3 Pdgf-pr mRNA expression is suppressed in response to Myc

induction.

3.4 Myc suppresses PDGF-PR expression in Rat-1 cells.

3.5 The suppression of endogenouspdgf-pr and c-myc mRNA

levels by Myc appear to be mediated by different pathways.

3.6 Pdgf--P, mRNA transcription is suppressed by Myc.

3.7 Deletion analysis of the mousepdgf-fir promoter.

3.8 Myc is required for the repression ofpdgf-fir mRNA levels in

both serum stimulated quiescent cells and in proliferating cultures.

Chapter 4

Figure 4.1 Serum stimulation of confluent, quiescent Rat-1 fibroblasts

results in the induction of c-n~yc mRNA expression followed

by a suppression ofgadd45 mRNA levels.

Ectopic Myc expression in primary and immortalized fibroblasts

results in the suppression ofgadd45 mRNA and protein levels.

gadd45 mRNA expression is suppressed with rapid kinetics in

response to Myc induction.

p53 mRNA and protein expression is not responsive to ectopic

Myc expression in Rat-1 cells.

The suppression ofgadd45 by Myc does not require wildtype

p53 activity.

Myc and p53 co-regulategadd45 transcription.

gadd45 transcription is suppressed in response to Myc.

Deletion analysis of the hamstergadd45 promoter.

Myc is an essential negative regulator of gadd45 expression.

LIST OF ABBREVIATIONS

AdMLP

AKT

aMEM

B

bFGF

Pgal

BSA

cad

cak

CAT

cdk

cdkl

C-fms

CKII

CSF-1 R

CTS

dhfr

D m 1

EGF

eIF-2a

eIF-4E

ERTAD

FBS

gadd

Adenovims major late promoter

Protein kinase B

Alpha modified Eagle's medium

Myc basic region (specific DNA binding domain)

Basic fibroblast growth factor

P-galactosidase gene

Bovine serum albumin

Carbamoyl-phosphate synthaselaspartate carbamoyl transferasel

dihydroorotase

Cyclin-dependent kinase activating kinase

Chloramphenicol acetyltransferase

Cyclin-dependent kinase

Cyclin-dependent kinase inhibitor

Colony stimulating factor receptor

Casein kinase I1

Colony stimulating factor receptor

Charcoal-treated serum

Dihydrofolate reductase

Cyclii D-interacting myb-like protein

Epidermal growth factor

Eukaryotic initiation factor-2a

Eukaryotic initiation factor-4E

Estrogen receptor transcriptional activation domain

Fetal bovine serum

Growth arrest and DNA damage-inducible gene

xvi

GAP

GAPDH

gas

GFP

GSK3

h

HLH

IGF-I

Inr

LDH-A

LTag

LZ

MAP Kinase

MBI

MBII

MEF

MIDb

NBD

NLS

NLS M1

NLS M2

odc

OH-T

PBS

PCNA

PDGF

PDGF BB

The GTPase-activating protein of Ras

Glyceraldehyde-3-phosphate dehydrogenase

Growth arrest specific gene

Green fluorescent protein

Glycogen synthase kinase 3

Hour(s)

Helix-loop-helix

Insulin growth factor-1

Initiator element

Lactate dehydrogenase gene-A

SV40 Large T antigen

Leucine zipper

Mitogen-activated protein kinase

Myc box I

Myc box I1

Mouse embryonic fibroblast

Myc-regulated DEAD box-related gene

Myc non-specific DNA binding domain

Nuclear localization signal

Myc nuclear localization signal 1

Myc nuclear localization signal 2

Omithine decarboxylase

4-hydroxy (Z) tamoxifen

Phosphate buffered saline

Proliferating cell nuclear antigen

Platelet-derived growth factor BB

Platelet-derived growth factor BB

xvii

pdgf-cu

pdgf-Pr

PI3K

PKA

PKB

PKC

PLcy

P R ~

REF

RB-1

sdr

TAD

TBP

TCR

TdT

TGF-P

wtMycER

Ah4ycER

W 1

Platelet-derived growth factor alpha receptor

Platelet-derived growth factor beta receptor

Phosphoinositol3' kinase

Protein kinase A

Protein kinase B

Protein kinase C

Phospholipase C-yl

Retinoblastoma susceptibility protein

Rat embryo fibroblast

The retinoblastoma susceptibility gene

Serum deprivation response gene

Transactivation domain

TATA-binding protein

T cell receptor

Terminal deoxy-transferase

Transforming growth factor P Human c-myc(wt)/ER

Human c-myc(A106-143)IER

Yin-Yang-1

Chapter 1

INTRODUCTION

1.1 The molecular basis of cancer

1.1.1 Normal cell signalling cascades in growth control

Cells are sensitive to both positive and negative growth stimuli in their

environment. Stimuli may take a number of forms including: growth factors, inhibitory

cytokines, cell-cell interactions, and cellular contacts with the extracellular matrix. These

extracellular changes are detected by specific receptors on the cell surface, which initiate

pathways that transmit signals into the cell, eliciting a cellular response to these stimuli. In

general, different signal transduction pathways are tasked with the transmission of specific

signals. Yet, several of these signalling pathways are organizationally related, insofar as

interaction of a ligand to its receptor elicits receptor phosphorylation; and the sequential

activation of a series of intermediate components of the signal transduction mechanism.

The nature of these intermediate components is diverse and includes: soluble factors, such

as calcium ions released from intracellular stores, membrane restricted proteins, such as

small GTPases, and serine/threonine protein kinases. The overall effect is the amplification

and transmission of a signal by a cascade of intermediate proteins to the nucleus (Figure

1.1).

At the nucleus, the endpoint of these pathways, the transduced signal elicits the

induction andlor post-translational modification of nuclear protein effectors. A variety of

nuclear effectors may be induced in response to the activation of a given signal transduction

pathway. As an example, the activation of a growth promoting signal transduction

pathway, following the exposure of cells to mitogenic growth factors, elicits the induction

of a class of genes called the immediate early response genes which include the proto-

oncogenes c-myc, c-fos, and c-jzm (Cochran et al., 1983; Greenberg et al., 1985; Kelly et

al., 1983; Lau and Nathans, 1987). The expression of these genes is up-regulated within 1

to 2 h following mitogen stimulation and occurs in the absence of protein synthesis. The

MITOGEN

MITOGEN RECEPTOR CASCADE OF

SECOND MESSENGERS

UIESCENCE (GO)

IMMEDIATE EARLY

RESPONSE GENES

Figure 1.1. Simplified schematic of r mitogenic signal transduction pathway. The interaction of a mitogenic growth factor with its receptor, elicits receptor phosphorylation and activation. The activated receptor initiates a signalling cascade, delivering a signal from the cell surface to the nucleus. The expression of immediate early genes such as c-myc, c-fos, and c-jtrr~ are induced, and they function to inhibit the expression or activity of growth arrest genes such as thegadd group of genes, as well as drive entry and progression through the cell cycle.

products of the immediate early genes function to drive cells out of a growth arrested

condition known as quiescence, into an actively proliferating state (Pledger et al., 1977;

Pledger et al., 1978). Conversely, the absence of growth factors results in the repression

of these growth promoting genes in conjunction with the induction of genes which serve to

either affect or maintain a variety of growth arrested states such as that associated with

quiescence and differentiation. These are the growth arrest genes, and include genes such

as: gax; sd,; as well as the gas and gadd group of genes. The activity and interplay

between the proto-oncogenes and the growth arrest genes evoke the appropriate

modifications in gene expression, allowing the cell to adapt to changes in its environment.

1.1.2 The cell cycle

Positive or negative growth stimuli activate distinct signaling pathways which

impact on the proliferation of cells. Therefore at a molecular level, both pathways must

feed into and modulate the cell cycle. The cell cycle is tasked with orchestrating the

accurate replication and segregation of the genome of a cell into the emerging daughter

cells, within the constraints of a strict temporal program. Duplication of the genome of a

cell occurs in S phase, and the subsequent segregation of the chromosome pairs into each

daughter cell occurs during mitosis or M phase. Each of these critical phases is separated

by a gap, the GI and G2 phases respectively. During these gaps, the cell synthesizes the

requisite machinery to effect the successful completion of the forthcoming phase (reviewed

in Hatakeyama et al., 1994; Heichman and Roberts, 1994).

The cell is sensitive to the inputs of signal transduction pathways initiated by

external stimuli predominantly in the G1 phase of the cell cycle. It was observed that

during the early part of this phase, cells receive information about the state of the extcrnal

milieu, and on the basis of these signals the cell will either commit to proceeding through

the cell cycle or enter quiescence. Transit through this phase and hence cell cycle

progression is dependent upon the presence of growth factors. The position at which cells

are mitogen independent, and are committed to progressing through the cell cycle, is called

the restriction point (Pardee, 1974; Temin, 1971). Beyond this point, cells are refractory

to the absence of growth promoting signals until their entry into the subsequent G1 phase.

Progression through the cell cycle is driven by the activity of cyclin-dependent

kinases (cdks). As such the activities of these proteins are common targets of nuclear

transcription factors, which are the endpoints of several signal transduction pathways. cdk

activity is regulated via a combination of three mechanisms: binding to a cyclin which is the

regulatory subunit, the phosphorylation state of the cdk, and the interaction of the cdk with

a cdk inhibitor (cdkI) (reviewed in Fisher, 1997; Harper, 1997; Nigg, 1995). The

expression of each cyclin and hence its associated kinase activity is generally restricted to

specific phases of the cell cycle (Figure 1.2). Cyclin abundance is a balance of

transcriptional induction versus rapid degradation via post-translational mechanisms, such

as the ubiquitination-proteosome pathway (reviewed in Hershko, 1997; Pagano, 1997).

Therefore each cyclidcdk complex is responsible for promoting the progression of cells

through that associated phase of the cell cycle. There are a number of cyclins which have

been shown to drive cell cycle progression, and they can be divided on the basis of when

their activity is maximal. The GI cyclins include the D-type cyclins, Dl, D2, D3, and

cyclin E. Cyclin A expression and likewise its associated kinase activity predominates in S

and early G2 phases, while cyclin B expression is restricted to the G2 phase (reviewed in

Nigg, 1995; Sherr and Roberts, 1995).

There are essentially four cdks whose phosphorylation capabilities are required for

cell cycle progression: cdk4, cdk6, cdk2, and cdc2 (cdkl). cdk4 and cdk6 interact with

and are r.sgulated by the D-type cyclins. cdk2 is regulated by cyclin E, as well as cyclin A,

while cdc2 (cdkl) is regulated by both cyclins A and B. Cyclin-associated kinase activity

is also regulated through phosphorylation. cdk activity is activated upon phosphorylation

by the cdk-activating kinase (cak), on a single threonine residue in both cdk2 and cdk4.

Cak itself is comprised of a cyclin, cyclin H, associated with a kinase subunit, cdk7

(reviewed in Sclafani, 1996). Conversely, the phosphorylation of cdks on specific

threonine and tyrosine residues by kinases such as weel, will inhibit kinase activity

(reviewed in Lew and Kombluth, 1996). These inhibitory phosphates are removed by the

cdc25 family of phosphatases (reviewed in Draetta and Eckstein, 1997; H o f i a n n and

Karsenti, 1994). One member of this family, cdc25A is active in G1 phase and it has been

suggested that the activity of this phosphatase is required for progression through the

restriction point ( H o f i a m et al., 1994; Jim0 et al., 1994).

The final mechmism through which cdk activity is regulated is via its interaction

with cdk inhibitors (cdkI). Based on sequence and functional similarities, there are two

families of cdkI: first, the INK4 family consisting of p16, p15, p18, and p19, which

specifically inhibit the activity of the D-type cyclin-associated cdks 4 and 6; second, the

cipikip family comprising p21, p27, and p57. These inhibitors interact with and inhibit all

cdks. In addition to the differences in binding specificities, these two groups of inhibitors

also interact with their respective cyclidcdk complexes via different mechanisms. Binding

of the INK4 family to cdk4 or cdk6 inhibits the interaction of the D cyclins with the cdk.

In contrast p21, p27, and p57 are found in ccmplexes with both the cyclin and cdk

subunits (Harper, 1997; Lees, 1995).

Activation of the cyclin-associated kinase cascade initiates a number of events

which stimulate the timely progression of cells through each phase of the cell cycle.

Among these the inactivation of the retinoblastoma tumour suppressor protein (pRb) in late

G1, appears to be the critical step for the transition through the G1 phase of the cell cycle

(reviewed in Bartek et al., 1997). The pRb protein is active in its hypophosphorylated

state. As the cell progresses through the cell cycle, pRb is sequentially phosphorylated by

successive cyclidcdk complexes, resulting in the inactivation of pRb activity. In late M

phase, pRb is dephosphorylated, and hence reactivated prior to entry into the subsequent

cell cycle (Figure 1.2). Hypophosphorylated pRb functions as a cell cycle arrestor by

binding to and inhibiting the activity of members of the E2F family of transcription factors.

The pRblE2F complex is an active repressor of E2F responsive genes whose activity is

required for progression through S phase. In GI phase, both cyclins D and E with their

associated cdks are required for the phosphorylation and inhibition of pRb function.

Therefore the inactivation of pRb permits the release of E2F ftom pRb/EZF complexes,

facilitating the transcription of S phase genes and S phase transition (reviewed in Nevins et

al., 1997; Reed, 1997).

1.1.3 Cancer: genetic aberration of growth control genes

Both growth stimulatory and inhibitory signals elicit modifications to gene

expression to drive either proliferation or growth arrest, in part through the modulation of

components of the cell cycle. Changes to the cell cycle are manifested in a number of

ways, through the regulation of either one or all of the following: cyclin expression, the

activity of cak and cdc25A, or the activity of cdkIs. Several players in signal transduction

pathways function either as proto-oncogenes conferring a strong proliferative impetus,

such as the c-myc proto-oncogene, or as growth inhibitors which guard against

uncontrolled cell proliferation; for example, the pRb tumour suppressor protein. Normal

cell proliferation entails a balance between proto-oncogenes and growth inhibitors, and

perturbations in this fine balance commonly lead to tumour formation. For example,

mutations which result in the deregulated expression of a component of a growth

promoting signal transduction pathway, such as a proto-oncogene, will enhance normal

cell proliferation and elicit unrestrained growth. A similar fate may arise if there is a

mutation in the expression or activity of growth inhibitors. In either case, there is the

potential that the increase in the number of cell divisions, with the associated elevation in

the number of DNA replication events, will facilitate the genesis of additional oncogenic

mutations which promote tumorigenesis. This model introduces one of the tenets of

turnorigenesis. A single genetic aberration may enhance the proliferative capacity of a cell,

yet will not induce turnour formation; additional cooperating oncogenic events are required

to elicit full cellular transformation. The reasoning for this cooperative relationship is still

unclear. On the basis of our knowledge of the functions of individual oncogenes which

have been reported to synergize to elicit tumour growth, we can hypothesize that each

oncogene within the partnership serves a complementary function. Each mutation is

required for full transformation. This may reflect the number of extracellular signals which

are needed by a normal cell to proliferate. The c-myc proto-oncogene is one well-described

example of an oncogene which is a component of many proliferative signal transduction

pathways and is also frequently mutated in a variety of tumours (reviewed in Field and

Spandidos, 1990; Garte, 1993; Schwab and Amler, 1990).

1.2 Myc activation and cancer

The v-gag-myc gene was the first member of the myc gene family to be isolated.

V-gag-myc is a potent transforming oncogene found in aggressive avian retroviruses, such

as MC29,OK10, CMII, and MH2. These viruses all induce acute lymphoid malignancies

in chickens with high frequency (reviewed in Cole, 1986; Graf and Beug, 1978). The

identification and analysis of the cellular homologues of this oncogene, revealed the strong

oncogenic potential of members of this gene family. C-myc, the first cellular homologue to

be identified, is frequently activated in human tumours originating from, but not restricted

to, cells of hematopoietic origin. Indeed, in humans the array of tumour types which

frequently exhibit alterations in Myc expression is extensive: hematopoietic malignancies

such as lymphoma and leukemia, connective tissue cancers such as osteosarcoma,

carcinomas derived from the epithelial layers of the breast, lung, cervix, ovary, stomach,

prostate, and colon, as well as squamous cell carcinomas of head and neck (reviewed in

Field and Spandidos, 1990; Garte, 1993; Schwab and Amler, 1990). Structurally and

functionally related family members, N-myc and L-myc also have a significant impact in

carcinogenesis. The N-myc gene is amplified in human neuroblastoma, retinoblastoma,

and small cell lung carcinoma (Brodeur et al., 1984; Brodeur et al., 1985; Kohl et al.,

1984; Schwab et al., 1983). L-myc expression is commonly elevated in small cell lung

carcinoma (Barren et al., 1992; Bernard et al., 1992; Kawashima et al., 1992; Makela et

al., 1992; Nau et al., 1985; Schreiber-Agus et al., 1993). Thus, the members of the myc

oncogene family possess a strong oncogenic potential and play a significant role in a large

number of human cancers arising from a wide variety of tissues.

In many turnours, Myc activation can be attributed to structural alterations affecting

the myc gene, including chromosomal translocations, retroviral promoter or enhancer

insertions, and gene amplification (reviewed in Cole, 1986; Kelly and Siebenlist, 1986;

Marcu et al., 1992). For example, B-cell neoplasias, including human Burkitt's

lymphoma, rat immunocytoma, and mouse plasmacytomas, consistently display a

reciprocal chromosomal translocation involving the c-myc locus with one of three

immunoglobulin loci (Kelly and Siebenlist, 1986). The chimeric gene consists of c-myc

coding sequences juxtaposed to transcriptional regulatory elements of one of the

immunoglobulin genes, resulting in a B cell-specific transcription unit driving the

constitutive expression of the c-myc oncogene. Although deregulated expression can be

associated with an elevation of Myc expression, many Burkitt's lymphoma cells exhibit c-

myc mRNA expression at levels comparable to physiological levels seen in proliferating B

cells (Keath et al., 1984; Taub et al., 1984). It has been shown that the translocated c-myc

allele frequently harbors point mutations in exon 11-coding sequences. However, the

individual mutations are not consistent among Burkitt's lymphoma cells, and they are not

correlated with specific phenotypes of the tumour (reviewed in Zimrnerman and Alt, 1990).

Thus, the primary event converting the proto-oncogene to its oncogenic form is

deregulation of the intact c-myc coding region, leading to continuous Myc protein

expression (Figure 1.4). Elevated expression or alterations in the protein product through

point mutations likely further potentiates the activity of the activated allele. It remains to be

determined whether deregulated, elevated Myc levels promote tumorigenesis by

constitutively activating the physiologically normal functions of Myc or through the

initiation of novel mechanisms which are specific to the turnongenic pathway.

Another c-myc activating mutation which commonly leads to deregulation of c-myc

expression is retroviral promoter or enhancer insertion (reviewed in Kelly and Siebenlist,

1986). The c-myc gene is a common site for integration of the avian leukosis virus in pre-

B cells of the bursa, resulting in the genesis of bursal lymphomas (Hayward et al., 1981;

Payne et al., 1982). Integrations occur predominantly 5' to e-myc coding sequences and in

the same transcriptional orientation as the c-myc gene. As a result of the juxtaposition of

the retroviral LTR and c-myc sequences, c-myc mRNA expression is enhanced due to

transcription from the retroviral LTR (Hayward et al., 1981; Payne et al., 1982). On

occasion, the proviral DNA integrates in the opposite transcriptional orientation to or

downstream of the c-myc gene (Corcoran et al., 1984; Li et al., 1984). In these instances,

elevated c-myc mRNA transcription appears to be due to the activity of retroviral enhancer

elements (Payne et al., 1982). T cell lymphomas resulting from the infection of newborn

mice with murine leukemia virus (Selten et al., 1984; Steffen, 1984) also exhibit proviral

integrations at the c-myc gene. In both T and B cell lymphomas, c-myc expression is

elevated in comparison to normal cells, yet it is not clear whether the increase in c-myc

expression is due to an elevation in the rate of transcription or due to the disruption of

transcriptional regulatory elements in the c-myc gene (Kelly and Siebenlist, 1986). The

consistent observation in these tumours of proviral integrations at the c-myc gene, suggests

that deregulated Myc expression may play an important role in the genesis of these

malignancies.

The third somatic cell aberration which leads to Myc activation is gene

amplification. Amplification does not alter the structure of the myc gene, it simply

increases the number of normal myc genes within the cell, resulting in an elevation in the

number of Myc protein molecules per cell (reviewed in Cole, 1986; Zirnmerman and Alt,

1990). The amplified myc genes are not necessarily deregulated, and retain their sensitivity

to growth regulatory agents in the external environment. For example, the promyelocytic

cell line HL60 exhibits enhanced c-Myc expression due to a 10 to 50 fold amplification of

the c-myc gene (Dalla-Favera et al., 1982), yet exposure to differentiation agents will

trigger the usual reduction in endogenous c-myc mRNA and protein levels associated with

myeloid cell differentiation (Reitsma et al., 1983). Myc amplification plays an important

role in the promotion of tumorigenesis, demonstrated by an analysis of a large number of

primary malignant tumours derived from a wide variety of different tissues, which revealed

that 10% contained an amplification of a c-myc allele (Yokota et al., 1986). Interestingly,

ntyc amplification appears to correlate with more aggressive stages of malignancies. In

neuroblastomas, N-my amplification is only observed in the more tumorigenic forms,

stages 111 and IV, of this disease (Brodeur et al., 1984; Brodeur et al., 1985; Seeger et al.,

1985). In addition, in small cell lung carcinoma, c-myc amplification is found only in the

more malignant variants of this cancer (Little et al., 1983). These observations suggest

Myc activation by amplification confers a growth advantage to a wide variety of cells, but

this mechanism of activation may be restricted to more genetically unstable transformed

cells. Thus, activation of Myc expression can arise from gross somatic mutations which

elicit either deregulated andlor constitutive Myc expression. Taken together the evidence

strongly suggests that temporally inappropriate Myc expression is the critical component of

Myc activation which promotes the tumorigenic program.

Gross genetic alterations may not be the only mechanism leading to Myc activation.

Myc activities may be enhanced through mutations which inactivate any one of the many

mechanisms controlling normal Myc expression, or the Myc protein may engage in novel

protein-protein interactions initiating transformation specific functions. These mechanisms

may potentially confer a growth advantage to the affected cell, and contribute to the

stepwise progression of tumorigenesis. If so, then the estimated frequency of Myc

activation in human cancer is presently grossly underestimated, as it is based on detection

of gross genetic abnormalities of the myc family members. The more subtle Myc activation

events which likely occur in cells from malignant transformations should be readily

ascertainable in the near future, primarily through the identification and characterization of

Myc-interacting proteins and the recent advances in diagnostic methods to measure changes

in gene expression, such as RNA-FISH and quantitative RT-PCR. By these approaches,

the absolute frequency of Myc activation, the specificity of tumour types affected and the

nature of collaborating mutations involved in human cancer will be understood. Moreover,

with these advances, the molecular mechanisms and the underlying cause of the more

subtle activation mechanisms can be better understood and key regulators of Myc

expression uncovered. Thus, quantitating subtle as well as gross Myc activation events in

human tumours will significantly advance both clinical and basic aspects of the field.

Whether gross or subtle mutations are involved, the net result of these somatic aberrations

is the activation of the myc proto-oncogene and the promotion of unrestrained cell

proliferation, leading ultimately to cellular transformation.

1.3 The c-myc proto-oncogene

1.3.1 The myc family of genes

The importance of the myc proto-oncogene is emphasized by its evolutionary

conservation. Homologues of c-myc have been identified in the genomes of a variety of

organisms including trout, zebrafish, frog, sea star, chicken, rat, mouse, and more

recently, the fruitfly, drosophila (Dalla-Favera et al., 1982; Dalla-Favera et al., 1982;

Eladari et al., 1992; Gallant et al., 1996; Hayashi et al., 1987; King et al., 1986; Schreiber-

A y s et al., 1993; Stanton et al., 1984; Van Beneden et al., 1986; Walker et al., 1992).

However, as yet no c-n~yc homologues have been detected in yeast or C. elegans, two

well-characterized organisms. In eukaryotic cells, the myc family members c-myc, N-

nyc, and L-myc are functionally related, executing similar biochemical activities. Yet they

do exhibit distinct temporal and spatial expression patterns in the developing embryo and in

adult tissues, suggesting that each member plays distinct roles during embryogenesis.

Indeed, transgenic and gene disruption studies in the developing mouse embryo have

revealed that each Myc family member plays a non-complementary role during

development. The loss of function of one Myc family member can only be partially

compensated through the expression of the other Myc proteins (Davis and Bradley, 1993;

Davis et al., 1993).

B-myc (Asker et al., 1995; Ingvarsson et al., 1988), and S-myc (Sugiyama et al.,

1989), are additional closely related genes which were both isolated From rat genomic

libraries. The importance of B-tyc and S-tnyc gene expression in normal cell growth is

not fully understood as their protein products remain to be detected in vivo. B-myc mRNA

is expressed in a variety of tissues with its highest expression in the brain (Ingvarsson et

al., 1988). The B-myc mRNA is homologous to exon 2 of the c-myc gene, and encodes a

putative 168 amino acid protein. The S-myc gene consists of sequences homologous to

exons 2 and 3 of N-myc, but lacks the intervening intron (Asai et al., 1994; Sugiyama et

al., 1989). The S-myc mRNA has only been detected in rat embryo chrondocytes.

Although the precise roles of the putative S-Myc and B-Myc proteins in the cell are not

known, some studies indicate that these proteins can antagonize the activity of Myc (Resar

et al., 1993). This may serve to fine-tune the activities of Myc and determine how Myc

responds to changes in the external environment.

1.3.2 Structure of the myc gene

The c-nzyc gene is located on chromosome 8 in humans and chromosome 15 in

mice (Dalla-Favera et al., 1982; Nee1 et al., 1982). The structure of the c-myc gene is

highly conserved between species, and among family members (reviewed in Lemaitre et

al., 1996). The c-myc gene consists of three exons; the first exon being largely

untranslated, with the remaining two highly conserved exons encoding the Myc protein

(Bernard et al., 1983; Colby et al., 1983; Wan et al., 1983). C-myc transcription is driven

from distinct promoters. The major promoters are PI and P2, P1 being located at the start

of exon I and P2, 150 bp downstream of P1. P1 accounts for only 10 % of total c-myc

mRNA, while P2 is responsible for over 85 % of the c-myc transcripts. There are also two

additional promoters, PO and P3; PO being found 550 bp upstream of PI, and P3 in intron

1. Less than 5 % of total c-myc mRNA originates from both of these promoters.

Transcription is also affected by the presence of two transcriptional attenuation sites, each

downstream of either P1 or P2, which are responsible for the pausing of RNA polymerase

11. Antisense c-myc transcripts originating from exon I1 and intron I have also been

reported; however, the importance of these transcripts to Myc expression or function is

unknown. Transcripts terminate at one of two polyadenylation signals in the untranslated

portion of exon 111; the 3' signal being the predominant site of transcriptional termination

(reviewed in Lemaitre et al., 1996). A schematic diagram of the genomic structure of the

human c-mjc gene is illustrated in Figure 1.3.

Figure 1.3. C-myc gene organization. The c-myc gene consists of three exons. The promoters (PO, PI, P2, P3), translation initiation sites (AUG, CUG), and the polyadenylation sites (AAAI, AAA2) are indicated. The translated sequences in each exon are represented by open rectangles.

1.3.3 Regulation of Myc expression

Expression of the myc proto-oncogene is induced as a consequence of signaling

pathways downstream of a variety of mitogens such as platelet-derived growth factor

(PDGF), epidermal growth factor (EGF), basic fibroblast growth factor @FGF), and 12-

0-tetradecanoylphorbol-13-acetate (Cutry et al., 1989; Dean et al., 1986; Kelly et al.,

1983). The components of the signal transduction pathways which drive myc transcription

have not been fully elucidated, yet studies have identified a few of the players such as

protein kinase C (PKC), protein kinase A (PKA), src, E2F, ets, and abl (Barone and

Courineidge, 1995; Cleveland et al., 1989; Coughlin et al., 1985; Hamel et al., 1992;

Kelly et al., 1983; Mudryj et al., 1990; Ran et al., 1986; Roussel et al., 1991; Roussel et

al., 1994; Wong et al., 1995).

A role for src in the induction of Myc expression, following mitogen stimulation,

was suggested in studies utilizing a mutant CSF-1 receptor (Roussel et al., 1991).

Activation of the mutant receptor elicited the induction of the immediate early genes. c-fos

and c-jlm, but c-myc induction was abrogated. In addition, activation of the src tyrosine

kinase was reduced, suggesting a possible link between src activation and Myc expression.

A definitive role for src in the signaling pathway responsible for Myc induction was

revealed upon the analysis of the PDGF-induced signaling mechanisms which drive

fibroblasts out of quiescence. Ectopic expression of dominant negative mutants of src, or

inhibitory antibodies to src inhibited c-Myc transcription and progression of cells into the

replicative phase (Barone and Courtneidge, 1995).

Evidence suggests that activated src induces the transcription of the c-myc gene by

regulating the activity of the ets family of transcription factors (Roussel et al., 1994).

There is a conserved ets binding site in the c-myc promoter between P1 and P2. Mutation

of this site will abrogate the src-dependent induction in c-myc expression. This

observation may suggest that inhibiting the binding of ets proteins will abrogate c-myc

transcription, yet the answer may not be so simple. The ets binding site is a part of a

highly conserved binding site for the E2F family of transcriptional regulators. The binding

of E2F to this site has also been shown to be required for the induction of c-myc

transcription in response to stimulation with mitogens such as serum (Mudryj et al., 1990).

In addition, the nonreceptor tyrosine kinase c-abl, another component of common signal

transduction pathways, also induces c-myc transcription through the E2F site (Wong et al.,

1995). Hence, mutation of the ets and consequently the E2F binding site, potentially

negates three separable signaling pathways. Thus, recent studies have identified a few

important regulators of myc transcription; however, further characterization is required to

determine the contribution of each of these mechanisms to the regulation of Myc expression

following exposure to external stimuli.

Given the strong proliferative capacity of an activated myc allele it is not surprising

that, in non-transformed cells, endogenous c-myc expression is tightly regulated at both the

mRNA and protein level (reviewed in Spencer and Groudine, 1991). Indeed, Myc

expression is tightly linked to the growth state of the cell, and is immediately responsive to

changes in the external environment (reviewed in Lemaitre et al., 1996). Myc mRNA and

protein expression are low in quiescent, growth arrested cells. Upon mitogen stimulation,

Myc mRNA and protein expression levels are rapidly elevated approximately 40 fold

within 2 h, concomitant with the entrance of cells into the cell cycle (Campisi et al., 1984;

Greenberg et al., 1986; Kelly et al., 1983). The induction in Myc expression is transient: 6

to 12 h following mitogen stimulation Myc expression decreases to levels approximately 10

fold above that observed in quiescent cells. Myc expression is maintained at this level

through the remainder of, and in subsequent cell cycles (Rabbitts et al., 1985; Rabbitts et

al., 1985). Removal of mitogens or induction to differentiate results in a rapid loss of Myc

expression and exit from the cell cycle (Figure 1.4) (Dean et al., 1986; Kelly and

Siebenlist, 1986; Rabbitts et al., 1985; Waters et al., 1991). As Myc expression is so

closely associated with the growth state of the cell, it is possible that Myc functions as an

internal barometer of conditions in the external mi!ieu, deciding when it is possible to

proliferate and maintaining the cell in this state until conditions are no longer favourable for

growth. The half-lives of the c-Myc protein and mRNA are short (Dani et al., 1984; Ham

and Eisenman, 1984), and are well suited to the role of Myc as a sensor and responder to

external changes. c-Myc protein and mRNA expression is controlled via a host of

processes which act at multiple stages: at the levels of mRNA transcription initiation,

elongation, and stability, as well as at the level of protein degradation (reviewed in Cole,

1986). The existence of multiple mechanisms to govern Myc expression emphasizes the

need to control the strong proliferative potential of Myc.

MYC mRNA and protein

EXPRESSION

t GROWTHARRESTED QUIESCENT PROLIFERATING

DIFFERENTIATED

TRANSFORMED CELLS

NON-TRANSFORMED

I CELLS

Mitogen Stimulation

Growth inhibitors or

Differentiation Agents

Figure 1.4. Myc mRNA and protein expression, in non-transformed and transformed cells, in response to external stimuli. In non-transformed primary and immortalized cells, Myc expression is highly responsive to changes in the external environment, with Myc expression being tightly linked to actively proliferating cells (solid line). In transformed cells, Myc expression is frequently deregulated, and is refractory to the nature of the extracellular milieau (hatched line). Levels of Myc expression in transformed cells vary, depending upon the nature of the activating mutation of the nlyc gene.

1.3.4 Structure of the Myc protein

The c-myc gene encodes two polypeptides with molecular weights of

approximately 64 and 67 kd (Hann et al., 1988). The half-life of the Myc proteins are very

short, approximately 20 to 30 mins, mimicking the half-life of the c-myc mRNA (Luscher

and Eisenman, 1990). Translation of the 64 kd (Myc-2) protein initiates from an AUG

start codon in exon 11, while the larger Myc-1 protein, containing 14 additional amino acids

at the amino-terminus, originates from a cryptic CUG at the end of exon I (Figure 1.3)

(Ham et al., 1988). Myc-2 consists of 439 amino acids and is more highly expressed in

most cell types, compared to Myc-1. At a functional level there is no apparent difference in

the activities of these two species (Blackwood et al., 1994). Yet it was observed that

expression of the p67Myc protein increases in quiescent cells, suggesting a potential

distinct role for this isoform of Myc in growth arrest (Hann, 1995; Ham et al., 1992).

Indeed, in support of this hypothesis, ectopic expression of p67Myc by Cos cells appeared

to induce growth arrest (Hann et al., 1994). Recently, it was recognized that smaller

proteins were also translated from the c-myc mRNA transcript, arising from conserved

internal translational initiation codons within the coding region of c-myc. C-Myc S

proteins lack the first 100 amino acids of the amino terminus of the full-length Myc-1

protein (Spotts et al., 1997). C-Myc S proteins are ubiquitously expressed, and are

evident in human, avian, and murine cells. Additional studies are required to fully

comprehend the relationship among the different Myc isoforms, and the role of these

species in the regulation of normal cell proliferation.

Computer analysis of the amino acid sequence of human c-Myc predicts that the

structure of the amino-terminus (amino acids 1 to 203) consists of a-helical and P-sheet

domains and the carboxyl end (amino acids 238 to 439) is composed primarily of a-

helices, while the intervening region appears to be an unstructured hinge (reviewed in Penn

et al., 1990). There is evidence for a number of functional domains, such as a

transcriptional regulatory domain, nuclear localization motifs, as well as sequences which

effect specific DNA binding, and protein dimerization (Figure 1 SA).

The transcriptional activation domains of Myc comprise the amino-terminal 143

amino acids and were identified through studies by Kato et al. (Kato et al., 1990). They

assayed the ability of different portions of Myc, fused to the DNA binding domain of the

yeast transcription factor Ga14, to transcriptionally activate a heterologous promoter

containing Gal4 DNA binding sites. This transactivation domain can be further divided

into three autonomous portions. The region spanning amino acids 1 to 41 is glutamine-

rich, and shares sequence homology with the transcriptional activation domains of the well-

characterized transcription factors Spl and herpesvirus VP16 proteins (Mitchell and Tjian,

1989). The second region, amino acids 42 to 103, is rich in prolines, similar to the

transcriptional regulatory domain of the CTFMFl family of transcriptional regulators

(Mitchell and Tjian, 1989). The final region, amino acids 104 to 143, is not homologous

to the transactivation domain of any known transcription factor (Kato et al., 1990). Myc

repression of gene expression has also been mapped to this region; however, the precise

sequences required for specific repression events vary. Amino acids 106 to 143 are

required for c-myc negative autosuppression; amino acids 92 to 106 are involved in the

suppression of cyclin Dl; while residues 122 to 143 effect repression of the adenovims

major late promoter (Li et al., 1994; Penn et al., 1990; Philipp et al., 1994). The existence

of this discrepancy suggests that there are multiple mechanisms for Myc repression.

Sequence analysis of the members of the Myc protein fami!? have highlighted regions

which are highly conserved among the members of the Myc protein family. Conservation

of these regions suggests that they may be critical for the functions of Myc. Two Myc

boxes, Myc Box 1 and 2, are found in the transcriptional activation domain; Myc Box 1

comprises amino acids 45 to 63 in humans, and Myc box 2 consists of amino acids 129 to

141 (reviewed in Ingvarsson, 1990). Myc box 2 is essential for all of the biological

activities of Myc such as cell cycle progression, inhibition of differentiation, exit

Figure 1.5. Functional and structural features of the human c-Myc protein.

A. Myc 1 (p67) initiates at a CUG in exon I, and contains 14 additional amino acids

compared to Myc 2 which initiates at an AUG in exon 11. Functionally important domains

are indicated: c-Myc harbours three independent transcriptional activation domains (I, 11,

111); a non-specific DNA binding domain (NDB); a basic region (B); a helix-loop-helix

motif (HLH); and a leucine zipper domain (LZ). Filled rectangles in the transcriptional

activation domains represent Myc Box I (MBI) and Myc Box I1 (MBII), two highly

conserved regions found in all Myc family members. At the carboxy terminus there are

two nuclear localization signals (NLS MI, NLS M2). Phosphorylation sites (P) and their

putative kinases are indicated at the top. B. The domains of Myc which are required for

Myc functions and involved in the interaction with other proteins are shown.

from quiescence, induction of apoptosis, and tumorigenesis (Eilers et al., 1989; Evan et

al., 1992; Freytag et al., 1990; Stone et al., 1987). The transcription regulatory domains

are also required for interactions with other proteins such as the transcription factor AP-2

(Gaubatz et al., 1995).

The c-Myc protein contains two nuclear localization domains, which facilitate the

transport and translocation of c-Myc into the nucleus (Dang and Lee, 1988; Stone et al.,

1987). The predominant nuclear localization signal (NLS) called NLS MI, comprises

amino acids 320 to 328 in human, and is homologous with the well characterized NLS of

the SV40 and polyoma virus T antigens (Kalderon et al., 1984; Richardson et al., 1986).

These sequences are able to direct the normally cytoplasmic protein pyruvate kinase into the

nucleus (Dang and Lee, 1988). In the absence of NLS MI, the weaker NLS M2 can

substitute partially for NLS Ml. NLS M2 consists of amino acids 364 to 374 in humans,

and coincides with the DNA binding domain (Dang and Lee, 1988).

c-Myc can bind nonspecifically to single-stranded and double-stranded DNA, in

vitro (Beimling et a]., 1985; Persson and Leder, 1984; Watt et al., 1985). The region

responsible for this activity is contained within amino acids 265 to 3 18 in human c-Myc

(NDB) (Dang et a]., 1989). It is believed that this activity allows c-Myc to b i d loosely to

and move along DNA, until it encounters a high affinity binding site. As yet, a role for this

domain in vivo has not been established; however, deletion of this region will abolish the

ability of c-Myc to transform cells (Stone et al., 1987).

Structure-function analysis of the carboxyl terminus of the c-Myc protein revealed

the presence of a number of conserved motifs which are common among well-characterized

transcription factors. There is a basic region (B) (amino acids 355 to 367 in humans),

which serves as a specific DNA binding domain. In addition, there are two protein-protein

interaction motifs, a helix-loop-helix domain (HLH) (amino acids 368 to 410 in humans),

and a leucine zipper motif (LZ) (amino acids 41 1 to 439) (Kerkhoff and Bister, 1991).

Deletion of this region will inhibit all of the biological activities of Myc, suggesting that

protein-protein interaction and DNA binding is required for Myc function. The

combination and contiguous arrangement of these domains is common within a family of

B-HLH-LZ-containing transcriptional regulators including USF, TFEB, AP-4, and TFE3

(Beckmann et al., 1990; Carr and Sharp, 1990; Gregor et al., 1990; Hu et al., 1990).

Both USF and TFE3 bind specifically to a consensus DNA element, CANNTG, called an

E-box, suggesting that Myc may also bind DNA through this site. This hypothesis was

substantiated in a variety of studies utilizing either the bacterially expressed c-Myc

carboxyl-terminus, chimeric proteins consisting of the c-Myc basic region linked to the

helix-loop-helix domain of either the drosophila El2 or the yeast pH04 proteins, or the full

length Myc protein (Blackwell et al., 1990; Fisher et al., 1991; Halazonetis and Kandil,

1991; Kerkhoff et al., 1991). All of these studies demonstrated that c-Myc binds

specifically to the sequence CACGTG in vitrv. Myc is also able to bind to specific

noncanonical sites related in sequence to the E-box, with high affinity (Blackwell et al.,

1993; Grandori et al., 1996). The identification of the specific DNA binding property of

Myc was a significant observation, as it suggested that Myc may effect its biological

activities through the regulation of gene expression. Myc homodimers are not observed in

vivo, due to steric interference between the protein-protein interaction domains of the c-

Myc proteins (Amati et al., 1993). Specific DNA binding by Myc is dependent upon

interaction with a ubiquitously expressed B-HLH-LZ protein, Max (Blackwood and

Eisenman, 1991; Prendergast et al., 1991). The formation of MycIMax heterodimers is

favoured over MadMax homodimers; and as a consequence Myc is predominantly found

complexed with Max (Blackwood and Eisenman, 1992; Kato et al., 1992; Littlewood et

al., 1992). Heterodimerization of Myc and Max is mediated through the protein

dimerization motifs, the helix-loop-helix and the leucine zipper (Blackwood and Eisenman,

1991).

1.3.5 Post-translational modification of the Myc protein

The c-Myc protein is phosphorylated at multiple serine and threonine sites in vivo,

suggesting that phosphorylation may play some role in the regulation of Myc activity. The

sites of phosphorylation include: Thr58, Ser62, Ser71, Ser82, Ser164, five sites between

residues 240 and 262, as well as Ser293, Thr343, Ser344, Ser347, and Ser348. In vitro,

the amino-terminus of Myc is phospholylated on three sites, threonine 58, serine 62, and

serine 71 by a variety of kinases including mitogen-activated protein kinase (MAP kinase),

glycogen synthase kiiase 3 (GSK3), cyclin-dependent kinase 1 (cdkl), and a pl07/cyclin

AIcdk2 complex (Alvarez et al., 1991; Gu et al., 1994; Henriksson et al., 1993; Hoang et

al., 1995; Lutterbach and Ham, 1994; Pulverer et al., 1994; Seth et al., 1991). The

importance of these phosphorylation events in Myc function is at present unclear, yet the

high incidence of mutations at these sites in tumours such as Burkitt's lymphoma suggests

that they may play a role in the capacity of Myc to promote hunour formation (Gupta et al.,

1993). Threonine 58 is also reported to be the target of glycosylation; however, whether

phosphorylation or glycosylation of this residue has any effect on Myc-mediated cellular

transformation is not known (Chou et al., 1995; Chou et al., 1995). Residues in the

carboxyl-terminus are phosphorylated by the ubiquitous kinase, casein kinase I1 (CKII), in

vitro (Luscher et al., 1989). CKII phosphorylates c-Myc at sites located behveen amino

acids 240 to 262, and amino acids 342 to 357 in the human c-Myc protein. The proximity

of the latter phosphorylation events to the basic region suggested that they may influence

Myc function by affecting DNA binding. However, studies have failed to attribute a

hnction to any of these phosphorylations (Street et al., 1990).

1.4 The biological activities of Myc

1.4.1 Myc in normal cell proliferation

Several studies have supported the model that c-Myc protein expression is essential

for the promotion of cell cycle progression and the maintenance of cell proliferation.

Constitutive ectopic Myc expression in fibroblast cells will abrogate the requirement for

growth factors to progress from the G1 to S phase of the cell cycle (Armelin et al., 1984;

Kam et al., 1989; Mougneau et al., 1984; Sorrentino et al., 1986; Stem et al., 1986).

Elevated expression will increase the growth rate, predominantly through the shortening of

the GI phase (Kam et al., 1989). Using MycER, an estrogen-inducible conditional allele

of Myc, it has been demonstrated that induction of Myc activity alone can induce cells to

exit growth arrest and progress through the cell cycle (Daksis et al., 1994; Eilers et al.,

1989). Indeed, microinjection of Myc alone into quiescent fibroblasts will induce cell cycle

entry (Kaczmarek et al., 1985). Moreover, an inhibition of Myc expression through the

use of antisense RNA will result in growth arrest (Heikkila et al., 1987; Holt et al., 1988).

Interestingly, a subclone of the immortalized cell line Rat-I, in which both alleles of the c-

myc gene are deleted through homologous recombination, is viable but exhibits a slower

growth rate and reduced ability to enter the cell cycle after serum stimulation (Mateyak et

al., 1997). The importance of Myc expression in effecting cell cycle entry and hence cell

proliferation in serum-stimulated quiescent cells, was illustrated via elegant studies by

Roussel eta[. (Roussel et al., 1991). Ectopic expression of the colony stimulating factor

receptor (CSF-I R) by the immortalized mouse NIH3T3 cell line renders these cells

responsive to stimulation with the cytokine, CSF-1. A Y809F substitution in the CSF-1

R, specifically abrogates the induction of c-myc expression inhibiting the growth

promoting affects of CSF-1 treatment. Ectopic expression of Myc in these cells will rescue

the CSF-1-mediated entry into cell cycle. Taken together, these observations suggest that

Myc activity is crucial for cells to exit growth arrest and enter the cell cycle.

As Myc protein is continuously expressed in asynchronously proliferating cells,

Myc may have additional functions at other points of the cell cycle. Indeed, a potential role

for Myc in the G2 phase has recently been suggested through the studies of c-myc null rat

fibroblasts. Disruption of Myc expression does not inhibit growth, but both the G1 and

G2 phases are noticeably lengthened while the duration of S phase is unaltered (Mateyak et

al., 1997). This was the first study to directly demonstrate a role for Myc in progression

through the G2 phase of the cell cycle, although its hnction in this period is unclear and

studies are currently underway which will shed light on this question.

Thus, considerable evidence exists to support a role for Myc in driving cells out of

quiescence and into the cell cycle. Yet the continued expression of Myc in asynchronously

proliferating cells suggests that additional roles for Myc in the promotion of cell cycle

progression remain to be defined. Studies in Myc-null cells hint at a role for Myc in the

progression of cells through the G2 phase; however, the function and the importance of

Myc in this phase has not been fully explored.

1.4.2 Myc: inhibitor of differentiation

Consistent with the role of Myc as a strong proliferative stimulus, Myc expression

can inhibit the differentiation program in a number of cellular systems. Indeed, in a variety

of cultured cells exposure to inducers of differentiation results in the repression of Myc

expression and a withdrawal from the cell cycle (Bianchi Scarra et al., 1986; Gonda and

Metcalf, 1984; Gowda et al., 1986; Griep and DeLuca, 1986; Lachman and Skoultchi,

1984; Reitsma et al., 1983). Myc downregulation is evident upon the differentiation of a

number of cell lines: 3T3L1 pre-adipocytes (Freytag, 1988; Freytag et al., 1990), F9

tetracarcinoma cells (Dony et al., 1985), U-937 monoblastic cells (Larsson et al., 1988),

murine myeloid M1 cells (Einat et al., 1985), and proerythroid K562 cells (Bianchi Scarra

et al., 1986). In other differentiating systems, Myc repression is associated with the

terminal stages of the differentiation process. In these cells, the repression of myc mRNA

is biphasic, exhibiting a transient reduction in myc mRNA expression before returning to

levels close to that observed in proliferating cultures prior to the loss of Myc expression

upon terminal differentiation. This biphasic pattern has been reported in the differentiation

of the human promyelocytic HL60 (Siebenlist et a]., 1988; Simpson et al., 1987), mouse

erythroleukemia MEL cells (Mechti et al., 1986; Nepveu et al., 1987), the mouse P19

embryonic carcinoma cell line, and L6E9-B rat muscle cells (Endo and Nadal-Ginard,

1986).

A direct role for Myc in inhibiting cellular differentiation was demonstrated in

several studies. Constitutive ectopic Myc expression will inhibit the differentiation of

MEL, U937, F9, and 3T3L1 cells (Coppola and Cole, 1986; Dmitrovsky et al., 1986;

Freytag, 1988; Kaneko-Ishino et al., 1988; Larsson et al., 1988; Onclercq et al., 1989;

Prochownik and Kukowska, 1986). The introduction of antisense c-myc RNA in HL60

cells will induce growth arrest (Griep and Westphal, 1988; Holt et al., 1988; Prochownik

et al., 1988). Myc can antagonize myogenic differentiation induced by myogenin and

MyoD, a process which is independent of Id, an inhibitor of both myogenin and MyoD

(Miner and Wold, 1991). Further evidence supporting a role for Myc in t!~e inhibition of

differentiation can be found in the analysis of B lymphoid cell development in mice

expressing the c-myc transgene governed by the immunoglobulin heavy chain enhancer

(Adams et al., 1985). These mice exhibit a B cell population consisting of a large

proportion of undifferentiated pre B cells. Thus, studies in a variety of cell types indicate

that Myc is a potent inhibitor of differentiation, inhibiting growth arrest and promoting the

expansion of an undifferentiated cell population.

Yet there are a few contradictory observations which seem to suggest that the role

of Myc in the inhibition of differentiation is dependent upon the cell type. Reexpression of

Myc in differentiated myoblasts can not reverse the differentiated phenotype (Endo and

Nadal-Ginard, 1986). Elevated ectopic Myc expression cannot inhibit the y-interferon

induced differentiation of U937 cells (Oberg et al., 1991). Myc expression promotes the

differentiation of human epidermal stem cells (Gandarillas and Watt, 1997). Similarly,

targeted expression of ectopic c-myc in the developing mouse lens did not inhibit the

elaboration of differentiation-specific markers, yet it did inhibit proliferative arrest

(Morgenbesser et al., 1995).

The molecular role of Myc in the inhibition of differentiation is not well

understood. It is still not clear whether Myc can directly inhibit both the elaboration of

differentiation-specific markers such as the mim-1 gene, andlor inhibit the growth arrest

program associated with cellular differentiation. Yet, in the pre-adipocyte cell line, 3T3-

L1, Myc appears to effect both pathways. Differentiation in these cells is accompanied at

the molecular level with a gradual decrease in Myc expression, and the induction of the

differentiation-specific transcriptional activator clebpa (Cao et al., 1991; Christy et al.,

1991; Christy et al., 1989). The upregulation in clebpa expression results in the

subsequent transcriptional induction of the growth arrest gene, gadd45 (Constance et al.,

1996), and differentiation-specific genes such as: senim albumin, lysozyme, and mim-1

(Burk et al., 1993; Christy et al., 1989; Friedman et al., 1989; Mink et al., 1996). The

upregulation and activity of clebpa is crucial and sufficient to induce the differentiation

process (reviewed in Lane et al., 1996; MacDougald and Lane, 1995, Lin and Lane, 1992;

Lin and Lane, 1994; Ron et al., 1992). Myc can inhibit the activity of clebpa, thereby

inhibiting the induction of differentiation-specific genes including gadd45. Myc effects this

activity in two ways: Myc has been reported to repress the transcription of clebpa;

alternatively, it has been shown in transcription promoter-reporter assays, that Myc can

inhibit the activity of debpa via an as yet uncharacterized mechanism (Antonson et al.,

1995; Constance et al., 1996; Li et al., 1994; Mink et al., 1996). Additional studies

support the view that clebpa is the critical target for Myc in the inhibition of adipocyte cell

differentiation, as introduction of ectopic clebpa into 3T3-L1 cells expressing ectopic Myc

protein will rescue the differentiation program. Therefore in differentiating 3T3-L1

adipocytes, Myc inhibits cellular differentiation by inhibiting both the elaboration of

differentiation-specific genes and the expression of growth arrest genes. As such, Myc is a

potent inhibitor of the differentiation program, serving a pivotal role in inhibiting growth

arrest and forcing cells into a proliferative state.

1.4.3 Myc and apoptosis

It is clear from both in vivo and in vitro studies that Myc confers a strong

proliferative stimulus, inhibiting growth arrest states such as quiescence, senescence, and

differentiation. Indeed, it can be universally stated that Myc expression and growth arrest

are mutually exclusive. The consequence of opposing this restrictive relationship is

illustrated in the Myc-dependent apoptotic mechanism. Ectopic Myc expression imparts

such a strong proliferative impulse upon cells that they are unable to arrest upon exposure

to growth arrest agents: instead they undergo apoptosis.

Expression of ectopic Myc in the IL3-dependent myeloid cell line 32D results in an

acceleration of apoptosis in cells cultured in the absence of IL3 (Askew et al., 1991).

Activation of a conditional Myc allele in serum starved rodent fibroblasts rapidly induces

apoptosis (Evan et al., 1992), and sensitizes rodent fibroblasts to apoptosis induced by

treatment with TNFa (Klefstrom et al., 1994). Identical results were observed upon Myc

activation in NIH3T3 cells and primary MEFS (Hermeking and Eick, 1994). A direct role

for Myc in promoting apoptosis was observed in studies of T cell receptor (TCR)-activated

T cell hybridomas (Shi et al., 1992). Activation of the TCR induces an apoptotic program

which requires Myc expression. The regions of Myc which are required to effect apoptosis

include MBII, DNA binding, and protein dimerization domains (Amati et al., 1993; Stone

et al., 1987). In addition, Myc-induced apoptosis is dependent upon the interaction of Myc

with its protein partner, Max (Amati et al., 1993). These results suggest that the ability of

Myc to transactivate gene expression is required for the induction of apoptosis. In support

of a role for transcriptional regulation by Myc in apoptosis, two Myc-regulated genesp53

(Hermeking and Eick, 1994), and odc (Packham and Cleveland, 1994; Packham and

Cleveland, 1995), appear to be required for Myc-induced apoptosis. Both these genes

contain Myc DNA binding sites, and are reported to be transcriptionally regulated by Myc . The importance of p53 expression in Myc-induced death, can be seen in studies performed

in cells lacking p53. Fibroblasts lacking p53 expression due to targeted gene disruption are

resistant to Myc-induced apoptosis (Hermeking and Eick, 1994). Similar studies

employing inhibitors of odc function have also implicated a role for odc in this mechanism.

For example, inhibiting odc expression via an antisense approach, abrogates the apoptotic

program in 32D cells (Packham and Cleveland, 1994). Ongoing dissection of the Myc-

induced apoptotic mechanism has revealed additional players in this pathway. Studies by

Evan et al, have elucidated that the interaction of Fas with its ligand, and the resultant Fas-

mediated signaling pathway is required for apoptosis (Hueber et al., 1997). In addition,

caspase 3, a cysteine aspartase, was shown to be required for Myc-induced apoptosis,

through the use of specific protease inhibitors (Kagaya et al., 1997; Kuchino et al., 1996).

Apoptosis can be inhibited by ectopic expression of bcl-2, or treatment with the growth

factors, insulin growth factor-1 (IGF-I), or platelet-derived growth factor BB (PDGF)

(Hamngton et al., 1994). IGF-1 or PDGF elicits a signaling pathway which involves

phosphoinositol 3' kinase (PI3K) and its downstream target AKT (Protein Kinase B)

(Figure 1.6) (Datta et al., 1997; Kauffinann-Zeh et al., 1997; Kennedy et al., 1997).

It is a paradox that Myc is a strong promoter of cell proliferation and apoptosis

(reviewed in Henriksson and Luscher, 1996; Packham and Cleveland, 1995). Yet, such

an arrangement may serve as an internal control responsible for inhibiting cell

transformation by the elimination of cells which have suffered c-rnyc activation as a

primary or early oncogenic hit in the step-wise progression of hunorigenesis.

1.4.4 Myc and cdkIs

The expression of the cdkIs is commonly elevated in a variety of growth arrest

states including quiescence, senescence, contact inhibition, exposure to growth inhibitory

agents. and in response to DNA damage. Thus, the cdkls could potentially be involved in

initiating or maintaining cells in a growth arrested state. A number of studies have clearly

shown that Myc can overcome many of these growth arrest states, although the precise

mechanism through which this is achieved is not known. Recent evidence has

demonstrated that Myc can overcome many of these growth inhibitory stimuli.

One of the first hints that Myc could overcome a cdkI-mediated block to cell

proliferation arose from the studies by Alexandrow et al. They demonstrated that Myc

circumvented a transforming growth factor P (TGF-P)-mediated growth arrest, which is

mediated by the activities of the cdkIs p27, p15, and p21 (Alexzndrow et al., 1995;

Alexandrow and Moses, 1995). More compelling evidence emerged from the studies of

Steiner et al. as well as Rudolph et al. It was demonstrated that Myc aciivation in quiescent

cells leads to the rapid induction in cyclin EIcdk2 activity and exit from growth arrest

(Rudolph et al., 1996; Steiner et al., 1995). The activation of cyclin EIcdk2 is due to the

absence of p27 activity in these Myc-activated cells. The mechanism effecting the loss of

p27 activity is controversial: two mechanisms have been proposed. First, the p27 within

cyclin EIcdk2lp27 complexes is phosphorylated by cyclin EIcdk2 followed by the

degradation of p27 possibly via a ubiquitin-dependent pathway (Clurman et al., 1996;

Muller et al., 1997; Sheaff et al., 1997; Steiner et al., 1995). Second, an alternate

mechanism was proposed by Vlach et a/., suggesting that p27 was converted into a

functionally inactive form which is incapable of biidiig to the cyclinlcdk complex (Vlach et

al., 1996). The reason for the dissimilar observations is puzzling, and cannot be explained

at this time. Rescue from p27-mediated growth arrest by Myc requires the amino-terminal

transcriptional regulatory domain (amino acids 7 to 145), the DNA binding domain, and

interaction with Max (Vlach et al., 1996). These domains are required for the

transactivztion function of Myc, suggesting that Myc may overcome the activity of p2i,

through the induction of gene expression. As p27 is bound predominantly to cyclin EIcdk2

complexes during quiescence, it was not surprising that the inhibition of p27 function by

Myc induces cyclin EIcdk2 kinase activity. Yet it is apparent that the induction in cyclin E-

associated cdk2 activity is not the sole function of Myc activation, as constitutive ectopic

expression of cyclin E alone in rat fibroblasts could not substitute for Myc. In addition,

elevated expression of the other G1 cyclins, cyclin A or the D-type cyclins, in rat

fibroblasts, could not circumvent the growth inhibito~y effects of p27. Neither can the p27

block be inhibited by overexpressing the cdk phosphatase, cdc25A. Interestingly, ectopic

expression of E2Fs alone cannot abrogate the p27 block. This supports an alternate role

for cyclin EIcdk2 activity distinct fiom pRb phosphorylation, and liberation of active E2F,

in the promotion of GUS transit (Vlach et al., 1996).

Thus, Myc induces exit from a G1 cell cycle block, potentially by antagonizing the

activity of the cdkI p27, resulting in the activation of cyclin EIcdk2 in late G1 phase

promoting entry into the cell cycle. Yet, while the induction in cyclin Elcdk activity is

important for the abrogation of the p27 cell cycle arrest by Myc, it clearly is not sufficient.

Cyclin EIcdk2 induction must be complemented by additional Myc-regulated activities

which remain to be identified and characterized (Vlach et al., 1996). It must be noted that

the story is not so simple. Ectopic expression of cyclin E in NIH3T3 cells can overcome a

p27-induced G1 block to cell growth (Sheaff et al., 1997). In addition, ectopic expression

of E2F-1 alone will overcome a p27-induced growth arrest in the immortalized rat cell line

REF52 (DeGregori et al., 1995). Both of these results contradict the observations made in

Rat-1 cells, hinting that the observed mechanisms which abolish the p27-dependent growth

arrest are influenced by unknown factors specific to individual immortalized cell lines.

Analyses in primary cell cultures may be the ideal system within which these discrepancies

may be resolved.

Myc activation can similarly bypass the inhibitory effects of either p21 or p16 in

fibroblasts. Myc can overcome a p2lcdkI-dependent G1 arrest by inducing the expression

or activity of an as yet uncharacterized heat labile inhibitor of this cdkI, abrogating its

inhibitory effect on cyclin associated kinase activity (Hermeking et al., 1995). An alternate

mechanism through which Myc may relieve a p21-induced growth arrest was proposed by

the observations of Saha et al. (Saha et al., 1997). Myc can transcriptionally induce the

expression of the phosphatase cdc25A. Both cdc25A and the p2lcdkI share the same

binding site on the cyclin/cdk dimer, hence binding by either of the two proteins is

mutually exclusive. Hence a Myc-dependent increase in the expression of cdc25A may

hinder the interaction and inhibition of cyclin E-dependent kinases by p21, allowing cell

proliferation.

In conjunction with the inhibition of p21 activity by Myc, Myc can also circumvent

a pl6-dependent growth arrest (Alevizopoulos et al., 1997). The precise mechanism

through which Myc abrogates the growth inhibitory effect of pl6 is unknown. However,

it is clear that this mechanism exhibits some similarities to the pathway via which Myc

circumvents the activity of the p27 cdkI. The end result ofboth of these mechanisms is a

loss of p27 activity concomitant with the activation of cyclin Wcdk2. Yet, there are notable

differences between these two mechanisms. In contrast to the p27-mediated growth arrest,

the pl6-dependent block to proliferation is also rescued by ectopic expression of cyclin E,

or E2Fs 1,2, and 3, indicating that these cell cycle effectors may function within the same

pathway as Myc. In addition, while Myc overcomes a p27-induced cell cycle arrest via a

pathway which involves the phosphorylation of the tumour suppressor pRb, the latter

pathway is dispensable for the Myc circumvention of the pl6-dependent growth arrest.

This discrepancy suggests that separable pathways are responsible for the Myc-induced

abrogation cf a p16- versus p27-mediated block to cell proliferation. Although our

understanding of these mechanisms is minimal, these pathways may represent the

mechanism through which Myc overcomes a variety of growth arrest states such as

senescence and quiescence and promotes cell proliferation. Hence, the elucidation of the

mechanisms through which Myc overcomes the growth inhibitory effects of the cdkIs will

aid in our understanding of how Myc confers a strong growth stimulus, and contributes to

tumour formation.

1.4.5 Myc Transformation in vitro and in vivo

Ectopic Myc expression in primary fibroblasts will abrogate the senescence

program and elicit cellular immortalization. Yet, immortalization does not render the cell

autonomous, as proliferation is still dependent upon culture in the presence of the

appropriate mitogens (reviewed in Henriksson and Luscher, 1996). Indeed in the absence

of mitogen, cells expressing ectopic Myc undergo apoptosis (Evan et al., 1992),

suggesting that the abrogation of the apoptotic program by mitogenic signals is critical to

the immortalization activity of Myc and permits immortalization to proceed unrestrained. In

combination with an additional cooperating oncogenic lesion such as an activated Ras, or

Raf allele, Myc will induce transformation in primaxy cells derived from a variety of tissue

types (Adams and Cory, 1992).

Transgenic mouse studies provide the strongest evidence for the role of Myc in the

genesis of tumours. Transgenic mice were generated which ectopically express human c-

Myc under the control of the immunoglobulin heavy chain enhancer, mimicking

translocation events observed in Burkitt's lymphoma. These transgenic mice exhibit

elevated levels of immature undifferentiated pre-B cells, suggesting that elevated ectopic

Myc expression can inhibit the normal differentiation program and enhance the proliferative

potential of the pre-differentiation lymphoid population. These mice develop monoclonal

B-lymphoid malignancies which are phenotypically characteristic of Bwkitt's lymphoma

(Adams et al., 1985). The monoclonal nature of these tumours indicate that secondary co-

operating mutations are required to elaborate the full transformed phenotype. In accord

with the functional similarity among the Myc family members, expression of an L-myc

transgene driven by an immunoglobulin enhancer induced T-lymphoid tumours in Ep-L-

niyc transgenic mice (Moroy et al., 1990). Ep-N-myc transgenic animals succumb to

clonal lymphoid malignancies, primarily B-cell lymphomas (Dildrop et al., 1989;

Rosenbaum et al., 1989). Importantly, targeted expression of the N-Myc protein in the

neuroectodermal cells of the developing mouse embryo results in the development of

neuroblastomas (Weiss et al., 1997). Therefore, it is clear from both the transgenic animal

and tissue culture studies that Myc can play a role in the initiation of cellular transformation

in a wide variety of cell types, and additional secondary genetic alterations are needed to

produce full transformation.

What is the contribution of Myc to the tumorigenic process? The work of

Tikhonenko et al., illustrates the conventional thinking regarding the characteristics of the

transformed phenotype which are thought to be directly controlled by Myc (Tikhonenko et

al., 1995). Avian embryo fibroblasts expressing a glucocorticoid-inducible v-gag-myc

allele, were transformed upon activation of Myc activity following treatment with the

glucocorticoid dexamethasone. Transformed cells exhibited an increased growth rate,

anchorage-independent growth and increased refractivity. However, removal of the

glucocorticoid, and the resulting loss in ectopic Myc activity, resulted either in growth

arrest or a reduced growth rate, while refractivity as well as anchorage-independent growth

were unaffected. Therefore it is apparent, at least in this Myc-dependent tumour model,

that escape from growth arrest and enhanced proliferation may be the primary effects of

Myc, and secondary mutations are responsible for the transformed morphology. The

ability of Myc to enhance the cellular proliferative potential, concomitant with the increased

number of DNA replications, may facilitate the emergence of additional genetic aberrations

which will permit full cellular transformation. Indeed, Myc has been reported to induce the

non-random amplification of the dihydrofolate reductase (dhfr) locus in a variety of cell

types originating from different species. Yet, it is not clear whether this activity is a direct

or indirect effect of Myc (Denis et al., 1991; Mai, 1994; Mai et al., 1996; Mai et al., 1996).

In essence Myc may promote cell transformation via two complementary

mechanisms: first, Myc inhibits growth arrest programs such as senescence and

differentiation; and second, Myc drives progression through the cell cycle. The provision

of both activities would confer an increased growth advantage to cells, facilitating the

emergence of additional mutations which permit the evolution of the fully transformed

phenotype.

1 .4 .6 Myc cooperativity in transformation

This past year has provided a wealth of information regarding how Myc interacts

with different components of the cell cycle machinery, particularly in the GI phase of the

cell cycle. These reports have provided new insights into the requirement of oncogene

cooperation for full cellular transformation by Myc. Myc is a component in two types of

cooperative relationships, these two categories are divided on the basis of the contribution

of the cooperating oncogene to the partnership. First, the cooperating oncogene within the

partnership may supply an independent and complementary function. The synergy of both

activities is sufficient to permit unrestrained cell growth. The most commonly studied of

these relationships is the synergy of activated Myc with Ras. Myc and Ras exist on

separable signal transduction pathways originating either i?om a single or multiple mitogen

activated receptors, depending upon the cell (Barone and Courtneidge, 1995). Hence the

ectopic constitutive expression of activated alleles of both c-myc and ras may circumvent

the necessity for mitogen stimulation in the promotion of continual cell proliferation.

Although it is well known that at a phenotypic level, Myc in conjunction with Ras

can elicit cellular transformation, the biochemical basis of this synergy is unclear. Studies

by Leone et al. sought to answer this question, by determining the contribution or the

impact each of these two oncogenes has on components of the cell cycle machinery. The

expression of both Myc and Ras is necessary for serum-stimulated quiescent cells to exit

growth arrest and enter the cell cycle. However, ectopic expression of activated Myc or

Ras in the immortalized cell line REF52 were ineffective in elaborating progression into S

phase. Moreover, ectopic Ras expression elicited growth arrest in G1 phase of the cell

cycle, likely the consequence of the absence of Gl cdk activity. Indeed, both cyclin E and

cyclin Dl-associated kinase activity was inhibited due to the activities of the cdkI p27.

Interestingly, coexpression of Myc with Ras permitted DNA replication, preceded by a

reduction in p27 levels and a concomitant induction in cyclin EIcdk2 activity. The

reduction in p27 levels is due to the synergistic effect of both Myc and Ras coexpression,

as expression of ectopic Myc or Ras did not affect p27 levels. The reduction in p27 was

not an indirect consequence of cell cycle progression as inhibition of cell proliferation

through the expression of the cdkI p21 did not abrogate the Myc, Ras-induced loss of p27

expression (Leone et al., 1997). Thus, the synergy of Myc with Ras in the induction of

cyclin EIcdk2 activity is a pathway, which may explain how Myc and Ras can cooperate to

elicit full cellular transformation in primary and immortalized fibroblasts.

The studies of Alevizopoulos et al. suggested another possible explanation for this

cooperative relationship (.4levizopoulos et al., 1997). Activated Myc can overcome a p16-

mediated cell cycle block, suggesting that the function of Myc within the Myc, Ras

partnership may involve inhibiting the activity ofp16. This hypothesis is supported by the

observation that while Myc and Ras are required for the transformation of wildtype MEF

cultures, Ras alone can transform pl6-null MEFs. Therefore, the loss of pl6 expression

precludes the requirement for Myc in cellular transformation. Irrespective of the identity of

the cdkI whose activity must be silenced to effect full transformation, the primary function

of Myc appears to be the inhibition of the activity of growth inhibitors which hinder cell

proliferation, facilitating cell transformation.

The second category is based on the observation that expression of activated Myc

promotes both cell proliferation as well as apoptosis. The ability of Myc to successfully

promote tumour formation is dependent upon the abrogation of the Myc-induced apoptotk

pathway. This can be achieved in two ways: first, the activation of inhibitors of apoptosis

such as bcl-2, and secondly, mutations in genes downstream of Myc, such as p53, which

effect the apoptotic mechanism. Hence, it was not surprising to discover that mutations in

bcl-2 or p53 can cooperate with Myc to promote tumour progression (reviewed in Adams

and Cory, 1992). A third of primary Burkitt's lymphoma isolates and most of the derived

cell lines exhibit point mutations inp53 (Farrell et al., 1991; Gaidano et al., 1991; Wiman

et al., 1991). In addition,p53 mutations were identified in some pre B and B-lymphomas

in Ep-myc mice (reviewed in Adams and Cory, 1992). The evidence for an interaction

between myc and bcl-2 originated primarily from transgenic mouse studies. Many of the

spontaneous tumours which arose in bcl-2 transgenic mice contained a aberrant myc allele

(McDonnell and Korsmeyer, 1991). In addition, crosses of Ep-myc and Ep-bcl-2 mice

produced progeny exhibiting an expanded population of pre B cells and rapidly succumbed

to B lymphoid malignancies (Strasser et al., 1990).

Both of these models are readily supported in experimental studies, indicating that

either pathway is equally likely and that the pathways are probably not mutually exclusive.

In addition, these observations support the view that multiple mechanisms can cooperate

with Myc to promote cell transformation. Yet what can we learn of the contribution of Myc

to transformation in both of these categories? In the first category, Myc activation will

overcome a G1 arrest resulting from Ras activation, allowing continued cell proliferation

and tumorigenesis. In the second category, Myc both enhances cell proliferation as well as

promotes apoptosis, thus there is an inherent suicide program within cells to inhibit Myc

activation from promoting the turnorigenic pathway. Activation of bcl-2 inhibits the Myc-

induced apoptotic program and permits Myc to drive cells through the cell cycle. Thus, in

both instances, we can hypothesize that the role of Myc is to confer enhanced proliferation,

which in conjunction with additional mutations facilitates tumorigenesis potentially via the

induction of genomic instability.

1.5 Mechanism of action

At a phenotypic level the biological effects of Myc expression are clear. Myc exerts

a strong proliferative impulse, inhibiting diverse growth arrest states such as quiescence,

senescence, or differentiation, and promoting cell proliferation. Yet at a molecular level, it

is not known how Myc executes these biological effects. It is known that Myc can bind to,

and influence the activity of components of the transcription machinery, as well as

companents of the cell cycle. In addition, Myc can activate as well as repress gene

transcription. Hence, Myc may utilize one or all of these activities to exert its strong

proliferative capacity. Further characterization of each of these Myc-dependent

mechanisms will help us to determine the importance of these pathways for Myc to drive

cells out of growth arrest and progess through the cell cycle.

1.5.1 Myc-interacting proteins

The identification of Max as a dimerization partner of Myc was a significant

breakthrough in the field of Myc research. It has contributed to our understanding of the

molecular mechanisms through which Myc effects its activities. Max was identified by

screening a human expression library with a radiolabeled carboxyl-terminal fragment of c-

Myc (Blackwood and Eisenman, 1991). This discovery was rapidly followed by the

identification of a murine homologue, nyn (Prendergast et al., 1991). The Max protein is

highly stable with a half-life of greater than 14 hours, and its expression is invariant

throughout all phases of the cell cycle (Berberich et al., 1992; Blackwood and Eisenman,

1992). There are two predominant isoforms of Max, resulting from alternative splicing,

p2 1 Max (1 51 amino acids) and p22 Max (160 amino acids) (Blackwood and Eisenman,

1991). They differ only in the presence of a 9 amino acid insertion amino-terminal to the

basic region. The reason for the two isoforms is unknown, as both proteins are

equivalently and ubiquitously expressed. Both isoforms bind to Myc with equal affinity

and facilitate the binding of Myc to DNA. The structure of Max is highly analogous to the

carboxyl-terminus of Myc. Max possesses a basic region, followed by HLH and LZ

domains; however unlike Myc, the NLS is located downstream of the LZ (reviewed in

Henriksson and Luscher, 1996). Alternate splicing is also responsible for additional

transcripts. These isoforms include AMax proteins p16 and p17 which lack the carboxyl

terminal NLS. The importance of these proteins is unclear as the respective endogenous

proteins remain to be isolated (Makela et al., 1992; Vastrik et al., 1995). In addition, there

is also a dMax variant, which has been identified in cell extracts. dMax lacks the basic

region and helix 1 and the loop of the helix-loop-helix domain (Arsura et al., 1995).

Ectopic expression of the AMax and dMax variants revealed that they are capable of

binding with high affinity to Myc, but MycIdMax heterodimers do not bind to DNA.

Thus, the dMax isoform may serve as an inhibitor of Myc function. Both Myc/Max and

MadMax dimers bind the same consensus DNA element, CACGTG. Some studies have

reported that binding is influenced by the nature of the sequences flanking the core.

MycIMax complexes appear to bind DNA with higher affinity compared to MadMax

homodimers, possibly due to the phosphorylation state of the MadMax homodimer

(Berberich and Cole, 1992).

Co-immunoprecipitation studies have demonstrated that Max is the predominant

binding partner of Myc. However, Myc has been reported to bind to a host of additional

proteins in vivo, including the transcription factors Yin-Yang-1 (YYl), AP-2, and Miz-1,

components of the basal transcription machinery, such as the TATA-binding protein

(TBP), regulators of the cell cycle such as p107, the putative tumour suppressor Bin-1, the

transformation-associated protein TR-AP, as well as cell structural elements such as a-

tubulin. Myc is also reported to bind in virro to TFII-I, a transcriptional initiator (Figure

1.5B) (reviewed in Henriksson and Luscher, 1996; Lemaitre et al., 1996).

YY-1, is a zinc-finger containing transcriptional regulator which regulates a number

of genes, inducing, repressing or initiating gene expression depending upon the context. A

search for proteins which interact with YY-1 using the yeast two hybrid approach yielded

c-Myc (Shrivastava et al., 1993; Shrivastava et al., 1996). YY-1 interacts with the

carboxyl terminus of cMyc, amino acids 250 to 439. As this region is also required for

binding to Max, the interaction of YY-1 or Max to c-Myc is mutually exclusive. Although

this interaction does not prevent YY-1 from binding to DNA, it inhibits the transcriptional

regulatory activity of YY-1, suggesting a DNA-independent mode of Myc action

(Shrivastava et al., 1993).

The carboxyl terminus of the c-Myc protein is also required for binding to the

transcription factor AP-2 (Gaubatz et al., 1995). Unlike YY-1, the interaction of AP-2

with Myc does not preclude Max binding; however, the AP-UMyc complex is incapable of

binding to DNA. Moreover, in some promoters, the Myc E box and the AP-2 binding site

overlap such as in the a-pr.othyrnosirz gene, and ectopic AP-2 expression can inhibit

MycIMax binding to DNA and consequently Myc-dependent transactivation. Hence the

interaction of Myc with AP2 results in either a direct or indirect inhibition of Myc

transcriptional activation function.

Miz-1, is a novel PO2 domain-containing protein which binds to the initiator in the

cycliit Dl and adenovims major late promoters (AdMLP), inducing transcription. Miz-1

was identified via a yeast two-hybrid approach, as a protein which can bind to the HLH

domain of c-Myc and not to Max or USF (Schneider et al., 1997). The association of Myc

with Miz-1 inhibits the transcription initiating activity of Miz-1, suggesting another

mechanism through which Myc may negatively regulate gene transcription. Interestingly,

ectopic expression of Miz-1 results in growth arrest and; as such, its activities are logical

candidates for Myc repression.

TFII-I is another example of an initiator-binding protein whose activity is inhibited

by Myc (Roy et al., 1993). TFII-I fulfills a unique role in transcription regulation; as a

component of the basal transcription machinery: TFII-I initiates transcription of TATA-less

promoters which possess initiator sites. In vitro, Myc HLH and LZ domains mediate the

interaction with TFII-I, and are responsible for the inhibition of TFII-I-dependent

transcriptional initiation. The recent cloning and characterization of TFII-I will allow an in-

depth characterization of the activities of this transcriptional regulator and permit an

analysis of the importance of the MycmFII-I interaction within the context of Myc function

and regulation. Myc has also been shown to interact with another component of the basal

transcription machinery. The amino-terminal transactivation domain of Myc can interact

with the TATA-binding protein (TBP), a subunit of the basal transcription factor, TFII-D,

in vivo and in vifro (Hateboer et al., 1993; Maheswaran et al., 1994). This interaction

substantiates a role for Myc in the regulation of transcription at core regulatory elements.

Myc can interact with p107, a member of the retinoblastoma susceptibility family of

tumour suppressors, in vivo and in vitro (Beijersbergen et al., 1994; Gu et al., 1994;

Hoang et al., 1995). p107 is a strong growth inhibitor and when ectopically expressed in

cultured cells, will induce growth arrest. The interaction between these two proteins is

mediated through the pocket region of p107 and amino acids 45 to 103 of Myc. The

binding of plO7 to Myc facilitates the formation of a quaternary protein complex consisting

of Myc, p107, and cyclin Ncdk2 (Gu et al., 1994; Hoang et al., 1995). The recruitment

of cyclin Ncdk2 to Myc facilitates the phosphorylation of Myc, hindering the ability of

Myc to transactivate gene expression. In support of this model, while p107 can bind to

both wildtype and mutant c-Myc proteins from Burkitt's lymphoma, the mutant c-Myc

proteins are not phosphorylated by cyclin Ncdk2, and p107 does not inhibit the

transactivation activity of these mutant Myc proteins. Thus, this correlative evidence

suggests that transcriptional activation by Myc and Myc-mediated transformation may be

linked, and that the phosphorylation of Myc by cyclin Ncdk2 appears to be a mechanism

through which Myc transcriptional activation function, and hence the tumorigenic potential

of Myc, is negatively regulated.

Bin-1, a novel Myc-interacting protein was isolated by screening a murine

embryonic cDNA library using the conserved Myc box I of Myc (Sakamuro et al., 1996).

Both MBI and MBII are required for binding to Myc. Bin-1 is a putative tumour

suppressor, its expression is absent in a variety of tumour cells, and ectopic Bin-1

expression antagonizes MycRas-dependent transformation (Sakamuro et al., 1996).

Consistent with its apparent role as a negative regulator of cell growth, the expression of

Bin-1 is induced upon the differentiation of C2C12 myoblasts (Wechsler-Reya et al.,

1998). Depending on the context, Bin-1 can inhibit Myc transactivation. Bin-1 can

abrogate the Myc-dependent induction of the omithine decarboxylase promoter, but is

ineffective against a promoter construct consisting of four CACGTGs upstream of a

chloramphenicol acetyltransferase (CAT) reporter gene. Thus, Bin-l may antagonize Myc

function by modulating the ability of Myc to regulate gene expression.

TR-AP, was identified as a Myc-binding protein via biochemical means (Brough et

al., 1995). TR-AP binds to the MBII region of Myc. As this region is also required for

Myc-mediated cellular transformation, TR-AP may play a role in effecting or enhancing the

tumorigenic activities of Myc. Indeed, this hypothesis was substantiated by studies which

demonstrate that dominant negative mutants of TR-AP will inhibit Myc-induced

transformation. It is not known how TR-AP functions. The carboxyl-terminus contains a

domain found in the phosphoinositol3' kinase (PI3K) family; however, TR-AP lacks the

catalytic kinase domain, hence it may not exhibit kinase activity. The model currently

under investigation proposes that TR-AP may function as a molecular scaffold recmiting

additional proteins which will execute the biochemical directives of Myc.

Thus, in addition to Max there are a host of Myc interacting proteins, which either

facilitate or impair Myc function. Miz-1, TBP, TR-AP, and TFII-I serve to enhance or

effect the activities of Myc; while AP2, YY-1, p107, and Bin-1 may antagonize Myc

function. The importance of each of these interactions within the context of the execution

of the biological activities of Myc is unknown, as the characterization of these novel

regulatory pathways is still in its infancy. Yet, these pathways may be the key to

unraveling the complexities that surround Myc hnction and regulation.

1.5.2 Transcriptional activation

One avenue of research which has been the primary focus for a number of

researchers in this field, involves determining how Myc can execute its biological functions

by regulating gene transcription. The evidence to support a role for Myc as a regulator of

gene transcription is considerable as well as convincing. This wealth of substantiation can

be attributed primarily to the simple fact that more effort has been devoted to this venture,

compared to any other, rather than a testament to the validity or importance of this

mechanism.

Structural analysis of the c-Myc protein revealed motifs commonly associated with

well characterized transcription factors, suggesting that Myc may function as a

transcriptional regulator (reviewed in Lemaitre et al., 1996). A number of studies support

this notion. First, Myc together with Max (Blackwood and Eisenman, 1991; Blackwood

and Eisenman, 1992; Blackwood et al., 1992; Blackwood et al., 1992; Prendergast et al.,

1991), can bind specifically to DNA at a consensus E-box element, CACGTG, as well as

to other related noncanonical sites (Blackwell et al., 1993; Fisher et al., 1993; Grandori et

al., 1996; Prendergast et al., 1991; Solomon et al., 1993). Second, in vitro transcription

assays using the Myc binding site linked in tandem to a reporter gene, have demonstrated

that Myc can activate transcription of the reporter gene upon binding to the Myc binding

site (Amati et al., 1992; Amin et al., 1993; Gu et al., 1993; Ham et al., 1994; Kretzner et

al., 1992; Kretzner et al., 1992). Third, a relatively small number of genes have been

identified as being transcriptionally induced in response to Myc activation (Bello-Fernandez

and Cleveland, 1992; Bello-Femandez et al., 1993; Benvenisty et al., 1992; Galaktionov et

al., 1996; Miltenberger et al., 1995; Rosenwald et al., 1993; Sears et al., 1997; Wagner et

al., 1993). Therefore, it is clearly evident that Myc can activate gene expression at a

transcriptional level. This led to the hypothesis that Myc promotes cell proliferation by

inducing transcription of genes which either promote or are required for progression

through the cell cycle.

The list of reported Myc-induced genes is small but contains a number of genes

whose cellular function may explain how Myc promotes cell growth (Figure 1.7). Myc-

regulated genes can be divided into distinct categories on the basis of their physiological

function. There are a subset of genes which are required for progression into S phase

consisting of ornithine decarboxylase (odc), the rate-limiting enzyme for polyamine

biosynthesis (Bello-Femandez and Cleveland, 1992; Bello-Femandez et al., 1993; Wagner

et al., 1993), and carbamoyl-phosphate syntltase/aspartate carbamoyl

transferase/dihydroorotase (cad), necessary for pyrimidine biosynthesis (Miltenberger et

al., 1995). E2F-2 (Sears et al., 1997), is required for the expression of genes such as

cyclin E, thymidim kinase, as well as the additional members of the E2F family of

proteins. A G1-specific phosphatase, cdc25A, activates cdk kinase activity (Galaktionov et

al., 1996). Other genes are involved in protein translation, such as eIF-4E and eIF-Za,

which encode enzymes that are involved in translation initiation (Rosenwald et al., 1993).

A DEAD box-related gene (MrDb), is a potential RNA helicase and mediator of RNA

stability (Grandori et al., 1996). Lactate dehydrogenase gened (LDH-A), which

functions in anaerobic glycolysis, is also a target of Myc (Shim et al., 1997). Lastly, Myc

has been reported to regulate genes of uncertain function: a-protl~ymosin (Desbarats et

al., 1996; Eilers et al., 1991; Gaubatz et al., 1994), and ECA39 (Benvenisty et al., 1992).

Both odc and c a d catalyze essential biosynthetic reactions producing

macromolecules which are required for DNA replication. The transcription factor E2F-2,

drives the expression of a host of genes whose expression is required for progression

odc + Polyamine biosynthesis \ /

DNA Replication cad -+ Pyrimidine biosynthesis

E2F-2 -- Gl and S phase genes A / Cell cycle progression ---8-

cdc25A + cdk activation

eIF-2a \ Initiation of protein translation =-

eIF-4E

MrDb -+ mRNA stability and transport m

LDH-A - Anaerobic glycolysis - Gmwth in hyporic conditions*

Figure 1.7. Possible mechanisms for the role of Myc in driving cell cycle progression, based on the induction of the indicated target genes bv Mvc. Abbreviations used: odc, omithine decarbox&G; cad, & r b ~ m ~ ~ l - ~ h o s ~ h a t c synthaselaspartate carbamoyl transferaseldihydroorotase; cdk, cyclin-dependent kinase; LDH-A, lactate

Cell Growth

through late G1 and S phase. Cdc25A is an intriguing target for Myc regulation, since it is

active in late G1 phase of the cell cycle and is required for transit from G1 to S phase.

Cdc25A may activate cdk activity via either of two mechanisms: first, it is a phosphatase

which can activate cdks by removing inhibitory phosphate groups (Jinno et al., 1994);

second, cdc25A can compete with the cdkI p21 for binding to cyclin E and A in vitro,

thereby abrogating the inhibitory effects of p21, permitting cell cycle progression (Saha et

al., 1997). EIF-4E, a translation initiation factor which recognizes the cap structure at the

5' end of rnRNAs, has been demonstrated to enhance specifically the translation of cyclin

Dl. Hence, this mechanism represents one mode via which Myc promotes cyclin Dl

expression. Thus, Myc induces a diverse array of genes whose finctions impact on a

number of proliferative systems. Myc promotes cyclin/cdk activation by enhancing the

expression or activation of cyclins, and Myc also induces genes which supply essential

reagents for the process of DNA replication. In addi.tion, Myc can elevate the expression

of growth promoting genes through the induction of eIF-4E and MrDb. All of these Myc-

initiated activities make a significant contribution to the progression of cells from GOIGl to

S phase of the cell cycle, enhancing cell proliferation.

Analysis of the promoters of these Myc target genes has provided further evidence

that Myc can induce the transcription of these genes. It was generally assumed that Myc

served a critical role in the induction of these genes upon serum stimulation, because of the

overwhelming evidence supporting a role for Myc in the exit from quiescence upon

mitogen stimulation. This is true for most of the induced genes; however, analysis of the

regulation of odc by Myc has refined and expanded this perception of its role in the

promotion of cell cycle progression. Packham et al. analyzed the role of Myc in the

induction of odc in quiescent murine myeloid progenitor cells, following stimulation with

the mitogen IL-3 (Packham and Cleveland, 1997). They reported that the transcriptional

regulation of ode occurs in two sequential stages. The first stage encompasses the initial

induction in odc transcription following IL-3 stimulation; it is effected via a pathway which

is independent of Myc and de novo protein synthesis. The second stage entails the

maintenance of odc expression throughout the cell cycle and is dependent upon Myc

activity and protein synthesis. In support of this model, inducible expression of a

dominant negative mutant of c-Myc inhibited the delayed second stage in odc expression

but did not affect the induction of odc following exposwe to IL-3. Thus, while Myc is

required for the transcriptional induction of odc, Myc appears to be responsible for the

maintenance of gene expression rather than the mitogen-stimulated transient activation of

gene transcription (Packham and Cleveland, 1997). This result proposes a novel role for

Myc, and prompts the question of whether the other Myc-induced genes are similarly

regulated.

1.5.3 Transcriptional repression

Recent studies have demonstrated that Myc can repress gene transcription, in

addition to activating gene transcription (Antonson et al., 1995; Facchini et al., 1997;

Inghirami et al., 1990; Jansen-Dun et al., 1993; Lee et al., 1997; Li et al., 1994; Marhin et

al., 1997; Penn et al., 1990; Philipp et al., 1994; Prendergast et al., 1990; Tikhonenko et

al., 1996; Versteeg et al., 1988). Characterization of the functions of the genes which are

repressed by Myc suggests that these Myc-mediated repression activities may play an

important role in the ability of Myc to control normal cell proliferation and consequently

tumorigenesis. In comparison with Myc-mediated transcriptional activation, Myc

repression is less well understood. Repression appears to be mediated through core

regulatory elements, and unlike Myc transactivation, repres~ion is not dependent upon the

presence of the conserved CACGTG within the gene. For some genes, repression appears

to be effected through the initiator element (Inr), where Myc is believed to inhibit the

binding and transcriptional activity of proteins which interact with the Inr. Two candidate

Inr-binding proteins whose activity has been reported to inhibited by Myc are TFII-I and

Miz-1. More than one mechanism of suppression may be orchestrated by Myc as

heterodimerization with its protein partner Max is dispensable for Myc repression of cyclin

Dl, yet is required for Myc autosuppression (Facchini et al., 1997; Philipp et al., 1994).

Indeed, structure/function analysis of the transcriptional regulatory domain of the Myc

protein has suggested that the regions which are important for Myc activation and

repression are separable. A few targets for Myc repression have been identified. Myc can

repress debpa (Antonson et al., 1995; Constance et al., 1996; Li et al., 1994; Mink et al.,

1996), cyclitl DI (Philipp et al., 1994), the adenovims 5 major-late promoter (AdMLP) (Li

et al., 1994), and c-myc (Facchini et al., 1997) promoter activities.

The importance of d e b p a to the differentiation program in some cell systems

makes it a logical candidate for Myc repression. Although it is clear that Myc can repress

the activity of this differentiation-specific transcriptional regulator, the precise mechanism

through which repression is effected is controversial. Using a transient transfection

approach in the immortalized cell line NIH3T3, it was demonstrated that Myc represses

transcription via a mechanism which is dependent upon the initiator element in the basal

promoter (Li et al., 1994). Repression requires both the MBII region and the B-HLH-LZ

of c-Myc. These domains coincide with the domains of Myc which are crucial to Myc-

dependent cell transformation, suggesting that Myc repression and Myc-mediated

transformation may be linked. In HIB-lB, a murine brown adipocyte cell line, ectopic

Myc expression did repress the transcription of debpa promoter reporter constructs;

however, mutation of the initiator element did not abolish the repression. Myc-dependent

repression appeared to be mediated through the core promoter, potentially through

interactions with the basal transcription machinely (Antonson et al., 1995). These

conflicting observations may be due to differences in cell type, or methodology, and need

to be further explored.

The relationship between Myc and cyclin Dl is complex, as in primary MEF

cultures, ectopic Myc expression does not regulate cyclin Dl expression. Yet, in some

immortalized fibroblasts, Myc can repress cyclin Dl expression, depending on the cellular

context. The precise nature of the discrepancy between primary cell cultures and

immortalized fibroblasts which facilitate the repression of cyclin Dl by Myc is unknown.

In sensitive cell lines, the repression of transcription of the cyclin Dl promoter by Myc

occurs via a mechanism which is dependent on the initiator site. Myc is suggested to effect

repression via its interaction with initiator-binding proteins such as Miz-1, and TFII-I,

thereby inhibiting the transactivation activities of these transcriptional initiators. The

domains in the human c-Myc protein which are required for the repression of cyclin Dl

mapped to amino acids 92 to 106, the carboxyl terminal DNA binding and protein

dimerization motifs. These regions do not coincide with the domains of Myc which are

crucial to cellular transformation. Hence, the relevance of cyclin Dl repression by Myc, to

the Myc turnorigenic program is questionable.

The regulation of the AdMLP by Myc is complex. The AdMLP possesses two

CACGTG motifs as well as an initiator site. In transient promoter reporter assays uing a

AdMLP-driven luciferase reporter construct, expression of low concentrations of ectopic

Myc protein results in the activation of transcription of the luciferase reporter. Myc

transactivation is dependent upon the consensus E boxes. Expression at high Myc

concentrations repressed transcription via a mechanism dependent upon the initiator.

Repression may be mediated through the interaction of Myc with the initiator-binding

protein, Miz-1, although this has yet to be demonstrated (Schneider et al., 1997).

The c-myc gene is one of the better characterized Myc-repressed target genes. C-

myc auto-suppression is evident in all primary and some immortalized cell lines. It is a

homeostatic regulatory mechanism which results in the repression of endogenous c-myc

expression at the level of transcription initiation. This feedback mechanism functions in a

Myc dose-dependent manner, and requires the interaction of Myc with Max (Facchini et

al., 1997; Grignani et al., 1990; Pem et al., 1990; Penn et al., 1990). Although there is an

initiator in the c-myc basal promoter, mutational analysis clearly demonstrated that it is not

required for Myc repression. Evidence to date suggests Myc represses gene transcription

through core regulatory elements via a mechanism which does not require the presence of

previously identified c-myc transcription regulatory sites (Antonson et al., 1995; Facchini

et al., 1997; Li et al., 1994; Lucas et al., 1993; Philipp et al., 1994; Roy et al., 1993).

The analysis of these few Myc-repressed genes has hinted at the potential

importance of Myc repression in ow understanding of Myc function. The identification

and analysis of additional Myc-repressed genes is essential to aid our understanding of the

mechanisms of Myc repression, and the significance of Myc repression in the context of

the biological activities of Myc.

1.6 The emerging significance of Myc repression

1.6.1 Correlation of repression with transformation

What is the significance of Myc repression of gene expression? R i s is a difficult

question given the small number of genes that have been identified as hlyc-repressed

targets. Characterization of the mechanisms through which these genes are regulated by

Myc has revealed that Myc repression may be as important as Myc transactivation in

effecting Myc function. Analysis of the Myc-mediated repression of the AdMLP

demonstrated that deletion of the MBII region of c-Myc will abrogate both transcription

repression, and the ability of Myc to cooperate with activated Ras, in a rat embryo

fibroblast transformation assay (Li et al., 1994; Stone et al., 1987). Importantly, this

mutant Myc protein can still transactivate, suggesting that Myc-mediated activation and

repression may be executed via distinct pathways (Li et al., 1994). This view was

substantiated through the characterization of mutant c-Myc proteins derived from Burkitt's

lymphoma cells (Lee et al., 1996). These mutants contained specific point mutations in the

Myc TAD, permitting a detailed analysis of this region to ascertain which residues in the c-

Myc protein were required for Myc-dependent transformation versus Myc activation or

repression. The results of this study suggested that Myc transactivation and Myc-mediated

repression are separable. Moreover, Myc repression seems to correlate with Myc-mediated

transformation (Lee et al., 1996; Li et al., 1994), suggesting that Myc repression of gene

transcription may serve an important role in Myc-dependent transformation. Further

evidence supporting the view that Myc transactivation was not sufficient to effect Myc

transformation arose in studies where the weak Myc TAD was replaced with the strong

TAD of the herpesvirus VP16 transcriptional activator (Brough et al., 1995). Although

this chimeric protein could transactivate, it was unable to repress gene transcription, nor

could it transform cells. Lastly, the domains required for Myc repression coincide with

those that are crucial to Myc transformation, supporting a role for Myc repression in

effecting cell transformation (Li et al., 1994; Penn et al., 1990; Philipp et al., 1994; Stone

et al., 1987).

1.6.2 Activation and Repression cooperate to promote cell proliferation

Given the potential importance of the mechanism of Myc repression in the

execution of the tumorigenic program, the conventional model depicting the role of Myc in

normal cell proliferation and tumorigenesis may need to be broadened. The ability of Myc

to promote cell proliferation may require the cooperation of Myc repression as well as

activation. For example, Myc may suppress the expression or activity of the products of

growth arrest genes, driving cells out of growth arrest. Myc transactivation will serve to

promote the progression through the GI phase into the DNA replicative phase, through the

induction of genes which are required for the passage through the cell cycle. Thus, both

pathways, Myc activation and Myc repression, are most probably essential for Myc-

mediated cell cycle progression, and tumorigenesis.

1.7 Objectives

Our appreciation of the potential role of Myc repression in facilitating enhanced cell

proliferation and tumorigenesis is hampered by the small number of Myc-repressed genes

that have been identified. The identification and characterization of additional genes which

are repressed by Myc will help us to understand both the mechanism of Myc-mediated gene

repression and how the repression of genes by Myc can stimulate cell proliferation and

ultimately promote tumour progression. To approach this objective, we attempted to

resolve the controversy surrounding the relationship between Myc, pRb, and cyclin Dl, by

analyzing the expression of cyclin Dl in response to Myc activation andlor pRb loss in

primary cell cultures. In addition, we have identified and characterized two genes which

are repressed in response to Myc activation: the PDGF-jR gene, and the growth arrest

gene, gadd45.

In a variety of tumour cell lines and selected immortalized cell lines, studies have

revealed a number of novel regulatory relationships among three key cell cycle regulators:

Myc, pRb, and cyclin Dl. For example, pRb and cyclin Dl are reported to exist in a

negative feedback loop, a mechanism proposed to regulate normal cell proliferation. In

addition, Myc activation has been reported to either repress, promote, or exert no effect on

the expression of cyclin Dl (Daksis et al., 1994; Marhin et al., 1996; Philipp et al., 1994;

Solomon et al., 1995). Clearly, the immortalized nature of these cell lines has influenced

the analysis of these putative normal regulatory mechanisms. Therefore, to assay the

validity of these reported pathways, primary MEF cultures were analyzed. Neither Myc

activation nor pRb loss in primary MEF cultures exerted an effect on cyclin Dl expression.

Interestingly, ectopic Myc expression in MEFs which lack expression of the retinoblastoma

tumour suppressor protein (pRb) exhibit a loss of cyclin Dl protein expression,

concomitant with an increase in cellular growth rate and a tumorigenic phenotype. Cyclin

Dl loss is mediated at both the mRNA and protein level, arguing that the suppression of

cyclin Dl is an indirect consequence of the transformed nature of these cells. Ow results in

nontransformed cells clearly contradict earlier studies conducted in turnow cell lines, and

support a novel cross-regulatory mechanism between Myc, cyclin Dl, and pRb.

Moreover, our results suggest a novel role for cyclin Dl in tumorigenesis (Marhin et al.,

1996).

The PDGF-PR is an important mitogenic receptor which initiates signal

transduction pathways required for cells to exit quiescence and enter the cell cycle. The

interaction of PDGF-PR with its ligand PDGF induces c-Myc expression and cell

proliferation. Following transmission of the signal, the ligand receptor complex is

internalized, and receptor expression is repressed in a putative negative feedback

mechanism. Recent studies have revealed some candidates for this inhibitory pathway, the

GTPase Ras, and the nonreceptor tyrosine kinase src (Vaziri and Faller, 1995). The

components downstream of these signal transducars responsible for the repression ofpdgf-

pr mRNA expression were unknown. We demonstrated that Myc is a potential candidate

in this pathway. Indeed, the expression of the pdgf-pr gene is repressed at the

transcriptional level in response to Myc. The Myc-mediated suppression in PDGF-PRs on

the cell surface represents a putative pathway through which Myc is able to regulate the

proliferative response of a cell to mitogens in the extracellular environment (WM,

submitted).

The growth arrest gene gadd45, functions as a negative regulator of cell growth

keeping cells in a non-proliferative, growth-arrested state. Gadd45 expression is elevated

in response to a variety of growth arrest signals, including contact inhibition, mitogen

withdrawal, and exposure to growth inhibitory agents. We demonstrate that the

transcription of gadd45 is repressed by Myc and as such this repression mechanism defines

a novel pathway in which Myc promotes cell growth by inhibiting the expression of a

growth inhibitor, driving cells out of growth arrest (Marhin et al., 1997).

Identification of these genetic targets of Myc repression will allow us to understand

the mechanism(s) by which Myc represses gene expression, and has elaborated as well as

defined novel roles for Myc in the control of normal cell proliferation md the promotion of

tumorigenesis.

Chapter 2

LOSS OF RB AND MYC ACTIVATION CO-OPERATE TO

SUPPRESS CYCLIN Dl AND CONTRIBUTE TO

TRANSFORMATION

This chapter is a modified version of the following manuscript: Loss of Rb and Myc activation co-operate to suppress cyclin Dl and contribute to transformation. 1996. Marhin, W.W., Hei, Y.-J., Chen, S., Rang, Z., Gallie, B., Philips, R.A. and Pem,

L.Z. Oncogene, 12,43-52.

2.1 Abstract

Cyclin Dl can bind and phosphorylate the product @Rb) of the retinoblastoma gene

(RE-I) and recent evidence suggests pRb, in turn, may regulate cyclin Dl protein

expression. In transformed cell lines, loss of pRb activity strongly correlates with a

decrease in cyclin Dl protein expression, and conversely, introduction of pRb can induce

cyclin D l promoter activity. Yet we show pRb does not regulate cyclin Dl directly as

basal and serum-stimulated levels of cyclin Dl protein and kinase activity are similar in

wildtype and pRb-deficient primary mouse embryonic fibroblasts (MEFs). These

observations suggest that the suppression of cyclin Dl in pRb-minus turnour cell lines

requires loss of pRb in conjunction with at least one additional genetic event. We have

determined that constitutive, ectopic Myc expression in pRb-deficient, but not wildtype,

MEFs suppresses cyclin Dl protein expression and kinase activity. The suppression of

cyclin Dl protein expression is mediated by a post-transcriptional mechanism.

Phenotypically, pRb-deficient MEFs consistently exhibited a delayed growth response in

comparison to wildtype MEFs. This growth delay is abrogated in pRb-deficient MEFs

which are expressing ectopic Myc protein, coincident with the loss of cyclin Dl protein

expression. Moreover, these cells exhibit an increased proliferative capacity, and they no

longer show contact inhibition. Our results support a cross-regulatory mechanism between

Myc, pRb and cyclin Dl and suggest a novel role for cyclin Dl in tumorigenesis.

2 . 2 Introduction

The retinoblastoma susceptibility gene is mutated in a variety of tumors including,

retinoblastomas, small cell lung carcinomas and osteosarwmas, implicating the loss of pRb

function as a key contributor to hunorigenesis (reviewed in Goodrich and Lee, 1993; Wang

et al., 1994; Weinberg, 1995; Zacksenhaus et al., 1992). This view was fhrther

substantiated by studies which showed that introduction of pRb into pRb-deficient tumour

cell lines, arrested cells in G1 phase of the cell cycle (Ewen et al., 1993; Fung et al., 1993;

Goodrich and Lee, 1993; Hinds et al., 1992; Huang et al., 1988; Templeton et al., 1991).

pRb is thought to play a critical role in the control of normal cell proliferation by negatively

regulating the progression of cells into the S phase of the cell cycle (DeCaprio et al., 1989;

Ewen et al., 1993; Goodrich and Lee, 1993; Hinds et al., 1992). The growth suppressive

activity of pRb is controlled at the level of phosphorylation as pRb protein levels are

invariant throughout the cell cycle. The hypophosphorylated form of pRb in GO and early

GI phase of the cell cycle is functionally active. Phosphorylation on serine and threonine

residues, in mid-GI and then throughout S and G2 phases, leads to the inactivation of pRb

which is then dephosphorylated following mitosis (Buchkovich et al., 1989; Chen et al.,

1989; DeCaprio et al., 1992; Lees et al., 1991; Lin et al., 1991; Ludlow et al., 1993;

Ludlow et al., 1990; Mihara et al., 1989; Mittnacht et al., 1994). pRb may exert its growth

suppressive properties by interacting with and inacrivating at least some members of the

E2F family of transcriptional transactivators (Chellappan et al., 1991; Helin and Harlow,

1992; Kaelin et al., 1992; Nevins, 1992; Shirodkar et al., 1992). It is believed that

phosphorylation of pRb in late GI disrupts complex formation with E2F allowing

expression of a subset of EZF-regulated genes required for progression into S phase

(Hamel et al., 1992; Hiebert et al., 1992; Weintraub et al., 1992). Thus, phosphorylation

of pRb is key to pRb regulation and cell cycle progression.

Thct phosphorylation sites on the pRb protein are consensus recognition sites for

cyclins and their catalytic partners, cyclin dependent kinases (cdk). The kinetics of pRb

phosphorylation suggest that the G1 cyclins, particularly the D-type cyclins (Baldin et al.,

1993; Motokura et al., 1990; Sherr, 1993; Xiong et al., 1991) together with their cdk

partners (Bates et al., 1994; Kato et al., 1993; Meyerson and Harlow, 1994; Tam et al.,

1994), function as pRb kinases. Indeed, the D-type cyclins are able to interact with and

phosphorylate pRb in vitro at sites identical to those phosphorylated in vivo (Dowdy et al.,

1993; Ewen et al., 1993; Horton et al., 1995; Kato et al., 1993; Matsushime et al., 1994;

Meyerson and Harlow, 1994). Moreover, ectopic expression of D-cyclins can overcome

the proliferation block imposed by pRb and stimulate the cells to enter S phase (Dowdy et

al., 1993; Ewen et al., 1993; Hinds et al., 1992).

Cyclin Dl is a delayed early response gene which is transiently induced following

mitogen stimulation, peaking late in G1 phase of the cell cycle. In human and rodent

fibroblasts, cyclin Dl is essential for the G1 to S phase transition (Baldin et al., 1993;

Lukas et al., 1995; Lukas et al., 1994; Roussel et al., 1995; Tam et al., 1994), and it is

believed that cyclin Dl regulates this step through phosphorylation and inactivation of pRb

(Lukas et al., 1995). In addition, it was demonstrated that pRb can induce transcription of

the cyclin Dl gene (Muller et al., 1994). Therefore, a cross-regulatory pathway involving

cyclin D l and pRb can be envisaged. Hypophosphorylated pRb induces cyclin Dl

expression, and in turn cyclin Dl together with its associated cdk can phosphorylate and

thereby inactivate pRb. A correlation between loss of pRb function and absence of cyclin

Dl protein in human tumour cells further supports such a cross-regulatory mechanism

(Lukas et al., 1994; Muller et al., 1994; Tam et al., 1994), yet recent results show the

suppression of cyclin D l is not a direct effect of pRb (Lukas et al., 1995). Therefore, the

molecular mechanism involved in the regulatory interaction of pRb and cyclin Dl remains

unknown.

Cyclin Dl expression is highly influenced by the concentration of cellular Myc

protein; however, the pattern of cyclin Dl expression in response to Myc is complex

(Daksis et al., 1994; Jansen et al., 1985; Philipp et al., 1994; Rosenwald et al., 1993).

Myc is a key regulator of normal cell proliferation whose expression is required for cell

cycle progression (reviewed in Dang, 1991; Penn et al., 1990). In its activated form,

deregulated Myc expression confers a strong cellular self-renewal potential and is a

common feature of a diverse array of tumors (reviewed in Spencer and Groudine, 1991).

Daksis et al., showed that induction of Myc activity in quiescent non-transformed Rat-1

cells transiently induced cyclin Dl mRNA and protein expression, and promoted the

transition from GI to S phase of the cell cycle. This induction is rapid and occurs at the

transcriptional level in the absence of de novo protein synthesis, suggesting Myc can

directly induce cyclin Dl gene transcription (Daksis et al., 1994). The work of Rosenwald

et al. also shows Myc can up-regulate cyclin Dl protein expression in NIH 3T3 cells at the

translational level by inducing the rate-limiting eukaryotic initiation factor-4E (eIF-4E)

(Rosenwald et al., 1993). Experimentally, constitutive Myc expression can lead to cyclin

Dl suppression in some (Jansen et al., 1985; Philipp et al., 1994), but not other fibroblast

cell lines (Rosenwald et al., 1993); Daksis and Penn, unpublished results). Thus, Myc

induction results in the elevation of cyclin Dl expression, whereas the effect of

constitutively expressed, activated Myc on cyclin Dl expression remains unclear.

Myc, cyclin Dl and pRb are highly regulated proteins which function as effectors

of cell proliferation in a wide variety of cells. To ascertain whether regulatory pathways

linking these molecules exist in non-transformed cells, we analyzed cyclin Dl protein

expression in mouse embryonic fibroblasts (MEFs) where pRb protein expression has

been abrogated through targeted gene disruption (Jacks et al., 1992). Neither basal nor

mitogen-induced cyclin Dl protein was suppressed in the pRb-deficient MEFs, and cyclin

Dl protein showed kinase activity. Expression of ectopic Myc in pRb-deficient, but not in

wildtype, cells suppresses cyclin Dl protein expression as well as its associated kinase

activity. The suppression of cyclin Dl protein in MEFs can occur by regulatory

mechanisms involving either RNA or protein expression.

2.3 Materials and Methods

2.3.1 Cell culture

129 mice heterozygous for the RB-I gene were obtained fiom Jacks et al. (Jacks et

al., 1992). Primary mouse embryonic fibroblasts (MEF) were prepared fiom embryos at

day 12.5 of gestation and derived fibroblast cell lines were cultured in alpha modified

Eagle's medium (&EM) supplemented with 10% fetal bovine serum (FBS, Gibco/BRL),

100 pg/ml kanamycin (Sigma) and 2 pg/ml gentamicin (Roussel). Early passage cell

cultures were used for all assays. Unless otherwise stipulated all analyses were performed

on asynchronous, subconfluent, exponentially growing cells. Growth kinetics were

conducted by trypsinizing near-confluent cells, seeding 1 x 103 cells per well of a 24-well

dish (Nunc) in 10% FBSIaMEM, then trypsinizing and counting the number of cells per

well at the time points indicated. Each time point represents an average of three wells. All

cell cultures were assayed in at least two independent experiments.

2.3.2 Genomic DNA extraction and mouse embryonic fibroblast

genotyping

Genomic DNA was extracted fiom primary MEFs using the QIAamp Blood Kit

(Qiagen). Genotype of the MEFs was detected by PCR analysis of 1 ng of genomic DNA

for the presence of the wildtype and mutant RB-1 alleles with Amplitaq (Perkin-Elmer-

Cetus). Primer sequences: 5' primer RX3 [AAlTGCGGCCGCATCTGCATCmATCG

C] (within R E - I exon 3); 3' primers, for the wildtype R E - I allele R13

[CCCATGTTCGGTCCCTAG] (within RB-1 intron 3), and for the mutant allele PGK3'

[GAAGAACGAGATCAGCAG] (within the 3' pgk promoter in the pgk neomycin

cassette). The PCR was performed for thirty cycles, each cycle consisting of 94"C, 1 min,

5 8 T , 1 min, and 72"C, 1 min, and the PCR products were resolved on a 1.5% agarose

gel.

2.3.3 Retroviral infection

The pBabeIpuro retroviral vector has been described previously (Morgenstem and

Land, 1990). The pBabelv-mycpuro vector was constructed by subcloning the 3 Kb Bgl I1

v-gag-myc fragment from the pRasImyc 9 retroviral vector (kind gift of Tim Thompson)

(Thompson et al., 1989) into the Barn HI site of the pBabe1puro plasmid. To produce

infectious replication-defective ecotropic retroviral particles, recombinant retroviral

constructs were trnnsfected, using calcium phosphate precipitation (Graham and Van der

Eb, 1973), into GP+E packaging cell lines (Markowitz et al., 1988), and selected in 2

y d m l puromycin (Sigma) or 150 ydml hygromycin B (Sigma). Drug resistant clones

were pooled and expanded for virus production. Primary MEFs were subsequently

infected with retrovirus and selected in either puromycin or hygromycin B. Individual

drug-resistant colonies were isolated and the remaining colonies combined, generating

pooled populations.

2.3 .4 RNase protection

RNA was prepared by the guanidinium isothiocyanate method (Chirgwin et al.,

1979) and assayed by RNase protection (Penn et al., 1990) essentially as described. The

probes were generated using T3 RNA polymerase (Stratagene) from linearized Bluescript

KS and SK cloning vectors (Stratagene) containing the following DNA fragments: rat c-

myc exon 1 (Penn et al., 1990); the gag gene derived from the avian myelocytomatosis

virus MC29 (Penn et al., 1990); and the murine glyceraldehyde-3-phosphate

dehydrogenase (gavdh) cDNA (Facchini et al., 1994). The probe for the mouse cyclin Dl

gene was transcribed using Sp6 RNA polymerase (Stratagene) from a linearized

pGEM7zf+ vector (Promega) containing the full-length murine cyclin Dl cDNA (kind gift

of M. Kiess and P. Hamel). The protected probes were resolved by electrophoresis on

6% denaturing polyacrylamide gels and visualized by autoradiography on X-OMAT film

(Kodak).

2.3.5 Immunoblotting and immunoprecipitation

Antibodies used in this study include the monoclonal antibodies: anti-cyclin Dl

HDI (kind gift of E. Lees and E. Harlow), anti-Rb 14001A (Pharmingen), and a

polyclonal pan-myc antibody (kind gift of G. Evan). Total cell lysates (5 x 105 cells) were

immunoblotted as previously described (Daksis et al., 1994). Cyclin Dl, v-Myc, and pRb

protein were detected by incubating the membrane with their respective antibody for 1 h in

20 mM Tris pH 7.6, 137 mM NaCI, 0.05 % Tween 20 (TBS-T) containing 1% non-fat

milk. The membrane was washed, and incubated with a 112000 dilution of either

horseradish peroxidase-conjugated goat anti-mouse IgG (Biorad) or swine anti-rabbit IgG

antibody (Dako) in TBS-T containing 1% non-fat milk for 0.5 h. After washing, signals

were detected using the ECL (Amersham) detection system. As a positive control for

cyclin Dl protein, we used whole cell extracts of confluent, quiescent BALBlc 3T3 cells

stimulated with serum for 10 h. (Lanahan et al., 1992). Immunoprecipitations using the

anti-pRb monoclonal antibody (14001A) were performed as described (Hamel et al., 1990)

and immune complexes were resolved on a 7.5% SDSlacrylamide gel.

2.3.6 In vitro kinase assay

Total cell lysates were prepared and in vitro kinase assays were performed as

previously described (Matsushime et al., 1994), except lysates were sonicated at 4 "C (full

microtip power 3 times for 15 secs each). Lysates were clarified by centrifugation at

13,000 rpm for 13 min., and cyclin Dl complexes were immunoprecipitated for 1 h at 4 'C

with 2 pg of Dl-72-13G, a monoclonal antibody specific for murine cyclin Dl (Santa

Cruz). Full-length pRb protein (2 ug; QED Adv. Res. Tech.) was used as substrate in the

kinase assays. Samples were boiled in Laemmli sample buffer and resolved on a 7.5%

SDSlacrylamide gel. Phosphorylated proteins were detected by autoradiogaphy.

2 . 4 Results

2.4 .1 Primary mouse embryonic fibroblasts lacking pRb protein express

normal levels of cyclin Dl protein

In a number of transformed cell lines, loss of pRb function correlates with an

absence of cyclin Dl protein expression (Lukas et al., 1994; Tam et al., 1994). In

addition, it was previously demonstrated that pRb can induce the transcription from the

cyclit~ Dl promoter (Mullet et al., 1994). Taken together these results suggest the

existence of a pRbIcyclin Dl cross-regulatory mechanism. To ascertain whether this

regulatory mechanism is present in non-transformed cell lines, we analyzed cyclin Dl

protein expression in primary MEFs lacking pRb protein. MEF cultures were prepared

using embryos derived from mating mice in which one RE-1 allele was mutated through

targeted gene disruption (Jacks et al., 1992). We analyzed three independent litters and

from each litter, cultures were derived from one wildtype embryo (MEF 4, MEF 40, MEF

53), and its pRb-deficient littermate (MEF 1, MEF 37, and MEF 47). The genotype of

each culture was verified by PCR (Figure 2.1A) and confirmed by assaying the level of

endogenous pRb protein expressed (Figure 2.1B). Analysis of cyclin Dl protein

expression revealed the level of cyclin Dl protein expressed in pRb-deficient cells was not

suppressed, and was similar to that seen in wildtype MEFs (Figure 2.2A). To determine if

the cyclin Dl protein expressed in the pRb-deficient cultures was functionally active, we

performed in vitro kinase assays. Our results indicate that, irrespective of the RE-1 status,

all six cultures exhibited readily detectable levels of cyclin Dl associated kinase activity

(Figure 2.2B). As pRb has been shown to induce cyclin Dl promoter activity [Miiller,

1994 #925], we determined whether pRb protein was required for the induction of cyclin

Dl protein following mitogen-stimulation of confluent, quiescent cells (Baldin et al., 1993;

Daksis et al., 1994; Lanahan et al., 1992; Lukas et al., 1994; Won et al., 1992). Serum-

nnnnnn PRIMERSET wt A wt A wt A wt A wt A wt A

mutant - wildtype -

LITTER 1 2 3 b i

MEF 4 1 40 37 53 47

Figure 2.1. Determination of RB-I gene status and pRb protein expression in primary mouse embryonic fibroblast (MEF) cultures derived from 3 Litters of embryos produced from the mating of RB-I heterozygotes. (A) Screening of genomic DNA extracted from 6 mouse embryonic fibroblast cultures was performed by PCR analysis of the disruption of the RB-I gene through the insertion of the neomycin cassette in intron 3. The wildtype allele was distinguished from the mutant allele through the use of specific primer sets. RX3 and R13 for the wildtype allele generated a 380 bp amplicon, RX3 and PGK3' for the mutant allele produced a 400 bp amplicon. MEF 4, MEF 40, MEF 53 are homozygous for the wildtype RB-1 allele and MEF 1, MEF 37, MEF 47 are homozygous for the mutant RB-I allele. (B) Total cell lysates of each of the MEF cultures in (A) were immunoprecipitated with an anti-Rb monoclonal antibody (14001A), resolved on a 7.5 % SDSlpolyactylamide gel, transferred to PVDF membrane and immunoblotted with the pRb antibody. Equivalent amounts of protein were loaded in each lane as determined by Coomassie blue staining.

L I r n R 1 2 3 -nn

MEF C 4 1 40 37 53 47

LITTER 1 2 3

MEF 4 1 40 37 53 47

Figure 2.2. MEFs lacking pRb protein express cyelin D l protein and cyclin Dl associated kinase activity at levels comparable to their wildtype littermates. (A) Immunoblot analysis of total cell lysates prepared from primary MEFs using a monoclonal antibody specific to mouse cyclin Dl. Equivalent amounts of total cell extracts were resolved on a 7.5% SDSIpolyacrylamide gel, transferred to PVDF membrane, and cyclin Dl protein expression detected with the monoclonal antibody HD1. MEFs lacking pRb protein expression (MEF 1, MEF 37, MEF 47) exhibit significant levels of cyclin Dl protein compared to that of their wildtype littermates (MEF 4, MEF 40, MEF 53). (C= Positive control) (B) Whole cell extracts of asynchronously growing MEFs were immunoprecipitated with a monoclonal antibody specific for mouse cyclin Dl and tested for kinase activity. Significant phosphorylation of the pRb protein substrate by cyclin Dl associated kinase activity was evident in lysates from both wildtype MEFs and MEFs lacking pRb protein expression.

stimulation of confluent, quiescent wild-type and pRb-deficient MEFs induces cyclin D 1

protein (Figure 2.3) suggesting that mitogen-induced cyclin Dl expression does not require

pRb protein. Therefore, in contrast to studies performed in transformed cell lines and in

agreement with recent studies in non-transformed cells (Lukas et al., 1995), loss of pRb

function in primary MEFs does not affect cyclii Dl protein expression or kinase activity.

2.4 .2 Constitutive Myc expression suppresses cyclin D l protein in pRb-

deficient but not wild-type MEFs

A second genetic event resulting from the transformation process may act in

conjunction with the lack of pRb expression to cause the observed suppression of cyclin

Dl in tumour-derived cell lines (Lukas et al., 1994; Tam et al., 1994). As Myc can

suppress cyclin D l expression in BALBlc 3T3 cells, Myc was a strong candidate for this

second event. To investigate this hypothesis, we constitutively expressed ectopic Myc

protein in both wildtype MEF 4 cells and in cells of the corresponding mutant sibling MEF

1. MEF cultures were infected with either a control retrovirus carrying only the puromycin

resistance gene (pBabepuro) or with a retrovirus canying the ectopic v-myc gene @Babe/v-

myc puro). Following selection, individual clones were isolated and the remaining clones

combined to generate pooled populations. Two forms of cyclin Dl were consistently

detected in immunoblots, as previously reported (Matsushime et al., 1991). Both forms

are phosphoproteins, and the biochemical difference between them has not been

characterized. Cyclin Dl protein expression in wildtype MEF 4 cells was not affected by

expression of ectopic ivIyc protein (Figure 2.4A, compare lanes 5-8 with lanes 1-4). In

contrast, elevated expression of ectopic Myc in pRb-deficient MEF 1 cells (Figure 2.4A,

lanes 13-16) resulted in a significant suppression in cyclin Dl protein compared to MEF 1

cells not expressing ectopic Myc (Figure 2.4A, lanes 9-12). Results obtained with clonal

populations were consistent with those obtained with pools. Indeed, the dose dependent

Cydin Dl--) -0

1 2 3 4 5 6 7 8

Figure 2.3. Cycl i D l protein expression is induced in quiescent wildtype and pRb-deficient MEFs in response to serum-stimulation. Imrnunoblot analysis of cyclin Dl protein levels was performed using whole cell extracts prepared from confluent MEF cultures rendered quiescent in 0.1% FBSIuMEM for 3 days and then stimulated with 10% FBSIuMEM at the times indicated. Cyclin Dl protein was detected using a monoclonal antibody (HDI).

effect of ectopic Myc protein expression on the degree of cyclin Dl suppression in the

pRb-deficient MEFs is particularly striking (Figure 2.4A, lanes 13 - 16). Introduction of

activated Myc in two additional wild-type and pRb-deficient MEFs resulted in an identical

pattern of cyclin Dl expression (data not shown). The abundant number of colonies

observed following selection of control or Myc-containing retrovirus indicated that Myc

suppression of cyclin Dl protein in a pRb-minus cell is a directed molecular event, and not

a selected rare event. As a further control, we assayed the protein lysates of the pooled

populations for in vifro cyclin Dl kinase activity and found the level of kinase activity

directly rtilects the level of cyclin Dl protein expressed (Figure 2.4B). Thus, suppression

of cyclin Dl occurs in response to Myc-activation in pRb-deficient MEFs.

To determine whether the lack of cyclin Dl suppression in the wild-type MEFs was

due to insufficient ectopic Myc activity, the expression and activity level of exogenous Myc

was measured. Ectopic Myc expression was clearly evident in wildtype and pRb-deficient

MEFs as assayed at the RNA level by RNase protection (Figure 2.4C) and at the protein

level by western blot analysis (Figure 2.4A). To determine whether this ectopic v-Myc

protein was functionally active in the MEF cultures, we assayed for Myc negative auto-

regulation. Through this homeostatic feedback mechanism, elevated expression of ectopic

Myc will suppress the transcription of the endogenous c-myc gene (Cleveland et al., 1988;

Grignani et al., 1990; Pem et al., 1990). RNase protection analysis of endogenous c-myc

mRNA expression showed that expression of ectopic Myc protein in both wildtype (MEF

4) and pRb-deficient (MEF 1) MEFs (Figure 2.4C) suppressed endogenous c-myc mRNA

levels. These results suggest that the lack of suppression of cyclin Dl protein in wildtype

cells expressing ectopic Myc is likely not due to a lack of Myc activity in these cells.

Figure 2.4. Constitutive ectopic Myc expression in primary MEFs lacking

pRb protein suppresses cyclin D l protein expression and activity. (A) Clonal

and pooled (P) populations of wildtype (MEF 4) and pRb-deficient (MEF 1) MEFs

infected with control retrovirus (lanes 1-4 and 9-12) or with retrovirus carrying the v-ntyc

gene (lanes 5-8 and 13-16), were assayed by immunoblot for cyclin Dl and v-Myc protein

expression. The weak signal in the control lanes of the v-Myc blot represents a cross-

reactive band evident with this batch of pan myc antibody. Equivalent amounts of protein

were loaded in each lane as determined by Coomassie blue staining. (B) Whole cell

extracts of asynchronously growing MEFs were immunoprecipitated with a monoclonal

antibody specific for mouse cyclin Dl and tested for kinase activity. Phospholylation of

the pRb protein substrate by cyclin Dl associated kinase activity was significantly reduced

in lysates from pRb-deficient MEFs which are expressing ectopic Myc protein, consistent

with the reduction in cyclin Dl protein expression. (C) The ectopic v-Myc protein is

functionally active in both wildtype and mutant fibroblasts, as Myc negative autoregulation

is evident in both cell types. RNase protection analysis of endogenous c-myc, ectopic v-

myc, and gapdh mRNA expression in wildtype (MEF 4) and pRb-deficient (MEF 1)

MEFs, showed endogenous c-niyc mRNA was clearly evident in control cells (lanes 1-4

and 9-12) and suppressed in cells expressing ectopic Myc protein (lanes 5-8 and 13-16).

(P= Pooled populations).

MEF 4 (+I+) MEF 1 (-I-) 1 U

CONTROL VMYC CONTROL VMYC I

CLONES CLONES CLONES CLONES I---

1 4 6 P l 4 6 P l 5 6 P 2 4 5 P

Cyclin Dl-+ *

a.ry*l).

. .

MEF4 MEFl (+I+) (-I-)

eRb - substrate

MEF 4 (+I+) MEF 1 (-I-) I a

CONTROL VMYC CONTROL VMYC I I

CLONES CLONES CLONES CLONES I---

1 4 6 P l 4 6 P 1 5 6 P 2 4 5 P

v-myc +

Endogenous- c-myc

2.4 .3 The suppression of cyclin D l protein in MEFs can occur by

regulatory mechanisms controlling either RNA or protein

expression

The suppression of cyclin Dl protein levels in pRb-deficient cells expressing

ectopic Myc is highly consistent as an identical pattern of regulation was observed among

MEFs derived from independent litters (Figure 2.5A). To determine whether the Myc-

induced suppression of cyclin Dl protein in pRb-deficient cells is the result of a repression

of cyclin Dl mRNA levels, we assayed endogenous cyclin Dl mRNA expression using

RNase protection. For both wildtype MEF 4 and pRb-deficient MEF 1 cells, pooled

cultures expressing ectopic Myc protein were compared to controls lacking ectopic Myc

expression. Expression of ectopic Myc protein had no effect on cyclin Dl mRNA levels in

either wildtype MEF 4 (Figure 2.5B, compare lanes 1 and 2) or pRb-deficient MEF 1

(Figure 2SB, compare lanes 3 and 4). Similar results were observed in an additional Rb-

deficient MEF culture (data not shown). However, analysis of another pair of wildtype

and Rb-deficient MEF cells gave a different result. Introduction of ectopic Myc did not

affect cyclin Dl mRNA expression in wildtype MEF 40 cells (Figure 2SB, compare lanes

5 and 6), yet resulted in suppression of cyclin Dl mRNA levels in pRb-minus MEF37 cells

(Figure 2SB, compare lanes 7 and 8). Thus, in wildtype MEFs, ectopic expression of

Myc does not suppress cyclin Dl protein (Figure 2.4A and 2.5A), kinase activity (Figure

2.4B) or mRNA (Figure 2.5B). By contrast, in pRb-deficient MEFs, introduction of

activated Myc consistently suppressed cyclin Dl protein (Figure 2.4A and 2.5A) and

kinase activity (Figure 2.4B). This regulation is at the level of RNA in MEF 37 cells

(Figure 2.5B, lanes 7 and 8) and at the protein level in MEF 1 cells (Figure 2.5B, lanes 3

and 4).

MEF 4 MEF 1 MEF 40 MEF 37 (+I+) (-1-1 (+I+) (-1-1 --

v-myc - + - $ - + - +

MEF 4 MEF I MEF 40 MEF 37 (+I+) (-I-) (+I+) (-I-) n-

v-myc - + - + - + - +

Cgclin 111 w 1

v-myc + ?

Figure 2.5. Cycl i D l expression in Myc-activated, pRb-deficient MEFs is suppressed either at the RNA or protein level. (A) Pooled populations of wildtype (MEF4, MEF40) and pRb-deficient (MEF 1, MEF 37) MEFs infected with control retrovirus (lanes 1,3,5 and 7) or with retrovirus carrying the v-myc gene (lanes 2,4,6 and 8), were assayed by immunoblot for cyclin Dl and v-Myc protein expression. The weak signal in the control lanes of the v-Myc blot represents a cross-reactive band evident with this batch of pan myc antibody. Equivalent amounts of protein were loaded in each lane as determined by Coomassie blue staining. A longer exposure of lanes 1 and 2 are shown for clarity. (B) Total RNA was extracted from control (lanes 1,3,5 and 7) and Myc-expressing (lanes 2,4,6 and 8) asynchronous pooled populations of both wildtype (MEF 4: lanes 1 and 2), (MEF40: lanes 5 and 6) and pRb-deficient (MEF 1: lanes 3 and 4), (MEF 37: lanes 7 and 8) cells. RNA was analysed by RNase protection for the expression of the endogenous cyclin Dl and gapdh, as well as ectopic v-myc mRNAs.

2.4.4 pRb-deficient MEFs exhibit a delay in growth compared to

wildtype MEFs

To examine the effect of pRb expression on the growth of primary MEF cultures,

we compared the growth of subconfluent cultures of pRb-deficient MEFs with that of

wildtype MEFs. It was immediately evident that subconfluent MEF cultures lacking pRb

protein exhibited a delayed growth response of approximately 1 to 2 days compared to

wildtype cultures (Figure 2.6, compare Ato E). This result was verified with two

additional pairs of wildtype and pRb-deficient MEF cultures, as well as with their

respective earliest-passage parental cultures derived prior to retroviral infection and

selection (data not shown). The delay can be attributed primarily to an initial lag, as

growth rate and saturation density were not affected (data not shown). Expression of

ectopic Myc in the pRb-deficient cell culture (0) significantly alleviated the delay in their

growth response and resulted in proliferation comparable to both control (A) and Myc-

expressing (A) wildtype cultures (Figure 2.6). This Myc-abrogated growth delay also

correlated with a loss of contact inhibition (Figure 2.7). The lack of contact inhibition was

not evident in control (panel A and E) or Myc-expressing (panel B and F) wildtype MEFs,

nor in control pRb-deficient MEFs (panel C and G), but was clearly visible in pRb-

deficient, Myc-activated MEFS (panel D and H). In summary, these results show that

cyclin Dl expression in pRb-deficient primary cells correlates with a reduced proliferative

capacity compared to wildtype MEFs. In pRb-deficient cells ectopic Myc expression can

lead to down-regulation of cyclin Dl and an increased proliferative capacity. The nature of

the additional factors which co-operate with a loss of pRb and Myc-activation in the

transformation process is the subject of further investigation.

J

: + MEF 4 (+I+) CONTROL - MEF4(+I+)MYC

& MEF I (-/-) CONTROL

-4- MEF 1 (-1-1 MYC

I I I a I I 1 I

0 1 2 3 4 5 6 7 8 9

TIME (days)

Figure 2.6. Proliferation profiles of wildtype (MEF 4) and pRb-deficient MEFs (MEF I), infected with control retrovirus (CONTROL) or with retrovirus carrying a v-riyc gene (MYC). Triplicate cultures of MEF 4 CONTROL cells (A), MEF 4 MYC cells (A), MEF 1 CONTROL cells (P), and MEF 1 MYC cells (0) were plated sub con fluent!^ at 1000 cells per well in 10% FBSlcLMEM and daily cell counts were performed. Mean values of the triplicates, together with standard errors, are shown plotted against time.

Figure 2.7. pRb-deficient MEFs constitutively expressing ectopic Myc protein exhibit an increased proliferative capacity. Photomicromaphs of confluent MEF cultures maintained in 10% FBS/&EM for 3 dais. MEF 4 C O N T R ~ L @ ~ ~ ~ I A and E), MEF 4 MYC (panel B and F), MEF 1 CONTROL (panel C and G), and MEF 1 MYC (panel D and H). Photomicrographs are shown at 40X (A - D), and 100X (E - H) magnification. The arrowheads point to examples of cells which have lost contact-inhibition, forming dense, refkctile foci.

2.5 Discussion

Our results in primary MEFs contrast with those conducted in established cell lines

and clearly show that loss ofpRb or Myc-activation alone is insufficient to suppress cyclin

Dl expression; yet in collaboration, loss of pRb and Myc activation can suppress cyclin Dl

and contribute to the transformation process. Previous analyses of human tumow cell lines

originating from a variety of tissues, showed a strong positive correlation between loss of

pRb function and a decrease in cyclin Dl protein expression (Lukas et al., 1994; Muller et

al., 1994; Tam et al., 1994). Under these conditions the residual cyclin Dl protein could

not associate with cdk4, its predominant catalytic partner in fibroblasts, resulting in loss of

cyclin Dl-associated kinase activity (Parry et al., 1995). In contrast, we have shown that

pRb-deficient MEFs express cyclin Dl protein and kinase activity further supporting Lukas

et al. (Lukas et al., 1995). The results presented here clearly show the down-regulation of

cyclin Dl expression and activity in transformed cells is complex and not dependent solely

on pRb. Downregulation of cyclin Dl activity likely depends upon other genetic changes

in addition to the loss of pRb function.

We reasoned that the additional transforming event may involve expression of the c-

myc proto-oncogene, as it is commonly activated in tumorigenesis and has recently been

shown to play a role in cyclin Dl regulation. Induction of Myc results in the rapid

elevation of cyclin Dl protein through transcriptional (Daksis et al., 1994) or post-

transcriptional (Rosenwald et al., 1993) mechanisms. Constitutive expression of Myc can

suppress cyclin Dl mRNA and protein through a transcriptional repression mechanism in

some (Jansen-Diirr et al., 1993; Philipp et al., 1994) but not other cell lines (Rosenwald et

al., 1993; Daksis and Penn, unpublished results). Ectopic expression of Myc in primary

MEFs, as shown in this thesis, does not result in a concomitant decrease in cyclin Dl

expression. Insufficient exogenous Myc activity cannot account for the apparent lack of

cyclin Dl suppression as exogenous Myc can trigger Myc negative autoregulation and

suppress endogenous MEF c-myc expression. Thus, in primary MEFs, constitutive

expression of Myc alone does not lead to the suppression of cyclin Dl expression or

activity.

To test whether the suppression in cyclin Dl observed in pRb-negative tumour cells

results from cooperative interaction between Myc-activation and pRb loss, we measured

their combined effect in early passage pr imw MEFs. Indeed, constitutive expression of

ectopic Myc protein in pRb-deficient, but not in wildtype MEFs, led to a suppression of

both cyclin Dl protein and its associated kinase activity. Thus, a second genetic event is

required in conjunction with the loss of pRb function to suppress cyclin Dl expression in

MEFs. It remains unclear whether Myc-activation is the only second transforming event

which can co-operate with pRb-inactivation to suppress cyclin Dl expression and whether

Myc-activation per se is evident in all or a limited subset of pRb-minus tumour cells.

Interestingly, Squire er a/. showed amplification of N-myc in retinoblastoma tumors,

demonstrating that this cooperativity can exist iiz vivo (Squire et al., 1986).

Cyclin Dl is a highly regulated molecule whose expression can be affected at both the

transcriptional and post-transcriptional levels. The complexity with which the activity of

this integral cell cycle regulator is modulated, is supported by our results showing the

suppression of cyclin Dl in response to pRb-loss and Myc-activation in MEFs can be

regulated at both the RNA and protein levels. Analysis of primary MEFs derived from

independent litters is key to hlly elucidating the multiple mechanisms which govern cyclin

Dl expression and activity. At the post-transcriptional level Myc may suppress cyclin Dl

protein expression by suppressing the translation of cyclin Dl or reducing the stability of

the cyclin Dl protein. e1F-4E, a translation initiation factor can be induced in response to

Myc and, in turn, specifically up-regulate cyclin Dl protein expression (Rosenwald et al.,

1993). It is tempting to speculate that e1F-4E may also serve as a control point for the

translational suppression of cyclin Dl in response to Myc in pRb-deficient MEFs, as

reported here. It is interesting that cyclin Dl mRNA was suppressed in the pRb-deficient

MEF 37 culture which expressed ectopic Myc protein. It will be important to ascertain

whether the suppression of mRNA expression in MEF37 is mediated at the transcriptional

level and whether pRb-dysfunction may be involved in Myc-repression of cyclin Dl

transcription as reported in (Philipp et al., 1994). Interestingly, the Myc negative

autoregulation mechanism remains intact in the pRb-deficient MEFs, and serves as a

positive control to show loss of pRb does not result in a general, non-specific disruption of

homeostatic regulatory mechanisms. Activation of c-myc expression in pRb-minus cells

reproducibly leads to downregulation of cyclin Dl activity. The mechanisms of this down-

regulation are complex involving RNA with some cells and protein in others: the different

mechanisms probably reflect the different secondary genetic changes in the cultured cell

lines.

Analysis of the growth of MEFs derived kom three independent litters of mouse

embryos revealed that pRb-deficient MEFs consistently exhibited a delay in growth

compared to wildtype MEFs. In contrast, Lukas el al. recently reported that pRb-deficient

MEFs have a shortened G1 phase compared to their wildtype counterparts (Lukas et al.,

1995). This difference in results may be attributed to our distinct experimental conditions.

The delay in growth, reported here, reflects an initial lag in proliferation following seeding

as subconfluent cultures. It remains unclear whether the lag in growth is a result of

decreased plating efficiency, an increased rate of apoptosis or a direct lengthening of the

cell cycle. Apoptosis is associated with pRb-inactivation (Clarke et al., 1992; Haas-Kogan

et al., 1995; Haupt et al., 1995; Jacks et al., 1992; Lee et al., 1992; Morgenbesser et al.,

1994), but is not likely contributing to the lag in population growth, as we did not observe

an increase in programmed cell death in pRb-deficient cells grown as subconfluent (Figure

2.6) or confluent (Figure 2.7) cultures (GW, WWM, Y-JH, LJZP, unpublished results).

Expression of Myc in pRb-deficient MEFs can suppress cyclin Dl, abrogate the

characteristic delay in cell growth and push the cells to a more transformed state. The

suppression of cyclin Dl in a pRb-deficient cell is not the only molecular event

downstream of Myc potentially involved in increasing cellular proliferative potential, but it

likely contributes and may be required in a pRb-deficient cell. These results are consistent

with observations reported in the literahue. Cellular transformation by other nuclear

oncogenes, adenovirus ElA or SV40 large T antigen, results in the suppression of cyclin

Dl expression as well as the inactivation of the product of the retinoblastoma gene (Bates et

al., 1994; Buchou et al., 1993; Lukas et al., 1994; Muller et al., 1994; Tam et al., 1994;

Xiong et al., 1993). Moreover, human cell lines harboring a loss of pRb uniformly show

a suppression in cyclin Dl protein expression (Bates et al., 1994; Tam et al., 1994; Xiong

et al., 1993). These human tumour cells also demonstrate an increase in the product of the

INK4IMTS1 gene, p16, which forms inhibitory complexes with cdk 4 or cdk 6 (Parry et

al., 1995), &her demonstrating that suppression of cyclin Dl kinase activity appears

crucial in pRb-deficient tumors.

Although cyclin Dl expression has been strongly implicated in tumour formation, the

precise role of cyclin Dl in tumorigenesis has not been elucidated. Cyclin Dl is

overexpressed in several types of tumors such as parathyroid adenomas (Motokura et al.,

1991), and centrocytic B-cell lymphomas (Motokura and Arnold, 1993), and is amplified

in breast cancers: (Motokura and Arnold, 1993). These observations suggested that cyclin

Dl may function as a promoter of cell proliferation. This view was supported by studies

demonstrating that cyclin Dl activity was required for progression through the cell cycle.

Microinjection of anti-cyclin Dl antibodies into wildtype MEFs (Lukas et al., 1995) or

human diploid fibroblasts (Baldin et al., 1993) blocks cells in the G1 phase. Furthermore,

expression of elevated levels of ectopic cyclin Dl shortens the G1 phase of the cell cycle

(Quelle et al., 1993; Resnitzky et al., 1994). However, in vivo its role as an oncogene is

not definitive. When constitutively overexpressed in transgenic mice, cyclin Dl provides

only a weak oncogenic stimulus (Bodrug et a]., 1994; Wang et al., 1994). Indeed, recent

evidence establishes a role for cyclin Dl as a negative regulator of cell proliferation

(Freeman et al., 1994; Lucibello et al., 1993; Quelle et al., 1993; Resnitzky et al., 1994).

Cyclin D l is induced in post-mitotic neurons (Freeman et al., 1994) and in senescent

h u m a cells (Lucibello et al., 1993). Constitutive expression of high levels of cyclin Dl in

a variety of mammalian cell lines inhibits cell growth (Quelle et al., 1993; Resnitzky et al.,

1994). Interestingly, microinjection of neutralizing antibodies to cyclin Dl in pRb-

deficient MEFs can accelerate S phase entry (Lukas et al., 1995), which agrees with our

results and further suggests suppression of cyclin Dl in an pRb-deficient cell can

contribute to cellular growth potential.

Clearly the regulation of cyclin Dl is highly dependent on the genetic context of the

cell. The tissue of origin, stage of differentiation and acquired genetic lesions will play a

significant role in determining the net effect of cyclin Dl expression on cell proliferation

and tumorigenesis.

2.6 Acknowledgments

We extend special thanks to R. Weinberg and T. Jacks for RB-I heterozygous

mice. We also thank S. Egan, P. Hamel, M. Kiess, L. Facchini and J. Lear for helpful

discussions and critical reading of the manuscript; E. Harlow and E. Lees for cyclin Dl

antibody; G. Evan for anti-myc antibody; M. Kiess and P. Hamel for murine cyclin Dl

cDNA; and T. Thompson for the pRasImyc 9 retroviral vector. This work was supported

by grants from the Medical Research Council of Canada (LJZP)( BLG), the National

Cancer Institute of Canada with funds from the Canadian Cancer Society (LJZP) and from

the Terry Fox Run (RAP & BLG), and by an Ontario Graduate Studentship Award

(WWM).

Chapter 3

M c

PLATELET-DERIVED GROWTH FACTOR

BETA RECEPTOR EXPRESSION

This chapter is a modified version of the following manuscript: Myc is an essential

negative regulator of platelet-derived growth factor beta receptor expression. 1998.

Wilson W. Marhii, Charlotte Asker, Shaojun Chen, Sara K. Oster, Linda M. Facchini, Patrick A. Dion, Martin Post, Akira Ishisaki, Keiko Funa, John M. Sedivy and Linda Z. Penn. Molecular and Cellular Biologv, submitted.

3.1 Abstract

Platelet derived growth factor BB (PDGF BB) is a potent mitogen for fibroblasts as

well as many other cell types. Interaction of PDGF BB with the PDGF P receptor (PDGF-

PR) activates numerous signaling pathways and leads to a decrease in receptor expression

on the cell surface. PDGF-PR downregulation is effected at two levels, the immediate

internalization of ligandlreceptor complexes and the reduction in pdgf-pr mRNA

expression. Our studies show thatpdgf-pr mRNA suppression is regulated by the c-myc

proto-oncogene. Both constitutive and inducible ectopic Myc protein can suppresspdgf-pr

mRNA and protein. Suppression of pdgf-pr mRNA in response to Myc is specific, as

expression of the related receptor, pdgf u receptor is not affected. We further show that

Myc suppresses pdgf-pr mRNA expression at the transcriptional level by a mechanism

which is distinguishable from Myc autosuppression. Analysis of c-Myc-null fibroblasts

demonstrates that Myc is required for the repression of pdgf-pr mRNA expression in

quiescent fibroblasts following mitogen stimulation. In addition, it is evident that the Myc-

mediated repression ofpdgf-jr mRMA levels plays an important role in the regulation of

basal pd&r expression in proliferating cells. Thus, our studies suggest an essential role

for Myc in a negative feedback loop regulaiing the expression of the PDGF-PR.

3 . 2 Introduction

The PDGF P receptor (PDGF-PR) has been extensively studied in the context of

wound healing and carcinogenesis where many of the biological activities of the PDGF-PR

clearly play a role (reviewed in Bennett and Schultz, 1993; Bomfeldt et al., 1995;

Claesson-Welsh, 1996; Claesson-Wdsh, 1994). The activated PDGF-PR can elicit

diverse and seemingly paradoxical biological activities, including cell growth, cell survival,

differentiation, and cellular transformation. Activation of the PDGF-PR occurs rapidly

following PDGF ligand-receptor interaction. PDGF A and B polypeptides can form

homodimers (AA and BB), or a heterodimer (AB), and bind two distinct PDGF receptors

(PDGF-aR, PDGF-PR) with differing affinity. In fibroblasts, the PDGF-aR is expressed

at low abundance and binds with high affinity to all three PDGF isofonns, whereas the

PDGF-PR is highly abundant and exhibits restricted binding to two isoforms; PDGF BB

with high affinity, and PDGF AB with lower affinity (reviewed in Westermark and Heldin,

1993). Ligand binding results in receptor dimerization which induces autophosphorylation

at multiple tyrosine residues and activates the receptor (Ullrich and Schlessenger, 1990).

The phosphorylated tyrosine residues serve as binding sites for a host of SH2-domain-

containing proteins which include: phospholipase C-yl (PLCy), the GTPase-activating

protein of Ras (GAP), the regulatory subunit of phophatidylinositol-3-kinase (PI3K), the

phosphotyrosine phosphatase Syp, Src-family members (Src, Yes and F p ) , Nck, Shc, as

well as additional uncharacterized proteins (see Claesson-Welsh, 1996). Following

receptor-binding, these proteins are activated through phosphorylation and subsequently

stimulate a number of signal transduction cascades. These signaling pathways include the

activation of MAP kinase and the induction of immediate early growth response genes such

as the c-myc proto-oncogene. The net outcome of receptor signaling is dependent upon the

combination of receptor-associated proteins recruited. For example, PI3K and PLCy are

required for transmission of the mitogenic signal of the PDGF-PR (Valius and Kazlauskas,

1993), but recruitment of GAP to the receptor can inhibit PLCy-mediated mitogenesis

(Valius et al., 1995). The mechanisms used to fine tune receptor-induced signaling and the

full diversity of the signaling potential of the receptor remain unclear.

The induction of Myc protein expression, following mitogen stimulation, is an

essential prerequisite for G1 to S phase progression of the cell cycle in non-transformed

cells (Campisi et al., 1984; Heikkila et al., 1987; Kaczmarek et al., 1985; Kelly et al.,

1983; Prochownik et al., 1988; Roussel et al., 1995; Shichiri et al., 1993). Consistent

with the role of Myc as a strong stimulator of cell proliferation, deregulated Myc

expression is a potentiator of tumorigenesis, and is a common hallnxk of a diverse array

of tumors (reviewed in Garte, 1993; Prins et al., 1993; Spencer and Groudine, 1991). In

addition to the well-established role of Myc as a positive growth regulator, Myc can also

induce the apoptotic death program (Askew et al., 1991; Evan et al., 1992; Shi et al.,

1992). Myc is thought to function as a central regulator of such diverse cellular activities

by regulating gene expression. Myc can function as a transcriptional activator when

heterodiierized with its partner Max, a basic helix-loop-helix leucine zipper protein (Amati

et al., 1992; Blackwell et al., 1993; Blackwell et al., 1990; Blackwood and Eisenman,

1991; Halazonetis and Kandil, 1991; Kretzner et al., 1992; Prendergast et al., 1991).

Transcription activation is dependent upon the binding of the Myc-Max heterodimer to a

canonical DNA sequence CACGTG as well as to specific non-canonical elements. In

addition to its role as a transcriptional activator, studies suggest Myc can also repress gene

transcription. The precise mechanism through which Myc represses gene transcription is

not clear, as only a relatively small number of Myc-repressed target genes have been

identified. Myc can repress d e b p a (Antonson et al., 1995; Li et al., 1994), cyclitt Dl

(Philipp et al., 1994), the adenovincs 5 major-late promoter (AdMLP) (Li et al., 1994), and

c-ntyc (Facchini et al., 1997) promoter activities. The c-myc gene is one of the better

characterized Myc repressed target genes. Myc autosuppression is a homeostatic

regulatory mechanism which results in the repression of endogenous c-myc expression at

the level of transcription initiation. This feedback mechanism functions in a Myc dose-

dependent manner, and requires the interaction of Myc with Max (Facchini et al., 1997;

Grignani et al., 1990; Penn et al., 1990). Evidence to date suggests Myc represses gene

transcription through core regulatory elements and does not require Myc-Max binding sites

(Antonson et al., 1995; Facchini et al., 1997; Li et al., 1994; Lucas et al., 1993; Philipp et

al., 1994; Roy et al., 1993). Identification of additional Myc-regulated target genes is

required to further delineate the molecular mechanisms through which Myc regulates gene

transcription, and help us to better understand how Myc modulates normal cell proliferation

in response to the external environment.

Given the potent mitogenic stimulus initiated by the ligand-activated PDGF-PR, it

is not surprising that the cell possesses negative feedback mechanisms to regulate receptoi.

expression. Proliferation of non-transformed cells following exposure to mitogen is

controlled through the down-regulation of growth factor receptors. Receptor down-

regulation involves the internalization and degradation of receptor-ligand complexes

(Nilsson et al., 1983; Rosenfeld et al., 1985; Sorkin et al., 1991). Additional mechanisms

also play a role in this process, as ligandheceptor interaction also leads to receptor mRNA

suppression, as shown for the CSF-1 (c-fms) receptor (Gliniak and Rohrschneider, 1990),

the c-kit receptor (Gliniak and Rohrschneider, 1990), and the PDGF-PR (Vaziri and Faller,

1995), all of which are members of the PDGF receptor family (Yarden et al., 1987). The

mechanism of receptor mRNA suppression is unclear; however recent studies have

provided clues as to the constituents of this pathway. Ectopic expression of activated Ras

or Src in fibroblasts results inpdgf-/3r mRNA down-regulation (Vaziri and Faller, 1995;

Zhang et al., 1995). As activated Src can target Myc (Barone and Courtneidge, 1995), the

latter may serve as an effector of~dgf-pr mRNA suppression. Further support for this

hypothesis comes from studies of small cell lung carcinoma by Plummer et al. (1993),

which showed an inverse correlation between the expression of Myc and the c-kit receptor.

In this thesis, we show that PDGF-PR expression is down-regulated in non-

transformed fibroblast cells following exposure to either serum, PDGF BB ligand or Myc-

activation. The suppression of PDGF-PR levels is specific as Myc activation has no effect

on pdgf-ffr expression. Myc suppression of pdgf-jr mRNA is effected at the

transcriptional level by a mechanism which appears to be uncoupled from Myc

autosuppression. We demonstrate that Myc is required for the repression of pdgf-pr

expression following serum stimulation. Moreover, we show that Myc repression ofpdgf-

j r mRNA levels plays an important role in regulating basal pdgf-fir expression in

proliferating cells. Our results support a role for Myc in the homeostatic regulation of

pdgf-pr mRNA levels in proliferating fibroblasts.

3 . 3 Materials and Methods

3 .3 .1 Cell culture, and somatic cell hybridizations

Primary rat embryo fibroblasts (REF) were prepared as described previously (Land

et al., 1983). Rat-1; Rat-1 pMV7hc-myc(wt)/ER (wtMycER), and Rat-1 pMV7hc-

myc(Al06-143)lER (AMycER) were previously described (Daksis et al., 1994; Evan et

al., 1992; Pem et al., 1990). The KPREF cell line is a population of spontaneously

immortalized rat embryo fibroblasts (Pem et al., 1990). Rat-1 wtMycERTM, and Rat-1

AMycERRVI cell lines were generated by infecting Rat-1 cells with replication incompetent

retroviruses either Babepuro c-mycERm or Babepuro AI06-143c-mycERm. Cells were

selected in medium supplemented with puromycin, and individual drug resistant colonies

were cloned. CIones were characterized for MycERTM expression, to ascertain that

expression was within physiological levels and that wtMycERm activation will induce

cells to progress from GOIG1 to S phase of the cell cycle. Clones of our variant NIH3T3

cell line, NIH3T3v3 and NIH3T3v10, were derived from and possess identical properties

to the parental population first described by Pem et al. (Pem et al., 1990). Wildtype

TGR-1 (+I+) and c-Myc null H015.19 (-I-) cell lines were previously described (Mateyak

et al., 1997). c-Myc null H015.19 gfp (-1- gfp) and c-Myc null H015.19 gfpmyc (-1-

gfpmyc) cells were generated by infecting c-Myc null H015.19 (-I-) cells with either the

retrovirus BabeMNIRESgfp or BabeMNIRESgfpmyc. Rat-1 wtMycER and Rat-1

AMycER cells were maintained in phenol red minus alpha modified Eagle's medium

((rMEM) supplemented with 10% charcoal treated serum (CTS)(Hyclone). Wildtype Rat-1

clone TGR-1 (+I+), c-Myc null H015.19 (-I-), H015.19 gfp (-1- gfp) and H015.19

gfpmyc (-1- gfpmyc) cells were grown in DMEM H21 supplemented with 10% calf serum,

as described (Prouty et al., 1993). A11 other fibroblast cell lines were cultured in aMEM

supplemented with 10% fetal bovine serum (FBS)(Gibco). Culture media was

supplemented with 100 pg/ml kanamycin and 2 pghl gentamycin or 100 pglml penicillin

and 100 pglml streptomycin sulfate. Somatic cell hybridizations were conducted as

previously described (Facchini et al., 1994; Penn et al., 1990). To analyze the response to

PDGF BB stimulation, subconfluent cells were cultured for three days in 0.3%

FBSIaMEM prior to stimulation with 40 nglml PDGF BB (Upstate Biotechnology

Incorporated). To activate ectopic Myc activity, Rat-1 wtMycER cells were exposed to 100

nM P-estradiol (Sigma) in ethanol, Rat-1 wtMycERTM cells were exposed to 100 nM 4-

Hydroxy (2) Tamoxifen (OH-T) in ethanol (Research Biochemical International, Natick,

MA), while controls were exposed to ethanol alone. To analyze the response to serum

stimulation, subconfluent wildtype TGR-1 (+I+), and c-Myc null H015.19 (-I-) cells were

maintained in 0.25% calf semm1DMEM HZ1 for 2 days, then stimulated with 10% calf

serum/DMEM H21. Unless otherwise stated, all cells were analyzed as subconfluent

proliferating cultures.

3.3.2 Retroviral vectors

The pDORheo, pDoklv-mycneo , pBabehygro , pBabelv-mychygro, pBabeIpuro,

pBabelv-mycpuro, retroviral vectors were constructed as previously described (Facchini et

al., 1994; Morgenstem and Land, 1990; Penn et al., 1990; Ridley et al., 1988). The

pBabepuro c-mycERTM, and pBabepuro A106-143c-mycERTM retroviral vectors were the

kind gift of G. Evan and T. Littlewood and were described previously (Littlewood et al.,

1995). To generate the pBabeMNIRESgfpmyc retroviral vector, human c-myc exon 11, I11

sequences excised from pBluescript KS+ Hc-myc were cloned into EcoRI, MtoI digested

pBabeMNIRESgfp (a kind gift of G. Nolan).

3.3 .3 Retroviral infection

To produce infectious replication-defective ecotropic retroviral particles,

recombinant retrovital constructs were transfected, using calcium phosphate precipitation

(Graham and Van der Eb, 1973), into either the GP+E packaging cell line (Markowitz et

al., 1988), or the Phoenix-Eco cell line (ATCC), and selected in 0.5 mg/ml G418 sulphate

(Sigma), 2 mglml puromycin (Sigma) or 300 pg/ml hygromycin B (Sigma). After drug

selection, drug resistant clones were pooled and expanded for virus production. Primary

rodent embryo fibroblasts and cell lines were infected with retrovirus and selected in either

G418, hygromycin B or puromycin. Individual drug-resistant colonies were either isolated

to produce clonal populations or combined to generate pooled populations. Green

fluorescent protein (gfp) positive c-Myc null H015.19 gfp (-1- gfp) and H015.19 gfpmyc

(-I- gfpmyc) cells were isolated with a Coulter 753 cell sorter using an absorption

wavelength of 488 nrn, and pooled.

3.3.4 RNase protection

RNA was prepared by the guanidinium isothiocyanate method of Chirgwin et al.

(Chirgwin eta]., 1979). RNaseprotection was conducted essentially as described (Penn et

al., 1990). The probes were generated using T3 RNA polymerase (Stratagene) from

linearized Bluescript KS and SK cloning vectors (Stratagene) containing the following

DNA fragments: rat c-myc exon I (Pem et al., 1990); thegag gene derived from the avian

myelocytomatosis virus MC29 (Penn et al., 1990); rat (Penn et al., 1990), and the murine

glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene (Facchini et al., 1994). The

mousepdgf-pr probe comprised nucleotides 1 to 227 of the mouse pdgf--pv cDNA. This

cDNA fragment was excised from h-N16 (kind gift of Y. Yarden) with EcoRI, and SmaI,

then cloned into a Bluescript KS vector (Stratagene). The Bluescript KS mousepdgf-pr

cDNA plasmid was linearized with BamHI, and thepdgf-fir RNase protection probe was

transcribed using T3 RNA polymerase (Stratagene). The aminoglycoside

phosphotransferasefileo) probe comprised nucleotides 1 to 245 of the neomycin resistance

gene. These sequences were excised from pBabeneo (Morgenstern and Land, 1990) with

Hind11 and ClaI, and cloned into a EcoRV and CIaI digested pBluescript KS+ vector

(Stratagene). The Bluescript KS+ aminoglycoside phosphotransferase plasmid was

linearized with NcoI, and the RNase protection probe transcribed with T3 RNA

polymerase (Stratagene). The protected probes were resolved by electrophoresis on 6%

denaturing polyacrylamide gels and visualized using a phosphoimager or by

autoradiography on X-OMAT film (Kodak). Densitometry was performed by

phosphoimager analysis.

3.3 .5 Northern blot Analysis

RNA was prepared by the yanidinium isothiocyanate method of Clrirgwin et al.

(Chirgwin et al., 1979). Northern Blot analysis was conducted as described previously

(Daksis et al., 1994). The ratpdgf-ar probe consisted of a 510 bp EcoRV fragment

derived from pCRlIPDGFaR (Souza et al., 1995). The probe for 36B4, a gene encoding

an acidic ribosomal protein, consisted of a 800 bp Pstl fragment derived from p36B4

(Masiakowski et al., 1982). Densitometry was performed by phosphoimager analysis.

3.3.6 PDGF BB binding studies

Subconfluent rodent fibroblasts were washed twice with ice-cold Phosphate

Buffered Saline containing 1% (w:v) bovine serum albumin (l%BSA/PBS). To determine

high-affinity saturable [ ~ ~ ~ I I P D G F BB binding, cells were incubated at 4OC in 500 yl of

l%BSAIPBS with increasing concentrations of [ ~ ~ ~ I I P D G F BB in the absence (total

binding) or presence (non-specific binding) of an 100-fold excess unlabelled PDGF BB.

After 2 hrs of incubation, the medium was aspirated and the cells were washed 5 times

with 1 ml ice-cold l%BSA/PBS. The remaining cells were extracted for 1 hour by adding

lml/well lysing buffer (1% (v:v) Triton X-100, 10 mM glycine and 20mM HEPES, pH

7.4), and total radioactivity determined by gamma spectrophotometry.

3.3.7 Luciferase assays

The mousepdgf-pr promoter luciferase constructs have been previously described

(Ballagi et al., 1995). Each plate was transfected with 10 pg of each of the pdgf-pr

promoter luciferase constructs, 0.1 pg of a vector carrying the P-galactosidase gene

(pCMVpgal), and 10 pg of pBluescript KS+ plasmid (Stratagene), using the calcium

phosphate method. Luciferase and P-galactosidase assays were performed as previously

described (Facchini et al., 1997).

3.4 RESULTS

3.4.1 PDGF P receptor mRNA expression is suppressed following

serum or PDGF BB stimulation of Rat-1 cells

PDGF BB is one of the primary growth factors for fibroblasts. Yet little is known

of the pattern or regulatory mechanisms which negatively govern the mRNA expression of

its key receptor, PDGF-PR, following ligandlreceptor interaction in normal and

transformed cells. Earlier reports have implicated Src and Ras as negative regulators of

pdgf-fir expression (Vaziri and Faller, 1995). As Myc has been placed downstream of src

in the PDGF BB signaling pathway (Barone and Courtneidge, 1995), we explored the

relationship between c-myc and pdgf-br expression following serum-stimulation of

quiescent cells. Subconfluent Rat-1 cells were incubated in low fetal bovine serum (0.3%

FBSlaMEM) for 3 days to reduce the levels of PDGF in the medium, and subsequently

exposed to either fresh 10% FBSIcdvlEM or PDGF BB growth factor. RNA was extracted

at the times indicated and analyzed by RNase protection to detect endogenous c-myc,pdgf-

pr and gapdh-specific transcripts. As expected (Campisi et al., 1984; Kelly et al., 1983),

exposure of serum-deprived Rat-1 cells to 10% serum induced a rapid and transient

increase of endogenous c-myc mRNA expression (Figure 3.1A). C-myc mRNA

expression peaked at approximately 2 h, then gradually decreased reaching steady-state

levels 12 to 18 h post stimulation. Exposure to PDGF BB alone also induced c-myc

mRNA expression, however, compared to serum stimulation, c-myc mRNA expression

was not sustained, as levels returned to basal approximately 4 h post-stimulation (Figure

3.1B). Basal pdgf-pr RNA is readily detectable in cells cultured under low serum

conditions, and is down-regulated approximately 3 to 4 h after exposure of cells to serum

or PDGF BB alone (Figure 3.1A, 3.1B). The sequential induction of Myc expression

Serum I I 0 1 2 3 6 121824 h

c-myc

pdgf-Pr

PDGF BB

Figure 3.1. Expression of c-myc andpdgf-pr mRNAs in quiescent Rat-1 cells stimulated with serum o r PDGF BB. Rat-1 cells were maintained in low serum 0.3% FBSIaMEM for 3 days then treated with either (A) 10% FBSIaMEM or (B) 40ndml PDGF BB. At the indicated intervals RNA was extracted and analyzed by RNase protection using single stranded probes complementary to endogenous c-nzyc, pdgf- p,; and gapdlz-specific sequences.

followed by the suppression ofpdgflpr RNA suggested Myc may play a role in initiating

receptor repression following growth factor stimulation.

3.4.2 PDGF P receptor RNA is down-regulated in Myc-activated non-

transformed cells

To determine whether constitutive ectopic Myc expression can regulate the

expression ofpdgf-br mRNA, primary rat embryo fibroblasts (REFS) and the immortal,

non-transformed cell lines, KPREF and Rat-1, were infected with control replication-

incompetent retrovims (DORIneo) carrying only the neomycin resistance gene, or with

retrovirus (Doklv-mycneo) canying the v-myc gene in addition to the neomycin resistance

gene. RNA from drug resistant, subconfluent proliferating cultures was analyzed by

RNase protection assay (Figure 3.2A). Pdgf-pr mRNA expression was readily visible in

control cells and clearly suppressed in the cells expressing ectopic Myc (Figure 3.2A;

compare lanes 1,3,5 with 2,4,6, respectively). As a positive control for exogenous Myc

activity, ectopic v-myc and endogenous c-myc mRNA expression were assayed. Our

results demonstrate ectopic v-myc protein was also expressed and functionally active as

endogenous c-myc mRNA levels are suppressed in v-Myc expressing cells, due to the Myc

negative feedback mechanism as previously reported (Figure 3.2B; compare lanes 1 ,3 ,5

with 2, 4, 6, respectively) (Cleveland et al., 1988; Grignani et al., 1990; Penn et al.,

1990). These results show that enforced Myc expression down-regulates pdgf-pr

expression in primary and non-transformed cells.

REF KPREF Rat-1 I nn

v-mnyc - + - + - +

REF JXPREF Rat-1 n

v-mnyc - + - +I I - +(

Figure 3.2. Suppression of endogenous c-myc and pdgf-pr mRNA levels in rodent cells constitutively expressing exogenous Myc. RNA (10 pg) fiom subconfluent rodent fibroblasts infected with a control retrovirus (-), or retrovirus carrying the v-myc gene (+) was analyzed by RNase protection assay to detect (A) endogenous pdgf-pr and control gapdh, (B) endogenous c- myc, ectopic v-myc, and control gapdh expression.

3.4.3 The suppression of PDGF P receptor mRNA levels following

Myc-activation occurs with rapid kinetics

To examine the kinetics ofpdgf-pr mRNA suppression we first employed an

estrogen-inducible MycER system in Rat-1 cells (Rat-1 wtMycER). These Rat-1 cells

constitutively express an inactive fusion protein consisting of the human c-Myc protein

fbsed to the estrogen binding domain of the estrogen receptor. This MycER fusion protein

is rapidly activated in response to P-estradiol or the P-estradiol antagonist 4-hydroxy-

tamoxifen (OH-T), allowing Myc activity to develop within minutes of ligand exposure

(Daksis et al., 1994; Eilers et al., 1989; Evan et al., 1992; Selvakumaran et al., 1993). As

a control, we analyzed the Rat-1 AMycER cell line, which expresses a AMycER fusion

protein containing a deletion of amino acids 106 to 143 within the Myc protein, rendering

the fusion protein inactive for all known biological activities of Myc (reviewed in Penn et

al., 1990). To determine the kinetics ofpdgf-jr suppression Rat-1 wtMycER and Rat-1

AMycER cell lines were analyzed during different states of cellular proliferation (Figure

3.3A). Subconfluent, proliferating Rat-1 wtMycER and Rat AMycER cells were exposed

to P-estradiol for the times indicated. In addition, confluent quiescent serum-deprived Rat-

1 wtMycER cells were either exposed to ethanol as a control or treated with P-estradiol

dissolved in ethanol, to induce Myc activity. Suppression of endogenouspdgf-jr mRNA

is clearly detectable in both subconfluent and confluent Rat-1 wtMycER cells at 6 h

following induction of Myc activity (Figure 3.3A. compare lanes 5 and 6; lanes 17 and

18). Thepdgf-jr mRNA remained suppressed in the P-estradiol-treated Rat-1 wtMycER

cells over the course of the experiment. Pdgf-pr expression is elevated in confluent

quiescent cells in comparison to subconfluent proliferating cells. Yet, despite the

difference in their basal level of RNA expression, the kinetics and degree of suppression

were similar in both subconfluent and confluent cultures. Suppression of the receptor was

not evident in control ethanol treated Rat-1 wtMycER cells (Figure 3.3A. lanes 13, 15,17,

19 & 21), and in Rat-1 AMycER cells which were or were not exposed to J3-estradiol

(Figure 3.3A. lanes 23 and 24). Receptor expression also remained invariant in

subconfluent Rat-1 AMycER cells in response to P-estradiol (Figure 3.3A. lanes 1,3, 5,

7, 9, 1 I) as apparent differences are due to lane loading variability. Thus our results

demonstrate that pdgf-pr mRNA expression is suppressed upon Myc induction in both

quiescent and proliferating cells with similar kinetics. To eliminate the possibility that the

intrinsic transactivation domain present in the estrogen receptor portion of the wtMycER

chimaera (ERTAD) could contribute to the suppression in pdgf-pr mRNA, we next

employed Rat-1 cells expressing a novel wtMycERm construct in which the ERTAD has

been deleted (Rat-1 wtMycERTM) (Littlewood et al., 1995). Confluent, quiescent Rat-1

wtMycERTM cells stably expressing the inactive wtMycERm protein were treated with

OH-T for the times indicated to induce Myc activity. The kinetics and overall level of

suppression observed with the wtMycERm construct are comparable to that observed in

Rat-1 wtMycER cells (compare Figure 3.3A, lanes 13 to 22 and Figure 3.3B). Hence, the

ERTAD in the wtMycER construct did not contribute significantly to Myc suppression of

endogenous pdgf-pr mRNA levels. As a positive control for exogenous wtMycERTM

activity, endogenous c-myc mRNA expression was assayed and showed the expected

suppression due to the Myc negative feedback mechanism as was previously described

(data not shown) (Facchini et al., 1997). Myc suppression ofpdgf-prmRNA levels is

specific, as induction of Myc activity has no effect on the mRNA expression of the related

growth factor receptor, Platelet-Derived Growth Factor a receptor (pdgf-ar) (Figure

3.3C). The suppression of endogenouspdgf-pr mRNA levels is not the result of a Myc-

induced autocrine negative feedback pathway, as MycER activation had no effect on the

expression of pdgf-B mRNA (data not shown). Therefore, the suppression ofpdgf-pr

mRNA by Myc represents a specific regulatory mechanism.

Figure 3.3. Pdgf-pr mRNA expression is suppressed in response to Myc

induction. (A)pdgf-jr mRNA expression in Rat-1 wtMycER (wt) and Rat-1 AMycER

(A) cells was analyzed under both subconfluent and confluent conditions. Cell lines were

grown either as asynchronous subconfluent populations and treated with P-estradiol (lanes

1-12) or as confluent quiescent cultures which were treated with either ethanol as control (-

) or with P-estradiol (+) (lanes 13-24). At the indicated intervals after P-estradiol

treatment RNA was extracted and total RNA (10 yg) was analyzed by RNase Protection to

detect the expression of endogenous pdgf-pr, and control gapdh genes. (B) Confluent

Rat-1 wtMycERm cells were maintained in low serum 0.1% FBSIaMEM for 2 days and

then exposed to 100 nM OH-T to induce exogenous MycERm activity. At the indicated

intervals after OH-T treatment RNA was extracted and 10 pg total RNA analyzed by

RNase Protection assay using single stranded probes complementary to endogenouspdgf-

pr, and gapdh-specific sequences. The plotted data indicates thepdgf-jr signal normalized

to gapdh, as determined by densitometry. (C) Northern blot analysis ofpdd-ar , and

36B4 expression in OH-T stimulated Rat-1 wtMycERTM cells. The constitutively

expressed 3684 mRNA codes for an acidic ribosomal protein, and serves as a measure of

rhe amount of RNA present in each lane. Confluent quiescent Rat-1 wtMycERTM cells

were exposed to 100 nM OH-T to induce exogenous MycERTM activity. At the indicated

intervals after OH-T treatment RNA was extracted and 10 yg total RNA analyzed by

Northern blot using gene-specific probes for endogenous pdgf-cr,, and 3684 specific

sequences. The plotted data is thepdgf-m- signal as normalized to 3684, as determined by

densitometry.

Subconfluent cells Confluent cells I Rat-1 Rat-l Rat-l Rat-l 1 I

AMycER Or wtMycER wtMycER AMycER I i I i n

0 3 6 9 1 2 2 4 1 3 6 9 2 4 2 4 h

m n n n n n n n n n n n A w t A w t A w t A w t A w t A w t - + - + - + - + - + - + - . - -

pdgf-Pr g b* wxl u c 4 &she 9 s. @ .Z . - ' . A -

. . . .

gffpdh

3.4.4 Myc-induced repression of cell surface PDGF P receptors

To determine whether Myc suppression ofpdd-pr mFWA affected receptor protein

expression, the number of PDGF-PRs expressed at the cell surface before and after ectopic

Myc-induction was measured in direct ligand binding assays. Specifically, subconfluent

Rat-1 wtMycER cells were exposed to ethanol as a control or to P-cstradiol in ethanol for

24 h and then incubated with increasing concentrations of [1251]-PDGF BB. Saturable

binding was observed for both control and P-estradiol treated cells at 12 nglml of [125~]-

PDGF BB. The binding capacity of control Rat-1 wtMycER cells was 5.0 h o l of [1251]-

PDGF BB per 3x105 cells (Figre 3.4, closed circles), whereas 24 h following MycER

activation Rat-1 wtMycER cells bound only 2.4 h o l of [1251]-PDGF BB per 3 x 1 0 ~ cells

at saturation (Figure 3.4, open triangles). Scatchard analysis revealed that the estimated

KD of binding for the PDGF ligand was 1.2 X 10-12M, and the estimated number of

binding sites was 12 X 103 for control cells. The specific binding affinity was not

significantly altered in response to Myc-activation, suggesting that our results reflect a

decrease in the number of receptors at the cell surface. Indeed, while the estimated KD of

binding for ligand in P-estradiol treated cells was 0.9 X 10-'ZM, the number of binding

sites was reduced approximately two-fold to 5 X 103. The results obtained from the

PDGF BB binding assays correlates with the decrease in pdgf-pr mRNA expression as

shown by RNase protection analysis. Thus, induced Myc expression leads to

approximately a 50% reduction in cell surface PDGF-PRs compared to control cells.

3.4.5 The mechanism of PDGF fi receptor suppression differs from Myc

autosuppression

A number of similarities exist between the mechanisms employed by Myc to

repress both the endogenous pdgf-pr as well as c-myc mRNA expression. First,

constitutive Myc expression resulted in the suppression of both endogenous pdgf-fir as

well as c-ntyc mRNA levels (Figure 3.2A and 3.2B) (Penn et al., 1990; Pem et al., 1990).

Second, the suppression of both endogenous c-myc (Facchini et al., 1997) andpdgf-pr

(Figure 3.3A and 3.3B) mRNA levels in response to Myc activation occurs with similar

kinetics, given that the half-life of c-myc mRNA is approximately 20 mins while the half-

life of pdgf-fir mRNA is 4 to 6 h. Taken together, these results suggested that the

repression of c-myc andpdgf-pr mRNA by Myc may be functioning through a common

pathway. To test this hypothesis, we employed a variant mouse NIH3T3v3 cell line. In

contrast to other NIH3T3 cells (K.F., unpublished data) and Rat-1 cells (Figure 3.2B),

expression of ectopic Myc in this variant NIH3T3v3 cell line, did not result in the

suppression of either c-myc orpdgf-fir mRNAs (Figure 3.5, lanes 1 and 2). Stable

intraspecies somatic cell hybrids were generated by fusion of the Rat-1 cell line with the

mouse NIH3T3v3 cell line. By this approach we can deterrn'w whether the trans-acting

factors in Rat-1 cells can complement the dysfunction in the mouse NIH3T3v3 cells for

Myc suppression of pdgf-fir mRNA, as we have previously shown for Myc

autosuppression (Penn et al., 1990).

To establish basal levels of endogenous mousepdgf-fir and c-nzyc mRNAs in the

control somatic cell hybrids, Rat-1 cells were fused with NIH3T3v3 cells in the absence of

ectopic Myc expression. Following drug selection, stable somatic cell hybrids were cloned

and assayed by RNase protection for the expression of mousepdgf-fir and c-ntyc mRNAs

(Figure 3.5, lanes 3-5). To distinguish whether the Myc suppression of endogenous c-

myc andpdgf-pr mRNAs is mediated by the same or different pathways, Rat-1 cells

expressing ectopic Myc were fused to Myc-activated mouse NIH3T3v3 cells. The

resultant stable hybrids were similarly analyzed (Figure 3.5, lanes 6-8). As previously

shown, endogenous mouse c-myc mRNA is readily detectable in the control hybrids, and

is suppressed in somatic cell hybrids expressing ectopic Myc (Figure 3.5; compare lanes 3-

5 to 6-8). In contrast, expression of pdgfpr mRNA in control and Myc-expressing

somatic cell hybrids is identical (Figure 3.5; compare lanes 3-5 with 6-8). Thus, trans-

acting components of Rat-1 cells were able to complement the Myc autosuppression, but

not thepdgf-pr repression mechanism in NIH3T3v3 cells. Identical results were obtained

in similar somatic cell hyhrids derived from another clone, NIH3T3v10, as well as the

original parental variant NIH3T3 cell line (Penn et al., 1990) (data not shown). Therefore,

the repression of mouse c-myc andpdgf-pr mRNAs in response to Myc appear to be

mediated by different pathways.

NIH3T3v3 Rat-lNH3T3v3 HYBRIDS - I I v-mvc - + - - - + + +

Figure 3.5. The suppression of endogenous pdgf$r and c-myc mRNA levels by Myc appear to be mediated by different pathways. Total RNA (10 pg) was analyzed by RNase protection to detect the expression of endogenous mouse c-myc, pdgf-pr and gapdh genes. The cells analyzed include: control NIH3T3v3 (lane l), NIH3T3v3 v-myc (lane 2), as well as three random, independent clones of the stable somatic cell hybrids, generated from the intraspecific fusion of Rat-1 x NIH3T3v3 cells (lanes 3-5), and Rat- 1 v-myc x NIH3T3v3 v-myc (lanes 6-8).

3.4.6 Myc represses transcription from the pdgf-fir promoter

To determine whether the pdgf-Pr promoter is responsive to Myc activity, we

performed in vitro transient-transfection-transcription assays. Subconfluent Rat-l

wtMycERTM and control Rat-l AMycERm cells were transfected with a 1.6 Kb, Sac1

reporter plasmid containing sequences between nucleotides -1994 and -396, relative to the

translation start site in the mousepdgf-pr gene (Figure 3.6A). To control for transfection

efficiency a vector canying the P-galactosidase gene, was co-transfected with the promoter

luciferase construct. Cells were treated with OH-T to induce ectopic Myc activity, and at

the indicated time intervals, whole cell lysates were prepared and then assayed for both

luciferase and P-galactosidase activity. Induction of Myc activity, in Rat-l wtMycERTM

cells, resulted in a clear and reproducible suppression of luciferase activity (Figure 3.6B),

reflecting the suppression of transcription from the mousepdgf-pr promoter by Myc. The

loss in luciferase activity is first evident approximately 6 h following Myc-activation. This

suppression is due solely to the activity of Myc as OH-T treated Rat-1 AMycERTM cells

exhibited no change in luciferase activity (Figure 3.6B). As the half-lives ofpdgj'+

mRNA and luciferase are both approximately 4 to 6 hours (Thompson et al., 1991; Vaziri

and Faller, 1995), the level and kinetics of repression as measured in the pdgf-pr

promoter-luciferase assay (Figure 3.68) closely mimicked the suppression of endogenous

pdgf-Pr mRNA in response to MycERm activation (Figure 3.3B). The repression of the

pdgf-jr promoter by Myc is specific, as induction of Myc activity had no significant effect

on transcription from the CMV promoter, as P-galactosidase activity remained invariant

(Figure 3.6C). Thus, Myc expression results in a suppression of transcription from the

pdgf-Pr promoter, and this suppression activity requires the sequences between nucleotides

-1994 and -396, relative to the translation start site of the mousepdgf-jr promoter.

Figure 3.6. Pdgf-fir mRNA transcription is suppressed by Myc. (A)

Schematic diagram of the genomic structure of the mousepdgf-pr promoter, showing

putative transcription factor binding sites. Numbers indicate position relative to the

translational start site (+l). Rat-1 AMycERTM and Rat-1 wtMycERTM cells were

transfected with a 1.6 Kb mousepdgfpr promoter luciferase construct, containingpdgf-pr

sequences fiom nucleotides -1994 to -396. To assay transfection efficiency, cells were co-

transfected with a plasmid carrying the p-galactosidase gene under the control of the CMV

promoter. Cell lines were subsequently treated with 100 nM OH-T for the times indicated

to induce ectopic MycERm activity. Whole cell lysates were prepared and analyzed for

luciferase and p-galactosidase activity. (B) Histograms represent the measured luciferase

activity. Data was normalized to 0-galactosidase activity to adjust for minor differences in

transfection efficiency amongst the samples. (C) Histograms represent the p-galactosidase

activity. Data are a representative out of 3 repeated experiments. Error bars denote

standard deviations.

Mouse pdg@r promoter

NFI SRF NFI

NF-Y APZ

GATA-I AP2 -1

0 1 3 6 9 1 2 1 8 2 4 TIME (h)

TIME (h)

0 1 3 6 9 1 2 1 8 2 4 TIME (h)

3.4.7 Characterization of the mouse pdgf-pr promoter.

The 1.6 Kb Sac I fragment of the mousepdgf-pr promoter contains consensus

sequences for a variety of known transcription factors including: GATA-1, SRF, NFl,

AP2, APl, and NF-Y. To determine which sequences within this fragment are responsible

for the repression of pdgf-pr mRNA transcription by Myc, we constructed a series of

deletion mutants within the Sac I mousepdgf-jr promoter luciferase construct (Figure

3.7). Thesepdgf-pr promoter constructs were transfected into Rat-1 wtMycERTM cells

and assayed for the responsiveness of the promoter to activation of Myc activity. Upon

activation of ectopic Myc activity in Rat-1 wtMycERTM cells, all of thepdgf-pr promoter

constructs, displayed a 2 to 3 fold reduction in luciferase activity, relative to the basal

luciferase activity exhibited by each promoter construct. Deletion of 558 bp at the 5' end of

the Sac I fragment, reduced basal luciferase activity to approximately 30% of that observed

with the full length Sac I fragment, suggesting the presence of a positive regulatory element

in this region. An additional deletion of 361 bp, further reduces basal luciferase activity to

about 17% of the full length Sac I fragment. Subsequent deletions between -975 and -745

do not impact upon basal transcription, however, deletion of sequences between -745 and

-430 results in a loss of basal transcription. Moreover, this region of the mouse pdgf-jr

promoter contains sequences which are responsive to Myc suppression.

NFI SRF NFI

NF-Y AP2

RELATIVE LUCIFERASE ACTIVITY

Figure 3.7. Deletion analysis of the mousepdsf-pr promoter. Constructs contain portions of the mousepdgf-j?rpromoter as indicated driving the transcription of a luciferase reporter gene. Positions indicated are relative to the translation start site. Rat-l w t ~ y c ~ ~ ~ cells were transfected with 10 ug of each promoter reporter construct. To measure transfection efficiency, cells were co-transfected with a plasmid carrying the P- galactosidase gene under the control of a CMV promoter. Cells lines were subsequently treated with 100 nM 4-Hydroxy-Tamoxifen in ethanol or ethanol alone as a control for 24 h. Histograms represent the measured luciferase activity, normalized to P-galactosidase activity, relative to the luciferase activity from the full length 1.6 Kb mousepdgf--P,promoter in untreated controls.

3.4.8 Myc is required for the suppression in pdgf-jr mRNA levels

following mitogen stimulation.

To examine the role of Myc in the repression ofpdgf-jr mRNA levels following

mitogen stimulation, we analyzedpdgf-jr mRNA expression in serum-deprived c-Myc-

null H015.19 (-I-) rodent fibroblasts upon stimulation with 10% serum. H015.19 (-I-)

cells, derivatives of parental Rat-1 fibroblasts, lack c-Myc expression due to targeted

disruption of both alleles of the c-myc gene with aminoglycoside transferase(neo) and

histidine-marked targeting vectors (Mateyak et al., 1997). Transcription of the disrupted c-

myc genes produces hybrid truncated transcripts consisting of c-myc exon I sequences

fused to the coding sequences of either the neo or histidine genes. c-Myc null (-I-) rodent

cells were cultured in 0.25% calf senun/DMEM H21 for two days then stimulated with

10% serum. RNA was extracted at the indicated time intervals and analyzed for

endogenous pdgf-jr and gapdh expression via RNase protection analysis. As shown in

Figure 3.1A, wildtype Rat-l (+I+) fibroblasts exhibit a gradual reduction in pdgf-fir

mRNA levels upon mitogen stimulation of serum-deprived cultures. In contrast, serum

stimulation of c-Myc null (-I-) fibroblasts failed to elicit a reduction in endogenouspdgf-jr

mRNA levels (Figure 3.8A). Indeed, pdgf-fir mRNA expression was invariant despite

prolonged exposure to serum for 48 h. Thus, Myc is essential for the repression ofpdgf-

j r mRNA levels in serum-deprived cells following serum stimulation.

Myc also plays a role in the regdation of basal pdgf-jr mRNA levels in

subconfluent, proliferating cells. Analysis of endogenous pdgf-jr mRNA levels in

asynchronously proliferating cultures of wildtype (+I+) and c-Myc null (-I-) fibroblasts

revealed that pdgf-pr mRNA levels in c-Myc null cells are elevated in comparison to

wildtype fibroblasts (Figure 3.8B, compare lane 2 to lane 1). Thus, in subconfluent,

proliferating cultures, Myc represses basal pdgf-jr mRNA expression. In addition to the

loss ofpdgf-jr mRNA repression due to the absence of endogenous Myc activity, there is

an abrogation of the c-myc autosuppression mechanism, resulting in an elevation in

expression of the disrupted c-myc allele as detected with a c-myc exon I specific probe

(Figure 3.8B, compare lane 2 to lane 1). The endogenous pdgf-pr and c-myc genes

remain responsive to Myc as repression of both genes can be rescued through the

reconstitution of c-Myc expression in H015.19 (-I-) cells. c-Myc null H015.19 gfpmyc (-

I- gfpmyc) cells constitutively expressing both ectopic human c-Myc, and green fluorescent

protein as a selectable marker, elicits a clear repression ofpd&3r mRNA levels compared

to the parental c-Myc null (4) cells as well as the control cell line H015.19 gfp (-1- gfp),

which constitutively expresses only the green fluorescent protein (Figure 3.8B, compare

lane 4 to lanes 2 and 3). In addition, there is also a rescue of the Myc autosuppression

mechanism, as detected by probes to either c-myc exon I or the neo sequences in the hybrid

transcripts from one of the disrupted c-myc alleles (Figure 3.8B, compare lane 4 to lanes 2

and 3). Analysis of human c-myc mRNA expression clearly demonstrated that ectopic

human c-myc is expressed in the appropriate cell populations (Figure 3.8C). Thus, studies

in c-Myc null fibroblasts have demonstrated that Myc is required for the suppression of

pdgf-/3r mRNA levels in serum-starved rodent fibroblasts following serum stimulation.

Moreover, c-Myc plays a role in maintaining basalpdgf-/3r mRNA levels by repressing

pdgf-/3r mRNA expression in asynchronously, proliferating cells.

Figure 3.8. Myc is required for the repression of pdgJ-jr mRNA levels in

both serum stimulated quiescent cells and in proliferating cultures. (A) Rat-1

cells, lacking c-Myc expression (-I-), were maintained in low serum 0.25% calf

serumJDMEM H21 for 2 days then treated with 10% calf semm/DMEM H21. At the

indicated intervals, RNA was extracted and analyzed by RNase protection using single

stranded probes complementary to endogenous pdgf-jr, and gapdh-specific sequences.

RNA(I0 yg), extracted from subconfluent cultures of wildtype rodent fibroblasts (+I+),

rodent fibroblasts lacking endogenous Myc expression (-I-), and c-Myc null fibroblasts

which have been stably infected with either a control retrovirus expressing gfp (-1- gfp) or a

retrovirus containing human c-myc exon 11,111 cDNA as well as gfp (-1- gfpmyc) was

analyzed by RNase protection for (B) endogenous pdgf-pr, c-myc, aminoglycoside

phosphotransferase (rteo) and gapdh (C) ectopic human c-myc, and endogenous gapdlt-

specific sequences. Similar results were obtained in three separate experiments.

3.5 Discussion

We show that the physiological down-regulation of cell surface PDGF-PRs

following mitogen stimulation involves the product of the c-myc proto-oncogene and

occurs at the RNA level. Constitutive or induced expression of Myc results inpdgf-pr

mRNA down-regulation in non-transformed cells by a mechanism which appears to be

distinct from that of the Myc negative feedback mechanism. The repression ofpdgf-Pr

levels by Myc occurs at the transcriptional level. This Myc-mediated repression ofpdgf-Pr

mRNA levels affects basal expression in subconfluent, proliferating rodent fibroblasts, as

well as mitogen-regulated expression. Taken together, our results show Myc is integral to

the regulation ofpdgf-pr expression.

The molecular mechanism ofpdgf-Pr RNA suppression in response to Myc protein

was explored using the inducible MycER system in Rat-1 cells. We show Myc induction

specifically down-regulates pdgf-pr RNA and does not affect RNA expression of the

related growth factor receptor, thepdgfa receptor. This effect is evident in both growth

arrested confluent, and asynchronous proliferating cells, suggesting receptor down-

regulation occurs in response to Myc per se and is not a consequence of the cellular

transition from GOlGl to S phase of the cell cycle. Ectopic Myc expression does not

induce the expression of PDGF B mRNA thus it is unlikely that Myc is suppressing

receptor expression via a ligand-induced autocrine feedback mechanism. Receptor

suppression is detectable within 3 to 6 h of either growth-factor stimulation or Myc-

induction. As the half-life ofpdgf-Pr RNA is 4 to 6 h, this is the earliest point at which

down-regulation of receptor RNA can be visualized (Vaziri and Faller, 1995). Indeed,

given the long half-life of the~dgf-Pr RNA, the repression of thepdgf-pr RNA following

induction of ectopic Myc activity is rapid, suggesting that Myc may exert a direct effect on

thepdgf-jr promoter.

The decrease in pdgf-pr mRNA expression in response to Myc activation is

mediated at the level of gene transcription. We demonstrate that Myc suppresses

transcription from a Sac I fragment of the mouse pdgf-pr promoter, consisting of

sequences between -1994 and -396, relative to the translational start site. Analysis of the

few Myc-repressed genes identified to date, suggests that Myc can repress gene

transcription through core regulatory elements such as initiator elements or TATA-boxes

(Desbarats et al., 1996; Facchini et al., 1997; Li et al., 1994; Philipp et al., 1994; Roy et

al., 1993). Myc can directly bind to components of the basal transcription complex such as

TFII-I and TBP, inhibiting the activity of these transcriptional initiators. However, the

upstream regulatoly region of the mousepdgf-pr gene is relatively simple, lacking both of

these elements. Yet, there are several putative binding sites for transcription factors such

as GATA-I, SRF, AP1, NF1, AP2, and NF-Y. Deletion analysis of the 1.6 Kb Sac I

fragment of the mousepdgf-pr promoter revealed that multiple elements found between

-1994 and -975 contribute to full transcriptional activity. Moreover, there exist sequences

within -745 to -396 which are required for the maintainence of basal transcription. This

region also contains elements which are responsive to Myc repression. Interestingly, this

fragment contains sequences which are binding sites for three well characterized

transcription factors: AP1, AP2, and NF-Y. It is possible that one or all of these sites are

required to mediate the repression ofpdgf-jr transcription by Myc. The NF-Y site is of

particular interest as mutational analysis of the mousepdgf-pr promoter by Ishisaki el al.,

has demonstrated that the NF-Y binding site is singularly crucial to maintaining basal

mRNA transcription (Ishisaki et al., 1997). 111 vivo studies have also demonstrated that

Myc can regulate the activity of the members of this CTF/NF-1 family of transcriptional

activators. Myc represses transcription of thepro-alpha 2 (1) collagen gene via an indirect

mechanism involving phosphorylation of NFI which inhibits its potential to activate

transcription (Yang et al., 1991; Yang et al., 1993). Hence this site is a likely candidate

through which Myc represses basal transcription of thepdgf-pr gene. The nature of this

Myc-mediated suppression of transcription of thepdgf-br gene, the DNA binding proteins

involved, and the DNA sequences required are currently under investigation.

Many characteristics of Myc-suppression of pdgf-pr are similar to features

associated with the Myc negative autoregulation mechanism (Cleveland et al., 1988;

Facchini et al., 1997; Grignani et al., 1990; Penn et al., 1990; Penn et al., 1990). Myc-

suppression ofpdgf-br and c-myc RNA expression are both evident in primary REFS and

established non-transformed cell lines. The critical Myc-box I1 domain of Myc which is

required for negative autoregulation and for all known biological activities of Myc is

mandatory forpdgf-Dr suppression. Both Myc autosuppression and Myc-suppression of

thepdgf-br occurs at the level of gene transcription. Interestingly, constitutive expression

of Myc in a variant NIH3T3v3 cell line does not lead to the suppression of either c-myc or

pdgf-pr RNA expression. Using a somatic cell hybridization approach with mouse

NIH3T3v3 and Rat-1 cell lines showed that rat cell factors restored suppression of the

mouse c-myc gene (Penn et al., 1990), however, the mousepdgf-br gene was unaffected

in the hybrid cellular background. These results suggest that the regulatory mechanisms

through which Myc suppresses the endogenous c-myc and thepdgf-br genes are distinct,

and further suggests that Myc repression of gene transcription may occur by multiple

mechanisms.

Interestingly, introduction of transforming ras or src oncogenes can lead to reduced

pdgf-pr RNA expression in rodent fibroblasts (Vaziri and Faller, 1995; Zhang et al.,

1995). Indeed, the characteristics of this suppression are similar to those seen in response

to Myc and suggests that Myc may lie in the same pathway, downstream of Ras and Src in

the repression of PDGF-PR. Studies by Barone et al., lend support to this model, as it

was demonstrated that Myc is the target of the Src signaling pathway following PDGF-PR

activation (Barone and Courtneidge, 1995). Thus pathway(s) downstream of Src and

possibly Ras likely signal through Myc to suppresspdgf--& RNA and protein expression,

suggesting Myc is a component of a homeostatic regulatoly mechanism controllingpdgf-jr

RNA expression.

Indeed, through the use of c-Myc null rodent fibroblasts, we are able to

demonstrate that Myc is an essential component of a regulatory pathway effecting

repression of pdgf-jr mRNA levels. While repression of pdgf-jr mRNA levels in

wildtype Rat-1 cells is maximal 12 h post mitogen exposure, c-Myc null fibroblasts did not

exhibit a reduction inpdgf-jr mRNA levels up to 48h after serum stimulation. Transit

through one full cell cycle was monitored in both wildtype and null Rat-1 cells, as the

doubling time is 17 to 22h and 45 to 60h, respectively. Thus the abrogation of the serum

induced repression ofpdgf-fir nJWA expression is due to the absence of Myc expression,

and not the indirect result of a reduced rate of transit through the cell cycle. This is further

demonstrated through our analysis of endogenous pdgf-jr mRNA expression in

proliferating c-Myc null fibroblasts. Basal expression ofpdgf-jr mRNA in asynchronous,

subconfluent, growing cells is elevated in null cells compared to wildtype Rat-1 cells.

Moreover, reconstitution of the null cells with ectopic human c-Myc protein elicited a

reduction inpdgf-jr mRNA levels, showing that thepdgf-jr gene is indeed responsive to

Myc.

The repression of PDGF-PR levels by Myc represents a negative feedback

pathway, which is functionally analogous to the Myc negative auto-regulation mechanism,

in that through both pathways Myc functions to curtail a proliferative stimulus. This

mechanism will serve to temper the growth impetus, guarding against deregulated cell

proliferation. Indeed, experimental evidence suggests that the inhibition of this negative

feedback loop can contribute to the formation of tumors. Glioblastomas and astrocytomas

constitutively express high levels of both the PDGF-PR and its ligand, which creates an

autocrine feedback pathway that contributes to the deregulated growth of these tumors

(Guha et al., 1995; Silver, 1992). The critical nature of receptor down-regulation is

supported by the many mechanisms which have evolved to ensure its successful execution;

internalization, intracellular degradation, and RNA suppression. We show that Myc can

suppress pdgf-pr expression at a transcriptional level, and propose that Myc is a

component of the pathway responsible for receptor suppression following ligand

stimulation.

3.6 Acknowledgments

We thank the members of the lab for helpful discussions and critical reading of the

manuscript; Y. Yarden, G. Evan, T. Littlewood, and G. Nolan for valuable reagents. In

addition, we extend our thanks to E. Fish, and C. Lingwood for their assistance with the

PDGF BB ligand binding assays.

Studentship support was kindly provided through the Ontario Graduate Scholarship

Program (W.W.M., S.K.O.) and the Medical Research Council of Canada (L.M.F.).

C.A. was supported by the Swedish Medical Research Council, the Karolinska-University

of Toronto Exchange Program, and a grant from the Swedish Cancer Society. K.F. was

supported by grants from the Swedish Medical Research Council, the Swedish Cancer

Society and the Barncancerfonden. 1% work was supported by a grant from the National

Cancer Institute of Canada with funds from the Canadian Cancer Society (L.Z.P.).

Chapter 4

MYC REPRESSES THE GROWTH ARREST GENE GADD45

This chapter is a modified version of the following manuscript: Myc represses the growth arrest gene gadd45. 1997. Marhin, W., Chen, S., Facchini, L., Fornace Jr., A., and Penn, L. Oncogene, 14 (23), 2825-2834.

4.1 Abstract

The c-Myc protein strongly stimulates cellular proliferation, inducing cells to exit

GOIGI and enter the cell cycle. At a molecular level, Myc prevents growth arrest and

drives cell cycle progression through the transcriptional regulation of Myc-target genes.

Expression of the growth arrest and DNA damage inducible gene 45 (gadd45) is elevated

in response to DNA damaging agents, such as ionizing radiation via a p53-dependent

mechanism, upon nutrient deprivation, or during differentiation. Gadd45 holds a vital role

in growth arrest as ectopic expression confers a strong block to proliferation. Exposure of

quiescent cells to mitogen stimulates a rapid increase in c-Myc expression which is

followed by the subsequent reduction in gadd45 expression. The kinetics of these two

regulatory events suggest that Myc suppresses the expression of gadd45, contributing to

entry into the cell cycle. Indeed, ectopic Myc expression in primary and immortalized

fibroblasts results in the suppression ofgadd45 mRNA levels, by a mechanism which is

independent of cell cycle progression. Using an inducible MycERTM system, rapid

suppression of gadd45 mRNA is first evident approximately 0.5h following Myc

activation. The reduction in gadd45 mRNA expression occurs at the transcriptional level

and is mediated by a p53-independent pathway. Notably, the repression in gadd45 mRNA

levels following serum-stimulation is absent in c-Myc null cells, demonstrating that Myc is

required for this activity. Moreover, we show that the Myc-mediated repression ofgadd45

is an essential regulator of basal gadd45 expression in proliferating fibroblasts. Thus, Myc

plays an essential role in the regulation of gadd45 expression; defining a novel pathway

through which Myc promotes cell cycle entry and prevents growth arrest of transformed

cells.

4 .2 Introduction

The ability of Myc to inhibit cellular growth arrest and promote GOIG1 to S phase

transition likely contributes to its strong oncogenic potential. Physiologically, c-myc

expression is tightly linked to the growth state of the cell (Dean et al., 1986; Kelly and

Siebenlist, 1986; Waters et al., 1991). Expression is nearly undetectable in growth-

arrested cells and is transiently elevated in response to mitogens, yet remains evident

throughout the entire cell cycle (Eilers et al., 1989; Eilers et al., 1991; Ham et al., 1985;

Kaczmarek et al., 1985). Exposure of non-transformed cells to growth arrest signals such

as se~m-withdrawal or inducers of differentiation, triggers a rapid drop in Myc expression

which results in cellular growth arrest. This responsiveness to extracellular signals is lost

with c-myc deregulation, such that a Myc-activated cell which encounters a growth-arrest

signal can no longer exit the cell cycle, and will undergo programmed cell death (apoptosis)

(Askew et al., 1991; Evan et al., 1992). Thus, Myc elicits its effects in a dose-dependent

manner; elevated levels stimulate cell proliferation and apoptosis, whereas the absence of

Myc may be required for growth arrest and differentiation. How Myc can so effectively

and universally control the balance between cell growth arrest and proliferation remains

unclear.

Evidence to date suggests c-Myc mediates such diverse activities by bctioning as

a transcriptional activator when complexed with Max, another basic region helix-loop-helix

leucine zipper protein (Amati et al., 1992; Blackwell et al., 1990; Blackwood and

Eisenman, 1991; Blackwood et al., 1992; Prendergast et al., 1991; Prendergast and Ziff,

1991). MycIMax heterodiiers bind to the canonical DNA hexamer CACGTG as well as to

specific non-canonical elements (Blackwell et a]., 1993; Blackwood and Eisenman, 1991;

Fisher et al., 1993; Grandori et al., 1996). Despite Myc being well-defined as an activator

of gene transcription, only a few direct genetic targets of Myc transactivation have been

identified in vivo, such as ornithine decarboxylase (odc) (Bello-Fernandez et al., 1993;

Wagner et al., 1993), carbamoyl-phosphate syt~tl~ase/aspartarecarbamoyltransferase/

dil~ydroorotase (cad) (Miltenberger et al., 1995), cdc25A (Galaktionov et al., 1996); eIF-

4E (Rosenwald et al., 1993), a DEAD box-related gene (MrDb) (Grandori et al., 1996);

a-prothymosin (Desbarats et al., 1996; Eilers et a]., 1991; Gaubatz et a]., 1995) and

ECA39 (Benvenisty et al., 1992).

As well as a transcriptional activator, Myc plays an important role as a repressor of

gene expression (Desbarats et al., 1996; Inghirami et al., 1990; Lee et al., 1996; Li et al.,

1994; Penn et al., 1990; Philipp et al., 1994; Versteeg et al., 1988; Yang et a]., 1993).

Recent structure-function analysis of the amino-terminus of the c-Myc protein shows Myc

repression and Myc kansactivation are separable, with Myc repression being more closely

associated with neoplastic transformation (Brough et al., 1995; Lee et al., 1996; Li et al.,

1994; Penn et al., 1990). Myc can repress c/ebpa(Antonson et al., 1995; Li et al., 1994),

c y c h D l (Philipp et al., 1994), the adenovims 5 major late promoter (AdMLP) (Li et al.,

1994), and c-myc (Facchini et al., 1997) promoter activities. Myc suppression appears to

be mediated through core regulatoly elements and does not require the consensus Myc/Max

binding sites within the target promoter sequence (Antonson et al., 1995; Facchini et al.,

1997; Li et al., 1994; Lucas et al., 1993; Philipp et al., 1994). Interestingly, Max

heterodimerization is not required for cyclin D l suppression (Philipp et al., 1994), yet it is

obligatory for Myc autosuppression (Facchini et a]., 1997) and remains unexplored for

d e b p a and AdMLP suppression. It remains unclear whether Myc repression of these

target genes occurs by direct or indirect pathways; however, Myc can clearly repress gene

expression by more than one mechanism (Marhin et al., 1996).

By identifying cellular Myc-regulated gene targets, the molecular mechanism(s) of

Myc as a regulator of gene transcription can be delineated. Moreover, the link between

Myc repression and transformation suggests the identification of genes whose expression

is suppressed in response to Myc will also prove crucial to understanding the role of Myc

in neoplastic transformation. Intuitively, candidate Myc-repressed genes could include

those whose pattern of expression contrasts that of c-Myc and are highly expressed during

cellular growth arrest or quiescence. Known genes belonging to this category include the

growth arrest-specific (gas) genes (Schneider et al., 1988), the serum deprivation response

gene (sdr) (Gustincich and Schneider, 1993), the homeobox gar gene (Weir et a]., 1995),

and the growth-arrest and DNA damage inducible (gadd) genes (reviewed in Fomace,

1992; Papathanasiou and Fomace, 1991). With the exception of gas1 andgadd45, the

hctional role of these genes in effecting growth arrest is not clear.

The gadd genes are coordinately expressed in response to growth arrest conditions

as well as to a variety of DNA damaging agents (Fomace et al., 1988; Fomace et al.,

1989). Elevated gadd45 expression has also been linked to growth arrest triggered either

by developmental signals (Constance et al., 1996) or to nutrient-deprivation associated

with GO phase of the cell cycle (Fomace et al., 1989; Smith and Fomace, 1996; Zhan et

al., 1994). Upon exposure to ionizing radiationgadd45 is induced (Hollander et al., 1993;

Papathanasiou et al., 1991), and this induction is dependent upon the p53 transcription

factor (Zhan et al., 1993). Functionally, Gadd45 con?ers a strong block to proliferation as

shown through ectopic expression in tumorigenic carcinoma cell lines such as HeLa and

RKO as well as non-transformed NIH3T3 cells (Vairapandi et al., 1996; Zhan et al.,

1994). Gadd45 is thought to mediate growth arrest through its interaction with two cell

cycle components: the cyclin dependent kinase inhibitor p21, and proliferating cell nuclear

antigen (PCNA) (Chen et al., 1995; Hall et al., 1995; Kearsey et al., 1995). The

mechanism to overcome the growth-inhibitory affects of genes such as gadd45 and exit

GO, in response to mitogen-stimulation or Myc-activation, remains unclear.

In this thesis we show gadd45 gene transcription is repressed in response to Myc

activation. Ectopic Myc expression in both primary and immortalized fibroblasts results in

a reduction in gadd45 mRNA expression. Myc suppression of gadd45 mRNA

corresponds to a similar reduction in Gadd45 protein levels in Rat-1 cells expressing

ectopic Myc protein. Although basal levels of gadd45 are low in subconfluent

asynchronous cultures, expression of ectopic Myc can further down-regulate gadd45

expression, demonstrating that Myc suppression ofgadd45 is not a simple consequence of

cellular transition fiom quiescence into the cell cycle. The suppression ofgadd45 by Myc

occurs with rapid kinetics and occurs at the level of gene transcription in a p53-independent

manner. Myc suppression affects the overall level of Gadd45 expression, as cells

expressing an activated c-myc allele do not show maximal induction of gadd45 expression

in response to gamma-radiation, We demonstrate that Myc plays an essential role in the

regulation of gadd45 expression in mitogen-stimulated and asynchronous proliferating

cells. The suppression ofgadd45 in response to Myc represents a novel and biologically

critical pathway, elucidating one of the mechanisms via which Myc stimulates cells to exit

growth arrest, enter the cell cycle and contribute to transformation.

4.3 Materials and Methods

4.3.1 Cell culture

Primary rat embryonic fibroblasts (REF) and primary mouse embryonic fibroblasts

(MEF) were prepared as previously described (Jacks et al., 1992; Land et al., 1983). Rat-

1 fibroblasts are a subclone of the Fischer rat embryo fibroblast line F2408 (Lania et al.,

1980). Wildtype TGR-1 (+I+) and c-Myc null H015.19 (-I-) cell lines were previously

described (Mateyak et al., 1997). c-Myc null H015.19 gfp (-1- gfp) and c-Myc null

H015.19 gfpmyc (-1- gfpmyc) cells were generated by infecting c-Myc null H015.19 (-I-)

cells with either the retrovirus BabeMNIRESgfp or BabeMNIRESgfpmyc.

Wildtype Rat-1 clone TGR-1 (+I+), c-Myc null H015.19 (-I-), H015.19 gfp (-1-

gfp) and H015.19 gfpmyc (-1- gfpmyc) cells were grown in DMEM HZ1 supplemented

with 10% calf serum, as described (Prouty et al., 1993). All other fibroblast cell lines

were cultured in alpha modified Eagle's medium (aMEM) supplemented with 10% fetal

bovine serum (FBS)(Gibco). Culture media was supplemented with 100 pdml kanamycin

(Sigma) and 2 pdml gentamycin (Roussel) or 100 pglml penicillin (Sigma) and 100 pglml

streptomycin sulfate (Sigma). To activate ectopic Myc activity, Rat-1 wtMycERTM and

AMycERm cells were exposed to 100 nM 4-hydroxy (2) Tamoxifen (OH-T) (Research

Biochemical International, Natick, MA). Rat-1 fibroblasts were exposed to 10 Gy of

ionizing radiation using a source at 1.13 Gylmin. To analyze the response to

serum stimulation, subconfluent wildtype TGR-1 (+I+), and c-Myc null H015.19 (-I-)

cells were maintained in 0.25% calf serudDMEM HZ1 for 2 days, then stimulated with

10% calf serudDMEM H21. Unless otherwise stated, all cells were analyzed as

subconfluent proliferating cultures. Early passage cell cultures were used for all assays.

4.3.2 Retroviral infection

The pDORheo, pBabePnygro, pBabeIpuro, pDoklv-mycneo, pBabelv-mychygro,

pBabelv-mycpuro, pSVX.1, pSVX.6, pBabepuro c-mycERTM, or pBabepuro 4106-143c-

mycERTM retroviral vectors were constructed as previously described (Facchini et al.,

1997; Littlewood et al., 1995; Morgenstem and Land, 1990; Ridley et al., 1988). To

generate the pBabeMNIRESgfpmyc retroviral vector, human c-myc exon 11,111 sequences

excised from pBluescript KS+ Hc-myc were cloned into EcoRI, XhoI digested

pBabeMNIRESgfp (a kind gift of G. Nolan).

To produce infectious replication-defective ecotropic retroviral particles,

recombinant retroviral constructs were transfected, using calcium phosphate precipitation

(Graham and Van der Eb, 1973), into either the GP+E packaging cell line (Markowitz et

al., 1988), or the Phoenix-Eco cell line (ATCC), and selected in 0.5 m g h l G418 sulphate

(Sigma), 2 mglml puromycin (Sigma) or 300 pglml hygromycin B (Sigma). Drug

resistant clones were pooled and expanded for virus production. Fibroblast cultures were

subsequently infected with retrovirus and stable drug-resistant colonies were combined

generating pooled populations. To generate clonal populations of Rat-1 wtMycERTM and

Rat-1 AMycERTM cells, individual drug resistant colonies were isolated. Green

fluorescent protein (gfp) positive c-Myc null H015.19 gfp (-1- gfp) and H015.19 gfpmyc

(-1- gfpmyc) cells were isolated with a Coulter 753 cell sorter using an absorption

wavelength of 488 nm, and pooled.

4.3.3 RNase Protection and luciferase assays

RNA was prepared by the guanidinium isothiocyanate method (Chirgwin et al.,

1979) and assayed by RNase protection assay as described (Penn et al., 1990). The

probes were generated using T3 RNA polymerase (Stratagene) from linearized Bluescript

KS and SK cloning vectors (Stratagene) containing: rat c-myc exon 1, thegag gene derived

from the avian myelocytomatosis virus MC29, murine and rat gapdh cDNA (Facchini et

al., 1994; Penn et al., 1990). The probe for the rat gadd45 gene was transcribed using

Sp6 RNA polymerase (Stratagene) from a linearized pGEM3ZF+ vector (Promega)

containing a 450 bp PstI fiagment of the rat gadd45 cDNA (kind gift of T. Yoshida). The

aminoglycoside phosphotransferase(neo) probe comprised nucleotides 1 to 245 of the

neomycin resistance gene. These sequences were excised from pBabeneo (Morgenstern

and Land, 1990) with Hind111 and ClaI, and cloned into a EcoRV and ClaI digested

pBluescript KS+ vector (Stratagene). The Bluescript KS+ aminoglycoside

phosphotransferase plasmid was linearized with NcoI, and the RNase protection probe

transcribed with T3 RNA polymerase (Stratagene). The protected probes were resolved by

electrophoresis on 6% denaturing polyacrylarnide gels and visualized by autoradiography

on X-OMAT film (Kodak). Luciferase and P-galactosidase assays were performed as

previously described (Facchini et al., 1997).

4.3.4 Immunoblotting and Immunoprecipitation

Antibodies used in this study include the monoclonal antibodies, anti-p53 Pab421

and anti-SV40 Large T antigen Pab419 (kind gifts of S. Benchiiol), as well as polyclonal

anti-gadd45 antibody Sc797 (Santa Cruz), and a polyclonal pan-myc antibody (kind gift of

G. Evan). Whole cell extracts were prepared as recommended (Santa Cruz), and total cell

lysates (5 x 10s cells) were immunoblotted as previously described (Daksis et al., 1994).

Proteins were detected by incubating the membrane with their respective antibody for 1 h in

TBS-T containing 1% non-fat milk. The membrane was washed, and incubated with a

112000 dilution of either horseradish peroxidase-conjugated goat anti-mouse IgG (Biorad)

or swine anti-rabbit IgG antibody (Dako) in TBS-T containing 1% non-fat milk for 0.5 h.

After washing, signals were detected using the ECL (Amersham) detection system and

visualized by autoradiography (Kodak). Imrnunoprecipitations using the anti-gadd45

polyclonal antibody (Sc797) were performed as recommended by the manufacturer (Santa

Cruz). Immune complexes were resolved on a 10% SDSlpolyacrylamide gel, transferred

to PVDF membrane (Millipore), developed with the same antibody, and then visualized by

autoradiography (Kodak).

4.4 Results

4.4.1 Sequential induction of c-myc and suppression of gadd45 mRNA

levels following serum stimulation of quiescent Rat-1 fibroblasts

To determine the relative kinetics of c-myc and gadd45 expression during cell cycle

progression, we assayed the endogenous mRNA expression of these two genes in

quiescent Rat-1 fibroblasts following serum stimulation. Confluent Rat-1 fibroblasts were

cultured in low serum (0.1% FBSIaMEM) for 2 days to induce quiescence and then

mitogen stimulated by changing the medium to 10% FBSIaMEM. RNA was harvested at

0, 0.5, 1, 2, 3, 6 , 12, 18, 24 hours post serum-stimulation and assayed by RNase

protection using single-stranded probes complementary to mRNA of endogenous rat c-

myc, rat gadd45 and rat glyceraldehyde-3-phosphate dehydrogenase (gapdh). Mitogen-

stimulation of quiescent Rat-1 fibroblasts resulted in a transient increase in c-myc mRNA

expression first evident at approximately 0.5 hours after serum-stimulation (Figure 4.1,

compare lane 2 with 1). This was followed by a gradual suppression in endogenous

gadd45 mRNA levels which was clearly evident approximately 1 hour post-serum (Figure

4.1, compare lane 3 with lane 1). To ascertain whether the suppression ofgadd45 mRNA

levels was dependent upon de novo protein synthesis, confluent quiescent Rat-1 cells were

treated simultaneously with 10% FBSIaMEM and 50 bg/ml cycloheximide for 3 hours.

Expression of the endogenous gadd45 gene was not suppressed in the presence of

cycloheximide, showing gadd45 suppression was dependent upon de novo protein

synthesis following serum stimulation. The timing of these two mechanistic events

suggested a role for Myc in effecting the suppression of gadd45 mRNA expression.

Figure 4.1. Serum stimulation of confluent, quiescent Rat-1 fibroblasts results in the induction of c-nryc mRNA expression followed by a suppression of gadd45 mRNA levels. Rat-1 cells were maintained in low serum 0.1% FBSIaMEM for 2 days then left untreated (lane 1) or exposed to 10% FBS (lanes 2 to 9) or 10% FBS and 50 pg/ml cycloheximide (lane 10). At the indicated intervals RNA was extracted and analyzed by RNase protection using single stranded probes complementary to endogenous c-myc, gadd45, and gapdh- specific sequences.

4.4.2 Ectopic Myc expression in primary cell cultures and immortalized

cell lines results in a suppression of endogenous gadd45 mRNA

levels

To determine whether ectopic Myc expression can suppress gadd45 mRNA levels,

primary rat embryo fibroblasts (REF) and the immortal non-tumorigenic Rat-1 cell line,

were infected with control retrovirus (DORheo) canying the neomycin resistance gene or

retrovirus (DoWv-mycneo) also carrying the v-myc gene. Primary mouse embryo

fibroblast cultures (MEF) were similarly infected with either a control retrovirus

(Babelpuro) or a retrovirus (Babelv-mycpuro) carrying the v-myc gene. Drug resistant

colonies were pooled and analyzed as asynchronous proliferating cultures. Subconfluent-

growing cells were analyzed to ensure changes in endogenous gadd45 gene expression

were not due to an indirect effect of Myc on cell cycle (Evan et al., 1992). Under the

conditions of these experiments, populations of both control cells and cells expressing

ectopic Myc protein showed similar rates of growth and a similar distribution of cells

throughout all phases of the cell cycle (data not shown). RNA was extracted and analyzed

by RNase protection assay for the expression of the endogenous gadd45 and gapdh

mRNAs. Expression of ectopic Myc protein in primary cultures as well as in Rat-1 cells

resulted in a 4 to 5 fold repression in endogenous gadd45 mRNA levels compared to

controls as determined by densitometry (Figure 4.2A, compare lanes 2,4,6 to lanes 1,3,5,

respectively). Suppression of gadd45 was also clearly evident in Myc-expressing cell

cultures which were grown to confluence (data not shown). The ectopic v-myc gene was

expressed as demonstrated by RNase protection assay (Figure 4.2B). Thus, constitutive

ectopic Myc expression leads to a suppression in gadd45 mRNA levels.

To determine if the suppression in gadd45 mRNA levels results in a corresponding

decrease in Gadd45 protein expression, whole cell lysates were prepared from

subconfluent asynchronous cultures of both control Rat-1 and Rat-1 cells expressing

Figure 4.2. Ectopic Myc expression in primary and immortalized

fibroblasts results in the suppression of gadd45 mRNA and protein levels.

(A) 10 pg of RNA from subconfluent proliferating rat embryo fibroblasts (REF), Rat-1

fibroblasts (Rat-I), and mouse embryo fibroblasts (MEF) infected with control retrovirus

(-) (lanes 1,3,5), or retrovirus carrying the v-myc gene (+) (lanes 2,4,6) was analyzed by

RNase protection assay to detect the presence of endogenous gadd45 and gapdh-specific

sequences; (B) 10 yg of RNA from subconfluent proliferating rodent fibroblasts infected

with control retrovirus (-) (lanes 1,3,5), or retroviruses canying the v-myc gene (+) (lanes

2,4,6) was analyzed by RNase protection assay to detect the presence of ectopic v-myc

and gapdh-specific sequences (C) 2 mg of total cell lysate from subconfluent Rat-1

fibroblasts infected with a control retrovirus (-), or retroviruses canying the v-myc gene

(+) was immunoprecipitated using a polyclonal anti-Gadd45 antibody (Sc797).

Immunoprecipitated proteins were resolved on a 10% polyacrylamide gel and Gadd45

protein detected by western blot analysis using antibody Sc797. The weak signal in the

control lanes of the western blot probed for exogenous Myc expression represents across-

reactive band evident with this batch of pan-myc antibody.

A. REF Rat-1 MEF

B. REF Rat-1 MEF + "--I - + '

I + I v-myc -

ectopic v-Myc protein and analyzed by immunoprecipitation followed by immunoblot

analysis. Rat-1 cells constitutively expressing ectopic Myc protein exhibited a 5 fold

decrease in Gadd45 protein expression compared to control cells (Figure 4.2C). This level

of suppression correlated with the observed 4 to 5 fold decrease in gadd45 mRNA

expression. Western blot analysis clearly demonstrated that the ectopic v-Myc protein is

expressed in Rat-1 Myc cells compared to the control Rat-1 cells. The weak signal in the

control lanes of the western blot probed for v-Myc expression represents a non-specific

cross-reactive band. Thus, constitutive ectopic Myc expression results in a suppression in

gadd45 mRNA levels with a corresponding decrease in Gadd45 protein expression.

4.4.3 The suppression of gadd45 in response to Myc-activation occurs

with rapid kinetics

To determine the kinetics of suppression of gadd45 in response to Myc we

employed an estrogen-inducible M ~ C E R ~ ~ system in Rat-1 cells (Littlewood et al., 1995).

Rat-1 cells were infected with a retrovirus carrying a chimeric gene consisting of the human

c-myc cDNA linked in frame to the estrogen binding regulatory domain of the estrogen

receptor, and individual drug-resistant clones were isolated. These Rat-1 wtMycERTM

cells constitutively express an inactive MycERTM fusion protein, which is rapidly activated

following exposure of cells to hydroxy-tamoxifen (OH-T). By this approach, Myc can be

induced in the absence of mitogen-stimulation. Confluent Rat-1 wtMycERTM cells were

cultured in low serum (0.1% FBSIaMEM) for two days to induce quiescence and then

treated with OH-T to induce ectopic Myc activity. RNA was extracted at 0,0.5, 1,2,3,6,

9, 12, 18, 24 hours following exposure of cells to OH-T and expression of gadd45 and

gapdh mRNA determined by RNase Protection assay (Figure 4.3). Induction of ectopic

Myc activity triggered a reduction ingadd45 mRNk levels, with a 2 fold reduction at 3 h

and a maximal 5 fold suppression after approximately 12 hours, as determined by

Figure 4.3. gadd45 mRNA expression is suppressed with rapid kinetics in response to Myc induction. Confluent Rat-1 wtMycERm cells were maintained in low serum 0.1% FBSIaMEM for 2 days and then exposed to 100 nM OH-T to induce exogenous MycERm activity. At the indicated intervals after OH-T treatment RNA was extracted and 10 pg total RNA analyzed by RNase protection assay using single stranded probes complementary to endogenous gadd45, and gapdh-specific sequences.

densitometry. Similar results were observed in multiple independent Rat-1 wtMycERm

cell clones. To determine whether Myc directly or indirectly suppresses gadd45, Rat-1

wtMycERm cells were exposed to cycloheximide at the time of MycER activation;

however, the short 20-30 minute half-life of MycER quickly depleted the cell of effector

protein making results uninterpretable (data not shown). As the half-life of gadd45 mRNA

is approximately 20 to 80 mins (Jackman et al., 1994), Myc repression of gadd45 RNA

was rapid and first evident as early as 0.5 h following OH-T treatment. gadd45 repression

was not evident in OH-T treated control Rat-1 AMycERTM cells expressing a non-

functional MycERTM fusion protein harbouring a deletion in amino acids 106-143 of the

Myc portion of the fusion protein (data not shown). This region of the c-Myc protein is

required for all known biological activities of Myc (Evan et al., 1992; Freytag et al.,'1990;

Garte, 1993; Penn et al., 1990; Stone et al., 1987). This cell line serves as a control for

the expression and hormone-activation of the M ~ C E R ~ ~ fusion protein. Thus, our results

show gadd45 mRNA expression is suppressed in response to Myc with rapid kinetics.

4.4.4 The suppression of gadd45 in response to Myc does not require

wildtype p53 activity

It is well established that induction of the gadd45 gene following exposure to

ionizing radiation, is mediated by the transcription factor p53 (Kastan et al., 1992; Zhan et

al., 1994; Zhan et al., 1993). To determine whether Myc may indirectly repress gadd45

expression by affecting wildtype p53 expression, RNA isolated from subconfluent control

Rat-l and Rat-l cells expressing ectopic v-Myc protein was assayed for endogenous p53

mRNA and protein expression. There was no change in endogenous p53 mRNA

expression in Rat-1 Myc cells compared to control Rat-1 cells (Figure 4.4A).

Furthermore, comparable levels of endogenous wildtype p53 protein were expressed in

Figure 4.4. p53 mRNA and proteh expression is not responsive to ectopic Myc expression in Rat-1 cells. (A) 10 pg of RNA from subconlluent Rat-1 cells infected with a control retrovirus (-), or retrovirus canying the v-myc gene (+) was analyzed by RNase protection assay to detect the presence of endogenous p53 and gapdh-specific sequences. (B) Immunoblot analysis of 200 pg of total cell lysates from subconfluent Rat-1 cells infected with a control retrovirus (-), or retroviruses canying the v-myc gene (+) to detect endogenous p53 protein expression.

control and Myc-expressing Rat-1 cells (Figure 4.4B). Thus, constitutive ectopic Myc

expression does not affect the expression of endogenous p53 mRNA or protein in Rat-1

cells.

To determine whether wildtype p53 activity is required for the suppression of

gadd45 by Myc, control Rat-1 and Rat-1 Myc cells were infected with either a control

retrovirus (SVX.1) carrying only the neomycin resistance gene, or a retrovirus (SVX.6)

also canying the SV40 LTag gene. Following selection, drug-resistant colonies were

pooled and analyzed as subconfluent proliferating cultures. The expression of SV4O LTag

results in the inactivation of wildtype p53 activity (Jiang et al., 1993; Lenahan and Ozer,

1996; Mietz et al., 1992). Constitutive expression of ectopic SV40 LTag had no

significant effect on gadd45 expression in control Rat-l cells or in Rat-1 Myc cells (Figure

4.5A). To ensure that the products of the ectopic genes were expressed, whole cell lysates

from each of the Rat-1 cultures were assayed by Western blot analysis for the expression

of ectopic v-Myc and LTag proteins. Ectopic v-Myc protein was clearly expressed in the

appropriate cell lines (Figure 4.5B, compare lanes 3 and 4 with 1 and 2). Ectopic SV40

LTag protein expression was also highly expressed in Rat-1 cells which were infected with

the SVX.6 retrovirus canying the LTag gene (Figure 4.5B, compare lanes 2 and 4 with 1

and 3). Thus, wildtype p53 activity was not required for either basal level gadd45

expression nor Myc suppression of gadd45.

Figure 4.5. The suppression of gadd45 by Myc does not require wildtype

p53 activity. (A) 10 pg of RNA from subconfluent Rat-1 cells infected with a control

retrovirus (-), retrovirus carrying the v-myc gene andlor retrovirus carrying the SV40 large

T antigen (+) was analyzed by RNase protection assay to detect the presence of

endogenous gadd45 and gapdh-specific sequences. Histograms represent gadd45 mRNA

abundance relative to the control as quantitated by densitometry. All data was normalized

to the level ofgapdh mRNA expressron. (B) Immunoblot analysis of total cell lysates from

subconfluent Rat-1 fibroblasts as described in (A), using a pan-myc polyclonal antibody to

detect ectopic v-Myc protein, and the monoclonal antibody Pab419 to detect ectopic SV40

LTag expression.

1 .o

0.8 RELATIVE

gadd45 0.6 infWA

ABUNDANCE 0.4

0.2

0.0

v-myc 6 I + + SV40LTag - + - +

Rat-1 I

v-myc I - + + I SV40LTag - + I. +

4.4.5 Myc and p53 co-regulate gadd45 transcription

gadd45 mRNA expression is suppressed in response to Myc activation and induced

by p53 following exposure to ionizing radiation (Kastan et al., 1992; Zhan et al., 1994;

Zhan et al., 1993). To determine whether these two pathways interact, subconfluent

proliferating control Rat-1 and Rat-1 Myc cells were exposed to 10 Gy of ionizing

radiation. Following treatment of cells, total RNA was extracted at 0, 1, 3, 8, 12 and 18

hours and expression of endogenous gadd45 and gapdh mRNA was analyzed by RNase

protection assay. gadd45 mRNA expression in unirradiated Rat-1 Myc cells was

suppressed compared to unirradiated control Rat-1 cells (Figure 4.6, compared lane 7 to

1 j. Furthermore, exposure of either of the two cell lines to ionizing radiation led to the

gradual induction of gadd45 mRNA expression (Figure 4.6, compare lanes 1 to 6 with

lanes 7 to 12). Importantly, maximal achievable levels ofgadd45 expression evident in the

control cells were not reached in the Myc-activated cells after 18 hours (Figure 4.6).

Extended time courses show similar results (data not shown). Hence both mechanisms;

the suppression of gadd45 by Myc and the induction of gadd45 by p53, are independent

regulatory mechanisms which combine to co-regulate gadd45 expression.

4.4.6 Myc regulates transcription from the gadd45 Promoter

To ascertain whether Myc represses gadd45 expression at the level of gene

transcription, we conducted transient-transfection reporter assays in both Rat-1

W ~ M ~ C E R T M and control Rat-1 AM~CERTM cells. Thegadd45 promoter lacks both

TATA and Inr elements and contains few putative transcription factor binding sites. There

are two potential OCT and two CAT sites located within 100 bp upstream of the

transcription start site, and an APl site as well as a p53 binding site in intron 3 which are

conserved in the human, mouse and hamster genes (Figure 4.7A) (Hollander et al., 1993).

As our results show that Myc suppression is p53 independent, we focused ow analysis on

upstream regulatory sequences using a hamstergadd45 promoter luciferase construct

consisting of the sequences from -1675 to +I49 relative to the transcription start site. This

construct was transiently transfected into subconfluent proliferating Rat-1 wtMycERTM

and control Rat-1 AM~CERTM cells. To control for transfection efficiency a vector

carrying the lacZ gene, was co-transfected with the promoter luciferase construct. Cells

were treated with OH-T and at 0,3,6, 9, 12, lg, 24 hours thereafter, whole cell lysates

were prepared and then assayed for both luciferase and P-galactosidase activity. Treatment

of Rat-1 AMycERm cells with OH-T had no effect on luciferase activity (Figure 4.7B).

However, OH-T induction of ectopic Myc activity in Rat-1 wtMycERm cells resulted in

the down-regulation of luciferase activity. As the half-life of luciferase is 4 to 6 hours

(Thompson et al., 1991), the level and kinetics of repression as measured in the gadd45

promoter-luciferase assay (Figure 4.7B) closely mimicked the suppression of endogenous

gadd45 mRNA in response to MycERTM activation (Figure 4.3). The repression of the

gadd45 promoter by Myc is specific, as induction of Myc aztivity had no significant effect

on P-galactosidase activity (Figure 4.7C). Thus, Myc can suppress transcription from the

gadd45 promoter, and this suppression activity can be localized to nucleotide sequences

found between -1675 and +149, relative to the transcription start site.

4.4.7 Deletion analysis of the hamster gadd45 promoter

The 1.8 Kb Xba I fragment of the hamster gadd45 promoter is responsive to Myc

activation. To determine which sequences in this promoter fragment are required for the

repression of transcription by Myc, consecutive deletions were made at the 5' end of the

Xba I fragment of the hamster gadd45 promoter, generating a series ofgadd45 promoter

luciferase constructs (Figure 4.8). These gadd45 promoter luciferase constructs were

transfected into Rat-1 wtMycERTM cells and assayed for the responsiveness of each

promoter to Myc activation. For each promoter construct, induction of Myc activity elicited

a 3 to 4 fold repression in luciferase activity relative to basal luciferase activity. Deletion of

419 bp at the 5' end of the Xba I hgment of the hamstergadd45 promoter elicited a 3 fold

elevation in basal luciferase activity suggesting that negative regulatory sequences are

present at the 5' end of the promoter. The subsequent deletion of 613 bp, truncating the

promoter to approximately 1 Kb, eliminates the elevation in basal transcription from this

promoter fragment, hinting at a positive regulator of transcription between nucleotides

-1256 and -643. Further deletions to position -222, relative to the transcription start site,

do not significantly impact on basal transcription. However deletion of a region of the

promoter consisting of nucleotides -222 to -72, which contains the putative transcription

factor binding sites, an Oct and a Cat site, results in a loss of basal luciferase activity.

Thus, sequences found between -222 and +I49 are required to maintain basal transcription

of the hamster gadd45 promoter, and contained within this region are sequences which

mediate the repression ofgadd45 transcription by Myc.

Figure 4.7. gadd45 transcription is suppressed in response to Myc. (A)

Schematic diagram of the genomic structure of the hamstergadd45 gene, showing putative

transcription factor binding sites. Numbers indicate position relative to the transcriptional

start site (+I). Rat-1 AMycERm and Rat-1 wtMycERW cells were transfected with a 1.8

Kb hamster gadd45 promoter luciferase construct, containing gadd45 sequences from

nucleotides -1675 to +149. To assay transfection efficiency, cells were co-transfected with

a plasmid carrying the Pgalacfosidase gene under the control of the CMV promoter. Cell

lines were subsequently treated with 100 nM OH-T for the times indicated to induce ectopic

MycERm activity. Whole cell lysates were prepared and analyzed for luciferase and P- galactosidase activity. (B) Histogmms represent the measured luciferase activity relative to

the untreated control. Data was normalized to P-galactosidase activity to adjust for minor

differences in transfection efficiency amongst the samples. (C) Histograms represent the

P-galactosidase activity. Data are a representative out of 3 repeated experiments. Error bars

denote standard deviations.

Hamster gadd45 promoter

TIME (h)

0 3 6 9 12 18 24 TIME (h)

0 3 6 9 12 18 24 TIME (h)

-- 0 3 6 9 12 18 24

TIME (h)

MycER TM Hamster gndd45 ~romoter ACTIVATION

OCT OCT

-1

RELATIVE LUCIFERASE ACTIVITY

Figure 4.8. Deletion analysis of the hamster gadd45 promoter. Constructs contain portions of the hamster gadd45 promoter as indicated driving the transcription of a luciferase reporter gene. Positions indicated are relative to the transcription start site. Rat-l w t ~ y c E R ~ M cells were transfected with 5 ug of each promoter reporter construct. To measure transfection efficiency, cells were co- transfected with a plasmid carrying the p-galactosidase gene under the control of a CMV promoter. Cells lines were subsequently treated with 100 nM 4-Hydroxy-Tamoxifen in ethanol or ethanol alone as a control for 24 h. Histograms represent the measured luciferase activity, normalized to p-galactosidase activity, relative to the luciferase activity from the full length 1.8 Kb hamstergadd45 promoter in untreated controls.

4.4.8 Myc is an essential negative regulator of gadd45 expression

Mitogen-stimulation of quiescent fibroblasts elicits a transient induction in

endogenous c-Myc protein expression; coincident with a reduction in gadd45 mRNA

expression. To determine if Myc is required for the repression in gadd45 mRNA levels

following serum-stimulation, we analyzed gadd45 mRNA expression in c-Myc null

H015.19 (-I-) cells, after stimulation with serum. H015.19 (-I-) cells, were cultured in

0.25% calf s e r u d M E M H21 for 2 days then treated with serum. At the indicated time

intervals, total RNA was extracted and analyzed for gadd45 and gapdh mRNA expression

by RNase protection assay. Serum-stimulation of quiescent wildtype Rat-1 (+I+)

fibroblasts elicits a clear repression in gadd45 mRNA levels (Figure 4.1). In contrast, in

serum-deprived c-Myc null H015.19 (-I-) rodent fibroblasts, gadd45 mRNA expression

was not repressed in response to serum-stimulation (Figure 4.9A). Moreover, gadd45

mRNA levels were invariant through the course of the experiment, up to 48 h post-

treatment. Thus, Myc is required for the suppression ofgadd45 mRNA levels following

mitogen-stimulation.

Analysis of exponentially growing cultures revealed that Myc also plays a role in

regulating basal gadd45 mRNA levels. In proliferating c-Myc null H015.19 (-I-) cells,

gadd45 mRNA levels are elevated in comparison to wildtype Rat-1 (+I+) fibroblasts

(Figure 4.9B). Hence, in addition to repressing gadd45 mRNA expression following

serum-stimulation, Myc also plays a key in regula!ing basal expression of the gadd45 gene

in proliferating cells. In conjunction with the elevated expression of the gadd45 gene, the

expression of the endogenous c-myc gene is also elevated (Figure 4.9B). Yet, both genes

remain responsive to Myc as the Myc-mediated repression of both genes can be rescued

upon expression of ectopic Myc in the c-Myc null (-I-) cells. Ectopic Myc expression in c-

Myc null H015.19 gfpmyc (-1- gfpmyc) cells results in a clear repression of endogenous

gadd45 as well as c-myc mRNA levels, in comparison with parental H015.19 (-I-) cells as

well as the control H015.19 gfp (-1- gfp) cells, which expresses only the green fluorescent

protein (Figure 4.9B). RNase protection analysis clearly demonstrates that exogenous

human c-myc mRNA is expressed in the appropriate cell cultures (Figure 4.9C). Thus,

Myc is required for the repression ofgadd45 expression in quiescent rodent fibroblasts,

following serum-stimulation. In addition, the repression ofgadd45 by Myc is an essential

regulatory mechanism maintaining basal gadd45 expression in proliferating fibroblasts.

Figure 4.9 Myc is an essential negative regulator of gadd45 expression (A)

Rat-1 cells, lacking c-Myc expression (-I-), were maintained in low serum 0.25% calf

serum/DMEM H21 for 2 days then treated with 10% calf serurn/DMEM H21. At the

indicated intervals, RNA was extracted and analyzed by RNase protection using single

stranded probes complementary to endogenous gadd45, and gapdh-specific sequences.

RNA(10 yg), extracted from subconfluent cultures of wildtype rodent fibroblasts (+I+),

rodent fibroblasts lacking endogenous Myc expression (-I-), and c-Myc null fibroblasts

which have been stably infected with either a control retrovirus containing the gfp cDNA (-

I- gfp) or a retrovirus containing human c-myc exon 11, 111 cDNA as well as gfp (-I-

gfpmyc) was analyzed by RNase protection for (B) endogenous gadd45, c-myc,

aminoglycoside phosphotransferase (neo) and gapdh (C) ectopic human c-myc, and

endogenous gapdlt-specific sequences. Similar results were ot:ained in three separate

experiments.

Human c-myc

gapdh

1 2 3 4

4.5 Discussion

Identifying the mechanisms by which Myc can overcome growth arrest and

stimulate cells to enter the cell cycle is crucial to our understanding of how Myc promotes

cell proliferation and contributes to cellular transformation. Our results show that the

suppression of the potent growth arrest gene gadd4S by Myc may constitute one such

mechanism. The reduction in gadd4S mRNA levels in response to mitogen stimulation of

quiescent cells coincided with the induction of endogenous c-Myc expression. Moreover,

simultaneous treatment of growth arrested cells with both cycloheximide and senun for 3h

abrogated the reduction in gadd4S mRNA expression, demonstrating that the down-

regulation of gadd4S mRNA levels was dependent upon de novo synthesis of a protein

which was expressed soon after serum stimulation. To investigate the role of Myc in

gadd4S suppression, we ectopically expressed Myc protein in primary cultures as well as

in immortalized non-tumorigenic cell lines. Our results showed a reduction of gadd4S

expression in Myc-activated cells. The 4 to 5 fold suppression of endogenous gadd4S

mRNA levels in Rat-1 cells expressing ectopic Myc protein, correlated with a similar

reduction in Gadd4S protein expression. Importantly, this suppression was evident in

subconfluent proliferating cells where the cell population was asynchronous, showing that

Myc suppression ofgadd45 was not an indirect result of the GOIGl to S phase transition of

the cell cycle.

To determine the kinetics ofgadd45 suppression in response to Myc induction we

employed Rat-1 MycERm cells. The MycERm fusion protein is composed of the human

c-Myc protein (Myc) fused in frame at its carboxyl terminus to the hormone regulatory

domain of the estrogen receptor (ERm). Rat-1 cells stably and constitutively express the

inactive MycERm hsion protein and exposure of cells to 4-Hydroxy-Taznoxifen (OH-T)

rapidly activates MycERm in the absence of the broader response to mitogen. Activation

of the MycERm protein resulted in the suppression of gadd4S mRNA levels; suppression

was first evident as early as 0.5 h following OH-T stimulation and half-maximal

suppression occurred after 3 hours. The kinetics of suppression were rapid, as the half-life

ofgadd45 mRNA is 20 to 80 mins (Jaclanan et al., 1994), showing gadd45 expression is

a downstream target of Myc regulation.

Transcription of thegadd45 gene is directly induced by the p53 transcription factor.

Exposure to ionizing radiation results in an increase in p53 protein levels which in turn

activates transcription through a p53 responsive element in intron 3 of the gadd45 gene

(Hollander et al., 1993; Kastan et al., 1992; Zhan et al., 1994; Zhan et al., 1993). Rat-1

cells express wildtype p53 protein as determined by immunoblot analysis, hence it is

plausible that Myc suppression of gadd45 is either mediated through p53 or requires

wildtype p53 activity. Our results demonstrated that constitutive expression of ectopic Myc

protein in Rat-1 cells did not affect the expression of the endogenous p53 mRNA or

protein. To determine if wildtype p53 activity is rcquired for the suppression ofgadd45 by

Myc, SV40 LTag was ectopically expressed in both control and Rat-1 Myc cells to inhibit

wildtype p53 activity (Jiang et al., 1993; Lenahan and Ozer, 1996; Mietz et al., 1992). We

observed that gadd45 mRNA expression was suppressed in Rat-1 cells which are

expressing ectopic Myc protein, irrespective of the p53 status of the cell. We further show

the suppression ofgadd45 by Myc and the induction ofgadd45 by p53 following exposure

to ionizing radiation were non-competitive regulatory mechanisms which co-regulated

gadd45 expression. Thus, the suppression ofgadd45 by Myc is p53 independent.

The results of the transient transfection reporter assays rlemonstrate that gadd45

transcription was suppressed in response to Myc activation. This response was specific to

Myc as it was not evident in the control Rat-1 AMycERm cells. The region of the gadd45

gene which was sufficient to confer suppression in response to Myc encompassed 371 bp

of the hamster gadd45 promoter containing nucleotides -222 to +149, relative to the

transcription start site. This region of the promoter also lacks the p53 binding site

(Hollander et al., 1993), further supporting our observation that the effects of Myc and p53

on gadd45 regulation are separable. Sequence analysis of the gadd45 promoter revealed

that this regulatory region lacks canonical Myc binding sites; a characteristic which is

common among genes which are repressed by Myc (Antonson et al., 1995; Facchini et al.,

1997). Recent studies suggest that Myc may repress gene expression either by directly

interacting with components of the basal transcription machinery or by inhibiting

transcription through an initiator element (Li et al., 1994; Philipp et al., 1994; Roy et al.,

1993). Interestingly, the gadd45 gene lacks both an initiator site and a TATA box. The

suppression ofgadd45 transcription by Myc appears to be universal, as this mechanism is

evident in both rat and mouse fibroblasts. This suggests that the suppression mechanism

may be effected through shared enhancer elements found in the promoters of the gadd45

gene from different species. Indeed this region of the gadd45 gene possesses only two

putative enhancers which are conserved among human, hamster, and mouse (Hollander et

al., 1993). These include two OCT sites and two CAT sites located within 100 bp

upstream of the transcription start site. The CAT sites are of particular interest as earlier

studies suggest Myc can repress gene transcription by inhibiting the activities of CAT-

binding transcription factors belonging to the CTF/NFl family and the clebp family of

trans-acting factors (Martin, 1991; Wedel and Ziegler-Heitbrock, 1995). Freytag and

colleagues suggest that Myc is able to mediate a change in the phosphorylation state of

CTF/NFl, modifying its potential to activate transcription of the pro alpha 2(I) collagen

promoter (Yang et al., 1991; Yang et al., 1993). Recently, transient transfection reporter

assays demonstrate that in 3T3-L1 cells, ectopic Myc expression can abrogate the clebpa

mediated activation of gadd45 transcription associated with adipocyte differentiation

(Constance et al., 1996). In addition, Mink et al. show ectopic v-Myc can suppress the

expression of myelomonocytic specific genes by inhibiting both the expression and the

fhction of clebpa transcription factors (Mink et al., 1996) Clebpa is predominantly

expressed in differentiated cells (Birkenmeier et al., 1989), hence the role of clebpa in the

GOIGl-associated up-regulation, or Myc-mediated repression of gadd45 in cycling cells,

remains unclear. Indeed, Myc may repress gadd45 directly by interacting with components

of the core transcription complex, indirectly in an enhancer-dependent manner or by a yet

unknown mechanism of action. With the identification of gadd45 as a Myc-suppressed

gene target, the molecular mechanism of Myc-repression of this uncomplicated promoter

can now be delineated.

Although the precise mechanism via which Myc represses transcription of the

gadd45 gene is not known, it is clear that Myc plays an important role in regulating the

expression of the gadd45 gene. Myc is required for the repression of gadd45 expression

following serum-stimulation of serum-deprived fibroblasts. The duration of the cell cycle

is prolonged in c-Myc null fibroblasts in comparison to their wildtype counterparts, 45 to

60 h, and 17 to 22 h, respectively. As such, in our serum-stimulation experiments, we

intentionally monitored gadd45 rnRNA expression through one full cell cycle, in both

wildtype and c-Myc null cultures. In wildtype Rat-1 fibroblasts, gadd45 mRNA levels are

maximally repressed approximately 9 h following serum-stimulation; approximately 4 h

prior to the GI to S transition (data not shown). Yet, in the c-Myc null cells, gadd45

mRNA expression is invariant despite prolonged exposure to serum for 48 h. Hence, the

abrogation of the serum-induced repression in gadd45 mRNA levels, in c-Myc null cells, is

due solely to the absence of c-Myc expression, and not the indirect consequence of a

reduced rate of transit through the cell cycle. The importance of the Myc-mediated gadd45

repression mechanism was further substantiated by the analysis of gadd45 mRNA

expression in subconfluent, proliferating cultures. Basal expression of endogenous

gadd45 mRNA is elevated in comparison with wildtype Rat-1 cells. Yet, the gadd45 gene

is responsive to Myc, as reconstitution of c-Myc null cells with ectopic human c-Myc will

result in the repression in gadd45 mRNA levels.

The identification of Myc-suppressed target genes is of fundamental importance, as

recent studies have linked Myc repression of gene transcription with Myc-induced

transformation (Brough et al., 1995; Lee et al., 1996; Li et al., 1994). Indeed, we show

that the region of the human c-Myc protein, amino acids 106 to 143, required for Myc-

suppression of gadd45, is also required for Myc-induced cell cycle progression, and

cellular transformation (Evan et al., 1992; Freytag et al., 1990; Garte, 1993; Pem et al.,

1990; Stone et al., 1987). Interestingly this region of the c-Myc protein was dispensable

for Myc suppression of cyclin Dl (Philipp et al., 1994), suggesting the molecular

mechanisms of Myc suppression of gadd45 and cyclin Dl are likely non-overlapping and

Myc may repress gene transcription by a number of pathways.

gadd45 expression is strongly associated with growth arrest, and identifying

gadd45 as a Myc-suppressed gene target contributes to our understanding of how Myc can

stimulate cells to exit growth arrest and enter the cell cycle. Myc suppression of gadd45

likely contributes to its turnongenic potential, precluding transformed cells from entering a

growth arrested state. In this report we propose thatgadd45 is a target of Myc repression,

but may not be the sole target of Myc repression to permit the GO to GUS transition.

Indeed we have observed that the growth arrest genes gasl , and gadd153 are also

suppressed in response to Myc, while the gas genes 3 and 6 are not regulated by Myc

(WWM, LZP, unpublished data). We propose that mitogen-stimulation induces Myc

which in turn plays an im,ortant role in co-ordinating the suppression of specific growth

arrest genes, thereby permitting cells to exit GOIGl and enter the cell cycle.

4 .6 Acknowledgments

We thank J. Lear, M. Shago, C. Ho, J. Dimitroulakos, and S. Lee for helpful

discussions and critical reading of the manuscript; S. Benchimol for SV40 large T antigen

and p53 antibodies; T. Yoshida for the rat gadd45 cDNA, and G . Nolan for the

pBabeMNIRESgfp retroviral vector.

Studentship support was kindly provided through the Ontario Graduate Scholarship

Program (W.M.) and the Medical Research Council of Canada (L.F.). This work was

supported by a grant from the National Cancer Institute of Canada with funds from the

Canadian Cancer Society (L.P.).

Chapter 5

FUTURE DIRECTIONS

5 . 1 Summary of research

Accumulating evidence suggests a role for Myc repression in Myc-mediated

tumorigenesis (Lee et al., 1996; Li et al., 1994; Stone et al., 1987). Yet the scarcity of

Myc-repressed cellular target genes has hampered the assessment of the significance and

contribution of Myc repression to enhancing cellular proliferation and tumorigenesis. This

concern is presently being addressed as a search of the literature illustrates the recent

interest in this avenue of Myc research. Realizing the potential of this subset of genes as

regulatory targets for Myc, we sought to identify and characterize genes whose expression

is repressed by Myc. We have pursued three avenues of investigation: we analyzed the

regulatory relationships between three key cell cycle regulators: Myc, cyclin Dl, and pRb

in primary embryonic cells. In addition, we have idxtified two genes pdgf-pr; and

gadd45 whose expression are repressed by Myc at the hanscriptional level. The analysis

of Myc-mediated repression events has contributed to our understanding of the

mechanism(s) of Myc repression, and the role these repression activities play in the

biological functions of Myc.

5 . 2 Analysis of the relationship between Myc, pRb, and cyclin D l

A variety of studies conducted in tumorigenic and immortalized cell lines have led

to the emergence of a number of conflicting regulatory pathways involving three key cell

cycle regulators: Myc, pRb and cyclin Dl. For example, in transformed cell lines, Myc

has been reported to suppress cyclin Dl expression. This repression activity is also

evident in a few selected immortalized cell lines but not others. Similarly, in a variety of

tumour cell lines, it has been demonstrated that hypo-phosphorylated pRb can transactivate

the expression of cyclin Dl. This observation is corroborated by the observation that in

many tumour lines pRb loss is associated with a loss in cyclin Dl expression. This led to

the hypothesis that a feedback mechanism exists between pRb and cyclin Dl .

Hypophosphorylated pRb transcriptionally induces the cyclin Dl gene, cyclin Dl with its

respective cdk, phosphorylates pRb, inactivating its activity. The universality of these

regulatory mechanisms and hence the importance of these findings to our understanding of

cell cycle regulation in non-transformed cells, was unclear. Conceivably, the transfonned

nature of these systems may taint many of these obsetvations. My goal was to assay the

validity of these reported mechanisms in a relatively normal cellular model, primary mouse

embryo fibroblasts.

We demonstrate that these three critical cell cycle regulators: Myc, pRb, and cyclin

Dl exist within a complex interdependent relationship (Marhin et al., 1996). Contrary to

the results obtained in transformed cell lines, in primary cells, pRb is not required for the

expression of cyclin Dl. In addition, Myc does not repress cyclin Dl transcription in

primary cells. Interestingly, cyclin Dl expression is lost in MEFs lacking pRb and

expressing ectopic Myc protein. The repression of cyclin Dl is comprehensive, as it is

evident in Myc-activated pRb-null MEFs derived fiom independent litters. The repression

of cyclin Dl expression can be effected at multiple levels, as we have described earlier.

Thus, irrespective of the mechanism of repression, the ultimate goal in these Myc-activated

pRb-null MEFs is the elimination of cyclin Dl expression. In the context of Myc-

activation and loss of pRb, what is the function of cyclin Dl? A survey of the literature

reveals a strong bias towards a singular function for cyclin Dl, namely the phosphorylation

and inactivation of pRb, facilitating cell cycle progression (reviewed in Sherr, 1996; Sherr

et al., 1994). This model was supported by studies which demonstrate that cyclin Dl

activity was dispensable for cell cycle progression in pRb-null MEFs (Baldin et al., 1993;

Lukas et al., 1995; Quelle et al., 1993). Yet, in pRb-null MEFs, loss of cyclin Dl

expression did affect cell growth, as there was a consistent acceleration into S phase

(Lukas et al., 1995). This observation suggested that cyclin D l expression may exert a

growth inhibitory function which is separable from its role as a pRb kinase in a pRb-null

MEF. Indeed, recent studies support the view that cyclin Dl exerts an activity independent

of the phosphorylation of pRb, and hint at additional novel functions for cyclin Dl. For

example, cyclin Dl can bind to and stimulate the estrogen receptor, activating the

transcriptional activity of the receptor. Importantly, this activity is independent of cdk4

activation (Neuman et al., 1997).

Thus, one can hypothesize that the repression of cyclin Dl in a pRb-null cell will

confer a selective proliferative advantage. This hypothesis is consistent with our

observation that the loss of cyclin D 1 expression in a pRb-null MEF, expressing ectopic

Myc, correlates with an increased proliferative potential (Marhin et al., 1996). Thus, the

cooperation of these two genetic events, Myc-activation and loss of pRb, may constitute a

novel mechanism for tumorigenesis, a mechanism which appears to require the repression

of cyclin Dl. This model is consistent with observations made in surveys of human

tumour cell lines, where there exists a strong correlation between the loss of pRb and the

absence of cyclin Dl expression. This hypothesis poses three questions: first, what is the

mechanism of cyclin Dl repression? Second, the cooperation of Myc-activation and pRb

loss is a novel pathway to enhance tumorigenesis. Is there evidence to support this

relationship in known tumors? Third, is cyclin Dl expression in apRb-null Myc-activated

cell growth inhibitory?

The question as to the mechanism of cyclin Dl repression is a difficult one to

answer. Repression is mediated via mechanisms which affect either mRNA or protein

expression, suggesting that secondaly genetic aberrations within each clone may influence

which pathway is adopted. As the ultimate goal appears to be the loss of cyclin Dl

activi~y, cyclin Dl repression may be effected by one of a multiple array of mechanisms,

depending upon the history of the cell. As such, it is not immediately apparent what can be

gained from characterizing one out of potentially multiple repression mechanisms in one or

two MEF cultures.

To determine if the association of Myc-activation and loss of pRb expression, with

the concomitant loss of cyclin Dl expression, is evident in vivo in known tumour samples

or hunorigenic cell lines, these cell sources will be surveyed to support the existence of this

relationship as a potentially new mechanism for tumorigenesis. In vivo evidence for the

existence of this regulatory association can be found in the cervical carcinoma cell line

HeLa, which possesses deregulated Myc expression, lacks pRb activity, and does not

exhibit cycli D 1 expression.

Our studies in cell culture have revealed a potentially novel mechanism for

tumorigenesis. What is the contribution of each player: Myc-activation and pRb loss, to

this mechanism? Ectopic expression of Myc did not impart an enhanced proliferative

potential to subconfluent asynchronously growing cultures of wildtype MEFs, yet ectopic

Myc expression in pRb-null cells resulted in a dramatic reproducible increase in the

proliferative potential of these cells. What is the reason for this phenomenon? This

question could be addressed by assaying the expression patterns of players in the cell cycle

machinery in control and Myc-activated pRb-null MEFs. In particular, the expression

patterns of cyclins, cdks, cdkls, the temporal activation of the cdks, and the activity of

activating and inhibitory kinases such as the cdc25 and wee1 group of kinases, throughout

the cell cycle will be ascertained. Earlier studies have demonstrated that the specific loss of

cyclin Dl expression will accelerate the transition of pRb null cells into S phase (Lukas et

al., 1995). Is this the reason for the increased proliferative and tumorigenic potential of

Myc-activated pRb-null MEFs? This question can be directly addressed by inhibiting the

expression of cyclin Dl in pRb-null MEFs, through the constitutive expression of

antisense cyclin Dl (Arber et al., 1997; Driscoll et al., 1997; Kornmann et a]., 1998;

Skotzko et al., 1995). These cells can then be assayed for their proliferative and

tumorigenic potential through live cell counts, and their ability to form foci and grow in

soft agar. This analysis will help to determine the molecular basis for the significant

difference in growth potential exhibited in these cells.

The increased expression of cyclin Dl in both senescent fibroblasts and upon

cellular differentiation in a variety of cell types suggests that cyclin Dl may exert a

heretofore uncharacterized function in growth arrest states, associated with senescence and

differentiation (Freeman et al., 1994; Fukami et al., 1995; Lucibello et al., 1993; Xiong et

al., 1997). Cyclin Dl expression is induced upon differentiation in the promyelocytic cell

line HL60, in C2C12 muscle cells, in the rat hippocampal cell line H19-7, and in PC12

pheochromocytoma cells (Horiguchi-Yamada et al., 1994; Jahn et al., 1994; Tamaru et al.,

1994; van Grunsven et al., 1996; Xiong et al., 1997; Yan and Ziff, 1995). This

mechanism appears to be distinct from its function as a regulator of pRb phosphorylation

(Skapek et al., 1996). These observations provide correlative evidence to suggest a role

for cyclin Dl in regulating growth arrest associated with cellular differentiation. Indeed,

cyclin Dl has been implicated in the inhibition of cell proliferation, as ectopic expression of

cyclin Dl in immortalized fibroblasts or mammary epithelial cells inhibits growth (Han et

al., 1995; Quelle et al., 1993; Resnitzky et al., 1994). What is the role of cyclin Dl in

instances of growth arrest such as differentiation and senescence? The answer is not clear

as recent reports have indicated that cyclin Dl can participate in an array of activities

independent of cdk activation. For example, cyclin Dl can interact with the estrogen

receptor inducing estrogen receptor transcriptional activity via a mechanism which is

independent of cdk activation (Neuman et al., 1997). Cyclin Dl can also bind to the

proliferating cell nuclear antigen (PCNA), and the novel transcription factor, cyclin D-

interacting myb-like protein (DMPI) (Hirai and Sherr, 1996; Inoue and Shen; 1998). In

addition, in differentiating neurons, cyclin Dl binds to cdk5, a cdk which is required for

neuronal differentiation, yet does not activate its intrinsic kinase activity (Xiong et al.,

1997). Thus, emerging evidence has expanded the biological activities of cyclin Dl.

Hence one of these activities, or an as yet unidentified cyclii Dl function, may explain the

role of cyclin Dl in growth arrest staks. Analysis of the role of cyclin Dl in differentiating

systems will potentially expose and delineate novel cyclin-dependent mechanisms.

Consequently, we may be able to translate these findings to aid our understanding of the

role of cyclin Dl not only in cell differentiation but also in the control of cell cycle

progression.

5.3 Characterization of the repression of PDGF-PR expression by

MY c

The activated PDGF-PR confers a strong proliferative response in a variety of cell

types. Moreover, deregulated activation of the PDGF-PR has been implicated in the

promotion of tumorigenesis (Guha et al., 1995; Silver, 1992). Hence, the suppression of

this gene by Myc represents a novel example of how Myc is tasked by the cell with

regulating a proliferative response through the inhibition of the proliferative stimulus at its

source, the cell surface. This is the second example of the involvement of Myc in a

negative regulatory pathway. The function of Myc in negatively regulating its own

transcription has been well described (Facchini et al., 1997; Penn et al., 1990; Penn et al.,

1990). However, in contrast to Myc negative auto-regulation, little is known of either the

mechanism or function of the Myc-mediated repression ofpdgf-PR mRNA expression.

The answers to these questions will be pursued in future.

5.3.1 Biological role of PDGF-PR suppression by Myc following ligand

stimulation

The repression of pdgf-pr mRNA by Myc potentially represents a negative

feedback mechanism through which the proliferative potential of PDGF-PR signalling can

be controlled. In addition, this mechanism will also serve to restrict PDGF-PR expression

to cellular growth states during which PDGF-PR expression is required to provide a

proliferative stimulus. Indeed, PDGF-PR expression is elevated in confluent, quiescent

fibroblasts compared to exponentially proliferating cells (Vaziri and Faller, 1995). The

elevated expression levels in growth arrested cells pennit the cell to respond quickly to the

presence of mitogens in the external environment, ensuring that the cell will be able to

quickly re-enter the cell cycle.

Is the mechanism of PDGF-PR repression by Myc necessary? Indeed, what are the

consequences of enforced PDGF-PR expression in a cell which is induced to enter the cell

cycle following mitogen-stimulation? To address this question, PDGF-PRs would be

ectopically expressed in Rat-1 cells, and the effect of deregulated PDGF-PR expression on

cell proliferation and turnongenesis in exponentially growing cells ascertained. Analysis of

tumors which exhibit deregulated PDGF-PR expression demonstrated that constitutive

PDGF-PR signaling constitutes a potent tumorigenic mechanism. This observation

provides correlative evidence to support the hypothesis that the repression of PDGF-PR by

Myc may serve to curtail a putative tumorigenic program.

5.3.2 Analysis of the mechanism of pdd-fir mRNA suppression by Myc

The repression of the pdgf-br by Myc occurs at the transcriptional level, and

requires sequences found between nucleotides -1994 and -396, relative to the translation

start site. The transcriptional start site as yet has not been precisely determined and lies

between nucleotides -421 to -41 1. The mousepdgf-pr promoter contains a variety of

putative transcription factor binding sites (Ishisaki et al., 1997). To determine which

sequences within this region are required for the repression of pdgf-pr by Myc, we

undertook deletion analysis of the mousepdgf-br promoter. The transcriptional repression

of thepdgf-pr gene by Myc appears to be mediated through sequences contained within

nucleotides -745 and -396 of the mouse pdgf-pr promoter. This region also contains

sequences necessary to maintain basal transcription. Repression is potentially effected

through one of the three putative transcription factor binding sites in this region. One of

these sites, the NF-Y site is particularly intriguing because of findings from two studies.

First, the NF-Y site is reported to be the sole element required for the maintenance of basal

transcription of thepdgf-jr gene in NIH3T3 cells (Ishisaki et al., 1997). Second, Myc can

repress the activity of the CTFNFI family of transcription factors, of which NF-Y is a

member (Yang et al., 1991; Yang et al., 1993). Myc can effect the phosphorylation of

NFl, another member of the CTFINFI family, inhibiting the ability of this transcription

factor to transactivate the expression of the pro-alpha 2(I) collagen gene. Hence, NF-Y is a

highly probable candidate through which Myc may repress transcription of thepdgf-Dr

gene.

Myc has also been reported to inhibit the activity of components of the basal

transcription machinery such as the Initiator binding proteins, Miz-1 and TFII-I, as well as

the TATA-binding protein (TBP), via direct association (Hateboer et al., 1993;

Maheswaran et al., 1994; Roy et al., 1993; Schneider et al., 1997). However, the absence

of both an initator element or a TATA-box in the mousepdgf--P,. promoter excludes these

pathways. To ascertain which sequences are cmcial for the Myc-mediated repression

mechanism, each of the putative transcription factor binding sites APl, AP2, and NF-Y in

this 31 5 bp region could be specifically altered via site directed mutagenesis. The effect of

these mutations on transcription from thepdgf-pr promoter will be assayed via promoter

luciferase assays in Rat-1 cells expressing an inducible Myc construct. Ascertaining

whether the NF-Y site is the site required for the repression of thepdgf-pr gene by Myc

will be difficult, as deletions in this site may abolish basal transcription in Rat-1 cells as

was reported in the NIH3T3 cell line.

An alternative approach, DNA-footprinting, will also be employed to analyze the

fragment of the mousepdgf-pr promoter comprising nucleotides -745 and -396. Using

nuclear lysates extracted from either control or Myc-activated rodent fibroblasts, the sites

on this promoter fragment which are protected with each nuclear extract will be compared

and consequently elements which are responsive to Myc identified. The responsiveness of

these elements to Myc can be assayed in promoter reporter assays, by fusing these

elements to heterologous promoters driving a reporter gene and performing transient

transfection promoter reporter assays. To identify sites which are specifically bound by

Myc or Myc containing complexes, electrophoretic mobility shift assays with nuclear

lysates from control and Myc-activated rodent fibroblasts will be performed. The presence

of Myc in DNA bound complexes can be assayed through the use of specific antibodies to

Myc; these will either produce a supershift in Myc containing complexes or will eliminate

these DNA bound complexes. Identification of the elements in thepdgf-P. promoter which

are responsive to Myc repression will allow us to better comprehend the mechanism via

which Myc represses gene transcription and consequently how Myc repression

contribute to cellular transformation.

5.4 Characterization of the mechanism of gadd45 repression by Myc

The repression of the gadd45 gene by Myc signals a change in direction in the M

field, as it reveals a novel function for Myc: the suppression of a growth arrest gene

(Marhin et al., 1997). Thus, Myc may promote cell proliferation by two pathways: first,

Myc induces genes which are required for progression through the cell cycle, and second

Myc represses the expression of genes which effect or maintain growth arrest, keeping

cells in cycle. Logically, both pathways must be equally essential, as a Myc enforced

initiation of a proliferative program in the presence of a strong block to growth, provided

by growth arrest genes, will induce apoptosis (Evan et al., 1992). Therefore, Myc-

mediated transcriptional repression and transactivation may both play an essential role not

only in enhancing cell proliferation, but at a more fundamental level, permitting normal cell

proliferation. Another member of the gadd group of genes, gadd153, and the fhctionally

related gene gasl, have also been shown to be repressed in response to Myc-activation

(Chen et al., 1996; Lee et al., 1997). However, whether the gaddgenes or gasl are direct

targets for Myc repression has to be ascertained. Not all of the known growth arrest genes

are suppressed by Myc. Preliminary studies indicate that the effect of Myc on the

expression of gas3 and gas6 is negligible (WM, unpublished results). Thus, Myc can

suppress the expression of specific growth arrest genes, potentially enhancing cell

proliferation.

5.4.1 Biological role of gadd45 suppression by Myc following mitogen

stimulation

Gadd45 is a physiologically important target for Myc repression. Gadd45

expression is associated with a variety of growth arrest states, suggesting that gadd45 may

play a role in the initiation or maintenance of growth arrest (reviewed in Fornace, 1992;

Papathanasiou and Fornace, 1991). Indeed, ectopic expression of gadd45 is growth

inhibitory in both immortalized fibroblasts and in tumorigenic cell lines, emphasizing its

strong growth inhibitory impetus (Vairapandi et al., 1996; Zhan et al., 1994). Yet, gadd45

is only one member of a group of growth arrest specific genes, which are functionally

highly related. Given that the growth arrest genes gadd153, and gas1 are also repressed by

Myc (Chen et al., 1996; Lee et al., 1997), what is the biological importance of the

repression of gadd45 by Myc? Is the repression of gadd45 by Myc required for cell cycle

progression?

To address this question, gadd45 will be ectopically expressed in control and Myc-

activated Rat-1 cells to ascertain the consequences of co-expression of a strong proliferative

with a strong growth inhibitory stimulus. I anticipate that, similar to the observations made

in NIH3T3 cells (Vairapandi et al., 1996), ectopic gadd45 expression in Rat-1 cells will be

growth inhibitory. Indeed, ectopic expression of the functionally related gene gadd153 in

rodent fibroblasts inhibited the propagation of these cells, hinting that ectopic gadd45

expression may elicit an analogous phenotype (Chen et al., 1996). Co-expression of

ectopic Myc and gadd45 in Rat-1 cells may elicit one of three outcomes: first, the growth

inhibitory effect of gadd45 expression will dominate, driving the cells into growth arrest.

Evidence to support this outcome arose from studies which have shown that ectopic

gadd45 expression in the hunorigenic Myc-activated cell line HeLa is growth inhibitory

(Zhan et al., 1994). Alternatively, the proliferative impetus of Myc may predominate,

overcoming a gadd45-induced block to cell proliferation. Again we could make some

predictions based on studies of the ectopic expression of Myc and gadd153 in rodent

fibroblasts (Chen et al., 1996). The gaddl53-induced block to cell proliferation was

rescued by ectopic Myc expression. Cells co-expressing Myc and gadd153 were able to

progress through the cell cycle and proliferate, yet the rescue was incomplete as these cells

exhibited prolonged GI and G2 phases. Another alternative outcome would be that the

conflict in growth signals may elaborate an apoptotic mechanism, a program distinct from

that dictated by gadd45 or Myc alone. A conflict between growth promoting and inhibitoly

stimuli is a well characterized trigger of a Myc-dependent apoptotic pathway (Evan et al.,

1992). Thus, there exist parallel studies which can support each of these hypothetical

outcomes, and as such each is equally possible.

Two of the three possible outcomes involve a gradual loss in cell viability, therefore

the growth of cells expressing ectopic gadd45 must be assayed quickly following

transduction of the gadd45 gene into Rat-1 cells. For this reason, control and Myc-

activated Rat-l cells will be infected with recombinant retroviruses which will deliver two

cDNAs into the cell, the gadd45 cDNA together with a cDNA encoding the green

fluorescent protein (GFP). Following infection, gadd45 expressing cells will be isolated

on the basis of GFP expression using a fluorescence based cell sorter. Pooled populations

will be derived, their growth assayed, and the growth response characterized.

5.4.2 Myc abrogates the stress induced upregulation in gadd45

expression

We have demonstrated that ectopic Myc expression can retard the induction of

gadd45 in Rat-1 cells exposed to ionizing radiation (Marhin et al., 1997). The biological

significance of this mechanism is unclear. Exposure of both control and Myc-activated

Rat-1 cells to ionizing radiation, resulted in apoptosis in both cell populations. However,

Rat-1 cells expressing ectopic Myc are more sensitive to exposure to ionizing radiation. At

a dose of 10 Gy, the Dl0 value (the dose at which only 10 % of the population survives)

for control Rat-1 cells is approximately 2 Gy greater than that of Myc-activated cells (WM,

unpublished data). The reason for the increase in sensitivity is not known. It is interesting

to hypothesize that the decreased expression of gadd45 following exposure to ionizing

radiation in Myc-activated cells may play a role in the initiation of the apoptotic mechanism.

One can envisage a model where the induction of gadd45 following exposure to DNA-

damaging agents such as ionizing radiation, serves to arrest cell growth, permitting the cell

to repair damaged DNA prior to progression into S phase. However, in a Myc-activated

cell, ectopic Myc expression confers a strong proliferativc stimulus, tempers gadd45

induction, inhibiting growth arrest, and DNA repair, driving the cells into the cell cycle.

The Myc-driven inability to arrest growth to repair damaged DNA prior to cell division may

trigger an apoptotic program.

To determine if the reduced expression of gadd45 in Myc-activated cells is

responsible for the increased sensitivity of these cells to ionizing radiation, control Rat-1,

Myc-activated Rat-1, Rat-1 cells expressing ectopic gadd45, and Rat-1 cells co-expressing

Myc and gadd45 will be exposed to 10 Gy of ionizing radiation. The growth of each of

these three cell culhlres will then be monitored to determine if the deregulated expression of

gadd45 could inhibit the apoptotic response. The caveat of this study is that one will be

capable of co-expressing Myc and gadd45 in Rat-1 fibroblasts without inducing apoptosis.

This concern will be addressed in the earlier described studies.

5.4.3 Analysis of the mechanism of gadd45 mRNA suppression by Myc

The suppression of gadd45 transcription by Myc is dependent upon sequences

located within a 1.8 Kb Xba I fragment of the hamster gadd45 gene, consisting of

nucleotides - 1675 to +149, relative to the transcription start site. Thegadd45 promoter is

relatively simple, possessing neither a TATA box, nor an initiator element. In addition,

there are only a few putative transcription factor binding sites. An analysis of thegadd45

promoters derived from human, hamster, and mouse species revealed the presence of a

conserved OCT, CAT, OCT, CAT motif located within 100 bp of the transcription start

site. To determine which sequences within the hamstergadd45 promoter are required for

the repression ofgadd45 expression by Myc, we employed deletion analysis of the 1.8 Kb

Xba I fragment of the hamstar gadd45 promoter. The sequences which are responsive to

Myc repression are located within a region of the promoter, consisting of nucleotides -222

to +149, which contains elements required for basal transcription.

Further analysis is required to reveal the sequences which are required for Myc

repression of gadd45 transcription. The relatively large region of the gadd45 promoter

consisting of nucleotides -222 to +I49 requires additional characterization via progressive

deletion analysis, and site-directed mutagenesis of the only conserved sequences within

this region, the OCT and CAT sites. Deletion or mutational analysis of this region is

potentially difficult, as the elements required for Myc repression may overlap with elements

which are required for mediating basal transcription. Thus, alternative approaches such as

DNA-footprinting, and electrophoretic mobility shift assays can be employed, via an

identical approach as proposed earlier for the analysis of thepdgf-B. promoter.

5.5 Models of Myc repression

The mechanism(s) through which Myc represses gene transcription have not been

fully determined. It is clear that contrary to Myc transactivation of gene expression, Myc

repression is not dependent upon the presence of a consensus E box, CACGTG, in the

target gene. Furthermore, analysis of the few Myc-repressed genes that have been

identified, has revealed that Myc-mediated repression may be effected through multiple

pathways. For example, repression of cyclin Dl by Myc is mediated through the initiator

element (Inr), and does not require heterodimerization with Max (Philipp et al., 1994).

Conversely c-myc negative autoregulation is not mediated through the Inr, yet binding to

Max is obligatory for this repression activity to occur (Facchini et al., 1997). What are the

possible mechanisms through which Myc represses gene transcription? Myc may repress

gene transcription via direct or indirect mechanisms.

Myc may repress gene expression by inhibiting the activity of enhancers or

initiators of gene transcription. Indeed, a variety of reports have demonstrated that Myc is

capable of fulfilling this role. Myc can interact with and inhibit the activity of the

transcriptional regulators AP2, YY1, Miz-1, TFII-I, and TBP (Gaubatz et al., 1995;

Hateboer et al., 1993; Maheswaran et al., 1994; Roy et al., 1993; Schneider et al., 1997;

Shrivastava et al., 1993; Shrivastava et al., 1996). Myc can inhibit the activity of the

enhancer AP-2 via two mechanisms (Gaubatz et al., 1995). First, as we have described

earlier, AP-2 is a Myc interacting protein. The interaction of Myc with AP-2 renders AP-2

incapable of binding to DNA and activating transcription. Alternatively, in the a-

prothymosin promoter there are overlapping AP-2 and Myc binding sites, hence both

factors compete for binding to this promoter. Thus, the interaction of Myc with AP-2 can

repress gene expression via both DNA-dependent as well as independent mechanisms.

Myc can also mediate the repression of gene transcription through Inr elements.

The list of promoters which belong to this category include: adenovirus 5 major late

promoter (AdMLP); cyclin Dl; clebpcr, serum albumin; terminal deoxy-transferme (Tdl);

and A5 (Antonson et al., 1995; Li et al., 1994; Mai and Martensson, 1995; Philipp et al.,

1994). Myc is reported to repress initiat0r-mfXbted transcription through the interaction

with Inr binding proteins including W 1 , Miz-1, and TFII-I (Roy et al., 1993; Schneider et

al., 1997; Shrivastava et al., 1993; Shrivastava et al., 1996). Both W1 and Miz-1 bind

specifically to Inr elements, initiating gene transcription. The interaction of Myc with either

of these factors directly curtails the transactivation role of these proteins, potentially

through the inhibition of contacts between YY1 or hypothetically Miz-1 with components

of the basal transcription machinery such as TBP or TFIIB (Shrivastava et a]., 1993;

Shrivastava et al., 1996). On TATA-less promoters, the transcription initiation factor

TFII-I, binds to the Inr and initiates formation of the RNA polymerase I1 pre-initiation

complex at the transcription start site. Myc complexes with and sequesters TFII-I,

preventing the initiation of transcription (Roy et al., 1993). In an analogous fashion, with

TATA-containing promoters, Myc can also directly bind to and sequester TBP, inhibiting

formation of the RNA polymerase I1 pre-initiation complex (Hateboer et al., 1993;

Maheswaran et al., 1994). These studies illustrate that Myc can interact with a variety of

proteins influencing the formation or activity of the basal transcription complex. Thus, this

may constitute the mechanism through which Myc effects transcriptional repression on a

variety of genes, yet it must be noted that all of these studies were conducted using isolated

promoters rather than the full genes. Hence, the significance of these mechanisms in Myc

repression and ultimately the biological activities of Myc need to be further characterized.

Myc can also indirectly inhibit the expression of genes by either repressing an

enhancer of transcription or inducing a repressor of transcription. Myc has been reported

to repress the transcriptional induction of specific genes through the inhibition of the

expression of transcriptional enhancers. During the differentiation of the pre-adipocyte cell

line, 3T3-L1, the transcription factor clebpa is induced (Cao et al., 1991; Christy et al.,

1991; Christy et al., 1989). Clebpa is responsible for the transcriptional induction of both

growth arrest genes such as gadd45 and differentiation specific genes such as mim-I

(Christy et al., 1989; Constance et al., 1996). Myc indirectly represses the induction of the

gadd45 gene by repressing the expression of clebpa (Constance et al., 1996). The Myc-

mediated repression of clebpa is mediated through either the Inr element or interactions

with components of the basal transcription machinery (Antonson et al., 1995; Li et al.,

1994). Myc is also capable of directly inhibiting the ability of clebpa to activate the

transcription of these genes via an unknown mechanism (Mink et al., 1996). Myc has also

been reported to modulate the phosphorylation state of the enhancer CTFMFI, inhibiting

the ability of this transcription factor to drive the expression of the pro alpha 2(I) collagen

gene (Yang et al., 1991; Yang et al., 1993). The most revealing approach to determining

the mechanism through which Myc represses the transcription of the gadd45 andpdgf-br

genes would be EMSAs as I have described earlier. EMSAs will demonstrate firstly, if

Myc represses gene expression through the regulation of complex formation on the

promoters of these genes and secondly, whether Myc effects these modulations via direct

or indirect means.

To date the analysis of the few genes which have been reported to be repressed by

Myc has exposed only a few components of Myc-dependent repression mechanisms. As

such, the delineation and understanding of how Myc represses gene transcription is still in

its infancy, primarily due to the limited number of genes that have been identified as targets

of Myc repression. It will be through the identification and characterization of additional

targets for Myc repression such as gadd45 and thcpdgf-br that we will be able to reveal

common themes from among seemingly different repression mechanisms to fully elaborate

the pathways of Myc repression.

5.6 Does Myc transactivation or Myc repression lead to cell

transformation?

The transforming domains of the c-Myc protein were initially identified via deletion

analysis in rat embryonic fibroblasts (Stone et al., 1987). The regions which were

required for cellular transformation include the amino ternlinal amino acids 106 to 143

(MBII), and the C-terminal89 amino acids. The amino terminal domain was subsequently

discovered to function as a transcriptional activation domain in transient transfection

reporter assays. When fused to the DNA binding domain of the yeast Gal4 transcription

factor, this domain can transactivate a reporter gene containing Gal4 DNA-binding sites

(Kato et al., 1990). Yet the link between Myc-mediated transformation and transactivation

is not concrete, as there are Myc mutants which can disassociate these two functions.

Ectopic expression of an N-terminal262 amino acid fragment of Myc will competitively

inhibit MycIRas cotransformation (Brough et al., 1995). Moreover, this inhibition is

dependent upon the presence of Myc box I1 amino acids 129 to 145. Yet, this Myc mutant

when fused to the DNA-binding domain of the yeast transcription factor GAL4, is able to

transactivate a heterologous reporter gene driven by GAL4 DNA binding sites. Thus,

MBII is required for Myc-dependent cell transformation, but is dispensable for the

transactivation of a synthetic promoter.

In the amino-terminus of Myc can also be found regions which are necessary for

Myc repression of gene expression. The domains required for repression are different for

each gene, mapping to amino acids 92 to 106 for cyclin Dl, but amino acids 122 to 143 for

the AdMLP (Li et al., 1994; Philipp et al., 1994). Further analysis of specific amino acid

mutations in Myc revealed a stronger correlation between Myc transformation and Myc

repression in comparison to Myc transactivation. For example, a Phe-115 mutation

enhanced both the transcriptional repression and transformation capabilities of Myc (Lee et

al., 1996).

Does Myc-mediated transcriptional repression or transactivation drive neoplastic

transfomatio-9 This question may be addressed in two ways. First, the identification of

additional g~netic targets for Myc repression may reveal novel functions of Myc which

directly contribute to cellular transformation. Second, preliminary mutational analysis of a

few mutants of the Myc protein has suggested that a structure-function analysis of the Myc

protein may be an ideal approach to dissect the regions of the Myc protein which are

required for Myc repression, transactivation, and transformation. Undertaking a structure

function analysis of the Myc protein with greater resolution, utilizing smaller deletions and

site directed mutagenesis may be the key to determining whether the domains required for

neoplastic transformation overlap with the regions required for Myc repression or Myc

transactivation.

5.7 Perspectives

The product of the c-myc proto-oncogene is a highly regulated protein which is

essential for cell proliferation, and has been implicated in the genesis and progression of

tumors arising from a wide variety of cell types. Delineating the mechanisms through

which Myc elicits cell proliferation in normal cells will aid in our understanding of the role

of Myc in tumorigenesis. In the last 18 years of Myc research, our progress in realizing

this goal has not been linear, but punctuated by bursts of discovery which have provided

us with novel views on the role of Myc.

More recently, the Myc field has embarked in a new direction centered around the

ability of Myc to abrogate molecular blocks to cell proliferation. Myc exerts this function

in three ways: directly associating with and inhibiting the activity of transcriptional

activators, bypassing the growth inhibitory effects of the cdkI proteins, and the repression

of gene transcription. Our understanding of these mechanisms is still in its infancy, the

role of the reported Myc binding proteins in the context of the biological functions of Myc

is uncertain. Likewise, the Myc-mediated circumvention of cell cycle blocks induced by

assorted cdkls has not been fully characterized. Yet, correlative evidence does suggest that

the ability of Myc to overcome a p27-induced arrest is indirectly dependent upon the

transcription regulatory activities of Myc. Myc repression is an intriguing puzzle which

holds a great deal of promise, due to the correlative evidence linking Myc-mediated

repression to tumorigenesis. Through my research I have expanded our knowledge of the

contribution of Myc repression to the activities of Myc in enhancing normal cell

proliferation. The repression ofpdgf-Pr expression by Myc represents a putative feedback

mechanism. Myc, as the endpoint of a potent proliferative signal transduction pathway,

can tightly control this stimulus by repressing the expression of the receptor which initiated

the signal, hence maintaining normal cell proliferation. In addition, Myc can also

contribute to an enhanced proliferative and tumorigenic potential. Myc-activation and pRb

loss appear to cooperate to repress cyclin Dl activity, and enhance turnongenic potential as

evidenced by loss of contact inhibition. Lastly, Myc represses the expression of growth

arrest specific genes, such as gadd45, gadd153, as well as gasl ; which are potent

inhibitors of cell proliferation and viability. The growth arrest genes have physiologically

important functions, antagonizing the activity of genes which drive cell proliferation.

Normal cell proliferation is achieved through the fine balance between these two opposing

stimuli, hence the repression of growth arrest genes by Myc helps to explain how Myc

promotes enhanced cell proliferation and consequently tumorigenesis. Yet it remains to be

determined if these genes are directly repressed by Myc and whether they are all repressed

via the same or different mechanisms.

The abrogation of growth arrest mechanisms by Myc expands our understanding of

the proliferative potential of Myc, that Myc may promote cell growth through both the

repression and activation of the functions of specific proteins. The circumvention of cell

cycle inhibitors by Myc will keep cells out of growth arrest, and the induction of growth

promoters such as: E2Fs; cdc25A; cad and ode promotes the transition of cells through G1

and into S phase of the cell cycle. These new developments arrive at a time when concerns

were emerging as to the part Myc transcriptional regulation plays in effecting the biological

activities of Myc. The chronic absence of biologically important targets for transcriptional

regulation by Myc, and the futility of current means of identification of Myc regulated

genes should prompt us to rethink conventional approaches and our views of how Myc

may function. Indeed the c-Myc deficient fibroblasts may represent a powerful tool to

screen and identify the biologically important genes from among the existing pool of

reported Myc regulated targets. In addition, this system may result in the identification of

novel targets for Myc, which wil: ultimately allow us to understand Myc function.

Eventually, with the adoption of novel -views and approaches we will develop the tools to

unravel the enigma that is Myc.

Chapter 6

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