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Cell. Vol.

69, 119-128,

April 3, 1992. Copyright

0 1992 by Cell Press

Induction of Apoptosis
by c-myc Protein

in Fibroblasts

Gerard I. Evan, Andrew H. Wyllie,t


Christopher
S. Gilbert,* Trevor D. Littlewood,
Hartmut Land, Mary Brooks,*
Catherine M. Waters,
Linda 2. Penn,
and David C. Hancock*
*Imperial Cancer Research Fund Laboratories
44 Lincolns Inn Fields
London WC2A 3PX
England
tDepartment of Pathology
University Medical School
Teviot Place
Edinburgh EH8 9AG
Scotland

Summary
Although Rat-l fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their
numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by
apoptosis.
Regions of the c-myc protein required for
induction of apoptosis overlap with regions necessary
for cotransformation,
autoregulation,
and inhibition of
differentiation,
suggesting that the apoptotic function
of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more
prone to cell death upon serum deprivation.
Finally,
we demonstrate
that deregulated
c-myc expression induces apoptosis in cells growth arrested by a variety
of means and at various points in the cell cycle.
Introduction
The c-myc gene is the cellular homolog of the viral oncogenev-myc, which is found in a number of avian and feline
retroviruses that induce leukemias and carcinomas. Recent evidence strongly suggests that the c-myc protein
(Myc) is a transcription factor (Blackwood and Eisenman,
1991). It possesses a number of functional domains found
in other proteins modulating transcription, specifically the
leucine zipper characteristic of the Fos-Jun-CREB
transcription factor families (Landschulz et al., 1988) and the
basic-helix-loop-helix
motif found in, for example, the
MyoD and E box enhancer-binding
proteins (Murre et al.,
1989). Recently, both a heterodimeric partner (Blackwood
and Eisenman, 1991) and a consensus DNA-binding sequence for Myc (Blackwell et al., 1990; Prendergast and
Ziff, 1991) have been identified. However, it is still unknown precisely which genes are regulated by Myc or to
what biological end.
The c-myc oncogene has been implicated in the control
of normal cell proliferation by many studies. In particular,
it is one of the immediate early growth response genes

that are rapidly induced in quiescent cells upon mitogenic


induction, suggesting that it plays some role in mediating
the transition from quiescence to proliferation. However,
unlike the majority of immediate early growth response
genes, expression of c-myc is not confined to a brief period
during the Go/G1 transition. Although a peak of c-myc expression in fibroblasts is observed some 3 hr after mitogenie stimulation, both c-myc mRNAand protein are continuously present at an appreciable level throughout the
cell cycle in proliferating cells. As both c-myc mRNA and
protein have very short half-lives in fibroblasts (Moore et
al., 1987; Persson et al., 1986; Waters et al., 1991), this
sustained presence of Myc protein can only result from
continuous synthesis. Ectopic induction of Myc activity is
sufficient to drive quiescent growth factor-deprived
fibroblasts into the cell cycle (Eilers et al., 1991). This argues
that Myc regulates genes mediating the mitogenic response, an idea consistent with the proteins rapid induction by mitogens in quiescent cells. In addition, sustained
expression of Myc can block both growth arrest and cell
differentiation programs, suggesting a role for Myc also in
regulating genes mediating both of these processes.
Untransformed fibroblasts respond to serum or mitogen
deprivation by growth arrest in a Gl-like state often termed
Go and can remain viable in this arrested state for extended
periods. Mitogen withdrawal is accompanied
by rapid
down regulation of c-myc expression at both the mRNA
and protein levels, irrespective of position within the cell
cycle (Dean et al., 1986; Waters et al., 1991). Because
cells deprived of growth factors eventually become quiescent, it has been suggested that Myc down regulation is
a requirement or even a signal for growth arrest (Freytag,
1988; Waters et al., 1991).
In tumor cells, elevated or deregulated expression of
c-myc (and occasionally other myc genes) is so widespread as to suggest a critical role for myc gene activation
in multi-stage carcinogenesis (Field and Spandidos, 1990;
Spencer and Groudine,
1991). Although it is unclear
whether it is deregulation or overexpression of c-myc that
makes up the major determinant in c-myc oncogene activation, it is nonetheless evident that c-myc activation disrupts the growth regulation of cells. For this reason, we
have investigated the consequences of deregulated and
elevated expression on the behavior of rodent fibroblasts.
In this report we show that deregulated c-myc expression
is a potent inducer of programmed cell death (apoptosis)
when combined with a block to cell proliferation. We discuss the implications of these findings in terms of the biological role of c-myc in normal and tumor cells.
Results
Constitutive
c-myc Expression Prevents Growth
Arrest in Serum-Deprived
Rat-l Fibroblasts
and Induces Cell Death
Growth arrest is accompanied by rapid down regulation

of

Cell
120

Rat- 1 /wt

Rat- 1 control

Figure 1. Growth Curves of Rat-l


Either with or without Constitutive
pression in Various Concentrations

myc

10%FCS

Tnplicateculturesofcontrol
Rat-l cellsor Rat-i
cells constitutively
expressing
wild-type c-myc
arc-mycmutantswereculturedin
mediumcontaining the various levels of FCS shown, and
live cells were counted at daily intervals, Mean
values of the triplicates, together with standard
errors, are shown plotted against time.

0.5% FCS
0.05% FCS

1 Rat-l/A106-143

Fibroblasts
c-myc Exof FCS

10% FCS

10% FCS

0.5% FCS
0.5% FCS

0.05% FCS

0.05% FCS

GROWTH

CURVES OF RAT-

1 FIBROBLASTS

EXPRESSING

VARIOUS

c-myc expression. We therefore investigated the ability of


Rat-l cells containing a normal human c-myc gene driven
by a constitutive promoter, and therefore unable to down
regulate c-myc expression, to arrest when deprived of mitogens. Control Rat-l cells (Rat-l Ineo) and Rat-l cells constitutively expressing wild-type c-myc (Rat-llmyc) or mutant c-myc were cultured at various serum concentrations,
and numbers of live cells were counted at daily intervals
(Figure 1). The c-myc deletion mutant Al 06-l 43 is inactive
in transformation
and autosuppression
assays, whereas
A145262 is active (Penn et al., 1990a; Stone et al., 1987).
Growth curves of all the cells appeared very similar, irrespective of whether or not the cells constitutively expressed active c-myc. This suggests that constitutive
c-myc expression has no effect on the ability of Rat-l cells
to slow their growth in low serum levels. To confirm this
finding, we examined the growth status of Rat-l cells and
Rat-llmyc cells in low serum for DNA content and bromodeoxyuridine (BrdU) incorporation by flow cytometry. The
results of this analysis are shown in Figure 2 and, paradoxically, appear to show that there is a complete block to
growth arrest in cells constitutively expressing c-myc. To
accommodate these two apparently conflicting results, we
examined the possibility that Rat-llmyc cells were dying
in low serum. Microscopic inspection of such cultures revealed this to be the case.
Myc-Induced
Cell Death Occurs by Apoptosis
The manner of cell death of Rat-llmyc cells in low serum
was examined by time-lapse cinemicroscopy. Logarithmic
Rat-llmyc cells were transferred into medium containing
0.1% fetal calf serum (FCS), and a field of about 120 cells
observed. Individual cell deaths occurred apparently randomly throughout the culture. The first cell deaths were
observed within 30 min of serum withdrawal, and cell
deaths then continued at a more or less constant rate,

MYC CONSTRUCTS

typically ending with death of the entire culture after about


6-9 days. Each death is rapid; the interval between apparent morphological normality and complete cell fragmentation is typically about 30 min (Figure 3A). Death begins
with loss of cell-cell contact. This is followed by nuclear
condensation, membrane blebbing, and cytoplasmic condensation and ends in cell fragmentation. All these characteristics were confirmed by transmission electron microscopy of dying cells (Figure 3C). DNA from dying cells was
analyzed and revealed fragmentation
of chromatin into
nucleosome
ladders (Figure 38). All the features described above are diagnostic of a form of programmed cell
death termed apoptosis (Bursch et al., 1990; Wyllie, 1987).
Identical apoptosis was seen in Rat-llmyc cultures in
which serum was replaced with adefined serum substitute
(data not shown) lacking mitogens and unable to support
fibroblast growth (Waters et al., 1991). Moreover, Rat-l/
myc fibroblasts could be viably maintained in serum-free
mediasupplemented
onlywith specificdefined
growth factors (data not shown). Both of these observations argue
that death is due to absence of necessary growth factors
rather than to depletion of metabolites.
Apoptosis
Is Dependent upon Active
Myc Expression
Thus far, all experiments had been conducted using independently isolated Rat-l clones. To confirm that cell death
was a property of c-myc expression rather than clonal variation, we examined cell death in Rat-l cells constitutively
expressing Myc-ER chimeras (Rat-l/myc-ER
cells). In
these cells, activity of the chimeric Myc protein is completely dependent
upon the availability of exogenous
6-estradiol (Eilers et al., 1989). In the absence of 8-estradiol, Rat-l/myc-ER
cells arrest in low serum in aGo/G1-like
state (data not shown) and remain viable for several
weeks. In the presence of 2 PM f3-estradiol, however, the

induction
121

of Apoptosis

by c-myc

Protein

A
Rat-llneo
0.1%

10%

serum

serm

control
for

far

Rat-l/my

48 hrs

0.1%

48 hrs

10%

serum

serum

Rat-llmyc cells, such REFlmyc fibroblasts fail to arrest


growth in low serum as determined by flow cytometry(data
not shown). As can be seen in Figure 5, substantial
apoptosis occurs within 24 hr of transfer into low serum.

c
for

for

48 hrs

48 hrs

ONA content -

Figure 2. Constituttve
Serum-Deprived
Rat-l

c-myc
Cells

Expression

Prevents

Growth

Arrest

in

Triplicate logarithmicculturesof
Rat-llneocontrol
and Rat-llmyccells
were transferred
into medium containing
either 10% or 0.1% FCS.
After 49 hr, cells were labeled for 1 hr with 2 mM BudR, trypsinized,
fixed in ethanol, and stained with propidium iodide and appropriately
confugated
anti-BudR antibody as described
(Ormerod,
1990). Flow
cytometric
analysis was carried out on a Beckton-Dickinson
FACSstar
plus. (A) DNA histograms
of Rat-l cells.(B) Percentage of cellsstaining
with anh-BrdU antibody (i.e., traversing
S phase) over a 1 hr perrod.

rapid onset of apoptosis is observed in low serum. In contrast, Rat-l fibroblasts expressing the transformationdefective mutant of Myc, A106-143
(Penn et al., 1990a;
Stone et al., 1987) fused to estrogen receptor (ER) (Rat-l/
A106-143-ER
cells) arrest in low serum and exhibit no
apoptosis irrespective of the presence of j3-estradiol (Figure 4). Thus, apoptosis of Rat-l/myc-ER
depends upon
the presence of active Myc protein and is not a trivial result
of the addition of j3-estradiol to the culture.
Deregulated
Expression
of Myc Induces Apoptosis
in Serum-Deprived
Rat Embryo Fibroblasts
Rat-l cells are an immortalized and established cell line.
We were therefore interested to determine how general
was Myc-induced apoptosis; in particular, whether it occurred in a nonestablished
primary fibroblast culture. Accordingly, primary rat embryo fibroblasts (REFs) constitutively expressing Myc were subjected to serum deprivation
and monitored microscopically over a 72 hr period. As with

Extent of Myc-Induced
Apoptosis
Is Related
to the Levels of Myc Protein in Cells
We next investigated whether there was any correlation
between intracellular Myc protein level and susceptibility
to Myc-induced
apoptosis in Rat-llmyc cells. Various
Rat-llmyc cell clones were selected, each of which expresses a different steady-state level of Myc protein (Penn
et al., 1990b). Each clone was then assayed for apoptosis
by two independent assays. First, cells were cultured in
various concentrations
of FCS, and the degree of cell
death was assessed by microscopic examination after 3
days. Results are shown in Figure 6A for the three Rat-l/
myc clones 21, 19, and 26, which representatively
span
the range of Myc protein levels investigated. Clone 26
expresses most Myc and exhibits significant apoptosis
even in serum levels as high as 2%. Apoptosis is even
more evident at lower serum levels. In contrast, clone 21,
which expresses a level of Myc protein similar to that found
in normal fibroblasts (Waters et al., 1991; Moore et al.,
1987), exhibits apoptosis only at the lowest serum levels.
Clone 19, with an intermediate Myc protein level, exhibits
an intermediate phenotype. Results obtained for four other
clones (2, 5, 12, and 1 l), each expressing a different Myc
level, were consistent with this trend (data not shown).
Second, the various Rat-llmyc clones were transferred
into 0.1% FCS, and fields containing identical numbers of
cells monitored for apoptosis by time-lapse cinemicroscopy. This type of analysis is complicated by the fact that
cell number is continuously varying because of both cell
division and cell death. For this reason, results were
pooled within each 2 hr time interval, and both cumulative
cell deaths and live cell numbers at the end of each 2 hr
interval were plotted against time. Data obtained using
clones 21, 19, and 26 is shown in Figure 6B. The rate of
apoptosis is highest in clone 26, intermediate in clone 19,
and lowest in clone 21. Again, results obtained with the
four other tested clones confirmed this positive correlation
between higher Myc levels and higher apoptotic rate (data
not shown).
Thus, we conclude that the sensitivity of Rat-l lmyc cells
to induction of apoptosis upon serum depletion and its rate
both depend upon the level of Myc protein expressed.
Even the low levels of Myc protein observed in normal
Rat-l fibroblasts are, however, sufficient to induce apoptosis in serum-deprived
cells if Myc expression is deregulated.
Regions of the Myc Protein Required for Apoptosis
Certain regions of the Myc protein are absolutely required
for its known activities in cotransformation,
autosuppression, and inhibition of differentiation (Freytag et al., 1990;
Penn et al., 1990a; Stone et al., 1987). These regions
include the basic-helix-loop-helix-leucine
zipper at the C
terminus and part of the N-terminal region. We examined
the ability of a range of Myc mutants to induce apoptosis

Cell
122

Figure 3. Myc Induces


Serum-Deprived
Rat-l

serum. Frame 1 shows a normal


myc cells at progressively
more

Rat-l /pMVG/wt

viable Rat-llmyc
cell. The nucleus is marked
advanced stages of apoptosis and exhibiting

c-myc-E

Rat-llpMV6/A106-143

N. Frames
cytoplasmic

c-myc-E

in

(A) Rat-l/c-myc
cells were transferred
to medium containing
0.1% FCS and observed
by
time-lapse
cinemicroscopy
at a rate of one
frame
every
30 seconds.
Representative
frames
from a typical apoptotic
event are
shown with the time in minutes given from the
last frame when the cell appeared normal.
(6) Cell death is accompanied
by nucleosome
laddering. Rat-l cells constitutively
expressing
either active A145-262
(lanes 2, 4, and 6) or
inactive A106-144
(lanes 3, 5, and 7) c-myc
mutants
were transferred
to medium
containing 0.1% FCS. Dying cells were harvested
at various times after transfer, by virtue of their
reduced adherence.
In cultures with no dying
cells, very few cells were harvested
by this
method. DNA was isolated and fractionated
on
a 1.5% agarose gel. Lane 1, standards;
Lanes
2 and 3, 0 hr; Lanes 4 and 5, 30 hr; Lanes 6
and 7, 40 hr; Lane 8, dexamethasone-treated
thymocytes.
(C) Electron microscopic
analysis of individual
Rat-llmyc
cells undergoing
apoptosis
in low

2 to 4 are representative
electron
and nuclear vesicularization.

Death by Apoptosis
Cells

micrographs

of Rat-11

Figure 4. Apoptosis in Serum-Deprived


Rat-II
myc Cells Is Dependent
upon Active Myc
Protein
Log-phase
Rat-l cells constitutively
expressing either full length c-myc protein or the deletion mutant A106-143
fused to ER were transferred to medium containing
0.1% FCS either
with or without 2 KM 8-estradiol.
After 3 days,
cultures were examined by phase microscopy.

Figure
duces
Cells

5. Deregulated
Myc Expression
InApoptosis
in Serum-Deprived
Primary

REFs constitutively
expressing
c-myc were cultured in medium containing 0.1% FCS for 72
hr. At various times, the culture was inspected
microscopically.
A, 0 hr; B, 24 hr; C, 48 hr; D.
72 hr.

inductron
123

of Apoptosis

by c-myc

Protein

B
CUMULATIVE

CELL DEATHS

when expressed constitutively in serum-deprived


Rat-l
cells. The results demonstrate a complete concordance
between those regions required for apoptosis and those
necessary for cotransformation,
autosuppression,
and inhibition of differentiation (Table 1). Thus, the ability of Myc
to induce apoptosis is mediated by similar domains of the
protein to those involved in other known functions attributed to the Myc protein.
Constitutive
Myc Expression
Induces Apoptosis
in
Rat-l Cells Arrested by Various Means at Various
Points in the Cell Cycle
Fibroblast proliferation can be temporarily blocked in a
number of mechanistically
different ways whilst maintaining viability (reviewed in Pardee, 1989). These include

three Rat-llmyc
clones is shown after transfer
to medium containing 0.1% FCS. Seventy-five
randomly picked live cells were selected at the
start of the experiment and these were followed
by time-lapse
cinemicroscopy
at a rate of 12
frames per hour. At the end of each 2 hr (24
frame) time interval, the total number apoptotic
events (top) and the total number of live cells
were summed and plotted against time.

Go arrest by serum deprivation, S phase block by thymidine excess, late G, block by isoleucine deprivation, interferon arrest in G,, and transient treatment with cycloheximide (Zetterberg
and Larsson, 1985). We examined
whether any of these procedures induced apoptosis in
Rat-l cells in a Myc-dependent
fashion. Rat-llmyc-ER
fibroblasts were maintained
in asynchronous
subconfluent logarithmic cultures for several days and then subjected to various types of proliferation block either in the
absence or presence of f3-estradiol. Cultures were then
examined for apoptosis at various appropriate time points
(Figure 7). Application of any of these growth-blocking
regimes activated apoptosis in a Myc-dependent
manner,
although the onset of apoptosis varied depending upon
the specific treatment. Significant apoptosis was visible

Cell
124

Table

1. Cotransforming

and Apoptosis-inducing

Activities

of Myc Mutants

Nature of
Mutation

Myc Mutant
wild type
A7-91
A41 -53

Transcriptional
modulation?
Loss of first Myc homology
box
Transcriptional
modulation?
No observed
effect
No observed
effect
Deletion of part of
helix-loop-helix
Deletion of leucine
zipper
interruption
of
leucine zipper

A106-143
A145-262
A26531 7
A371-412
A41 4-433
in414

within only 4 hr of treatment with cycloheximide


in cells
containing active Myc. In contrast, appreciable apoptosis
was visible only after 24-48 hr in cells starved of serum or
isoleucine or blocked with thymidine.
We next investigated whether Myc activation would induce apoptosis in cells already arrested and, if so, how
rapidly. Rat-l/myc-ER
cells were growth arrested by serum deprivation (Go), isoleucine starvation (G,), or thymidine block (S) for 48 hr in the absence of f3-estradiol.
Growth arrest and cell cycle position was confirmed by
flow cytometric analysis and by BrdU incorporation (data
not shown). 8-estradiol was then added, and the cultures
monitored for apoptosis by time-lapse cinemicroscopy
(Figure 8). Apoptosis is evident within 60 min of Myc activation in serum-starved cells and within 3-4 hr of Myc activation in isoleucine-starved
or thymidine-blocked
cells.
We conclude that Myc activation combined with any
tested growth-arrest procedure, either before or after the
late G, commitment point, leads to the rapid onset of
apoptosis. Moreover, it is not necessary for cells to be
actively cycling for Myc to activate programmed cell death.

Rat-IhI

myc-ER

no fl-rsrradiol

Rat-l/W

myc-ER

plus fl-cslmdiol

Rat-l/D106-143

Active in
Cotransformation

Active in Inducing
Apoptosis

+
+
-

+
+
-

Discussion
Deregulated
Myc Expression
Induces Apoptosis
in Fibroblasts Blocked in Proliferation
Our results demonstrate that c-myc can be a potent inducer of apoptosis in immortalized Rat-l fibroblasts and
primary REFs when combined with a block to proliferation.
We also see similar behavior in analogous experiments
with Swiss 3T3, NIH 3T3, and mouse embryo fibroblasts
(G. I. E., M. B., and T. D. L., unpublished data). Somewhat
similar observations have been recently made in a bone
marrow-derived
cell line (Askew et al., 1991).
Myc expression induces apoptosis both in proliferating
cells upon which a proliferation block is imposed and in
cells already arrested and in which Myc is subsequently
activated. In both cases, the effect isobserved irrespective
of the method used to implement growth arrest, whether
it be by growth factor or metabolite depletion or by the
action of a drug or chalone. Moreover, the fact that rapid
initiation of apoptosis by Myc occurs in cells arrested in
either G, or S phase argues that cells can enter a pro-

Figure 7. Myc-Dependent
Apoptosis
in Rat-l
Fibroblasts
Growth Arrested by Various Means

myc-ER

plus 0.eslradiol

Logarithmically
growing subconfluent
Rat-l/
myc-ER
and Rat-l/A106-143
myc-ER
cells
were growth blocked by the following means,
either in the presence
of absence
of 2 uM
6-estradiol as indicated. Thymidine block, DME
containing 10% stripped FCS plus 2mM thymidine. lsoleucine
starvation,
isoleucine-free
DME containing
10% dialyzed and stripped
FCS. Interferon, DME containing 10% stripped
FCS plus 2,000 U/ml recombinant
rat y-interferon. Cycloheximide,
DME containing
10%
stripped FCS plus 50 uglml cycloheximide.

Thymidine
Blocked
(48 hrs)

lsoleucine
starved
(60 hrs,

2Kulml
interferon

I60

y
hrs,

SOpglml
rycloheximide
(4 hrs)

Inductron
125

of Apoptosrs

EFFECT

A. Serum

by c-myc

Protein

OF MYC ACTIVATION
RAT- I /myc-ER

~starved

(GO)

GROWTH-ARRESTED
CELLS

C. Thymidtne-blocked

B. Isoleucine-starved

Figure 8. Ectopic Myc Activation Induces Apoptosis in Rat-llmyc-ER


Fibroblasts Already Growth Arrested by Serum Deprivation, Thymidine
Block, or lsoleucine
Starvation
Subconfluent
Rat-l/myc-ER
fibroblasts
were growth arrested either
by serum deprivation
(0.1% FCS for 48 hr), thymidine block (2 mM
thymidine
for 48 hr). or isoleucine starvation
(60 hr). Cells were observed by time-lapse cmemicroscopy
for the last 40 hr of this starvation
period, which revealed essentially a complete absence of cell division.
Growth arrest was further confirmed
by flow cytometry
(data not
shown). The medium was then changed and replaced with the same
growth-blocking
medium either with or without 2 pM !3-estradiol. The
cells were then observed
for a subsequent
35 hr, and apoptotic ceil
deaths recorded and summed for each 1 hr period. The cell number
indicated at start is the actual number of live cells followed from the
time of the start of recording.

grammed cell death pathway both before and after the


commitment point in late G, (Pardee, 1989).
Whenever we observe Myc-dependent
apoptosis, cell
deaths proceed over a fairly extended time span. This
may suggest that, although promoted by Myc, the exact
moment of commitment to apoptosis of any individual cell
is partially determined by certain stochastic factors. In the
case of serum-starved Rat-llmyc cells, those cells not dying continue to proceed through the cell cycle, consistent
with established mitogenic properties of c-myc (Eilers et
al., 1991). In proliferating asynchronous flat-llmyc cells,
the time of onset of apoptosis varies depending upon the
nature of the proliferation block imposed. We presume
that this reflects the different rapidities with which various
proliferation blocks exert their effect, a presumption consistent with flow cytometric analyses (G. I. E., unpublished
data). For example, isoleucine deprivation arrests cells
only after about 48 hr, the time we presume it takes to
exhaust endogenous isoleucine stores. Apoptosis also becomes evident around this time. On the other hand, serum

deprivation tends to arrest fibroblasts when they next enter


GI. As Rat-l cells have a cell cycle time of about 15 hr
(G. I. E. and T. D. L., unpublished data), we expect virtually
all cells in an asynchronous culture would pass through G,
and encounter a signal to arrest within that time, although
some would do so much sooner. Consistent with this,
apoptosis is first detectable within an hour of serum withdrawal in asynchronous Rat-llmyc cultures, after which it
continues at a more or less uniform rate (G. I. E., unpublished data). A similarly rapid onset of apoptosis is seen in
asynchronous cultures of Rat-llmyc cells in which DNA
blocked in S phase with thymidine. Thus, the combination
of constitutive Myc expression and any tested block to
proliferation appears to be lethal.
Almost all tested tumor cells possess a deregulated myc
gene and this may explain why they undergo apoptosis so
readily in the presence of cytotoxic and growth-inhibiting
drugs (Cotter et al., 1990; Lennon et al., 1990). A similar
mechanism may be responsible for the reported induction
of apoptosis by exogenous wild-type p53 recently observed in the Ml myeloid leukemic cells (Yonish-Rouach
et al., 1991). Given that Ml cells express high levels of
c-myc (Resnitzky et al., 1986) and that the probable action
of wild-type ~53 is to slow or block proliferation, our model
would predict apoptosis as the likely outcome. Equally,
other anti-oncogenes
may interact similarly with Myc.
The rapidity with which Myc can induce programmed
cell death is most dramatically displayed in Rat-lImycER cells that have been previously arrested and are then
subsequently stimulated with 6-estradiol to activate Myc.
In the instance of Go arrest by serum deprivation, Myc
activation leads to substantial apoptosis within 1 hr, long
before overt entry into the cell cycle. Such rapidity argues
that Myc is acting directly to activate the programmed cell
death pathway and, moreover, demonstrates that cells do
not need to be cycling in order for Myc-induced apoptosis
to occur. Others have reported that ectopic activation of
Myc in serum-starved fibroblasts is mitogenic (Eilers et al.,
I 991). In our own analogous experiments with Rat-llmycER cells, we note that many cells survive the first wave of
apoptosis, enter S phase after about 12 hr, and subsequently divide. Within this population, however, apoptosis
continues, and the fate of the culture is ultimately determined by the relative rates of Myc-induced death and Mycinduced proliferation. In isoleucine-starved
cells, arrested
in late G,, and in thymidine blocked cells, arrested in S,
activation of Myc leads to the onset of significant apoptosis
after 3-4 hr. In both cases, flow cytometric analysis demonstrates that the cells concerned exhibit a complete cell
cycle block despite Myc activation. Even so, apoptosis
proceeds over an extended time; typically it takes several
days before all the cells in the culture are dead. We suggest that, although Myc activates the apoptotic pathway,
the commitment of an individual cell to undergo apoptosis
is governed by as yet unidentified stochastic factors. The
observation that Myc activation kills S phase thymidineblocked cells is particularly intriguing because, by definition, all the cells are beyond the late G, restriction point
in the cell cycle, after which it is usually assumed that
regulatory components like Myc no longer exert any effect.

Cell
126

Myc and the Mechanism of Apoptosis


The complete dependence of the apoptosis we describe
upon the presence of active Myc is shown by the absolute
requirement for exogenous P-estradiol in Rat-llmyc-ER
cells. Moreover, both the rate of apoptosis and its sensitivity to induction by serum deprivation depend on the
amount of Myc protein present in a cell. In addition, deletion mapping shows that the same regions of Myc are
involved in inducing apoptosis as are involved in cotransformation, autoregulation,
and inhibition of differentiation
(Freytag et al., 1990; Penn et al., 1990a; Stone et al.,
1987). This includes regions mediating heterodimerization
with Max and sequence-specific
DNA binding (Blackwell
et al., 1990; Blackwood and Eisenman, 1991) and those
thought to be involved in transcriptional
modulation (Kato
et al., 1990). Given the above, together with the rapidity
with which Myc induces programmed cell death, we deem
it likely that Myc is directly involved in initiating apoptosis,
presumably by the regulation of specific apoptotic genes.
This hypothesis can be tested now that probes for some
apoptosis-associated
genes are available (Owens et al.,
1991).
Our results further raise the intriguing possibility that
c-myc may comprise a general component of the apoptotic
pathway. Such an idea is consistent with results from a
number of studies of apoptosis in which c-myc expression
has been examined. For example, in a study of experimentally induced rodent tumors, Wyllie et al. (1987) reported
an elevated level of apoptosis specifically associated with
c-myc expression. In two well characterized instances of
normal tissue involution through apoptosis, estrogen ablation of MCF-7 breast carcinoma xenografts and androgen
ablation of rat prostate, c-myc is the only major oncogene
whose expression is consistenUy raised during the period
of cell death (Buttyan et al., 1988; Kyprianou et al., 1991;
Quarmby et al., 1987). Finally, apoptosis of embryonic
thymocytes during thymic censorship is initiated by activation of the T cell receptor (Smith et al., 1989) and such
activation is known to cause substantial induction c-myc
in immature CD4*8+ thymocytes (Riegel et al., 1990). Notwithstanding such circumstantial evidence, the notion that
myc genes comprise general components
of the programmed cell death pathway, in addition to regulating proliferation, is radical and will require further substantiation.
Myc, Proliferation,
and Apoptosis
What might be the evolutionary sense in having the c-myc
oncogene drive both proliferation and programmed cell
death, two apparently contradictory processes? Antisense
oligonucleotide
experiments confirm that Myc is essential
for cell proliferation (Heikkila et al., 1987; Loke et al., 1988;
Prochownik et al., 1988), whereas it has been shown that
induction of Myc activity alone is sufficient to drive quiescent cells into the cell cycle and maintain them there (Eilers
et al., 1991). Myc is therefore both necessary and sufficient
for cell proliferation. Clearly, this is a potentially very dangerous state of affairs because any mutation that deregulated c-myc expression would in principle be oncogenic.
If, however, in addition to regulating genes mediating pro-

liferation, Myc also activates genes mediating apoptosis,


then all cells expressing Myc would necessarily be primed
for programmed cell death. Successful proliferation would
then presumably occur only if apoptosis were actively inhibited, perhaps by activation of complementary
signal
transduction pathways.
Fibroblasts in the adult are mesenchymal cells in which
proliferation is confined to maintenance and repair. Proliferation and quiescence are regulated by mitogen availability and topoinhibition
and are accompanied and perhaps
mediated by appropriately
regulated c-myc expression.
In contrast, when many other cell types are deprived of
necessary cytokines, they undergo apoptosis (Araki et al.,
1990; Williams et al., 1990) and only appear able to enter
a quiescent state upon differentiation. We suggest that the
reason such cells die is that they are not configured to
down regulate c-myc in response to cytokine removal and
consequently undergo apoptosis. In this model, the role of
differentiation-inducing
factors is to turn off c-myc expression, so allowing both differentiation
and stable growth
arrest. Such a hypothesis is eminently testable in a wide
range of experimental systems.
Myc and Carcinogenesis
If c-myc is a potent potential inducer of apoptosis, why is
its deregulation
and overexpression
so pervasive, and
thus so strongly positively selected, during carcinogenesis? We propose that the proliferative advantage afforded
by c-myc activation is essential for carcinogenesis
but,
because the proliferative and apoptotic functions of Myc
are tightly coupled, it is impossible to select for one without
also selecting for the other. According to this argument,
deregulation of c-myc is an essential step in carcinogenesis, but it is lethal should the cell concerned ever be unable
to proliferate due to lack of nutrients, space, or necessary
cytokines. If this hypothesis is correct, one can envisage
three obvious mechanisms that might allow a cell with
deregulated c-myc to survive. The first involves acquisition
of a second mutation that renders the affected cell independent of previously obligate cytokines. We suggest that
activation of an appropriate cooperating oncogene might
exemplify this. The second involves acquisition of a lesion
in the programmed cell death pathway. Indeed, an example of such a mechanism is already known. The activated
bcl-2 proto-oncogene
cooperates with c-myc and is known
to block programmed cell death in lymphoid tissues (Hockenbery et al., 1990). Third, a cell with deregulated myc
might survive if subjected to continuous mitogenic stimulation. This might occur in chronic degenerative, hyperplastic, and inflammatory situations, all of which are known to
predispose to neoplasia.
Conclusion:
a Model for Myc Function
in the Determination
of Cell Fate
On the basis of ourdataand the arguments detailed above,
we venture to propose the following model. Myc drives
two coupled functions: proliferation and programmed cell
death. Successful proliferation in normal cells requires the
active suppression of programmed cell death, thereby pro-

InductIon
127

of Apoptosts

by c-myc

Protein

viding an inbuilt failsafe to guard against uncontrolled proliferation and so allowing the same basic machinery to
regulate the two necessarily linked processes of proliferation and programmed cell death. Given that c-myc expression is necessary for proliferation and that down regulation
of myc appears necessary for growth arrest and differentiation, the c-myc proto-oncogene
would appear to be an
important central regulator determining the various fates
of a cell: proliferation, arrest, differentiation,
and death.
Experimental Procedures
Cell Culture

and Cell Lines


The preparation
of recombinant
retroviruses
directing constitutive
expression of c-myc, various c-myc mutants, and myc-ER chimeras has
already been described,
as has the isolation of appropriately
infected
Rat-l cells (Eilers et al., 1989. 1991; Penn, et al., 1990a, 1990b). Cells
were assayed
for constitutive
expression
of both c-myc mRNA by
RNAase protection
and Northern blot analysis (Penn, et al., 1990a,
1990b) and for c-myc protein by ELISA and semiquantitaive
immunofluorescent
confocal microscopy
(Moore et al., 1987; Waters et al.,
1991). All cells were maintained
in Dulbeccos
modified E4 medium
(DME) supplemented
with 10% FCS and 1 mglml Geneticin.
Cells
were passaged
by standard trypsinization
and seeded directly onto
tissue culture plastic. Ecotropic viruses directing expression
of chimeras between Myc and truncated
ER were a kind gift from Dr. Martin
Eilers and Professor
J. Michael Bishop (University
of California, San
Francisco).
Rat-l cells were infected with viruses encoding Myc-ER
chimeras,
and Rat-l lines expressing
wild-type Myc-ER
and A106143 Myc-ER were isolated as described for Rat-llmyc
lines (Penn, et
al., 1990a, 1990b). Myc-ER
and A106-143
Myc-ER
clones were
maintained
in phenol red-free
DME supplemented
with 10% charcoal-dextran
stripped FCS and 1 mglml Geneticin.
Myc was functionally activated by the addition of b-estradiol
to the medium at a final
concentration
of 2 uM.
Biochemical
and Analytical
Techniques
To examine nucleosome
laddering, equal numbers of cells were established in 25 cm3tissue
culture flasks. Medium was then changed in
each flask, as indicated, and at various time points dying cells were
harvested
by virtue of their reduced adherence.
As a consequence,
very few cells were obtained from nondying
cultures,
with consequently little DNA. The experiment
was thus normalized
according
to
starting cell number rather than to amount of DNA extracted. We chose
this method of normalization
because the proportion of laddered chromatin at any one time is quite small relative to intact chromatin present
in those nonapoptotic
cells in the culture, and this large excess of intact
DNA obscures any ladders present. The assay therefore shows when
chromatin laddering occurs, but the results are in no way quantitative.
Instead, quantitation of apoptosis was carried out by time-lapse cinemicroscopy
(see below). DNA extraction
and fractionation
on 1.5% agarose gels were performed
as described
(Smith et al., 1989).
Standard electron microscopic
procedures
were used.
Time-lapse
cinemicroscopy
was conducted
using a Olympus
inverted phase contrast microscope,
and images were collected on 16
mm monochrome
tine film with a tine camera regulated by an external
timer. Cell division events were scored at the time at which septa
formed between two daughter cells. Apoptotic cell death events were
scored midway between the last appearance
of normality and the point
at whtch the cell became fully detached and rounded. This corresponds
to about t+ 15 min in Figure 3A.
Acknowledgments
We are Indebted to Derek Davies for his invaluable and expert help in
flow cytometric
analysis.
We are grateful to all our many scientific
colleagues for their advice and help, and in particular to Drs. Richard
Treisman, Mike Owen, Mair Churchill, Mary Collins, Ron Laskey. Paul
Nurse, and Kathy Weston. Dedicated to Tam and Theo.
The costs of publication
of this article were defrayed
in part by

the payment of page charges. This article must therefore be hereby


marked advertisement
in accordance
with 16 USC Section 1734
solely to indicate this fact.
Received

October

22, 1991; revised

January

14, 1992

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Note
Please

Added
address

in Proof
correspondence

to Dr. G. I. Evan.