Professional Documents
Culture Documents
69, 119-128,
Induction of Apoptosis
by c-myc Protein
in Fibroblasts
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
of
Cell
120
Rat- 1 /wt
Rat- 1 control
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
MYC CONSTRUCTS
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
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
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
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
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
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
Cell
126
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
October
January
14, 1992
References
Araki, S., Shimada, Y., Kaji, K., and Hayashi, H. (1990). Apoptosis
of vascular
endothelial cells by fibroblast
growth factor deprivation.
Biochem. Biophys. Res. Commun.
768, 1194-1200.
Askew, D., Ashmun, R., Simmons, B., and Cleveland, J. (1991). Constitutivec-mycexpression
in IL-9dependent
myeloid cell linesuppresses
cycle arrest and accelerates
apoptosis.
Oncogene
6, 1915-1922.
Blackwell, T. K., Kretzner. L., Blackwood,
Weintraub,
H. (1990). Sequence-specific
protein. Science 250, 1149-I 151.
Blackwood,
E. M., and Eisenman, R. N. (1991).
zipper protein that forms a sequence-specific
with Myc. Science 251, 1211-1217.
Max: a helix-loop-helix
DNA-binding
complex
S., and Bishop, J. M. (1991). The Myc protein actiof the alpha-prothymosin
gene. EMBO J. 70, 133-
An aminoactivates
of
di-
Cell
128
Loke, S. L., Stein, C., Zhang, X., Avigan, M., Cohen, J., and Neckers,
L. M. (1988). Delivery of c-mycantisensephosphorothioateoligodeoxynucleotides to hematopoietic
cells in culture by liposome fusion: specific reduction in c-myc protein expression
correlates
with inhibition of
cell growth and DNA synthesis.
Curr. Top. Microbial. Immunol. 741,
262-289.
Moore, J. P., Hancock,
D. C., Littlewood, T. D., and Evan, G. I. (1987).
A sensitive and quantitative
enzyme-linked
immunosorbence
assay
for the c-myc and N-myc oncoproteins.
Oncogene
Res. 2, 65-80.
Murre, C., McCaw, P. S., and Baltimore, D. (1989). A new DNA binding
and dimerization
motif in immunoglobulin
enhancer binding, daughterless, &fyoD, and myc proteins. Cell 56, 777-783.
Ormerod,
Approach,
M. G. (1990). Analysisof
DNA. In Flow Cytometry,
A Practical
M. G. Ormerod, ed. (Oxford:
IRL Press), pp. 69-87.
A. B. (1989).
246, 603-608.
GI events
and regulation
of cell proliferation.
Persson, H., Gray, H. E., Godeau, F., Braunhut, S., and Bellve, A. Ft.
(1986). Multiple growth-associated
nuclear proteins immunoprecipitated by antisera raised against human c-myc peptide antigens, Mol.
Cell Biol. 6, 942-949.
Prendergast,
G., and Ziff, E. (1991). Methylation-sensitive
specific DNA binding by the c-myc basic region. Science
189.
sequence251, 186-
Prochownik,
E. V., Kukowska,
J., and Rodgers, C. (1988). c-myc antisense transcripts
accelerate
differentiation
and inhibit GI progression
in murine erythroleukemia
cells. Mol. Cell. Biol. 8, 3883-3695.
Ouarmby, V., Beekman, W., Wilson, E., and French, F. (1987). Androgen regulation of c-myc messenger
ribonucleic acid levels in rat ventral
prostate. Mol. Endocrinol.
7, 865-874.
Resnitzky,
D., Yarden, A., Zipori, D., and Kimchi, A. (1986). Autocrine
p-related interferon controls c-myc suppression
and growth arrest during hematopoietic
cell differentiation.
Cell 46, 31-40.
Riegel, J. S., Richie, E. R., and Allison, J. P. (1990). Nuclear events
afler activation of CD4+8+ thymocytes.
J. Immunol. 744, 361 l-3618.
Smith, C. A., Williams, G. T., Kingston, R., Jenkinson,
E. J., andowen,
J. J. T. (1989). Antibodies to CDS/T-cell receptor complex induce death
by apoptosis in immature T cells in thymic cultures. Nature 337, 181184.
Spencer, C. A., and Groudine,
M. (1991). Control of c-myc regulation
in normal and neoplastic
cells. Adv. Cancer Res. 56, l-48.
Stone, J., de Lange, T., Ramsay,
G., Jakobvits,
E., Bishop, J. M.,
Varmus, H., and Lee, W. (1987). Definition of regions in human c-myc
that are involved in transformation
and nuclear localization.
Mol. Cell.
Biol. 7, 1697-1709.
Waters, C., Littlewood, T., Hancock, D., Moore, J., and Evan, G. (1991).
c-myc protein expression
in untransformed
fibroblasts.
Oncogene
6,
101-109.
Williams, G. T., Smith, C. A., Spooncer,
E., Dexter, T. M., and Taylor,
D. R. (1990). Haemopoietic
colony stimulating
factors promote cell
survival by suppressing
apoptosis.
Nature 343, 76-79.
Wyllie, A. H. (1987). Apoptosis:
cell death under
Arch. Toxicol. Suppl. 7 1, 3-10.
homeostatic
control.
Wyllie, A. H., Rose, K. A., Morris, R. G., Steel, C. M., Foster, E., and
Spandidos, D. A. (1987). Rodent fibroblast tumours expressing
human
myc and raas genes: growth, metastasis
and endogenous
oncogene
expression.
Br. J. Cancer 56, 251-259.
Yonish-Rouach,
E., Resnitzky.
D., Lotem,
J., Sachs,
L.. Kimchi,
A.,
Zetterberg,
A., and Larsson, 0. (1985). Kinetic analysis of regulatory
events in GI leading to proliferation
or quiescence
of Swiss 3T3 cells.
Proc. Natl. Acad. Sci. USA 82, 5365-5369.
Note
Please
Added
address
in Proof
correspondence
to Dr. G. I. Evan.