© 1995 American Heart Association, Inc.
Articles |
Apoptosis of Rat Vascular Smooth Muscle Cells Is Regulated by p53-Dependent and -Independent Pathways
From the Department of Pathology (M.R.B., S.M.S.), University of Washington, Seattle, and Biochemistry of the Cell Nucleus Laboratory (G.I.E.), Imperial Cancer Research Fund, London, UK.
Correspondence to Dr M.R. Bennett, Unit of Cardiovascular Medicine, University of Cambridge School of Clinical Medicine, Department of Medicine, Level 5, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2QQ, UK. E-mail mrbennet@u.washington.edu.
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Abstract Apoptosis of vascular smooth muscle cells has recently been described in culture and also in remodeling of the artery after birth. However, the genes that regulate apoptosis in smooth muscle cells are mostly unknown. We studied the regulation of apoptosis in rat smooth muscle cells stably infected with retrovirus constructs containing c-myc, adenovirus E1A, bcl-2, and a temperature-sensitive mutant of the tumor suppressor gene p53. Apoptosis was verified by electron microscopy and quantified by time-lapse videomicroscopy. Death was induced by c-myc and E1A when cells were deprived of serum survival factors. bcl-2 suppressed apoptosis of cells infected with c-myc and E1A and also normal smooth muscle cells. Overexpression of wild-type p53 induced apoptosis of cells infected with E1A and c-myc but not normal cells. In contrast, expression of mutant p53, which blocks wild-type p53 function, suppressed apoptosis of cells infected with E1A or c-myc but not normal cells. Both adenovirus E1A and c-myc increased the expression of endogenous p53 protein but not p53 mRNA. Although bcl-2 suppressed apoptosis induced by E1A and c-myc, upregulation of p53 protein induced by these agents was unaffected. We conclude that apoptosis of vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Death induced by c-myc and E1A is mediated by, and dependent on, p53. However, the suppression of apoptosis by bcl-2 is not mediated by changes in p53 expression, and the low level of apoptosis seen in normal VSMCs upon removal of survival factors is independent of p53.
Key Words: apoptosis • smooth muscle • c-myc • bcl-2 • p53 • adenovirus E1A
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Introduction |
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Cell death is a prominent component of atherosclerotic plaques both in animals1 2 and in humans.3 4 Cell death has previously been considered to be due to toxic insult (eg, as a reaction to free radicals or oxidized lipids).5 However, recent evidence has indicated that all the component cells of the atherosclerotic plaque can, at least in culture, undergo apoptosis (programmed cell death).6 7 8 9 Cell death occurs after the removal of serum survival factors, with deregulated expression of specific gene products, or after exposure to oxidized lipids or drug therapy. Furthermore, apoptosis has been identified in normal rat and human VSMCs in culture9 10 and in remodeling of the arterial wall after birth in animals.11
Apoptosis is an evolutionarily conserved physiological form of cell death that regulates cell mass and architecture in many tissues. The precise occurrence of apoptosis in development has fostered the idea that there is a genetic program regulating cell death via the activation and inactivation of specific genes. Many of these genes have now been identified and have been shown to regulate apoptosis in a wide variety of cell types and a diverse selection of organisms. In particular, dominant proto-oncogenes such as c-myc and bcl-2, adenovirus proteins such as E1A and E1B, and tumor suppressor genes such as p53 have been shown to be potent regulators of apoptosis (see Reference 1212 for review). However, the role of these genes in regulating apoptosis of cells of the vessel wall has been less well defined. We have studied the action and interaction of c-myc, bcl-2, E1A, and p53 in apoptosis of VSMCs by creating stable cell lines expressing these genes via retroviral transfer. In addition, to investigate whether there is a final common path involving c-myc, bcl-2, or p53 in VSMC apoptosis, the endogenous expression of these genes was also analyzed. We show that apoptosis in VSMCs is induced by c-myc and E1A and blocked by expression of bcl-2. In addition, overexpression of wild-type p53 induces apoptosis in E1A-infected and c-myc–infected VSMCs in low-serum conditions; this overexpression is blocked by the expression of mutant p53. E1A and c-myc also increase the expression of p53 protein. Thus, c-myc–induced and E1A-induced apoptosis of VSMCs is mediated by and dependent on p53. However, the suppression of apoptosis by bcl-2 in normal VSMCs or cells infected with c-myc or E1A appears to be independent of p53.
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Materials and Methods |
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Cell Culture
VSMCs were isolated from thoracic aortic explants of 6-week-old Sprague-Dawley rats. Cells were cultured in Waymouth's medium containing 10% FCS (GIBCO) and 20 mmol/L HEPES (Flow) and equilibrated with 95% air/5% CO2. Subconfluent cells were passaged by trypsinization in 0.05% trypsin in PBS and reseeded in Waymouth's medium+10% FCS (normal culture medium). VSMCs were identified by their typical hill-and-valley morphology in culture and their characteristic immunocytochemical staining for
-smooth muscle
actin (monoclonal anti–smooth muscle -actin antibody, Sigma
Chemical Co). Cells at passage 5 were used for experiments and
retrovirus infections.
Production of Retrovirus-Infected Cell Lines
The retrovirus constructs used to create VSMC cell lines
constitutively expressing c-myc, bcl-2, and E1A
were based on the pDORneo and pBabepuro retroviral
vectors13 and contained full-length cDNAs of each species.
c-myc and bcl-2 used human
sequences of each gene and have already been
described.14 15 The adenovirus E1A sequence encoded the
12S subunit (obtained from Dr H. Land, ICRF). In these retrovirus
vectors, expression of the gene of interest is driven by the Moloney
murine leukemia virus long terminal repeat and expression of the
neomycin or puromycin resistance gene selectable marker from the simian
virus 40 early promoter. Use of different antibiotic genes for
different vectors allowed selection for each gene of interest
independently. In addition, we used the temperature-sensitive mouse
mutant of p53, p53val135,16 under the control of the Rous
sarcoma virus long terminal repeat. This mutant expresses wild-type p53
activity at 32°C17 18 19 but mutant p53 activity at 37°C,
thus acting as a dominant negative suppressor of wild-type p53 function
at 37°C.17 20 21
Infection of VSMCs With Retrovirus Vectors
Infectious retrovirus was harvested from 
or GP+E packaging
cells transfected with pDORneo or pBabepuro retrovirus DNA containing
the gene of interest as previously described.13 14 22
VSMCs were infected with retrovirus vectors also as previously
described.9 The temperature-sensitive p53 mutant was
introduced by calcium phosphate transfection13 and
cotransfected with pBabepuro. Twenty-four hours after infection or
transfection, resistant cells were selected in 500 µg/mL of
G418 (Geneticin, GIBCO) or 2.5 µg/mL of puromycin (Sigma).
Resistant cell populations were used for experiments at least 6
weeks after infection. Cells were maintained in medium containing
antibiotics at all times.
RNA Isolation and Northern Hybridization
Normal rat VSMCs and retrovirus-infected cell lines were grown
in medium+10% FCS until 50% confluent. Total RNA was isolated,
electrophoresed, and probed for c-myc, p53,
bcl-2, or the 28S ribosome subunit as previously
described.23 24 The riboprobe used to study p53 expression
was generated from php53-wt-c (a generous gift from Dr Denise Galloway,
Fred Hutchison Cancer Center, Seattle, Wash), which contains human p53
cDNA with a cap-independent translation enhancer present at the 5'
end.
Time-Lapse Videomicroscopy
Cells were prepared for videomicroscopy, as previously
described, by using nonconfluent cells, which did not overlap or form
multiple layers.9 The microscope was enclosed in a plastic
environment chamber and maintained at 37°C by an external heater. For
experiments involving the temperature-sensitive p53 mutant, the chamber
temperature was kept at either 37°C or 32°C. Flasks were gassed
with 95% air/5% CO2 every 24 hours and sealed. The
time-lapse equipment consisted of a Dage-MTI camera (Dage-MTI Inc) and
a Panasonic 8050 time-lapse video recorder with a Colorado video
synchronization system (Colorado Video). Films were analyzed
for cell death rates14 25 by using an observer blind to
cell type and treatment conditions. Cell divisions were scored at the
time at which septa appeared between two daughter cells. Apoptotic cell
death events were scored midway between the last appearance of
normality and the point at which the cell became fully detached and
fragmented, an interval of typically 60 to 90 minutes. Each individual
cell culture was analyzed in triplicate as a minimum.
Flow Cytometry
Cell lines growing in 10% FCS for at least 48 hours were
prepared for flow cytometry as previously described.9
Cells demonstrating less than the diploid content of DNA were excluded
from the measurement of the percentages of cells in each cell-cycle
phase.
Western Blotting
Western blots were prepared by lysis of cells cultured in medium
containing 10% FCS. Protein isolation, electrophoresis, and blotting
were performed as previously described26 by using Pab1
(Oncogene Science), which recognizes both rat and human p53.
Immunocytochemistry
Immunocytochemistry of VSMCs for p53 was as previously
described.23 The primary p53 antibody (Pab1) was used at
1:500 dilution, a biotinylated anti-mouse secondary antibody was used
at 1:200, and bound antibody was detected by using a standard
avidin-peroxidase detection system (Vector Laboratories) with
diaminobenzidine used as a chromogen.
Electron Microscopy
Cells undergoing apoptosis were prepared for electron
microscopy as previously described.24
Statistical Analyses
The means of numbers of cells undergoing apoptosis were
analyzed by ANOVA for multiple comparisons. Paired
analysis between two groups (eg, between normal VSMCs and
retrovirus-infected cell lines) was performed by Student's
t test when ANOVA indicated significance for the multiple
comparison.
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Results |
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Rat VSMCs derived from the thoracic aortic media were transfected or infected with retrovirus vectors containing cDNAs encoding c-myc, TSp53, bcl-2, adenovirus E1A, or the retrovirus vector alone. Cell lines were designated as normal VSMCs (uninfected), VSM-myc cells, VSM-p53 cells, VSM-bcl-2 cells, VSM-E1A cells, and VSM-vector cells, respectively. The TSp53 construct allows overexpression of mutant-type p53, which suppresses endogenous wild-type p53 activity, or wild-type p53 itself. Stable infections that resulted in expression of the gene product up to 3 months in culture after infection were performed. The use of vectors with different antibiotic resistance genes allowed the isolation of cells expressing more than one gene of interest. Integration and expression of the infected gene was confirmed by Southern and Northern blotting of DNA and RNA from resistant cell lines (not shown). In view of the number of gene interactions we studied and to avoid clonal variation, pooled populations of each cell line rather than individual clones were used in all experiments. Cells were tested for their ability to undergo apoptosis after the removal of serum growth factors. In addition, the presence of apoptosis in cells containing the TSp53 construct was analyzed at the permissive and restrictive temperatures in medium containing 10% or 0% FCS.
Cell Death in VSMCs Occurs by Apoptosis
Apoptosis of VSMCs was first confirmed in each cell line
by a combination of time-lapse videomicroscopic appearances and
electron microscopic morphology. Time-lapse analysis of cell
death is a sensitive measure of apoptotic events, because it identifies
the mode of death by morphological criteria and also measures cell
death independent of cell proliferation. In each cell line, death was
associated with the characteristic sequence of apoptosis,
namely, cell shrinkage with retraction away from its neighbors,
condensation of the nuclear chromatin, intense membrane activity with
the formation of blebs and vesicles, and formation of a dense apoptotic
body (Figs 1
and 2). We have previously
shown that these morphological changes on time-lapse videomicroscopy
and electron microscopy correlate well with other features of
apoptosis, such as DNA fragmentation in
VSMCs.9 24
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Effect of c-myc, bcl-2, E1A, or p53 on
Apoptosis of VSMCs
We next assessed the rate of apoptosis in normal
(uninfected) VSMCs and in cells infected with c-myc,
bcl-2, E1A, or p53 in 0% FCS. Cells were
cultured in medium containing 10% FCS, then transferred to low-serum
conditions, and filmed. Normal VSMCs exhibited a small amount of cell
death in 0% FCS (
5% of cells) over 24 hours; this low level of
apoptosis was also seen in cells infected with the retrovirus
vector alone (Fig 3). Apoptosis of normal VSMCs
was suppressed completely by expression of bcl-2
(Fig 3) (P<.05 versus normal VSMCs). Cells infected with
c-myc showed a markedly increased frequency of
apoptosis (36.4±4.8%, mean±SEM) over 24 hours, as we have
previously reported.9 Cells infected with adenovirus E1A
also underwent significantly greater apoptosis than uninfected
cells, both in the presence and absence of serum (Fig 3 and Table 1). In contrast, cells infected with p53 alone, either
in the mutant or wild-type conformation, showed no difference in rates
of apoptosis from normal (uninfected) cells (Fig 3 and Table 1).
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Effect of p53 and bcl-2 on
Apoptosis Induced by c-myc and E1A
To analyze the interaction between p53 or
bcl-2 and c-myc or E1A in the
mediation of apoptosis, cell lines were isolated from double
infections. These cell lines expressed c-myc and
bcl-2, c-myc and p53, E1A and
bcl-2, or E1A and p53 and were designated
VSM-myc-p53, VSM-myc-bcl-2, VSM-E1A-p53, or VSM-E1A-p53 cells. Cells
were again cultured in medium containing 10% FCS and then transferred
to medium containing 0% FCS, and rates of apoptosis were
determined. Coexpression of bcl-2 with
c-myc markedly suppressed the apoptotic action of
c-myc. A similar effect was observed with coexpression of
bcl-2 with E1A (Fig 4). However,
the effects of p53 on apoptosis induced by c-myc or
E1A were dependent on the conformation of the p53 protein. p53 in the
mutant conformation suppressed both c-myc–induced and
E1A-induced apoptosis, whereas overexpression of wild-type p53
increased apoptosis in both c-myc–infected and
E1A-infected cells in 10% or 0% FCS (Table 1
).
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Effect of p53 on Cell Proliferation
Wild-type p53 has been shown to exert profound effects on cell
replication, in particular, by induction of a G1 arrest in
cells that have DNA damage. We assayed the effect of overexpression of
wild-type p53 or mutant p53 on the proliferation of normal VSMCs or
cells infected with the retrovirus vector, c-myc, or E1A in
10% FCS. The presence of cell death precluded the use of conventional
measures of cell proliferation (cell counts or
3[H]thymidine incorporation). Rather, proliferation was
assessed by quantifying the percentage of cells going through mitosis
over 24 hours by time-lapse videomicroscopy in cells that did not
undergo apoptosis over this period. This gave an estimate of
proliferation independent of cell death. Table 2 shows
that 41% to 43% of normal rat VSMCs underwent mitosis in 24 hours.
This percentage was not altered by expression of wild-type or
mutant-type p53. Similarly, although the percentage of VSM-myc or
VSM-E1A cells undergoing mitosis was greater than the percentage of
normal cells undergoing mitosis, this percentage did not change
significantly with the expression of p53 in either the wild-type or
mutant-type conformation. Infection of VSMCs with the retrovirus vector
alone or in combination with p53 also did not affect the proliferation
of cells (Table 2). Cell-cycle distribution of each cell line was also
assessed by flow cytometry (Table 3). This indicated
that there was no evidence of cell-cycle arrest in the live cells of
any of the cell lines infected with p53 in either conformation. This is
evidenced by the fact that the S-phase percentage of any cell line did
not change significantly at 37°C or 32°C compared with cells not
infected with p53, and there was no accumulation of cells in
G1.
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Effect of E1A, p53, and bcl-2 on
c-myc Expression
E1A is a functional homologue of c-Myc in a number of assays,
specifically replacing the action of c-Myc in the promotion of
proliferation and apoptosis.27 In addition, there
is some evidence that E1A downregulates c-myc28
and that the proteins of each are structurally related.29
We examined whether the effects of E1A on inducing apoptosis or
the effects of p53 and bcl-2 in regulating
apoptosis induced by c-myc were mediated by changes
in endogenous c-myc expression. The expression
of c-myc mRNA was examined by Northern hybridization in
cells infected with E1A, p53, or bcl-2 (Fig 5). E1A expression had no effect on levels of
c-myc mRNA in cells cultured in medium containing 10% FCS.
Infection of VSMCs with p53 or bcl-2 alone also
had no effect on c-myc mRNA expression. Similarly,
bcl-2 overexpression did not suppress
c-myc mRNA levels in VSM-myc cells or VSM-E1A cells (Fig 5).
In 0% FCS, c-myc mRNA was not detectable on Northern
hybridization at 24 hours in any VSMC line that was not infected with
c-myc (not shown). This is consistent with previous
observations showing that c-myc is downregulated rapidly in
VSMCs after the removal of serum.30
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Effect of c-myc and E1A on p53 Expression
Because changes in p53 expression markedly affected
apoptosis induced by c-myc and E1A, we examined
whether expression of E1A or c-myc regulated the
endogenous expression of p53. p53 mRNA and protein levels
were assayed in VSM-myc and VSM-E1A cells by Northern and Western
blotting and compared with levels of p53 expression in normal VSMCs and
cells infected with the retrovirus vector alone. Figs 5 and 6 indicate that p53 protein expression was increased in
VSM-myc and VSM-E1A cells compared with normal VSMCs and VSM-vector
cells, although levels of p53 mRNA were unchanged. This finding was
confirmed by immunocytochemistry for p53, which demonstrated
undetectable p53 staining in normal VSMCs or VSM-vector cells but
obvious staining in VSM-myc or VSM-E1A cells (Fig 7). In
contrast, although bcl-2 suppressed
apoptosis induced by E1A and c-myc, it did not
suppress the increased levels of p53 seen in VSM-myc or VSM-E1A cells
or endogenous levels of p53 in VSM-bcl-2 cells (Fig 6).
bcl-2 mRNA expression was undetectable by
Northern hybridization in any cell line not infected with
bcl-2 (Fig 5).
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Discussion |
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The finding that normal VSMCs, both rat and human, undergo apoptosis in culture9 10 indicates that normal VSMCs possess the machinery to undergo apoptosis. Thus, apoptosis may be one method of regulating cell number in the vessel wall. Apoptosis has also been demonstrated to occur with increased frequency in cells derived from atheromatous plaques24 and in plaques themselves.31 Apoptosis may thus contribute to the clinical sequelae of atherosclerotic plaques, such as plaque rupture and thrombosis. However, although we have shown that apoptosis of human VSMCs can be blocked by expression of bcl-2,24 the genes that regulate apoptosis in VSMCs are mostly unknown. Therefore, we have investigated the regulation of apoptosis of VSMCs by a number of known proapoptotic and antiapoptotic genes and the interactions between these genes. We have specifically focused on the genes c-myc, bcl-2, p53, and E1A.
The c-Myc protein is a nuclear phosphoprotein with structural motifs consistent with an action as a transcription factor. In earlier studies, we have demonstrated that c-myc can induce apoptosis in growth factor–deprived VSMCs and fibroblasts.9 14 15 25 In the present study, we demonstrate that apoptosis of both normal VSMCs and cells expressing deregulated c-myc upon removal of serum is suppressed by coexpression of bcl-2. The effect of bcl-2 is not mediated by a reduction in c-myc expression in either cell type and is consistent with earlier studies in fibroblasts demonstrating that bcl-2 blocks c-myc–induced apoptosis without affecting its mitogenic capacity and does not suppress overexpression of c-myc.15 In addition, our data indicate that bcl-2 suppresses apoptosis without suppressing p53 expression in normal VSMCs or in VSM-myc cells, which have upregulated p53 protein levels. Although bcl-2 probably does not play a major role in regulating apoptosis of VSMCs (since we cannot demonstrate significant levels of bcl-2 expression in these cells and very low levels are demonstrable in VSMCs in smooth muscle cells in vivo),32 33 Bcl-2 belongs to a larger family of structurally related proteins whose function and presence in the vessel wall are little explored.
Adenovirus E1A is the principle early gene of the virus and is responsible for inducing host cell proliferation, encoding a multifunctional polypeptide that interferes with basal transcriptional machinery, including the retinoblastoma gene product and associated proteins p107 and p300.34 Although the adenovirus E1A gene product is not part of the normal mammalian genome, the use of E1A in the present study allows us to assess whether apoptosis induced by two different proapoptotic gene products, E1A and c-myc, acts via a similar pathway. We demonstrate that similarities exist between apoptosis induced by either E1A or c-myc. Apoptosis in both instances is suppressed by bcl-2 and promoted by removal of serum survival factors. In addition, both E1A-induced apoptosis and c-myc–induced apoptosis appear to be dependent on p53 expression, because apoptosis is promoted by p53 in the wild-type conformation but suppressed by p53 in the mutant conformation. Both E1A and c-myc also increase the expression of p53 protein but have little effect on p53 mRNA levels. Although we cannot exclude the possibility that induction of p53 protein is due to some DNA damage in the E1A-infected and c-myc–infected cells, E1A has been shown to stabilize p53 protein.35 Although the precise mechanism of action of this effect is unclear, the stabilized p53 protein is indistinguishable from wild-type protein.35 Furthermore, recent evidence suggests that p53 protein and, to a much lesser extent, p53 mRNA may be induced by c-myc.36 Although an induction of p53 by c-Myc is not universally found,37 the p53 promoter has been shown to possess a c-Myc consensus DNA binding site and be transactivated by c-Myc.38
A recent study has found that the wild-type p53 protein is overexpressed in 30% of human angioplasty restenosis sites.39 Although requiring confirmation, this study has raised interest in the role of p53 in the proliferation and death of vascular smooth muscle cells. The introduction of wild-type p53 into p53-negative cells has previously been shown to induce apoptosis,40 41 and the finding that p53 levels are upregulated in some cases of hormone-dependent apoptosis42 43 indicates that p53 may act as a physiological regulator of apoptosis. In the present study, we show that introduction of p53 in the wild-type or mutant-type conformation into normal VSMCs (which presumably have normal p53 regulation) does not affect apoptosis of cells upon removal of serum survival factors. Introduction of p53 also had no effect on the rate of cell proliferation of normal VSMCs, VSM-vector cells, VSM-myc cells, or VSM-E1A cells, measured by direct analysis of mitotic events or by cell-cycle distribution of the cell lines. Our findings are contrary to findings in many tumor cell lines (in which p53 is frequently absent or mutated) that undergo a G1 arrest or apoptosis upon expression of high levels of wild-type p53. However, our data are consistent with findings that cells with a functional p53 gene are more resistant to the introduction of the addition of an extra wild-type p53 gene.44 45 46 Thus, unless there is a second lesion (in a proto-oncogene, for instance) in cells composing the sites of angioplasty restenosis, our findings do not elucidate a direct role for the upregulated p53 expression observed in restenosis, whether this upregulated p53 is functional or not. In this regard, it is intriguing to note that upregulation of c-myc mRNA expression has been observed in human VSMCs cultured from plaques compared with normal arteries47 and that neonatal VSMCs, a cell type thought to share many features with VSMCs from sites of restenosis, may also show upregulated c-myc expression.48
As mentioned above, our data indicate that overexpression of wild-type p53 does not appear to suppress the proliferation of VSM-E1A or VSM-myc cells. However, a word of caution is necessary in the interpretation of these data. We show that even though there is apoptosis in cultures of VSM-myc-p53 and VSM-E1A-p53 cells, particularly when the p53 is in the wild-type conformation, there is no significant difference in the percentage of cells that undergo mitosis over 24 hours and do not die or the cell-cycle distribution of these live cells. An alternative explanation for this observation should be considered, namely, that there are two subpopulations of cells within each cell line, one that undergoes apoptosis and one that undergoes mitosis. We have addressed this question by subjecting VSM-myc-p53 and VSM-E1A-p53 cells to repeated rounds of apoptosis in 0% FCS and then in 10% FCS (not shown). These experiments indicate that there is no reduction in the rate of apoptosis with each round of killing, as would be predicted if an apoptotic subpopulation were being progressively lost from the system. Furthermore, we have demonstrated that the regions of the c-Myc protein that induce apoptosis and those that induce proliferation are identical49 and also that cells expressing deregulated c-myc undergo both processes.9 Thus, we consider the possibility of two subpopulations unlikely.
Our observations indicate that apoptosis in VSMCs may occur via p53-dependent and -independent pathways. Apoptosis is induced by deregulated expression of c-myc. E1A can functionally replace c-myc in inducing apoptosis but does not act by means of an increase in c-myc expression. Rather, both c-myc and E1A may act on a common downstream target to induce apoptosis; this target may be p53, because both E1A and c-myc increase p53 protein expression and death induced by E1A and c-myc is p53 dependent. However, the fact that upregulation of p53 alone in VSMCs does not induce death without coexpression of c-myc or E1A implies that p53 may be a necessary component of the death pathway but is not the only proapoptotic target of E1A and c-myc. Furthermore, the low level of apoptosis seen in normal VSMCs when transferred to low-serum conditions is not altered by suppressing p53, although it is suppressed by bcl-2. Thus, the apoptosis occurring in VSMCs without deregulated c-myc or E1A is p53 independent. The fact that expression of wild-type p53 increases apoptosis in VSM-myc and VSM-E1A cells without inducing a growth arrest indicates that the function of p53 in inducing apoptosis may be distinct from that inducing cell-cycle arrest. This is consistent with recent findings indicating that apoptosis induced by p53 does not require de novo protein synthesis or induction of the cyclin-dependent kinase inhibitor p21waf1/cip1,37 which is thought to mediate p53-dependent growth arrest.50 51 The finding that bcl-2 blocks death induced by both E1A and c-myc without altering p53 expression indicates that the ability of bcl-2 to suppress apoptosis is not mediated through p53.
In conclusion, our observations that apoptosis in VSMCs is regulated by dominant proto-oncogenes, antiapoptotic genes such as bcl-2, and tumor suppressor genes such as p53 suggest that the mechanisms regulating apoptosis may be as complex and diverse as those regulating proliferation and differentiation. However, identification of mechanisms that regulate apoptosis may reveal novel targets for therapeutic intervention in vascular disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-47151. Dr Bennett is supported by a British Heart Foundation Clinical Scientist Fellowship. We would like to thank Stephanie Lara and Rene Collman for the electron microscopy.
Received February 16, 1995; accepted May 5, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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