© 2000 American Heart Association, Inc.
Vascular Biology |
p53, p21WAF1/CIP1, and MDM2 Involvement in Proliferation and Apoptosis in an In Vitro Model of Conditionally Immortalized Human Vascular Smooth Muscle Cells
From Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology, and Medicine, St Mary’s Hospital, and the Ludwig Institute, St Mary’s Hospital, London, UK (J.-K.H.); and the Institute of Biology, N.C.S.R. Demokritos, Athens, Greece (D.K.).
Correspondence to Dr A.D. Hughes, Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology, and Medicine, St Mary’s Hospital, London W2 1NY, UK. E-mail a.hughes{at}ic.ac.uk
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Abstract |
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Abstract—Using an in vitro model of a conditionally immortalized cell line, we investigated how human vascular smooth muscle cells (VSMCs) are affected by the expression of simian virus 40 (SV40) large T antigen (LT antigen), which binds to cell cycle regulators, such as the tumor suppressor protein p53. Cells were obtained after infection of saphenous vein–derived VSMCs with a nonreplicative retroviral vector containing a temperature-sensitive (ts) mutant of SV40 LT antigen and were shown to have maintained some characteristics and responses of VSMCs. Under permissive temperature conditions (36°C), the increased rate of cell proliferation was shown to be associated with expression of LT antigen and with LT-antigen binding to and inactivation of p53. p53 inactivation failed to block apoptosis induced by serum withdrawal or by UV irradiation. Downregulation of LT-antigen expression at the nonpermissive temperature (39°C) was shown to be associated with growth arrest, increased expression of the cell cycle inhibitor p21WAF1/CIP1, increased MDM2-promoter activity, and differential expression of MDM2 gene products, suggesting that p53-induced transcription/transactivation may be involved in VSMC cell cycle control but not necessarily apoptosis. The established SMC line HVTs-SM1 may be a useful model for the study of processes involved in myointimal hyperplasia and cellular aging, as well as for the study of cell cycle control in general.
Key Words: vascular smooth muscle • SV-40 • cell line • p53 • MDM2 • p21WAF1/CIP1
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Introduction |
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Vascular smooth muscle cell (VSMC) proliferation has long been considered to be a key event in the remodeling of the vascular wall during development and following vascular injury in such diseases as atherosclerosis and vascular restenosis after invasive intervention.1 2 More recently, the precise role of proliferation in atherogenesis has received more critical attention,2 and other processes, such as contraction, cell migration, and apoptosis, have also been proposed to be involved. The mechanisms regulating cell cycle control in VSMCs remain largely unknown.2 Primary VSMCs, especially of human origin, are difficult to study in vitro: their availability is limited, they grow slowly, and they have a limited life span. Furthermore, cell responses may vary between cell lines depending on the donor, disease, or tissue.2 3 Recently, there has been interest in in vitro models of established VSMC lines obtained from animal or human primary cells by transfection with SV40 LT antigen or other viral oncogenes.4 5 6 7 However, although there are some reports of immortalized cell lines derived from animal cells, human cells in general have been more difficult to immortalize.
We describe here some of the cellular characteristics of a SMC line derived from human saphenous vein that was established by infection with a temperature-sensitive (ts) SV40 LT antigen. Rapid proliferation of cells was associated with LT antigen expression and LT antigen–dependent inactivation of G1/S checkpoint control via inactivation of such cell cycle regulators as p53.8 Downregulation of LT antigen expression under nonpermissive temperature conditions was shown to be associated with growth arrest, the appearance of morphological characteristics typical of senescent cells, and upregulation of the expression of growth regulator(s) downstream of p53, such as p21WAF1/CIP1.9 Transformed cells showed high levels of apoptosis that increased after serum withdrawal or DNA damage by UV irradiation. Because this cell line maintains some characteristics of human VSMCs, it may provide a useful in vitro model to study the regulation of phenotype and to investigate changes in cell cycle control involved in cellular aging, cell death, and other processes contributing to vascular disease.
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Reagents, Antibodies, and Plasmids
All culture media were from Gibco Life Technologies/BRL. [Methyl-3H]thymidine (740 GBq/mmol) was from ICN Flow. Unfractionated porcine heparin (Paynes & Byrne) was a gift from Dr B. Mulloy, National Institute of Biological Standards and Control, South Mimms, UK. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor (EGF), and transforming growth factor (TGF)-ß were from Life Technologies. All other reagents used, unless otherwise specified, were from Sigma. Primary and secondary antibodies were from DAKO A/S. The p53 (non–cross-reactive) human specific monoclonal antibody (mAb) DO-1,10 the p21WAF1/CIP1 specific mAb pAb53G817,11 the MDM2-luciferase reporter plasmid, and the independent reporter plasmid CMB–ß-gal, were kindly donated by Dr X. Lu (Ludwig Institute for Cancer Research, London, UK) and the SV40 LT antigen specific mAbs 419 and 42312 by Dr T. Kamalati (Haddow Laboratories, Institute for Cancer Research, Sutton, UK). The mAb SM-E713 for smooth muscle myosin heavy chains type 1 and 2 was a gift from Prof P. Pauletto (Clinica Medica I, Padova, Italy).
Cell Culture Conditions: Transfection and Isolation of
Conditionally Immortalized Human VSMCs
Human SMCs were isolated from explants derived from normal
saphenous vein from patients undergoing coronary artery bypass
as previously described.3 Cells that migrated from these
explants were subcultured at a split ratio of 1:2 in DMEM containing
15% FCS. Equal numbers of cells from 12 separate lines derived from
human saphenous vein–derived SMCs with similar proliferative responses
were pooled at passage 2, subcultured, and used at passage 3 to 4 and
are referred to in this study as normal or untransformed VSMCs.
Actively growing primary VSMCs obtained from a single middle-aged
patient were infected at passage 4 with conditioned medium from the
amphotropic packaging PA317 cell line with Polybrene (8 mg/mL). The
PA317 cell line produces the replication-defective helper-virus–free
vector pZipSV40-U19-5, which was constructed by insertion at the single
BamHI restriction site of the vector pZipNeoSV(X), a
full-length SV40 LT antigen carrying both the ts A58 and U19
mutations.14 The ts A58 mutation confers temperature
sensitivity for LT-antigen expression, and the U19 mutation disables
replication of the retroviral vector.14 15 Seven days
after infection, the cells were put under G418 (500 µg/mL) selection.
The infection was carried out by Dr M.J. O’Hare at Haddow
Laboratories, Institute of Cancer Research (Sutton, Surrey, UK).
Transformed cells that survived G418 selection were subcultured in DMEM
containing 10% FCS and 200 µg/mL G418. Infection, selection, and
maintenance of cells were carried out at the permissive
temperature of 36°C. After 
100 population doublings (PDs), the
cells were subcloned by serial dilution, because there was no apparent
crisis. The various clones obtained had similar morphologies, but only
20% survived subculturing for an additional 10 passages. One,
designated here as HVTs-SM1, has been in continuous culture for >2
years, exceeding 200 PDs.
Plasmid Transfection and Luciferase Assay
HVTs-SM1 cells plated in dishes 3 cm in diameter
(104/dish) in 10% FCS/DMEM were transfected 24
hours later with 1 to 2 µg of MDM2-luc reporter plasmid by the
calcium phosphate precipitation method.9 An equivalent
amount of CMV–ß-gal plasmid was cotransfected for normalization of
transfection efficiency. The transfected cells were incubated at 36°C
or 39°C, and at the indicated time points, they were washed 3 times
with PBS, lysed in 100 µL of lysis buffer (Promega UK Ltd), and
assayed for reporter activity of luciferase and
ß-galactosidase.16
DNA Synthesis and Cell Proliferation
Normal and HVTs-SM1 cells were plated at 36°C in 96-well
plates at 104 cells/well in 10% FCS/National
Cancer Tissue Culture (NCTC)-109. Twenty-four hours later, the
cells were washed 3 times with PBS and growth-arrested (72 hours) in
serum-free (SF) medium. DNA synthesis induced by the various stimulants
was measured 30 hours after stimulation with 5 µCi
[methyl-3H]thymidine that was added 6 hours
before the DNA was harvested, as previously described.17
Cell growth rates were measured by plating 5x104
cells/cm2 in 6-well plates in 10% FCS/NCTC-109
medium. Twenty-four hours later, the cells were growth-arrested in SF
NCTC-109 medium for 72 hours. Then the medium was replaced with SF or
10% FCS-containing medium in the presence or absence of 150 µg/mL
heparin, and the cells were incubated at 36°C or 39°C with medium
changes every 3 days. At the indicated time points, cells were
trypsinized and counted with a Coulter counter. In addition, in some
studies, cells synthesizing DNA were also identified after dual
labeling with bromodeoxyuridine (BrdU) and Hoechst 33258 dye. Briefly,
cells plated on glass coverslips at different densities in 24-well
plates overnight were growth-arrested as above and labeled with 50
µmol/L BrdU added in fresh SF medium for the last 24 hours. The cells
were fixed in freshly prepared 4% paraformaldehyde/PBS
(10 minutes), permeabilized with 0.2% Triton X-100/PBS
(5 minutes), treated with 2N HCl (10 minutes), incubated with
fluorescein-conjugated anti-BrdU mAb/PBS (30 minutes), and
then stained with 1 µg/mL Hoechst/PBS (30 minutes) in the dark at
room temperature. Cells were washed 3 times with PBS at each step.
Hoechst- and BrdU-positive nuclei were counted on a Zeiss
fluorescence microscope with a x20 objective; a field
containing 200 cells was used for quantification purposes.
Immunofluorescence Microscopy
Cells were plated on glass coverslips and were grown in 10%
FCS/DMEM. They were washed with PBS, fixed in ice-cold ethanol or
methanol/acetone solution (10 minutes), washed 5 times with PBS, and
air-dried. Then, they were incubated with primary antibody (diluted
1:50 or 1:100 in 10% serum) overnight at 4°C, washed 3 times for 10
minutes with PBS, and incubated with FITC-conjugated anti-mouse or
anti-rabbit IgG (1:50) in PBS at 37°C (1 hour). After being washed,
the cells were stained with Hoechst 33258/PBS (1 ng/mL, 10 minutes) and
were observed under a fluorescence microscope.
Immunoprecipitation and Immunoblotting
Subconfluent cultures of normal or HVTs-SM1 cells grown at
36°C were growth-arrested in SF NCTC-109 medium, which was replaced
after 72 hours with either fresh SF or 10% FCS medium. After
incubation at 36°C or 39°C for the indicated periods, the cells
were washed 3 times with ice-cold PBS and lysed with 100
µL/106 cells lysis buffer. Proteins in cell
lysates were resolved by electrophoresis on 10% or 12.5%
SDS-polyacrylamide gels, followed by Western blotting on
nitrocellulose membranes (Schleicher and Schuell). Briefly, the
membranes were blocked with 10% nonfat milk in 10 mmol/L Tris-HCl
(pH 7.4), 150 mmol/L NaCl, 0.05% Tween-20 (TTBS) buffer, washed
with TTBS, and incubated with the primary antibody at room temperature
(1 hour) or at 4°C overnight. Then they were incubated (1 to 2 hours)
with horseradish peroxidase–conjugated second antibody (1:1000) in
TTBS at room temperature. Immunoreactive bands were visualized on Kodak
X-OMAT AR film by chemiluminescence (ECL kit) according to the
manufacturer’s (Amersham) instructions.
For immunoprecipitation, cell lysates (1.0 µg) were precleared with protein G Sepharose beads (10 µL) and mixed with 5 to 10 µg of purified antibody or 1 to 2 mL of ascites at 4°C overnight. Antibody complexes were pulled down with 10 µL of protein G Sepharose beads at 4°C (1 to 2 hours). After the beads had been washed 3 times with lysis buffer and twice with the same buffer without NP-40, bound immunocomplexes were extracted by boiling (3 minutes) in 20 µL of 2x concentrated SDS-PAGE sample buffer, electrophoresed, and immunoblotted.
UV Irradiation and Cell Apoptosis
Normal and HVTs-SM1 cells were irradiated at 50
mJ/cm2 in an XL-1000 UV crosslinker (Spectronics
Corp) and then incubated at 36°C in 10% FCS/DMEM. At the indicated
time points, they were trypsinized and counted. In parallel, the
detached and adherent cells from each of 2 additional wells were
collected for flow cytometry, washed with PBS, fixed in 70% ice-cold
ethanol, and centrifuged at 2000 rpm (10 minutes, 4°C). Cells
and apoptotic bodies in each pellet were resuspended in PBS
containing 50 µg/mL propidium iodide and 12 µg/mL RNase A and
incubated in the dark (30 minutes, room temperature). Flow
cytometry was performed in a FACSORT (Becton Dickinson), and
fluorescence signals were plotted with Cell Quest software.
Statistics and Data Analysis
All data are presented as mean±SD of n observations.
Statistical comparisons were made with a Student’s t test;
a value of P<0.05 was considered significant.
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Results |
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Conditionally Immortalized Human Vein–Derived SMCs
Normal VSMCs (passaged at 1:3 split ratio) stopped dividing after 10 to 15 passages and became senescent. The transformed HVTs-SM1 cells, however, have been in continuous culture at the permissive temperature for >200 PDs. The transformed cells lost the elongated, spindle-like shape of normal VSMCs and were smaller, with a morphology ranging from polygonal epithelioid to fibroblast-like (Figure 1A
versus 1B). When HVTs-SM1 cells were
transferred to a temperature of 39°C, after 5 to 7 days they
acquired morphological characteristics more similar to those of normal
cells (Figure 1C), except that cells were more enlarged and
flattened, a characteristic of in vitro senescent
cells.18 19
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We investigated whether the transformed HVTs-SM1 cells retained
cytoskeletal proteins typical of SMCs. The majority of transformed
cells stained positive for smooth muscle 
-actin. The decorated
filaments appeared to be more cytoplasmic and irregular in the
transformed cells, whereas in normal cells they traversed the cell
along its long axis (Figure 2, A1 and
A2). There were no observable differences in the strength of smooth
muscle -actin staining and filament distribution when either normal
or transformed VSMCs were incubated at 39°C for 24 to 48 hours.
Staining for vimentin was positive in both cell types, but compared
with normal cells, the filaments of the transformed cells were more
sparse, less organized, and spread from perinuclear bundles to the
periphery of the cell (Figure 2, B1 and B2). Antibody staining
for von Willebrand factor and desmin was negative for either
normal or transformed cells. The transformed cells showed weak and
mainly perinuclear rod-like staining for smooth muscle myosin heavy
chain (Figure 2, C2), whereas in normal VSMCs, the staining was
observed in organized filaments along the long axis of the cell (Figure 2, C1).
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LT-Antigen Expression and Expression of Cell Cycle Regulatory
Molecules in HVTs-SM1 Cells
Previous reports on cells immortalized with SV40 have identified
that the expression of a functional LT antigen is required for the
maintenance of the immortalized
phenotype.15 Those studies have also established
that SV40 LT antigen binds to wild-type p53 and extends its
half-life.8 20 We therefore investigated whether HVTs-SM1
cells expressed LT antigen conditionally and whether LT-antigen
expression had an effect on p53 protein level. Indirect
immunofluorescence with anti–LT-antigen or with
anti-human specific wild-type p53 antibodies in cells growing at 36°C
showed positive nuclear staining for both antigens in all cells (Figure 3). LT-antigen and p53 nuclear
immunofluorescence staining were reduced to almost
background levels when cells were transferred to 39°C (Figure 3, A2 and B2), and normal cells grown under similar conditions
showed no nuclear staining with either antibody. Similarly,
immunoblotting studies showed elevated levels of LT
antigen and p53 in cells growing at 36°C but not in cells cultured at
39°C (Figure 4). Levels of LT antigen
and p53 were unaffected by the presence or absence of 10% FCS, and p53
was found to coimmunoprecipitate with LT antigen (Figure 4). It
should be noted that the HVTs-SM1 cells had to be cultured for 5 to 7
days at 39°C before the protein levels of LT antigen and p53 were
reduced below detection limits of the antibodies used.
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We also examined the transcription of molecules that are known to be
regulated by p53, such as p21 and MDM2, because it has been documented
that LT-antigen binding to p53 may adversely affect its
functions.20 The specific mAb to the cell cycle
inhibitor p21 detected a 21-kDa protein band only when the
transformed cells were cultured at 39°C (Figure 4
). Taking
into account the observed temperature-dependent LT-antigen and p53
protein expression profiles and the coimmunoprecipitation of LT antigen
with p53, these results are in keeping with the proposal that
p53-induced p21 expression may be inhibited by LT-antigen binding to
p53.9
To investigate the effect of LT-antigen expression on the
transcriptional regulation of MDM2 by p53, HVTs-SM1 cells were
transfected with an MDM2-luciferase reporter plasmid. The luciferase
activity profile obtained showed a 4-fold increase in MDM2-promoter
activity when the cells were cultured at 39°C rather than at 36°C
(Figure 5). Furthermore, Western blot
analysis of HVTs-SM1 lysates from cells cultured at the 2
temperatures indicated that MDM2-protein levels were also reduced in
HVTs-SM1 cells cultured at permissive temperature (Figure 5, inset).
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Growth and Survival of HVTs-SM1 Cells
We then investigated the growth rate and survival of HVTs-SM1
cells. At 36°C, the serum growth-response curves of transformed
(Figure 6A) and normal (Figure 6D)
cells plated at 5x104
cells/cm2 appeared sigmoidal. The transformed
cells, however, grew more rapidly (doubling time, 49±9 hours; n=4;
P<0.001) and reached a density 6-fold higher than that
of normal cells (doubling time, 132±36 hours [n=12]; passage 3).
Under permissive temperature conditions, the serum-dependent
proliferation of HVTs-SM1 cells, like that of normal cells, appeared to
slow down through contact inhibition (Figure 6A versus 6D).
Anti-BrdU antibody labeling and acridine orange staining showed that
cell division as well as cell death by apoptosis was taking
place in HVTs-SM1 cells concurrently during this period (data not
shown). After a long incubation period (3 to 4 months), normal VSMCs
continued to proliferate very slowly and form a densely packed
multilayer, whereas the transformed cells did not form such a
multilayer.
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Unlike nontransformed cells, serum withdrawal under permissive
conditions did not cause the HVTs-SM1 cells to growth-arrest (>90% of
the cells were BrdU-positive 24 hours later). However, the cells were
not able to sustain continuous proliferation; their number declined
(Figure 6C), and acridine orange staining demonstrated many
apoptotic cells in this period (Figure 6, E and F). In
contrast, serum withdrawal from normal SMCs induced only a small loss
of cells within the first 24 to 48 hours. The remaining cells appeared
to survive in a growth-arrested state for at least a week (Figure 6D).
The temperature change to 39°C appeared to have no effect on the
serum-dependent proliferation of HVTs-SM1 cells in the first few days.
However, cell number declined 5 to 7 days later (Figure 6B), and
acridine orange staining indicated increased apoptosis. The
fate of surviving cells depended on their density. If the cells did not
become very sparse after the period of rapid cell death, they continued
to proliferate very slowly and remained viable for at least 3 to 4
months. Flow cytometry indicated that the majority of cells accumulated
at G1 as well as G2 phases
of the cell cycle (Table), in
agreement with previous reports.15 With time, there was a
significant increase of cell size and appearance of perinuclear
vacuoles, and these cells resembled in vitro senescent or
atherosclerotic cells.18 19 Provided that the temperature
was shifted back to 36°C before the cells became very sparse, they
were able to quickly resume as vigorous a growth in response to serum
as that of cells that had not been exposed to the nonpermissive
temperature.
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We also investigated whether HVTs-SM1 cells retained responses to
smooth muscle growth factors and heparin. As shown in Figure 7A, under permissive temperature
conditions, induction of HVTs-SM1 cells by 10% FCS had a large,
positive effect on thymidine incorporation, whereas induction with PDGF
and EGF had only a small positive effect; TGF-ß had no apparent
effect. Heparin (150 µg/mL) inhibited thymidine incorporation induced
by serum by 50% and that induced by PDGF and EGF to a lesser extent
(Figure 7A). Heparin inhibition of serum-induced DNA synthesis
was broadly comparable to that we have previously observed in normal
VSMCs,3 and no further effect was seen after an increase
in the heparin concentration up to 5-fold. Serum-induced cell
proliferation was inhibited by 25% to 30% when cells were
cultured in the presence of 150 µg/mL heparin for 7 days (Figure 7B). When cells were cultured in the presence of 0.5% serum,
addition of heparin (150 µg/mL) resulted in a small net reduction in
cell number (Figure 7B versus Figure 6C).
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LT-Antigen Transformation Increases SMC Sensitivity to
UV Irradiation
Flow cytometry of UV-irradiated (50 mJ/cm2),
normal SMCs cultured at 36°C showed no significant time-dependent
increase in the percentage of apoptotic cell bodies (control, 0
time: 10%; 24 hours after irradiation: 7%), suggesting that the cells
are resistant to UV irradiation. The majority of cells remained
in G1 phase of the cell cycle, with only a small
proportion of cells traversing S phase (Table), in keeping with
the slow proliferation rate of normal SMCs. In contrast, the
transformed cells showed a 6-fold increase in the percentage of
apoptotic bodies as early as 6 hours after irradiation
(control, 0 time: 6%; 6 hours after irradiation: 34%). This was
accompanied by a time-dependent increase in degraded DNA and a decrease
in the percentage of cells in G1 phase. UV
irradiation had no significant effect on the proportion of cells
traversing S phase. However, 24 hours after irradiation, there was an
increase in the proportion of cells in G2 phase
(Table). Compared with nonirradiated HVTs-SM1 cells, there was
40% to 50% reduction in cell numbers 48 to 50 hours after
irradiation; this was accompanied by a period of growth arrest and then
recovery (Figure 8). Early
apoptosis of UV-irradiated HVTs-SM1 cells was also confirmed by
detection of DNA fragmentation 4 hours after irradiation (data not
shown).
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Discussion |
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In vitro aging and senescence limits the life span of primary human VSMCs.18 19 This and the interindividual and intraindividual variability of cellular responses to growth factors/inhibitors3 emphasizes the need for established cell lines. However, in contrast to rodent cells, only a few such human cell lines have been established by viral infection.21 In a previous study that used SV40 infection of human VSMCs, the maximum life span reported was
60
PDs.22 Using a ts replication-defective SV40 vector, we
have been able to establish an immortal (life span >200 PDs) human
VSMC line that we have named HVTs-SM1. These cells are conditionally
immortalized, because their proliferative phenotype is
expressed only under the permissive temperature. Under nonpermissive
temperature conditions, the cells growth-arrest and eventually lose
viability in a density-dependent manner, implying the presence of an
autoregulatory mechanism for survival. The transformed cells are
smaller and have lost the typical spindle morphology that characterizes
normal VSMCs. Nevertheless, the cells continue to express smooth
muscle–specific cytoskeletal proteins, including myosin heavy chain
and -actin. We have also shown that at permissive temperature,
HVTs-SM1 cells retain, to some extent, their response to such SMC
growth factors as PDGF-BB and to such growth inhibitors as
heparin.3 23 However, DNA synthesis in response to PDGF-BB
was 2- to 3-fold lower than in normal cells, possibly as the result of
LT-antigen–induced downregulation of PDGF receptor
expression.24 SMCs play an important role in the pathogenesis of atherosclerosis, vascular graft occlusion, and restenosis after angioplasty. Although the precise role of smooth muscle proliferation and apoptosis in the development of these diseases, particularly atherosclerosis, is debated,2 25 it is likely that these processes contribute, at least at some stage, to their pathogenesis. HVTs-SM1 cells may be a useful model to study the role of growth regulators in normal and pathological VSMC proliferation, cell death, and other aspects of cell cycle control.
SV40 LT antigen is known to have transforming and tumorigenic potential, because it is able to target and inactivate several molecules involved in the control of cell cycle progression, including the tumor suppressor molecules Rb and p53.20 p53 mediates the transcription of the cyclin-dependent kinase inhibitor p21WAF1, and this is a key event in cell growth arrest and in cell apoptosis due to DNA damage.9 p21 regulates the activity of Rb by inhibiting its phosphorylation, the subsequent release of the E2F family members, and finally the cell’s entry to S phase.26 p53 is also known to interact with the tumor progression factor MDM2,27 28 which promotes p53 degradation and can inhibit p53 transactivation and transrepression.29 30 Interestingly, both p53 and MDM2 have been reported to be expressed in SMCs and macrophages in human atherosclerotic tissue,31 and high levels of p53 in association with cytomegalovirus infection were seen in a study of coronary restenosis.32 Our results indicate that the rapid and continuous proliferation of HVTs-SM1 cells under permissive temperature conditions is associated with the expression of SV40 LT antigen and with LT-antigen binding and inactivation of p53. This was based on the evidence that the expression of p21WAF1 protein and of MDM2-promoter activity was inhibited under these conditions. Because Rb activity is regulated by p21-dependent phosphorylation, deregulation of p21 expression would also be expected to affect Rb activity. In addition, as previously reported, direct binding of LT antigen to Rb may release E2F transcription factor(s) and thus contribute further to the deregulation of cell growth arrest and to apoptosis.33 Deregulation of cell cycle control at the level of p53 and at the level of growth regulators that depend on p53 activity in HVTs-SM1 cells is further suggested by the fact that when LT antigen is no longer functional because of a temperature shift to 39°C, the cells were able to growth-arrest. Under these conditions, the cells express high levels of p21, as is observed in senescent cells in vitro.34 Furthermore, cells remained viable and could be rescued and resume proliferation even several weeks later after return to the permissive temperature.
Apoptosis of VSMCs has been observed in vascular remodeling during development and may be another important mechanism in the modulation of cellularity in vascular disease.35 We observed that proliferation of HVTs-SM1 cells at the permissive temperature was accompanied by apoptosis. Compared with normal cells, apoptosis of transformed cells was increased by serum withdrawal, suggesting that the cells are more dependent on serum factors for survival than for proliferation. It was recently proposed that p53 is a potent inducer of apoptosis when Rb is inhibited in smooth muscle.22 However, the data from our system, in which both Rb and p53 are inactivated by SV40 LT antigen (at the permissive temperature), imply the presence of a p53-independent pathway for apoptosis. This inference is supported by the conclusions of a recent study that examined the effect of p53 inactivation on atherogenesis in apolipoprotein E–knockout mice, an animal model for atherosclerosis.36 However, the cell death observed on transfer of cells to the nonpermissive temperature may be attributable to the slow loss of SV40 LT-antigen expression. UV irradiation induced rapid apoptosis in HVTs-SM1 cells grown at the permissive temperature, whereas normal cells did not undergo detectable apoptosis after UV irradiation. The reason for this is unclear, but it may be a result of their slow proliferation rate, which allows DNA repair to take place. This question requires further study, and examination of the effects of more pathophysiologically relevant mediators, such as oxidized lipoproteins, oxysterols, and nitric oxide, may be of particular interest. Apoptosis after DNA damage by UV irradiation has been also associated with increased p53 activity,37 but from the results of the present study, it appears that p53-induced transcriptional activity is not required for UV-induced apoptosis. Overall, a possible hypothesis is that in HVTs-SM1 cells, E2F1 overexpression (due to inactivation of Rb by LT antigen) can induce apoptosis independently of p53, as previously shown in Saos-2 cells,27 which are null for p53 and lack a functional Rb. Further studies will be necessary to confirm this hypothesis.
In conclusion, we have shown that the established HVTs-SM1 cell line can be used as an in vitro model to study specific perturbations of cell proliferation. This model of human VSMCs offers opportunities to establish a better understanding of the processes and molecules involved in the aging and senescence of the vasculature and in the control of the cell cycle in human VSMCs.
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Acknowledgments |
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This work was supported by the Thrombosis Research Trust (UK), the British Heart Foundation (UK), the British Council, the UK Home Office, and the Greek-Britain Scientific and Technological Collaboration. D. Kletsas was supported by a FEBS fellowship. We are grateful to M.J. O’Hare for the transfection of cells and K. Gallagher for assistance with tissue culture. We also thank H. Pratsinis and members of Clinical Pharmacology, Imperial College, for their helpful comments and assistance.
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Footnotes |
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C. Demoliou-Mason, J.-K. Hsieh, and D. Kletsas contributed equally to this work.
Received March 11, 1999; accepted September 13, 1999.
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References |
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