© 2001 American Heart Association, Inc.
Vascular Biology |
Mitogen-Induced p53 Downregulation Precedes Vascular Smooth Muscle Cell Migration From Healthy Tunica Media and Proliferation
From the Cardiovascular Research Center, IIBB-CSIC, HSCSP, UAB, Barcelona, Spain. Dr Rodríguez-Campos is now at Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.
Correspondence to Prof Lina Badimon, IIBB-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain. E-mail lbmucv{at}cid.csic.es
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Abstract—The tumor suppressor protein p53 plays an important role in the cell-cycle G1 and G2 checkpoints. In response to DNA damage, p53 can induce the transcription of p21, which inhibits the activation of various G1 cyclin/cyclin-dependent kinase complexes. It is not known whether p53 plays a role in the initial migration of vascular smooth muscle cells from the arterial tunica media (mVSMCs). In this study, we have investigated whether mVSMC migration from healthy tunica media of young pigs and proliferation are regulated by p53. After 6 hours of incubation in mitogen-rich medium, explanted porcine tunica media tissue showed complete downregulation of p53 protein and p53 mRNA. The blockage of gene activity was not due to DNA methylation at the 5' control region of the gene. The mVSMC outgrowth did not show p53 expression. Mitogen-depletion of cultured p53-/mVSMCs did not restore p53 expression. Incubation of explanted porcine tunica media tissue in mitogen-deprived medium increased p53 protein content and blocked mVSMC outgrowth from the explant. As in p53-deficient rodent cells, mVSMCs incubated with colcemid overrode the spindle-dependent checkpoint, giving polyploidy and chromosomal pairing. UV-induced DNA damage in mVSMCs incubated with mitogen-free medium induced p53 expression and apoptotic cell death showing DNA nucleosomal laddering. However, UV-irradiated mVSMCs incubated in mitogen-rich medium did not express p53 and did not show cell death. In conclusion, our results demonstrate that early mVSMC migration from the tunica media requires mitogen-induced suppression of p53 that is highly expressed in contractile mVSMCs residing in the healthy vessel wall.
Key Words: p53 • cell proliferation • vascular disease • restenosis • UV irradiation
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Introduction |
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Atherosclerosis is responsible for cardiovascular and cerebrovascular diseases, the first cause of morbidity and mortality in Western countries.1 Mitogens, growth factors, cytokines, and vasoregulatory substances are some of a large number of elements involved in the chronic evolution of the disease.2 In response to multiple stimuli, vascular smooth muscle cells (VSMCs) from the arterial tunica media (mVSMCs) are activated, and migration to the intima and proliferation seem to be early steps at the onset of the atherosclerotic process.1 2 3
Studies of human carotid atheromatous plaques have shown p53 immunoreactivity in the nuclei of the different cell types present in the nonproliferative atheroma core, suggesting that the expression of p53 may be involved in the control of cellular proliferation in advanced human atherosclerotic plaques.4 Additionally, cell proliferation, senescence, and apoptosis in intimal VSMCs from atherosclerotic plaques has shown to be regulated by p53.5 p53 has multiple functions, including cell-cycle control in response to DNA damage, DNA repair, and induction of apoptosis. Mutations on the p53 gene, promoting the loss or loss of function of p53, seem to be a common feature in the development of most human cancers.6
In response to stresses such as hypoxia, nucleotide depletion, or DNA damage, p53 is activated to preserve the integrity of the genome either by arresting cell-cycle progression in G1 through induction of p21 or by directing apoptosis by the induction of specific target genes that differ from those that induce growth arrest.7 p53 is not detectable immunohistochemically in either balloon-injured rabbit carotid arteries or in cultured bovine VSMCs8 ; however, the content of endogenous p53 in VSMCs in culture remains controversial because rat VSMCs seem to express p53 mRNA.9 On the other hand, Yonemitsu et al,8 who did not find an explanation for the loss of endogenous p53, observed a strong effect of transfected wild-type p53 on VSMC growth control. However, the role of p53 content in cells of the healthy tunica media (contractile phenotype) of a young artery has yet to be determined, and whether there is a p53 regulation of the mVSMC outgrowth, migration, and proliferation has not yet been investigated. It is our hypothesis that p53 is involved in the maintenance of mVSMC quiescence in the healthy tunica media of young vessels and that the early proatherosclerotic changes in the vascular wall with mVSMC migration from the tunica media to the intima are due to mitogen-induced suppression of p53.
The objective of the present study has been to assess whether the triggering factor in smooth muscle cell migration from the media, in conditions associated with early atherosclerotic lesion formation, is related to regulatory changes in the cell level of the tumor suppressor protein p53. The present study has demonstrated through cellular, molecular, and functional evidence that mVSMC migration from the explanted tunica media (ETM) occurs after mitogen-induced p53 downregulation of the contractile VSMCs.
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Methods |
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Explants and Cell Cultures
Primary VSMCs were obtained as previously described.10 Porcine arteries from healthy 6-month-old pigs were excised immediately after euthanasia, the tunica media was carefully dissected and isolated, and small pieces (1- to 5-mm2 square) were explanted and incubated at 37°C and 5% CO2 in growth medium supplemented (20%) or not with FCS. The first migrating mVSMCs were seen between 7 and 10 days after the initiation of the incubation in FCS-supplemented growth medium. Outgrowing mVSMCs reached subconfluence 15 to 20 days later. We did not observe mVSMC migration or proliferation in explanted tissue incubated with FCS-depleted growth medium. Pieces of ETM before and after incubation with or without FCS were frozen in liquid nitrogen, homogenized in a mortar with liquid nitrogen, dissolved in lysis buffer, and processed as in cultured mVSMCs (see below). Monoclonal cell cultures were prepared as follows: In brief, polyclonal mVSMC culture B87 (passage 9, from the tunica media of a pig coronary artery) and B136 (passage 6, from the carotid artery) were trypsinized, counted, and replated in a 24-well plate (Falcon) at 0.5, 1, 2, 5, 10, and 30 cells per well. One week later, dispersed well-separated colonies (derived from a single cell) were observed; they were carefully isolated with a sterile tip of an automatic pipette and replated on a fresh multiwell plate (1 colony per well); and 5 clonal cultures, named B87 and B136 mVSMC L1 to L5, were obtained. When the cells reached 70% to 80% confluence, they were trypsinized and continuously subcultured. Monoclonal and polyclonal mVSMC lines were checked by immunostaining with anti–
-actin antibodies; furthermore, when confluent, the cells
displayed the typical "hill-and-valley" shape characteristic of
smooth muscle cells. Lysates from cultured mVSMCs were prepared from
cell pellets. After they were pelleted, the cells were washed 2 times
in cold PBS, diluted in 0.5 mL lysis buffer (50 mmol/L HEPES [pH
7.4], 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl
fluoride [Sigma], and 2 U/mL RNasin [Promega]), and
incubated for 30 minutes on ice. Lysates were then cleared for 15
minutes at 14 000 rpm at 4°C, and supernatants were stored at
-80°C. Protein concentration was measured by using the BCA protein
assay reagent (Pierce). Animal care was in accordance with
institutional guidelines.
Immunodetection
Approximately 30 µg of each extract was
electrophoresed in 8% SDS-PAGE, followed by electrotransfer to
nitrocellulose (Bio-Rad). Immunodetection was performed at room
temperature by incubation with mouse monoclonal antibodies against
human p53 (Pab 240, No. sc-99, or DO-1, No. sc-126, Santa Cruz
Biotechnology Inc) diluted at 1:2000 in BLOTTO (5% fat-free dry milk
and 0.05% Tween 20 [Fluka] in PBS). Filters were washed in PBS and
0.05% Tween 20 and incubated with a rabbit antimouse conjugate (DAKO;
P0161, diluted at 1:10 000 in BLOTTO). As a positive control, 0.1 µg
of full-length human p53 fused with glutathione S-transferase [80 kDa;
p53(1-393), No. sc 4246, Santa Cruz Biotechnology Inc] was used.
Western blots were assayed by use of ECL detection reagents (Amersham
Pharmacia Biotechnology)
RNA Isolation and Northern Blot
Small pieces of tunica media were dissected as
described for the whole-cell extracts, frozen in liquid nitrogen,
homogenized, and dissolved in an ULTRASPEC RNA Isolation
System (Biotecx Laboratories, Inc) according to the manufacturer’s
instructions. Approximately 10 µg of total RNA was separated in 1%
formaldehyde-MOPS denaturing agarose gel, transferred to Hybond N+
membrane (Amersham), and probed with a human p53 cDNA. After exposure,
the filter was washed and reblotted with a probe of
GAPDH.
Scanner Quantification
To analyze the differences in band
intensities, Western and Northern blots were scanned with an
AlphaImager system (Alpha Innotech Corp).
Analysis of 5' Control Region of p53
Gene
We used a technique that was based on the ability of
sodium bisulfite to convert cytosine residues, but not the
5'-methylcytosine, to uracil; this conversion was followed by
polymerase chain reaction (PCR) amplification to yield a fragment in
which all the uracil and thymine residues were amplified as
thymine and only the 5'-methylcytosine residues were
amplified as cytosine; thus, when cytosines are not
methylated, the 5'-cytosine-phosphate-guanine-3' (CpG)
dinucleotides are converted to
5'-uracil-phosphate-guanine-3' (5'-cytosine-phosphate-adenine-3' in the
complementary strand) after the bisulfite-PCR
process.11
Genomic DNA from the polyclonal cell line B136 (P6) and from
fresh aortic tunica media was isolated, and the 5' control region of
the swine p53 gene was obtained by PCR amplification using 2 primers
designed on the basis of the high degree of homology between rats,
mice, and humans.12 Primers
sequences were as follows: 5'-AGTTGCCCTTACTTGT-3' and
5'-GGGAAGCGTG-TCACCGT-3'. PCR amplification was performed with 100
ng of genomic DNA as a template. Sequencing revealed 
80% homology
with the human sequence. Bisulfite modifications were performed as
described, and the modified DNA was PCR-amplified with use of the
following primers: 5'-TAGTTGTTTTTATTTGTGATGGTGATTTTG-3' and
5'-TAAAAAACATATCACCATTCTATTCTAAAA-3'. Fragments of the expected size
were purified from a 2% agarose gel as described and directly
sequenced with the 70770 Sequenase DNA Sequencing Kit Version 2.0
(US&B, Amersham). The primer was 5' end-labeled with T4
polynucleotide kinase (Promega) and
[
-33P]ATP.
Cytogenetics
Cells were plated on 100-mm dishes containing 4
sterile glass cover slides; when cells reached 50% to 60% confluence,
they were synchronized by the addition of thymidine (2.5 mmol/L
final concentration) to the growth medium. After 6 to 8 hours, medium
was removed, cells were washed with sterile PBS, fresh medium
containing 0.2 µg/mL of colcemid (Boehringer-Mannheim) was
added, and the plates were incubated at several different time
intervals (6 to 72 hours). Swelling of the mitotic cells was carried
out by the addition of 6 mL of 75 mmol/L potassium chloride for 30
minutes at 37°C. Finally, the cells were fixed with 6 mL of
fresh-made methanol–glacial acetic acid (3:1); to ensure complete
fixation, the fixative was changed 4 times before
drying.
UV Irradiation of Cultured mVSMCs
mVSMC cultures from the monoclonal cell line B87 (L1,
derived from the coronary artery) were plated and grown in
100-mm dishes with medium supplemented with 20% FCS until 60% to 70%
confluence. Half of the plates were arrested by serum
deprivation for 48 to 72 hours; before UV irradiation, the
medium was removed, and the plates were irradiated at different doses
(from 25 to 600 J/m2) by UV-C light
(254 nm) with use of a Stratagene UV Stratalinker 2400. Growth medium
was added, and the plates were incubated at 37°C for 8 hours. At 1,
3, and 8 hours after irradiation, the cells were trypsinized and washed
with PBS, and each sample was divided into equal aliquots for protein
extracts and DNA purification.
DNA Purification
The pelleted cells were resuspended in DNA lysis
buffer (10 mmol/L Tris-HCl [pH 7.5], 1 mmol/L EDTA, 0.5%
SDS, and 10 µg/mL boiled RNase A [Boehringer-Mannheim]) and
incubated for 3 hours at 37°C. Deproteinization was performed by the
addition of powdered proteinase K (Boehringer-Mannheim) up to a
final concentration of 0.1 mg/mL and by incubation overnight at 55°C.
DNA was extracted twice with a phenol:chloroform (1:1) mixture and
finally dialyzed against 10 mmol/L Tris-HCl and1 mmol/L EDTA
(pH 8.0). After DNA measurement, the samples were electrophoresed in
1.5% agarose/1x Tris-borate EDTA gel.
Cytology
At 1, 3, and 8 hours after UV irradiation, the same
preselected areas of living cells were examined with an Olympus IMT-2
inverted microscope with a phase-contrast ULWCD 0.3 and photographed
without light filter with use of Kodak T-Max 100 negative film;
positives were performed in AGFA BN312 RC#3 B/W paper. Positives of
UV-irradiated cells (displaying a surface of 292 000
µm2) were used for cell counting.
Quantitative data is expressed as percentage of cell
survival.
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Results |
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p53 in Tissue and Primary mVSMC Lines
Porcine p53 was immunodetected by means of 2 monoclonal antibodies that recognize the human wild-type form. p53 was present in all tissues analyzed (aortic tunica media, whole aorta, and liver); however, after the incubation in FCS-supplemented growth medium, ETM and its migrating mVSMC outgrowth were negative for p53 (Figure 1A
, lanes 1 and 3, respectively). Similarly, the
proliferating polyclonal (B136) and monoclonal (B87, L1 to L5) cell
cultures did not show detectable levels of p53
(Figure 1B). To assess whether the loss of protein was time
dependent, we incubated ETM in growth medium supplemented with 20% FCS
for different time periods. There was a time dependence in the
disappearance of p53 in the explanted tissues. After 6 hours of
incubation, ETMs contained an average 40% of the p53 content in the
original tissue not incubated
(Figure 1C, lanes 2 and 3); when the mVSMC outgrowth reached
80% confluence, 15 to 20 days after the onset of the incubation,
p53 was undetectable
(Figure 1C, lane 8). A similar time-dependent pattern of loss
of p53 protein was observed when another monoclonal anti-p53 antibody
(DO-1, Santa Cruz Biotechnology Inc) was used (data not
shown).
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Because mitogens from the serum seem to be responsible for
the p53 loss, we examined the effect of serum deprivation
on aortic ETMs incubated in growth medium without FCS. As shown in
Figure 1D
, there was a time-dependent increase of p53 in the
tissue; 6 days after the onset of the incubation in FCS-depleted growth
medium, the p53 level reached a 70% average increase over baseline
values in nonincubated tissue
(Figure 1D, lanes 1 and 4). Interestingly, in the ETMs with
increasing p53 protein, there was no detectable mVSMC migration and
outgrowth.
p53 Synthesis and mRNA Expression After
Serum Deprivation
Northern blots showed that the loss of protein in
mitogen-incubated tissue was associated with a loss of mRNA
(Figure 2A and top blots in 2B, lanes 1 to 4). To assess the
reversibility of p53 downregulation after FCS incubation, ETMs
incubated in 20% FCS medium were transferred at different time points
to the arresting (FCS-depleted) medium; however, ETMs did not recover
either p53 protein or gene expression
(Figure 2A and top blots in 2B, lanes 5 to 7). Similarly,
mVSMCs from the polyclonal cell culture B136 were totally unable to
recover p53 synthesis after cell arrest by incubation in serum-depleted
medium
(Figure 2C) or when 100% confluence was reached with growth
in FCS-supplemented medium (data not shown). Cell death was not
observed even after 72 hours of serum withdrawal.
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Sequencing of the 5' Control Region of the
p53 Gene
To determine whether the p53 gene inactivation in
growing mVSMCs was due to methylation at the promoter in p53-negative
cells, we used the technique that was based on the ability of sodium
bisulfite to convert cytosines, but not
5'-methylcytosines, to uracil; the conversion was followed by
PCR amplification.11 As shown
in Figure I (which can be accessed online at
http://atvb.ahajournals.org), sequencing of the PCR products with
use of the bisulfite-converted genomic DNA from fresh aortic tunica
media or the polyclonal cell line B136 as template demonstrated that
the 4 CpG dinucleotides analyzed in the 5' region
of the p53 gene were not methylated and, therefore, that methylation
was not the mechanism involved in blocking the p53 expression in
growing mVSMCs.
Functional Assay of Effects of p53
Suppression
As a functional assay of the absence of p53 in mVSMC
cultures, we performed the experiment depicted in
Figure 3. mVSMCs were grown until subconfluence and exposed
to the spindle-assembly inhibitor colcemid. The observation
of metaphase spreading demonstrates how the mVSMCs overrode the
p53-dependent spindle checkpoint and, without sister chromatic
separation, entered into a new DNA replication round promoting the
observed longitudinal arrangement of the homologous chromosome pairs
(endomitosis). As in p53-deficient murine embryonic
fibroblasts,13 proliferating
mVSMCs overrode the p53-dependent spindle checkpoint and continued
their progression through the cell cycle regardless of the presence of
drug
(Figure 3B). Therefore, the absence of p53 can explain the
development of polyploidy in cultured mVSMCs; in fact, even in the
absence of the spindle inhibitor, our cell cultures became
aneuploid or polyploid at a high number of
passages.
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p53 Levels in Cultured Monoclonal mVSMCs After
UV Irradiation
To analyze whether p53 synthesis could be
recovered after DNA damage, the effect of UV irradiation on p53 levels
was assessed by Western blot analysis of protein extracts from
a cultured monoclonal B87 mVSMC line incubated with FCS in exponential
growth and from the same cells that had been previously arrested by FCS
depletion. UV light irradiation (from 25 to 600
J/m2) was followed by incubation for 8
hours. Two mouse monoclonal antibodies against wild-type human p53 (Pab
240 and DO-1) were used. Cells grown with 20% FCS did not show
detectable levels of p53 on UV irradiation with either Pab 240 or DO-1
antibodies (Figure II, top and middle panels, +FCS, which can
be accessed online at http://atvb.ahajournals.org). An equal amount of
total protein from intact vessel tunica media cell extract was p53
positive with both antibodies (online Figure II, top and middle panels,
+FCS, lane 1). However, cells incubated in FCS-depleted medium
displayed a clear induction in their p53 level at all tested
UV-irradiation doses with respect to the cells that were not irradiated
when assayed with DO-1 but not with Pab 240 (online Figure II; compare
top and middle panels, -FCS). In cultured human fibroblasts incubated
in 20% FCS-supplemented growth medium, we have observed, in agreement
with other authors,14 that
low UV-irradiation doses trigger elevations of p53 protein (data not
shown). In the same conditions, only arrested mVSMCs show p53 protein
elevation (online Figure II, middle panel, -FCS). In fibroblasts,
exposure to UV doses >50 J/m2 promotes a
decrease in p53 content, probably by the inhibition of total protein
synthesis,15 which we did not
observe in arrested mVSMCs. On the other hand, UV-induced p53 displays
an electrophoretic mobility higher than that of p53 from aortic tunica
media tissue, suggesting a different posttranslational modification in
each of them (online Figure II, middle panel,
-FCS).16 17
DNA Analysis After UV
Irradiation
Apoptosis promotes, among other events, DNA
fragmentation; this process is activated by caspases that
cleave the ICAD/DFF-45, an
inhibitor of the caspase-activated
deoxyribonuclease (CAD) responsible for DNA fragmentation as a
nucleosomal ladder.18 We
analyzed nucleosome laddering in DNA extracted from irradiated
cells. mVSMCs incubated with FCS did not show DNA laddering regardless
of the UV dose; their DNA integrity was similar to control DNA from
nonirradiated cells (online Figure II, bottom panel, +FCS, lane
2). A little smearing in the samples from cells irradiated at 300 and
600 J/m2 could be seen (online Figure II,
bottom panel, +FCS, lanes 6 and 7); this was the result of a high
number of nicks produced at the high UV doses. In fact, when those
samples were melted at 95°C before the gel loading, there was a UV
dose-dependent smearing rather than nuclease-dependent DNA
fragmentation (data not shown). On the contrary, a clear DNA
nucleosomal ladder was observed in UV-irradiated mVSMCs incubated in
FCS-depleted medium (online Figure II, bottom panel,
-FCS).
Cytological Analysis of UV-Irradiated
Cells
Cultured monoclonal B87 L1 mVSMCs were cytologically
analyzed on UV irradiation to morphologically ascertain the
rate of cell mortality. The morphology of living cells was
time-dependently analyzed after UV exposure. UV irradiation at
a high UV dose (600 J/m2) killed VSMCs
incubated in medium containing FCS (Figure III, +FCS, which can
be accessed online at http://atvb.ahajournals.org); within 3 to 8 hours
after irradiation, roughly half of those cells were dead
(online Figure III, +FCS, 3h and 8h). However, neither the
control (not irradiated) nor the irradiated cells at 75
J/m2 showed major external signs of cell
death (online Figure III, -UV and +FCS, 1h, 3h, and 8h); after 8
hours of UV irradiation at 75 J/m2, 90%
of the cells incubated with FCS were still alive. mVSMCs showed
cytoskeletal and migratory changes, as revealed by the modification in
their shape and position, suggesting that the molecular machinery and
components involved in this process remain functional despite the
strong DNA damage. By contrast, mVSMC cultures incubated without FCS
showed a clear cell death (37% of survival) 8 hours after a UV dose of
75 J/m2 (online Figure III, -FCS) that
induced the synthesis of p53 and DNA fragmentation (online Figure II).
It is noteworthy that 8 hours of serum withdrawal did not induce either
DNA nucleosomal laddering (online Figure II, -FCS, lane 2) or cell
death (online Figure III, -UV and
-FCS).
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Discussion |
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Biochemical or mechanical injury to the healthy vessel with exposure of the underlying tunica media cells to mitogens might trigger a signal that induces the rapid degradation of endogenous mVSMC p53 and stops its mRNA synthesis. Because p53 has a short half-life (5 to 20 minutes),19 the downregulation or suppression of the gene activity can explain the decrease in the protein level. Tunica media VSMC migration and cell proliferation are then initiated. mVSMC outgrowth is preceded by mitogen-dependent suppression of p53 in the cells residing in the healthy tissue before they can migrate out of the explant and initiate proliferation. In culture, mVSMCs do not reexpress p53, even when mitogens are removed. In vivo, during normal tissue regeneration in response to cutaneous injury, the skin cells downregulate p53 in parallel with the autocrine expression of both c-sis/platelet-derived growth factor-B mitogen and its receptor ß; during healing, mitogen expression drops, and p53 reemerges.20 In that situation, skin cells express platelet-derived growth factor-B and its receptor in an autocrine regulation of response against injury. In the present study, we describe another situation in which cells from healthy vascular tissue increase or decrease its p53 level depending on external mitogen signals. Our results indicate that quiescent contractile VSMCs cannot migrate and proliferate out of the medial tissue in the absence of p53 blockade by mitogenic signals. We have also seen that it is not downregulation but an increase in its p53 content that likely may initiate mVSMC apoptosis (Figure 1D
). In fact, in mVSMCs incubated in mitogen-rich
medium, DNA damage does not restore p53 expression; only after
mitogen-depletion do the UV-damaged mVSMCs synthesize p53 and undergo
DNA fragmentation. Recently, it has been reported that wild-type (but
not the mutant) p53 can induce apoptosis in cancer cell lines
after DNA damage through the proapoptotic CD95/APO-1/Fas
receptor-ligand system.21
After DNA damage promoted by -irradiation or UV irradiation, the
most common mechanism for DNA repair is nucleotide excision
repair, which removes UV-induced lesions that operate as structural
blockers to processes such as replication or transcription. These
processes may also result in mutation if they are not repaired
correctly. p53-homozygous mutant fibroblasts are severalfold more
resistant to UV cytotoxicity and exhibit a decreased UV-induced
apoptosis with respect to the primary heterozygous mutant or to
normal skin fibroblasts expressing wild-type
p53.14 It has been suggested
that DNA strand breaks (nicking) are sufficient and probably necessary
for p53 induction in cells with wild-type p53 alleles when exposed
to DNA-damaging agents,15 but
the precise mechanism of p53 induction remains unclear. In the
present study, UV irradiation promoted a marked increase in p53
content in serum-deprived arrested cells but not in those incubated in
medium containing 20% serum. DNA analysis of irradiated mVSMCs
incubated with serum did not show nucleosome laddering, characteristic
of apoptosis, at any UV dose. On the other hand, when serum was
absent, cells displayed de novo p53 synthesis and DNA fragmentation,
suggesting a link between apoptosis and p53 synthesis after DNA
damage. Cytological examination of irradiated cells incubated with
serum revealed cell death only at very high UV doses (600
J/m2) within 3 to 8 hours after irradiation;
the absence of induced p53 and nucleosomal DNA laddering suggest a
nonapoptotic cell death. On the contrary, cells incubated in
FCS-depleted medium showed cell death in parallel with p53 induction
and DNA fragmentation at much lower UV-irradiation doses. These results
indicate that exposure of mVSMCs to mitogen-rich medium abrogates p53,
disabling its induction after DNA damage. On the other hand, FCS
deprivation enables p53 synthesis and DNA fragmentation
after DNA damage and mVSMC death. Serum withdrawal has been described
as an apoptotic inducer, but p53-induced apoptosis
seems to be driven by transcription-dependent or -independent
mechanisms determined by cell type and apoptotic
stimulus7 ; in this regard, in
our experimental conditions, serum-deprived arrested mVSMCs did not
show either DNA laddering (online Figure II, -FCS, lane 2) or
cell death (online Figure III, -UV and -FCS, 8h). Our data reveal that the UV-induced p53 cannot be recognized by the Pab 240 antibody, whose epitope is a short stretch of 6 amino acidic residues located at the center of the DNA binding core domain of the wild-type human p53, suggesting posttranslational modifications that can mask its recognition. In contrast, the DO-1 antibody, which recognizes the amino-terminal (transactivation domain) epitope mapping between amino acidic residues 11 and 25 of human wild-type p53, is able to detect the UV-induced p53 with a higher electrophoretic mobility, suggesting that the UV-induced form is posttranslationally modified to a lesser extent than the p53 form of the differentiated steady-state cells in the tissue (online Figure II).
The results reported in the present study indicate that mitogen-induced migration of tunica media VSMCs is modulated by p53. Hence, the loss of p53 and its gene expression might be an earlier step in VSMC migration from the vessel tunica media and proliferation in the intima at the onset of atherosclerotic plaque development. Furthermore, our experimental observations suggest that when cultured mVSMCs (p53-) are arrested, they might still mimic postinjury cells rather than quiescent p53-expressing cells in the healthy vessel wall because p53 expression is not restored.
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Acknowledgments |
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This work was funded in part by MARATO-TV3-1996, FIS98/0715, FIS99/0907, and FIC-Catalana Occidente. The authors thank Dr E. May (p53 cDNA probe), M.T. Pallicé (VSMC cultures), and Dr F. Posas (editorial help).
Received May 9, 2000; accepted June 14, 2000.
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