Mitogen-Induced p53 Downregulation Precedes Vascular Smooth Muscle Cell Migration From Healthy Tunica Media and Proliferation
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.
- Received May 9, 2000.
- Accepted June 14, 2000.
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.
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–α-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.
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.
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 [γ-33P]ATP.
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.
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/1× Tris-borate EDTA gel.
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.
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).
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.
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.
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).
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 β; 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 γ-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.
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).
Schwartz SM, Majesky MW, Murry CE. The intima: development and monoclonal responses to injury. Atherosclerosis. 1995;118:125–140.
Ihling C, Menzel G, Welens E, Montig JS, Schaefer HE, Zeiher AM. Topographical association between the cyclin-dependent kinase inhibitor p21, p53 accumulation, and proliferation in human atherosclerotic tissue. Arterioscler Thromb Vasc Biol. 1997;17:2218–2224.
Bennet MR, Macdonald K, Chan SW, Boyle JJ, Weissberg PL. Cooperative interactions between RB and p53 regulate cell proliferation, cell senescence, and apoptosis in human vascular smooth muscle cells from atherosclerotic plaques. Circ Res. 1998;82:704–712.
Evan G, Littlewood T. A matter of life and cell death. Science. 1998;281:1317–1322.
Yonemitsu Y, Kaneda Y, Tanaka S, Nakashima Y, Komori K, Sugimachi K, Sueishi K. Transfer of wild-type p53 gene effectively inhibits vascular smooth muscle cell proliferation in vitro and in vivo. Circ Res. 1998;82:147–156.
Bennett MR, Evan GI, Schwartz SM. Apoptosis of rat vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Circ Res. 1995;77:266–273.
Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 1994;22:2990–2997.
Cross SM, Sánchez CA, Morgan CA, Schimke MK, Ramel S, Idzerda RL, Raskind WH, Reid BJ. A p53-dependent mouse spindle checkpoint. Science. 1995;267:1353–1356.
Ford JM, Hanawalt PC. Expression of wild-type p53 is required for efficient global genomic nucleotide excision repair in UV-irradiated human fibroblasts. J Biol Chem. 1997;272:28073–28080.
Nelson WG, Kastan MB. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol. 1994;14:1815–1823.
Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW, Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev. 1998;12:2831–2841.
Kapoor M, Lozano G. Functional activation of p53 via phosphorylation following DNA damage by UV but not gamma irradiation. Proc Natl Acad Sci U S A. 1998;95:2834–2837.
Oren M, Maltzman W, Levine AJ. Post-translational regulation of the 54K cellular tumor antigen in normal and transformed cells. Mol Cell Biol. 1981;1:101–110.
Antoniades HN, Galanopoulos T, Neville-Golden J, Kiritsy CP, Lynch SE. p53 expression during normal tissue regeneration in response to acute cutaneous injury in swine. J Clin Invest. 1994;93:2206–2214.
Müller M, Wilder S, Bannasch D, Israeli D, Lehlbach K, Li-Weber M, Friedman SL, Galle PR, Stremmel W, Oren M, et al. p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs. J Exp Med. 1998;188:2033–2045.