Inhibition of the p53 tumor suppressor gene results in growth of human aortic vascular smooth muscle cells. Potential role of p53 in regulation of vascular smooth muscle cell growth.
Loss of activity of the p53 tumor suppressor gene product has been postulated in the pathogenesis of human restenosis. Although the antioncogenes p53 and retinoblastoma (Rb) susceptibility gene have been reported to play a pivotal role in cell cycle progression in various cells, the role of p53 and Rb in the growth of human vascular smooth muscle cells (VSMC) has not yet been clarified. We used antisense strategy against p53 and Rb genes by the viral envelope-liposomal method. Transfection of antisense p53 oligodeoxynucleotides (ODN) alone resulted in an increase in DNA synthesis compared with control (P<0.01). Similarly, transfection of antisense Rb ODN alone resulted in a higher DNA synthesis rate than control (P<0.01). Moreover, increase in VSMC number was only induced by transfection of antisense p53 ODN alone or cotransfection of p53/Rb ODN (P<0.01), whereas a single transfection of antisense Rb ODN had little effect on cell number. Therefore, we hypothesized that this discrepancy is due to the induction of apoptosis mediated by p53. Interestingly, apoptotic cells were markedly increased in VSMC transfected with antisense Rb ODN alone, accompanied by the induction of p53 protein. The number of apoptotic cells was attenuated by cotransfection of antisense p53 ODN (P<0.01). We finally examined the molecular mechanisms of apoptosis induced by the absence of Rb. In VSMC transfected with antisense Rb ODN, bax, a promoter of apoptosis, was significantly increased in VSMC transfected with antisense Rb ODN (P<0.01), whereas bcl-2 and Fas did not play a pivotal role in the induction of apoptosis. Overall, these data first demonstrated that the antioncogenes p53 and Rb negatively regulated the cell cycle in VSMC, suggesting that the modulation of their activity may mediate VSMC growth such as that in restenosis and atherosclerosis. The presence of p53 plays a pivotal role in the regulation of apoptosis in human VSMC growth, probably through the bax pathway. These results provide evidence that p53 is a functional link between cell growth and apoptosis in VSMC.
Aoki
M
M
Department of Geriatric Medicine, Division of Gene Therapy Science, Osaka University Medical School, Suita, Japan.
Morishita
R
R
Matsushita
H
H
Hayashi
S
S
Nakagami
H
H
Yamamoto
K
K
Moriguchi
A
A
Kaneda
Y
Y
Higaki
J
J
Ogihara
T
T
D000704:Analysis of Variance; D001011:Aorta / Q000166:cytology; D017209:Apoptosis; D001161:Arteriosclerosis / Q000473:pathology; D001483:Base Sequence; D015153:Blotting, Western; D002452:Cell Count; D002453:Cell Cycle; D002478:Cells, Cultured; D003470:Culture Media; D004247:DNA / Q000096:biosynthesis; D053938:DNA Fragmentation; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry; D016161:Genes, Retinoblastoma / Q000235:genetics / Q000502:physiology; D016158:Genes, p53 / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D018180:Thrombomodulin / Q000032:analysis; D014162:Transfection / Q000235:genetics / Q000379:methods; D014404:Tubulin / Q000032:analysis
D003470:Culture Media; D016376:Oligonucleotides, Antisense; D018180:Thrombomodulin; D014404:Tubulin; D004247:DNA