p53 acts as a tumor suppressor by inducing both growth arrest and apoptosis. p53-induced apoptosis can occur without new RNA synthesis through an unknown mechanism. In human vascular smooth muscle cells, p53 activation transiently increased surface Fas (CD95) expression by transport from the Golgi complex. Golgi disruption blocked both p53-induced surface Fas expression and apoptosis. p53 also induced Fas-FADD binding and transiently sensitized cells to Fas-induced apoptosis. In contrast, lpr and gld fibroblasts were resistant to p53-induced apoptosis. Thus, p53 can mediate apoptosis through Fas transport from cytoplasmic stores.

Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105RB (retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.

—Increasing evidence indicates that the control of cell proliferation and apoptosis are linked. The c- myc proto-oncogene is induced early after cell-cycle entry in vascular smooth muscle cells (VSMCs) in vitro and after arterial injury and regulates both cell proliferation and apoptosis. Although both proliferation and apoptosis are likely to be mediated via transcriptional activation of target genes, few c- myc targets have been identified. Therefore, the recent identification that cdc25A, a cell-cycle phosphatase involved in G 1 progression, is transcriptionally activated by c- myc and regulates c- myc –induced apoptosis has suggested that cdc25A may be the principal mediator of c- myc in VSMCs. We examined cdc25A regulation of c- myc –induced proliferation and apoptosis by expressing cdc25A or antisense cdc25A in primary rat VSMCs or in VSMCs expressing deregulated c- myc or adenovirus E1A. Ectopic c- myc increased cdc25A expression, but cdc25A was still responsive to serum components, which indicated that c- myc alone is not the main determinant of cdc25A expression. Antisense cdc25A inhibited c- myc –induced proliferation and apoptosis; however, drug and metabolic blocks indicated that this effect was limited to G 1 . Ectopic cdc25A augmented the proproliferative and proapoptotic action of c- myc but did not increase cell proliferation or apoptosis in the absence of ectopic c- myc . In contrast, E1A/E2F-induced apoptosis was independent of cdc25A. We conclude that cdc25A expression modulates the ability of c- myc to induce apoptosis in G 1 . However, cdc25A alone does not induce apoptosis and cannot substitute for c- myc in VSMCs. Additional targets of c- myc are therefore involved in apoptosis of both G 1 and post-G 1 VSMCs.