p53/p21(CIP1) cooperate in enforcing rapamycin-induced G(1) arrest and determine the cellular response to rapamycin

The relationship between G, checkpoint function and rapamycin-induced apopt osis was examined using two human rhabdomyosarcoma cell lines, Rh1 and Rh30 , that express mutated p53 alleles. Serum-starved tumor cells became apopto tic when exposed to rapamycin, but were completely protected by expression of a rapamycin-resistant mutant mTOR. Exposure to rapamycin (100 ng/ml) for 24 h significantly in creased the proportion of Rh1 and Rh30 cells in G, p hase, although there were no significant changes in expression of cyclins D 1, E, or A in drug-treated cells. To determine whether apoptosis was associ ated with continued slow progression through G, to S phase, cells were expo sed to rapamycin for 24 h, then labeled with bromodeoxyuridine (BrdUrd). Hi stochemical analysis showed that >90% of cells with morphological signs of apoptosis had incorporated BrdUrd. To determine whether restoration of G, a rrest could protect cells from rapamycin induced apoptosis, cells were infe cted with replication-defective adenovirus expressing either p53 or p21(CIP I). Infection of Rh30 cells with either Ad-p53 or Ad-p21, but not control v irus (Ad-beta -gal), induced G, accumulation, upregulation of p21(CIPI), an d complete protection of cells from rapamycin-induced apoptosis. Within 24 h of infection of Rh1 cells with Ad-p21, expression of cyclin A was reduced by >90%. Similar results were obtained after Ad-p53 infection of Rh30 cell s. Consistent with these data, incorporation of [H-3]thymidine or BrdUrd in to DNA was significantly inhibited, as was cyclin-dependent kinase 2 activi ty. These data indicate that rapamycin-induced apoptosis in tumor cells is a consequence of continued G(1) progression during mTOR inhibition and that arresting cells in G, phase, by overexpression of p53 or p21(CIPI), protec ts against apoptosis. The response to rapamycin was next examined in wild-t ype or murine embryo fibroblasts nullizygous for p53 or p21(CIPI). Under se rum-free conditions, rapamycin-treated wild-type MEFs showed no increase in apoptosis compared to controls. In contrast, rapamycin significantly induc ed apoptosis in cells deficient in p53 (similar to2.4-fold) or p21(CIPI) (s imilar to5.5-fold). Infection of p53(-/-) MEFs with Ad-p53 or Ad-p21 comple tely protected against rapamycin-induced apoptosis. Under serum containing conditions, rapamycin inhibited incorporation of BrdUrd significantly more in wild-type murine embryo fibroblasts (MEFs) than in those lacking p53 or p21(CIPI). When BrdUrd was added 24 h after rapamycin, almost 90% and 70% o f cells lacking p53 or p21(CIPI), respectively, incorporated nucleoside. In contrast, only 19% of wild-type cells incorporated BrdUrd in the presence of rapamycin. Western blot analysis of cyclin levels showed that rapamycin had little effect on levels of cyclins D1 or E in any MEF strain. However, cyclin A was reduced to very low levels by rapamycin in wild-type cells, bu t remained high in cells lacking p53 or p21(CIPI). Taken together, the data suggest that p53 cooperates in enforcing G, cell cycle arrest, leading to a cytostatic response to rapamycin. In contrast, in tumor cells, or MEFs, h aving deficient p53 function the response to this agent may be cell cycle p rogression and apoptosis.