p53/p21CIP1 Cooperate in Enforcing Rapamycin-induced G1 Arrest and Determine the Cellular Response to Rapamycin

The relationship between G 1 checkpoint function and rapamycininduced apoptosis was examined using two human rhabdomyosarcoma cell lines, Rh1 and Rh30, that express mutated p53 alleles. Serum-starved tumor cells became apoptotic 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 increased the proportion of Rh1 and Rh30 cells in G 1 phase, although there were no significant changes in expression of cyclins D1, E, or A in drug-treated cells. To determine whether apoptosis was associated with continued slow progression through G 1 to S phase, cells were exposed to rapamycin for 24 h, then labeled with bromodeoxyuridine (BrdUrd). Histochemical analysis showed that >90% of cells with morphological signs of apoptosis had incorporated BrdUrd. To determine whether restoration of G 1 arrest could protect cells from rapamycin-induced apoptosis, cells were infected with replication-defective adenovirus expressing either p53 or p21 CIP1 . Infection of Rh30 cells with either Ad-p53 or Ad-p21, but not control virus (Ad-β-gal), induced G 1 accumulation, up-regulation of p21 CIP1 , and 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 cells. Consistent with these data, incorporation of [ 3 H]thymidine or BrdUrd into DNA was significantly inhibited, as was cyclin-dependent kinase 2 activity. 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 1 phase, by overexpression of p53 or p21 CIP1 , protects against apoptosis. The response to rapamycin was next examined in wild-type or murine embryo fibroblasts nullizygous for p53 or p21 CIP1 . Under serum-free conditions, rapamycin-treated wild-type MEFs showed no increase in apoptosis compared to controls. In contrast, rapamycin significantly induced apoptosis in cells deficient in p53 (∼2.4-fold) or p21 CIP1 (∼5.5-fold). Infection of p53 −/− MEFs with Ad-p53 or Ad-p21 completely 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 CIP1 . When BrdUrd was added 24 h after rapamycin, almost 90% and 70% of cells lacking p53 or p21 CIP1 , 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, but remained high in cells lacking p53 or p21 CIP1 . Taken together, the data suggest that p53 cooperates in enforcing G 1 cell cycle arrest, leading to a cytostatic response to rapamycin. In contrast, in tumor cells, or MEFs, having deficient p53 function the response to this agent may be cell cycle progression and apoptosis.