P53 STATUS AFFECTS THE RATE OF THE ONSET BUT NOT THE OVERALL EXTENT OF DOXORUBICIN-INDUCED CELL-DEATH IN RAT-1 FIBROBLASTS CONSTITUTIVELY EXPRESSING C-MYC

To better understand the effects of p53 on the process of DNA damage-i nduced cell death, we examined the influence of p53 status on the rate of tile onset and the overall extent of cell death induced by doxorub icin. We performed this study with Rat-1 fibroblasts, with Rat-1/myc c ells which constitutively express c-Myc, and with Rat1/myc/p53His175 c ells derived from Rat-1/myc cells, which, in addition, express the ful l-length dominant-negative p53His175 mutant gene, The p53His175 mutant suppresses the transactivation function of endogenous p53 in these ce lls. In contrast to the parental Rat-1 cells, which exhibited only low levels of apoptosis within the first 24 h of treatment with 0.4 to I mu M doxorubicin, similarly treated Rat-1/myc cells underwent massive and rapid apoptosis. Introduction of p53His175 into Rat-1/myc cells re versed this effect, indicating that Myc-accelerated doxorubicin-induce d apoptosis requires functional p53. However, when the overall extent of cell death was measured using clonogenic assays, we found that grea ter than 90% of ems did not survive upon a 24-h pretreatment with doxo rubicin at a concentration as ion as 0.1 pit. Moreover, tile effect of doxorubicin all all three cell lines was similar, irrespective of the ir p53 or c-Myc status. Taken together, our experiments indicate that: (a) constitutive expression of c-Myc accelerates the onset of doxorub icin-induced apoptosis in Rat-1 fibroblasts; (b) wild-type p53 functio n is necessary for this acceleration; and (c) neither overexpression o f c-Myc nor the p53 status influences the overall extent of doxorubici n-induced cell death.