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-induced cell death, we examined the influence of p53 status on the rate of the onset and the overall extent of cell death induced by doxorubicin. We performed this study with Rat-1 fibroblasts, with Rat-1/myc cells which constitutively express c-Myc, and with Rat-1/myc/p53His175 cells derived from Rat-1/myc cells, which, in addition, express the full-length dominant-negative p53His175 mutant gene. The p53His175 mutant suppresses the transactivation function of endogenous p53 in these cells. In contrast to the parental Rat-1 cells, which exhibited only low levels of apoptosis within the first 24 h of treatment with 0.1 to 1 microM doxorubicin, similarly treated Rat-1/myc cells underwent massive and rapid apoptosis. Introduction of p53His175 into Rat-1/myc cells reversed this effect, indicating that Myc-accelerated doxorubicin-induced apoptosis requires functional p53. However, when the overall extent of cell death was measured using clonogenic assays, we found that greater than 90% of cells did not survive upon a 24-h pretreatment with doxorubicin at a concentration as low as 0.1 microM. Moreover, the effect of doxorubicin on all three cell lines was similar, irrespective of their p53 or c-Myc status. Taken together, our experiments indicate that: (a) constitutive expression of c-Myc accelerates the onset of doxorubicin-induced apoptosis in Rat-1 fibroblasts; (b) wild-type p53 function is necessary for this acceleration; and (c) neither overexpression of c-Myc nor the p53 status influences the overall extent of doxorubicin-induced cell death.