CELL-CYCLE ANALYSIS OF P53-INDUCED CELL-DEATH IN MURINE ERYTHROLEUKEMIA-CELLS

A temperature-sensitive mutant of murine p53 (p53Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression or p-53. While the transfected cells grew normally in the presence of mutant p53 (37.5-degrees-C), wild-type p53 (32.5-degrees-C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5-degrees-C was seen to be fragmented into a characteristic ladder consistent with cell death due to apoptosis. Following synchronization by density arrest, transfected cells released into G1 at 32.5-degrees-C were found to lose viability more rapidly than did randomly growing cultures. Following release into G1, cells became irreversibly committed to cell death after 4 h at 32.5-degrees-C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G, prior to being placed at 32.5-degrees-C continued to cycle until subsequently arrested in G1; loss of viability occurred following G1 arrest. In contrast to cells in G1, cells cultured at 32.5-degrees-C for prolonged periods during S phase and G2/M, and then returned to 37.5-degrees-C, did not become committed to cell death. G1 arrest at 37.5-degrees-C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5-degrees-C, these G1 synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5-degrees-C (G0) lost viability at a much slower rate than did cells released into G1. Taken together, these results indicate that wild-type p53 induces cell death in murine erythroleukemia cells and that this effect occurs predominantly in the G1 phase of actively cycling cells.