Cell Cycle Analysis of p53-Induced Cell Death in Murine Erythroleukemia Cells

Abstract

A temperature-sensitive mutant of murine p53 (p53^Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression of p53. While the transfected cells grew normally in the presence of mutant p53 (37.5°C), wild-type p53 (32.5°C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5°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°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°C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G₁ prior to being placed at 32.5°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°C for prolonged periods during S phase and G₂/M, and then returned to 37.5°C, did not become committed to cell death. G₁ arrest at 37.5°C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5°C, these G₁ synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5°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.