Expression of Wild-Type p53 Increases Etoposide Cytotoxicity in Ml Myeloid Leukemia Cells by Facilitated G2 to M Transition: Implications for Gene Therapy'

We have evaluated the role of p53 in the induction of cell death by the DNA topoisomerase II inhibitor etoposide in Ml myelold leukemia celia. Three different clones of Ml cells were used: S6, which lacks p53; Phe 132, which expresses mutant p53 constitutively; and LTR-13, which ex presses mutant protein at 37°Cand wild-type p53 at 32°C. As described previously, LTR-13 cells undergo rapid apoptosis upon Inductionof wild type p53 at 32°C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 gag/mI) of all three cell lines at 37°C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferen tially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss ofcells in late S and G2-Mphase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type pS3 and etoposide) leads to sup.. pression of the etoposide-Induced G2 block. To determine the effect of p53 on the G2to M transition, LTR-13 cells were incubated with etoposide for 24hat37°C andtheneither maintained foranadditional 12hat37°C or shifted to 32°Cto activate wild-type p53. The expression of wild-type p53 resulted in an increase in mitosis-specific phosphorylation, as determined by the MPM-2 antibody as well as the formation of mitotic spindles. This was associated with an Important augmentation of the cytotoxic effect of etoposide. In contrast, a similar temperature shift of Phe-132 cells, which express mutant p53, had no effect on either lmmunostaining with MPM-2 or the cytotoxicity. Taken together, our results indicate that wild-type p53 can override the etoposide-Induced G2 block in at least some cell types. These data propose a new role for p53 in the cell death induced by chemotherapeutic agents and may have important Implications for gene therapy.