Expression of wild-type p53 increases etoposide cytotoxicity in M1 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 M1 myeloid leukemia cells. Three different clones of M1 cells were used: S6, which lacks p53; Phe-132, which expresses mutant p53 constitutively; and LTR-13, which expresses mutant protein at 37 degrees C and wild-type p53 at 32 degrees C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32 degrees C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 microg/ml) of all three cell lines at 37 degrees C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferentially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss of cells in late S and G2-M phase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type p53 and etoposide) leads to suppression of the etoposide-induced G2 block. To determine the effect of p53 on the G2 to M transition, LTR-13 cells were incubated with etoposide for 24 h at 37 degrees C and then either maintained for an additional 12 h at 37 degrees C or shifted to 32 degrees C to 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 immunostaining 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.