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Expression of Wild-Type p53 Increases Etoposide Cytotoxicity in M1 Myeloid Leukemia Cells by Facilitated G₂ to M Transition: Implications for Gene Therapy¹

Department of Structural Biology and Pharmacology, CNRS URA 147, Institut Gustave Roussy PR2, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France [A. S., A. K. L.], and Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Narutowicza St 11/12, 80-952 Gdansk, Poland [A. S.]

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°C and wild-type p53 at 32°C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32°C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 µg/ml) of all three cell lines at 37°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°C and then either maintained for an additional 12 h at 37°C or shifted to 32°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.

¹ This work was sponsored by la Ligue National contre le Cancer, Association pour la Recherche sur le Cancer and F.E.G.E.F.L.U.C. (France). A.S. is a fellow of Fondation pour la Recherche Medicale (France).

² To whom requests for reprints should be addressed. Phone: (331) 4211 4593; Fax (331) 4211 5276.

Received 12/18/96. Accepted 1/17/97.

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