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Inhibition of Etoposide (VP-16)-induced DNA Recombination and Mutant Frequency by Bcl-2 Protein Overexpression¹

Departments of Medicine and Biochemistry and Cancer Center, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4937

Bcl-2 has been shown to inhibit apoptosis induced by several anticancer agents and to cause a dissociation between etoposide (VP-16)-induced protein-cross-linked DNA strand breaks and VP-16-induced cell death. We suggested previously that VP-16-induced cytotoxicity is mediated by a series of events leading from cleavable complex formation to aberrant DNA recombination, as measured by sister chromatid exchange (SCE) and Southern blot analysis of the hypoxanthine phosphoribosyl transferase (hprt) gene mutations. To further evaluate this hypothesis and to determine whether Bcl-2 could affect any steps leading to the aberrant DNA recombination process, we stably transfected an expression vector containing human Bcl-2 cDNA into V79 Chinese hamster cells. This transfection resulted in overexpression of the Bcl-2 gene product. We subsequently quantitated the relationship between VP-16-induced cytotoxicity, DNA strand breaks, SCE, and mutant frequency at the hprt locus in these Bcl-2-overexpressing cells. Two independent Bcl-2-overexpressing cell lines, BCL2/2 and BCL2/4, showed 3–5 times higher survival at 15 µM VP-16 compared with parental V79 cells or control NeoR cells that were obtained by transfecting V79 cells with the expression vector containing the G-418 resistance gene only. DNA single-strand breaks induced by VP-16 were similar in parental V79, control NeoR, BCL2/2, and BCL2/4 cells. In contrast, VP-16 induced significantly less SCE in Bcl-2-overexpressing cell lines compared with parental V79 and control NeoR cells. The SCE/chromosome induced by 15 µM VP-16 were 0.65, 0.42, 0.09, and 0.10, respectively, in V79, NeoR, BCL2/2, and BCL2/4. In addition, there was an excellent correlation between VP-16-induced SCE and cytotoxicity in all cell lines. Furthermore, VP-16-induced mutant frequencies at the hprt locus were 5–10 times less in BCL-2/2 and BCL-2/4 cells than those observed in the V79 or NeoR control cells. These results indicate that overexpression of Bcl-2 is associated with reduction in VP-16-induced genetic recombination, mutation, and cytotoxicity. Moreover, they suggest that Bcl-2 modulates cytotoxicity of VP-16 between cleavable complex formation and subsequent induction of DNA recombination events. Thus, our results provide important support for the hypothesis that VP-16-induced cytotoxicity is associated with aberrant recombination events, including gene deletions and rearrangements.

¹ This study was supported in part by National Cancer Institute Grants R29 CA65920, P01 CA51183, and P30 CA43703.

² To whom requests for reprints should be addressed, at Case Western Reserve University, Biomedical Research Building 3 West, 10900 Euclid Avenue, Cleveland, OH 44106-4937.

Received 10/31/94. Accepted 7/13/95.

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