Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines

Mutant Chinese hamster V79 cells selected for alterations in poly(ADP-ribose) metabolism were shown to be resistant to epipodophyllotoxin (VP-16)-induced cytotoxicity. Cell lines ADPRT 54 and ADPRT 351 have reduced activity of poly(ADP-ribose) polymerase. N2, N3, and N4 cell lines grow in the absence of nicotinamide, with total NAD levels 1.5-3% of those found in parental V79 cells grown in complete medium. When grown in complete medium, the mutant cell lines are 2.3- to 9.6-fold resistant to VP-16-induced cytotoxicity. All of the cell lines respond to VP-16 treatment by formation of protein-cross-linked DNA strand breaks. Upon drug removal, all the cell lines reverse the DNA strand breaks at similar rates. Our studies show a clear dissociation between induction of DNA strand breaks and cytotoxicity. However, there is a good correlation between drug-induced sister chromatid exchanges and cytotoxicity. Thus, N3 cells, with low levels of VP-16-induced sister chromatid exchanges, show reduced levels of cytotoxicity relative to parental V79 cells, despite the fact that both cell lines show similar levels of VP-16-induced protein-cross-linked DNA strand breaks. Additional studies show that the time course of VP-16-induced cytotoxicity correlated better with the time course of sister chromatid exchange formation than with protein-cross-linked DNA strand break formation. These studies provide strong support for the proposal that VP-16-induced cytotoxicity involves the induction of sister chromatid exchanges. Thus, we suggest that drug-induced stabilization of topoisomerase II-DNA complexes stimulates induction of sister chromatid exchanges, which consequently lead to cell death.