Activation of a cysteine protease in MCF-7 and T47D breast cancer cells during beta-lapachone-mediated apoptosis

beta-Lapachone (beta-lap) effectively killed MCF-7 and T47D cell lines via apoptosis in a cell-cycle-independent manner, However, the mechanism by whi ch this compound activated downstream proteolytic execution processes were studied. At low concentrations, beta-lap activated the caspase-mediated pat hway, similar to the topoisomerase I poison, topotecan; apoptotic reactions caused by both agents at these doses were inhibited by zVAD-fmk. However a t higher doses of beta-lap, a novel non-caspase-mediated "atypical" cleavag e of PARP (i.e., an similar to 60-kDa cleavage fragment) was observed. Atyp ical PARP cleavage directly correlated with apoptosis in MCF-7 cells and wa s inhibited by the global cysteine protease inhibitors iodoacetamide and N- ethylmaleimide. This cleavage was insensitive to inhibitors of caspases, gr anzyme B, cathepsins B and L, trypsin, and chymotrypsin-like proteases. The protease responsible appears to be calcium-dependent and the concomitant c leavage of PARP and p53 was consistent with a beta-lap-mediated activation of calpain, beta-Lap exposure also stimulated the cleavage of lamin B, a pu tative caspase 6 substrate, Reexpression of procaspase-3 into caspase-3-nul l MCF-7 cells did not affect this atypical PARP proteolytic pathway. These findings demonstrate that beta-lap kills cells through the cell-cycle-indep endent activation of a noncaspase proteolytic pathway. (C) 2000 Academic Pr ess.