Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: Evidence for cytotoxic mechanisms different from inhibition of protein synthesis

Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death ar e not well understood. In this work, we identify such determinants of the c ytotoxicity induced by onconase, an amphibian cytotoxic RNase, Onconase dis played a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA app arently undamaged. Onconase-treated cells displayed apoptosis-associated ce ll blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmen tation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we prese nt evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibit or benzyloxycarbonyl-VaI-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at con centrations that completely prevent apoptosis and caspase activation induce d by ligation of the death receptor Fas, had only a partial protective effe ct on onconase-induced cell death. The proapoptotic activity of the p53 tum or suppressor protein and the Fas ligand/Fas/Fas-associating protein with d eath domain (FADD)/caspase-8 proapoptotic cascade were not required for onc onase-induced apoptosis, Procaspases-9, -3, and -7 were processed in oncona se-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis, However, the onconase-induced act ivation of the caspase-9/caspase-3 cascade correlated with atypically littl e release of cytochrome c from mitochondria, In turn, the low levels of cyt ochrome c released from mitochondria correlated with a lack of detectable t ranslocation of proapoptotic Bar from the cytosol onto mitochondria in resp onse to onconase, This suggests the possibility of involvement of a differe nt, potentially Bax- and cytochrome c-independent mechanism of caspase-9 ac tivation in onconase-treated tells. As one possible mechanism, we demonstra te that procaspase-9 is released from mitochondria in onconase-treated cell s. A detailed understanding of the molecular determinants of the cytotoxic action of onconase could provide means of positive or negative therapeutic modulation of the activity of this potent anticancer agent.