title: Molecular determinants of apoptosis induced by the cytotoxic ribonuclease onconase: evidence for cytotoxic mechanisms different from inhibition of protein synthesis.
abstract: Cytotoxic endoribonucleases (RNases) possess a potential for use in cancer therapy. However, the molecular determinants of RNase-induced cell death are not well understood. In this work, we identify such determinants of the cytotoxicity induced by onconase, an amphibian cytotoxic RNase. Onconase displayed a remarkable specificity for tRNA in vivo, leaving rRNA and mRNA apparently undamaged. Onconase-treated cells displayed apoptosis-associated cell blebbing, nuclear pyknosis and fragmentation (karyorrhexis), DNA fragmentation, and activation of caspase-3-like activity. The cytotoxic action of onconase correlated with inhibition of protein synthesis; however, we present evidence for the existence of a mechanism of onconase-induced apoptosis that is independent of inhibition of protein synthesis. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone (zVADfmk), at concentrations that completely prevent apoptosis and caspase activation induced by ligation of the death receptor Fas, had only a partial protective effect on onconase-induced cell death. The proapoptotic activity of the p53 tumor suppressor protein and the Fas ligand/Fas/Fas-associating protein with death domain (FADD)/caspase-8 proapoptotic cascade were not required for onconase-induced apoptosis. Procaspases-9, -3, and -7 were processed in onconase-treated cells, suggesting the involvement of the mitochondrial apoptotic machinery in onconase-induced apoptosis. However, the onconase-induced activation of the caspase-9/caspase-3 cascade correlated with atypically little release of cytochrome c from mitochondria. In turn, the low levels of cytochrome c released from mitochondria correlated with a lack of detectable translocation of proapoptotic Bax from the cytosol onto mitochondria in response to onconase. This suggests the possibility of involvement of a different, potentially Bax- and cytochrome c-independent mechanism of caspase-9 activation in onconase-treated cells. As one possible mechanism, we demonstrate that procaspase-9 is released from mitochondria in onconase-treated cells. 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.
authors.affiliation: Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland 97201, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Iordanov
authors.forename: O P
authors.initials: OP
authors.lastname: Ryabinina
authors.forename: J
authors.initials: J
authors.lastname: Wong
authors.forename: T H
authors.initials: TH
authors.lastname: Dinh
authors.forename: D L
authors.initials: DL
authors.lastname: Newton
authors.forename: S M
authors.initials: SM
authors.lastname: Rybak
authors.forename: B E
authors.initials: BE
authors.lastname: Magun
mesh_terms: D000970:Antineoplastic Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003513:Cycloheximide / Q000633:toxicity; D003574:Cytochrome c Group / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism* / Q000633:toxicity*; D004640:Emetine / Q000633:toxicity; D006367:HeLa Cells; D006801:Humans; D007930:Leucine / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D011500:Protein Synthesis Inhibitors / Q000633:toxicity*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism* / Q000633:toxicity*; D013379:Substrate Specificity; D051028:bcl-2-Associated X Protein
chemical_list: D000970:Antineoplastic Agents; C494826:BAX protein, human; D003574:Cytochrome c Group; D004527:Egg Proteins; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D012343:RNA, Transfer; D003513:Cycloheximide; D012260:Ribonucleases; D007930:Leucine; D004640:Emetine; C058703:ranpirnase