Caspase-dependent apoptosis by 2',5'-oligoadenylate activation of RNase L is enhanced by IFN-beta.
The 2',5'-oligoadenylate (2-5A) system is an interferon (IFN)-regulated RNA decay pathway that provides innate immunity against viral infections. The biologic action of the 2-5A system is mediated by RNase L, an endoribonuclease that becomes enzymatically active after binding to 2-5A. RNase L is also implicated in mediating apoptosis in response to both viral and nonviral inducers. To study the cellular effects of RNase L activation directly, 2-5A was transfected into the human ovarian cancer cell line, Hey1B. Activation of RNase L by 2-5A resulted in specific 18S rRNA cleavage and induction of apoptosis, as measured by TUNEL and annexin V binding assays. In contrast, the dimeric form of 2-5A, ppA2'p5'A, neither activated RNase L nor caused apoptosis. Treatment with IFN-beta prior to 2-5A transfection enhanced cellular RNase L levels (< or = 2.2-fold) and increased the proportion of cells undergoing apoptosis (by < or =40%). However, rRNA cleavages after 2-5A transfections were not enhanced by IFN-beta pretreatments, indicating that basal levels of RNase L were sufficient for this activity. Apoptosis in response to RNase L activation was accompanied by cytochrome c release from mitochondria. Induction of apoptosis by either 2-5A alone or by the combination of 2-5A and IFN-beta was effectively blocked with either the pancaspase inhibitor, Z-VAD-fmk, or with the caspase 3 inhibitor, DEVD-fmk. Therefore, activation of RNase L by 2-5A leads to cytochrome c release into the cytoplasm and then to caspase activation and apoptosis. These results suggest potential uses for 2-5A in augmenting the anticancer activities of IFN.
Rusch
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Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Zhou
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A
Silverman
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RH
D000227:Adenine Nucleotides / Q000494:pharmacology*; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis; D020169:Caspases / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D004347:Drug Interactions; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D016899:Interferon-beta / Q000494:pharmacology*; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D009843:Oligoribonucleotides / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012335:RNA, Ribosomal / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000227:Adenine Nucleotides; D017304:Annexin A5; D003574:Cytochrome c Group; D007155:Immunologic Factors; D009843:Oligoribonucleotides; D011500:Protein Synthesis Inhibitors; D012335:RNA, Ribosomal; C023505:2',5'-oligoadenylate; D016899:Interferon-beta; D012259:Ribonuclease, Pancreatic; D020169:Caspases