title: Role of RNase L in apoptosis induced by 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine.
abstract: OBJECTIVE
  1-(3-C-Ethynyl-beta-D: -ribo-pentofuranosyl)cytosine (ECyd), a ribonucleoside analog, has a potent cytotoxic activity against cancer cells. The present studies have been performed to elucidate the overall mechanisms of ECyd-induced apoptotic cell death.
  
  
  METHODS
  Cultured cells of mouse mammary carcinoma FM3A and human fibrosarcoma HT 1080 lines were used. The efficacy of RNA synthesis inhibition by ECyd was assessed by kinetic analysis using nuclei isolated from FM3A cells. RNA status in ECyd-treated cells was investigated by Northern blots, and the cleavage sites of RNA were identified by rapid amplification of 5' cDNA ends (5'-RACE). The effect of protein functions on the ECyd-induced apoptotic pathway was analyzed by siRNA and immunohistochemical techniques. Apoptotic cells were detected by TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assay.
  
  
  RESULTS
  ECyd induces inhibition of RNA synthesis in vitro and in vivo, which appears to be a major cause for the apoptosis. It is known that ECyd is converted inside the cell into its 5'-triphosphate (ECTP). We have now found in test-tube experiments that ECTP strongly inhibits the activity of RNA polymerase I by competing with CTP. In the absence of robust RNA synthesis, the cellular RNAs would be destined to break down. RNase L was found to be playing a role in the breakdown: thus, the 28S rRNA-fragmentation pattern observed for the ECyd-treated cells was very similar to that observable in an in vitro treatment of the 28S ribosomes with RNase L. Association of RNase L with the cytotoxic action of ECyd was confirmed by use of the siRNA-mediated suppression of the cellular RNase L. Thus, the cells in which the RNase L was knocked-down were highly resistant to the cytotoxic action of ECyd. Further events, downstream of the RNase L action that can lead to the eventual apoptosis, would conceivably involve the phosphorylation of c-jun N-terminal kinase and subsequent decrease in mitochondrial membrane-potential. Evidence to support this flow of events was obtained by siRNA-experiments.
  
  
  CONCLUSIONS
  The results from this study demonstrated that RNase L is activated after the inhibition of RNA polymerase, and induces mitochondria-dependent apoptotic pathway. We propose this new role for RNase L in the apoptotic mechanism. These findings may open up the possibility of finding new targets for anticancer agents.
authors.affiliation: Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushimanaka, Okayama, 700-8530, Japan.
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Naito
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Yokogawa
authors.forename: Satoshi
authors.initials: S
authors.lastname: Takatori
authors.forename: Kazato
authors.initials: K
authors.lastname: Goda
authors.forename: Akiko
authors.initials: A
authors.lastname: Hiramoto
authors.forename: Akira
authors.initials: A
authors.lastname: Sato
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kitade
authors.forename: Takuma
authors.initials: T
authors.lastname: Sasaki
authors.forename: Akira
authors.initials: A
authors.lastname: Matsuda
authors.forename: Masakazu
authors.initials: M
authors.lastname: Fukushima
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Wataya
authors.forename: Hye-Sook
authors.initials: HS
authors.lastname: Kim
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects*; D003562:Cytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005354:Fibrosarcoma / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D012339:RNA, Ribosomal, 28S / Q000378:metabolism; D014162:Transfection
chemical_list: C103084:1-(3-C-ethynylribopentofuranosyl)cytosine; D004791:Enzyme Inhibitors; D012339:RNA, Ribosomal, 28S; D003562:Cytidine; D004722:Endoribonucleases; C031956:2-5A-dependent ribonuclease