TY - JOUR
T1 - Role of RNase L in apoptosis induced by 1-(3-C-ethynyl-β-d-ribo- pentofuranosyl)cytosine
AU - Naito, Tomoharu
AU - Yokogawa, Tatsushi
AU - Takatori, Satoshi
AU - Goda, Kazato
AU - Hiramoto, Akiko
AU - Sato, Akira
AU - Kitade, Yukio
AU - Sasaki, Takuma
AU - Matsuda, Akira
AU - Fukushima, Masakazu
AU - Wataya, Yusuke
AU - Kim, Hye Sook
N1 - Funding Information:
Acknowledgments The authors thank Dr. Hikoya Hayatsu (Faculty of Pharmaceutical Sciences, Okayama University) for helpful discussions. This research was partially supported by a grant for Exploractory Research (18659029, Y.W.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2009/4
Y1 - 2009/4
N2 - Purpose: 1-(3-C-Ethynyl-β-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. Conclusion: 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.
AB - Purpose: 1-(3-C-Ethynyl-β-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. Conclusion: 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.
KW - 28S rRNA fragmentation
KW - Apoptosis
KW - ECyd
KW - JNK
KW - RNA synthesis
KW - RNase L
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U2 - 10.1007/s00280-008-0810-y
DO - 10.1007/s00280-008-0810-y
M3 - Article
C2 - 18668243
AN - SCOPUS:61449095627
VL - 63
SP - 837
EP - 850
JO - Cancer Chemotherapy and Pharmacology
JF - Cancer Chemotherapy and Pharmacology
SN - 0344-5704
IS - 5
ER -