Cell cycle arrest triggered by conjugated eicosapentaenoic acid occurs through several mechanisms including G1 checkpoint activation by induced RPA and ATR expression |
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Authors: | Yuko Kumamoto-Yonezawa Ryohei Sasaki Yosuke Ota Yoko Suzuki Shoji Fukushima Takahiko Hada Keisuke Uryu Kazuro Sugimura Hiromi Yoshida Yoshiyuki Mizushina |
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Affiliation: | 1. Laboratory of Food and Nutritional Sciences, Department of Nutritional Science, Kobe-Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan;2. Division of Radiology, Kobe University Graduate School of Medicine, Chuou-ku, Kobe, Hyogo 650-0017, Japan;3. Faculty of Pharmaceutical Sciences, Kobe-Gakuin University, Chuou-ku, Kobe, Hyogo 650-8586, Japan;4. Cooperative Research Center of Life Sciences, Kobe-Gakuin University, Chuo-ku, Kobe, Hyogo 650-8586, Japan;5. Department of Research and Development, Bizen Chemical Co. Ltd., Akaiwa, Okayama 709-0716, Japan |
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Abstract: | BackgroundConjugated eicosapentaenoic acid (cEPA) containing conjugated double bonds, which is prepared by alkaline treatment of eicosapentaenoic acid (EPA), selectively inhibited the activities of both mammalian DNA polymerases (pols) and human DNA topoisomerases (topos).MethodsHuman colon carcinoma cell line, HCT116, was cultured and performed drug and small interfering RNA (siRNA) treatment, flow cytometry analysis, BrdU incorporation analysis, and western blot analysis.ResultsThe levels of bromodeoxyuridine (BrdU) incorporation labeling during DNA synthesis were decreased in time- and dose-dependent manners in HCT116 cells, treated with cEPA. The level of chromatin association of RPA70, a subunit of the single-stranded DNA (ssDNA)-binding protein, was increased following cEPA exposure, suggesting that the replication forks were stalled in response to inhibition of replicative pol activity by cEPA in the cells. cEPA also induced the activation of ataxia-telangiectasia and Rad3-related (ATR) protein in HCT116 cells, and activated the G1 checkpoint pathway in the cells, which was down-regulated by a small interfering RNA (siRNA) against ATR protein. Moreover, caffeine, a known ATR kinase inhibitor, abrogated the cEPA-induced G1 checkpoint in HCT116 cells.General significancecEPA could inhibit the activity of replicative pols, such as pols α, δ and ?, affect the DNA replication fork including ssDNA, and then activate the G1 checkpoint pathway by the induction of RPA and ATR expression levels in cancer cells. |
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Keywords: | EPA, eicosapentaenoic acid cEPA, conjugated EPA PUFA, polyunsaturated fatty acid pol, DNA polymerase (EC 2.7.7.7) topo, DNA topoisomerase ATR, ataxia-telangiectasia mutated- and Rad3-related protein kinase ATRIP, ATR-interacting protein RPA, replication protein A DSB, double strand breaks Cdk, cyclin-dependent kinase Chk1, checkpoint kinase 1 Chk2, checkpoint kinase 2 Cyc E, cyclin E dTTP, 2&prime -deoxythymidine 5&prime -triphosphate siRNA, small interfering RNA MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide DI, DNA Index BrdU, bromodeoxyuridine dsDNA, double-stranded DNA ssDNA, single-stranded DNA CPT, camptothecin |
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