論文 - 詳細
| RRC ID | 64907 |
|---|---|
| 著者 | Yamashita S, Tanaka M, Sato T, Ida C, Ohta N, Hamada T, Uetsuki T, Nishi Y, Moss J, Miwa M. |
| タイトル | Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells. |
| ジャーナル | Biochem Biophys Res Commun |
| Abstract |
Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24-72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX, PAR could be a sensitive marker for DNA single- and double-strand breaks. These two molecular markers provide evidence of physiological changes occurring within cells. |
| 巻・号 | 476(4) |
| ページ | 594-599 |
| 公開日 | 2016-8-5 |
| DOI | 10.1016/j.bbrc.2016.06.001 |
| PII | S0006-291X(16)30919-6 |
| PMID | 27262441 |
| PMC | PMC6118343 |
| MeSH | Animals Benzamides / pharmacology CHO Cells Cricetulus DNA Breaks, Double-Stranded DNA Breaks, Single-Stranded Enzyme Activation Glycoside Hydrolases / metabolism HeLa Cells Histones / metabolism* Humans Poly (ADP-Ribose) Polymerase-1 / antagonists & inhibitors Poly (ADP-Ribose) Polymerase-1 / metabolism Poly Adenosine Diphosphate Ribose / metabolism* Poly(ADP-ribose) Polymerase Inhibitors / pharmacology Temperature |
| IF | 2.985 |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 各媒体での言及数の合計 | 0 |
| リソース情報 | |
| ヒト・動物細胞 | CHO-K1 |