Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells.
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.
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Sachiko
S
Yamashita
Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010, Japan.
Masakazu
M
Tanaka
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Teruaki
T
Sato
Department of Applied Life Studies, College of Nagoya Women's University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610, Japan.
Chieri
C
Ida
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Narumi
N
Ohta
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Takashi
T
Hamada
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Taichi
T
Uetsuki
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan.
Yoshisuke
Y
Nishi
Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590, USA.
Joel
J
Moss
Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829, Japan. Electronic address: m_miwa@nagahama-i-bio.ac.jp.
Masanao
M
Miwa
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D016466:CHO Cells; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D000071137:Poly (ADP-Ribose) Polymerase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000378:metabolism*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000494:pharmacology; D013696:Temperature
D001549:Benzamides; C468783:H2AX protein, human; D006657:Histones; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011064:Poly Adenosine Diphosphate Ribose; C025160:3-aminobenzamide; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D006026:Glycoside Hydrolases; C022407:poly ADP-ribose glycohydrolase
DNA double-strand break; DNA repair; DNA single-strand break; Poly(ADP-ribose); Thermal environment; γH2AX