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Title: Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

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 signalsmore » 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. - Highlights: • Physiological level of poly(ADP-ribose) was determined during mild temperature shift. • Poly(ADP-ribose) level in HeLa and CHO-K1 cells significantly increased. • γH2AX signals increased during culture at 40.5 °C as compared to 37.0 °C. • Poly(ADP-ribose) polymerase inhibitor potentiated γH2AX signals at 40.5 °C. • γH2AX and poly(ADP-ribose) level provide evidence of physiological changes in cells.« less

Authors:
 [1];  [2];  [1];  [3]; ; ; ;  [1];  [4];  [1]
  1. Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)
  2. Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010 (Japan)
  3. Department of Applied Life Studies, College of Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610 (Japan)
  4. Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590 (United States)
Publication Date:
OSTI Identifier:
22606140
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 476; Journal Issue: 4; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADP; CELL CULTURES; CELL PROLIFERATION; CONTINUOUS CULTURE; DNA REPAIR; HELA CELLS; POLYMERASES; RIBOSE; STRAND BREAKS; TOXICITY

Citation Formats

Yamashita, Sachiko, Tanaka, Masakazu, Sato, Teruaki, Ida, Chieri, Ohta, Narumi, Hamada, Takashi, Uetsuki, Taichi, Nishi, Yoshisuke, Moss, Joel, and Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells. United States: N. p., 2016. Web. doi:10.1016/J.BBRC.2016.06.001.
Yamashita, Sachiko, Tanaka, Masakazu, Sato, Teruaki, Ida, Chieri, Ohta, Narumi, Hamada, Takashi, Uetsuki, Taichi, Nishi, Yoshisuke, Moss, Joel, & Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells. United States. doi:10.1016/J.BBRC.2016.06.001.
Yamashita, Sachiko, Tanaka, Masakazu, Sato, Teruaki, Ida, Chieri, Ohta, Narumi, Hamada, Takashi, Uetsuki, Taichi, Nishi, Yoshisuke, Moss, Joel, and Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp. Fri . "Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells". United States. doi:10.1016/J.BBRC.2016.06.001.
@article{osti_22606140,
title = {Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells},
author = {Yamashita, Sachiko and Tanaka, Masakazu and Sato, Teruaki and Ida, Chieri and Ohta, Narumi and Hamada, Takashi and Uetsuki, Taichi and Nishi, Yoshisuke and Moss, Joel and Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp},
abstractNote = {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. - Highlights: • Physiological level of poly(ADP-ribose) was determined during mild temperature shift. • Poly(ADP-ribose) level in HeLa and CHO-K1 cells significantly increased. • γH2AX signals increased during culture at 40.5 °C as compared to 37.0 °C. • Poly(ADP-ribose) polymerase inhibitor potentiated γH2AX signals at 40.5 °C. • γH2AX and poly(ADP-ribose) level provide evidence of physiological changes in cells.},
doi = {10.1016/J.BBRC.2016.06.001},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 476,
place = {United States},
year = {Fri Aug 05 00:00:00 EDT 2016},
month = {Fri Aug 05 00:00:00 EDT 2016}
}