Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Aug 5;476(4):594-599.
doi: 10.1016/j.bbrc.2016.06.001. Epub 2016 Jun 2.

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

Sachiko Yamashita  1 , Masakazu Tanaka  2 , Teruaki Sato  1 , Chieri Ida  3 , Narumi Ohta  1 , Takashi Hamada  1 , Taichi Uetsuki  1 , Yoshisuke Nishi  1 , Joel Moss  4 , Masanao Miwa  5
Affiliations

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

Sachiko Yamashita et al. 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.

Keywords: DNA double-strand break; DNA repair; DNA single-strand break; Poly(ADP-ribose); Thermal environment; γH2AX.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Mild temperature shift decreased cell proliferation and viability, which was enhanced by a PARP inhibitor. (A) Growth of HeLa cells was determined at indicated temperatures with or without 7 mM 3AB for 24 h, 48 h and 72 h. (B) Cell viability, expressed as a percentage, was calculated as the number of cells that did not stain with trypan blue, divided by the total number of cells. Cells that did not stain with trypan blue were counted on a hemocytometer. (C) Morphology of HeLa cells cultured for 48 h was shown. (D) Flow cytometric analysis of HeLa cells cultured for 24 h was performed. (E) The percentage of cells in each phase of the cell cycle was determined using DNA profile shown in (D). The results are the means ± standard deviations from two (A, B) and four (D, E) independent experiments. *p < 0.05, **p < 0.01 vs. 37 °C.
Fig. 2
Fig. 2
Mild temperature shift induced phosphorylation of H2AX in HeLa cells and CHO-K1 cells, and 3AB potentiated this effect. (A) HeLa cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX, PARP1, PARG and p53 antibodies. β-Actin was used as a loading control. n.s.; nonspecific band. (B) CHO-K1 cells were incubated at 37.0 °C or 40.5 °C with or without 7 mM 3AB for 24 h. Cells were subjected to Western blotting by using anti-phosphorylated H2AX antibody. β-Actin was used as a loading control. (C) HeLa cells were grown on coverslips, incubated for 24 h at 37.0 °C or 40.5 °C with or without 7 mM 3AB, fixed, and were examined for γH2AX foci by immunofluorescence. (D) Quantification of γH2AX foci-positive HeLa cells cultured for 24 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (E) Quantification of γH2AX foci-positive HeLa cells cultured for 12 h was performed. For each point, 200 cells were analyzed. This experiment was repeated thrice and the mean number of foci was plotted. (D, E) **p < 0.01, n.s.; not significant.
Fig. 3
Fig. 3
Mild temperature shift induced PAR in HeLa cells and CHO-K1 cells. (A) A typical dose response curve for purified PAR was generated using 10H antibody [15] as the capturing antibody and rabbit anti-PAR polyclonal antibody [16] as the detection antibody. (B) The level of PAR in HeLa cells at indicated temperature with and without 7 mM 3AB for 24 h. (C) The level of PAR in CHO-K1 cells at indicated temperature with or without 7 mM 3AB cultured for 24 h. (D) The level of PAR in HeLa cells at indicated temperature with or without 7 mM 3AB cultured for 12 h. HeLa cells and CHO-K1 cells were treated with TCA during cell lysis. The results are the means ± standard deviations from three (B) and two (C, D) independent experiments. (B, C, D) *P < 0.05, **P < 0.01. n.s.; not significant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Lepock JR. How do cells respond to their thermal environment? Int J Hyperth. 2005;21:681–687. - PubMed
    1. Lepock JR. Cellular effects of hyperthermia: relevance to the minimum dose for thermal damage. Int J Hyperth. 2003;19:252–266. - PubMed
    1. Oei AL, Vriend LE, Crezee J, Franken NA, Krawczyk PM. Effects of hyperthermia on DNA repair pathways: one treatment to inhibit them all. Radiat Oncol. 2015;10:165. - PMC - PubMed
    1. Tansey EA, Johnson CD. Recent advances in thermoregulation. Adv Physiol Educ. 2015;39:139–148. - PubMed
    1. Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–5868. - PubMed