Evaluation Of The Possible Genotoxicity Of Heat Shock By γH2AX Foci Formation

Heat shock can induce changes in many nuclear events related to DNA semi-conservative replication, including the initiation of new replication, the extension of replication fork, the synthesis and degradation of histone, and the recognition of the newly synthesized DNA strand. Heat shock can also cause cell death, especially when the temperature is over 42℃. However, the mechanism is still unclear, although it was reported that DNA damage might be involved. On the other hand, there were also reports indicating that heat shock could not induce DNA damage. Therefore, it is necessary to re-evaluate whether heat shock can induce DNA damage.The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) is a potent carcinogen and mutagen which can be found in tobacco smoke, and it is involved in the development of gastric and colorectal cancer in both animals and humans. It generally targets DNA and proteins to generate adducts, which can eventually lead to chromosomal aberrations, point mutations, and cell death. In addition, MNNG can also induce DNA strand breaks detected by using comet assay.Recently, researchers have found that histone H2AX is phosphorylated (denoted as γH2AX) by phosphatidylinositol 3-like kinase in responses to DNA double strand breaks(DSBs). γH2AX could recruit many other DNA repair proteins to DSBs and form "foci", then repair the damaged DNA together. It has been shown that afterexposure to ionizing radiation (IR), γH2AX foci form quickly, and the number of γH2AX foci detected by immunofluorescence is quantitatively the same as that of DSBs, suggesting that γH2AX is an indicator for DSBs induced by IR. Some other genotoxic agents, such as cisplatin, camptothecin, which do not induce DSBs directly but nonetheless cause DSBs indirectly have also been shown to induce γH2AX foci formation. Since both direct and indirect induction of DSBs can lead to γH2AX foci formation, γH2AX foci now is used as an indicator in the detection of DSBs.In the current study, using this indicator, we systematically studied the possible genotoxicity of heat shock.Heat shock decreases the survival rate of cellsAfter heat shock (45℃, 30 min), CHL and HeLa cells were further incubated at 37℃ for various intervals (0.5 h, 2 h, 8 h, and 24 h), and cell survival was examined by MTT test.We found that heat shock could decrease the survival of CHL and HeLa cells. Further incubation back to 37℃ did not reverse the trend as the relative survival rate decreased steadily for the heat-treated cells. After heat shock 0.5 h, 2 h, 8 h and 24 h, the relative survival rate of CHL and HeLa cells were decreased compared with cells without heat shock treatment.Immunofluorescent microscopy demonstrates that heat shock per se does not induce γH2AX foci formation in CHL and HeLa cellsAfter heat shock (45℃, 30 min), CHL and HeLa cells were further incubated at 37℃ for various intervals (0.5 h, 2 h, 8 h, and 24 h). Compared with the blank group, the number of γH2AX foci in CHL and HeLa cells was not changed significantly, i.e., majority of the cells showed no γH2AX foci existence, while a small percentage of cells contained 1-10 foci.Heat shock could not induce γH2AX foci formation in p53 deficient cellsp53 is involved in the maintenance of genome stability, thus we investigated whether p53 deficiency might render cells more sensitive to heat shock. for this purpose, U2OSE6tet24 cells were used. The expression of p53 could be regulated by tetracycline in this cell line, e.g., with the presence of tetracycline, the cells are p53 proficient, while without the presence of tetracycline, the cells are p53 deficient. We found that heat shock could decrease the survival rate of this cell line, however, heat shock still could not induce the formation of γH2AX foci in these cells.Heat shock could not induce γH2AX foci formation in FEN1 deficient cellsFEN1 plays an important role in DNA replication and repair. We also found that the deficient of FEN1 could lead to genome instability, and it could also enhance the genotoxicity of the MNNG So we chose this cell line to evaluate the impact of FEN1 on heat shock induced γH2AX foci. It was found that the number of γH2AX foci in heat shocked FEN1 deficient cells was not increased compared to that of the control.Heat shock pre-treatment decreases MNNG-induced γH2AX foci formationIn order to understand whether heat shock could possibly influence the process of γH2AX foci formation, the effect of heat shock on MNNG, which is known to induce DSBs and γH2AX foci, was examined. 1 μg/ml MNNG treatment for 2 h induced strong γH2AX foci formation in CHL and FL cells. However, pre-heat shock cells at 45℃ for 30 min followed by MNNG (1 μg/ml) for 2 h, the number of γH2AX foci was decreased compared to the that of cells with MNNG treatment only.Heat shock does not interfere with already formed γH2AX foci induced byMNNGSince heat shock pretreatment could inhibit MNNG-induced γH2AX foci formation, we next examined whether heat shock could interfere with already formedγH2AX foci induced by MNNG It was found that heat shock after MNNG treatment did not reduce the number nor the intensity of γH2AX foci.Conclusion: First, heat shock is cytotoxic as heat shock can decrease the survival rate of cells. Second, heat shock does not induce γH2AX foci formation as revealed by immunofluorescent microscopy and flow cytometry. In addition, heat shock cannot induce the formation of γH2AX in p53 and FEN1 deficient cells, suggesting that p53 and FEN1 were not involved in this process. Third, heat shock pre-treatment reduced MNNG-induced γH2AX foci, indicating that heat shock could protect cells from the genotoxicity of MNNG Forth, heat shock treatment could not reduce the number of γH2AX foci which were already induced by MNNG. Taken together, these data indicated that heat shock does not induce γH2AX foci formation, however, it does influence the γH2AX foci formation process induced by other agents.