The Role And Molecular Mechanism Of JWA Involved In Oxidative Stress In K562 And MCF-7 Cells

A lot of human diseases are caused by or associated with various environmental exposures. A series of environmental responsive genes in cells will actively regulated their expression levels to adapt the changed environmental factors when human exposed to physical, chemical and biological harmful agents. Harmful environmental factors can directly induce damage of genomes, specific distribution of chemical substances, or interfere with signal pathways in cells, and finally lead to diseases.JWA gene was primarily cloned from human trachea-bronchial epithelial cells using mRNA differential display-reverse transcription polymerase chain reaction (DD-RT-PCR) technique by Zhou, et al in 1998 (NCBI, AF070523.1) . Several cis-elements was found within the identified 3000 bp of its promoter region, such as heat shock response element (hRE), stress response element (SRE), and the retinoic acid response element (ARE^※) semi-site and so on. During recent years, our preliminary studies have shown that JWA gene might be regulated by avariety of inducers and environmental stresses including oxidative stress. However, how the molecular mechanism of JWA gene involved in signal pathways in oxidative stress exactly operates is still unknown.In this study, we use different doses of hydrogen peroxide (H₂O₂) induced K562 (chronic myelogenous leukemia cells) and MCF-7 (human breast carcinoma cells) cells DNA damage and/or apoptosis by different exposure time, and to investigate the role of JWA under oxidative stress process. Furthermore, we try to explore the possible interaction mechanisms of among JWA, heat shock proteins (hsp70, hsp27), HSF1 and p53, thus to explore the role and molecular mechanism of JWA involved in signal pathways in oxidative stress in K562 and MCF-7 cells.1. The effect of hydrogen peroxide on cell viabilityTo evaluate the effect of H₂O₂ on the growth of K562 and MCF-7 cells in vitro, both time- and dose-course models were designed to exposure cells at 10-180 min and at 0.01-1 mmol/L of H₂O₂, respectively. The cell growth status was evaluated by MTT assay. The results showed that H₂O₂ displayed significant cytostatic and cytotoxic effects on both cell lines. The role of cell growth inhibition by H₂O₂ was enhanced with the increasing of H₂O₂ concentrations and with delayed exposure times of cell culture. These results indicated that the inhibitory effect of H₂O₂ on cell viability was a dose- and time-dependent manner. There was obvious DNA damage (DNA tail) in K562 and MCF-7 cells after H₂O₂ exposure at short time (10-180 min). And the damage was in dose-dependent manner. However, when exposure time was lengthened to 24 and 48 hours, H₂O₂ induced K562 cells apoptosis.In order to evaluate DNA damage and formation of 8-OH-dG induced by H₂O₂, we complete the modified comet assay by detection of oxidized bases and use of bacterial repair endonucleases (endonuclease â…¢, formamidopyrimidine glycosylase). DNA originated from K562 and MCF-7 cells was incubated with H₂O₂ at every concentration andfollowed by treatment of FPG and Endo III. The result showed a significant change (p < 0.01 or p < 0.001) in comet tail length compared with the cells without enzymatic treatment. There were also some changes (p < 0.05 or p < 0.01) on percentage of tailed DNA in both cells between treatment with and without the enzymes. And we also found that the higher of concentration H₂O₂ treated, the more serious DNA damages were observed. The tail length was obviously lengthened and the percentage of tailed DNA was increased after enzymes digestion. It indicated that H₂O₂ increased the level of DNA strand breaks, oxidative purine and pyrimidine, which were the recognized site of FPG and Endo III. And the formation of 8-OH-dG was dose-dependent. In addition, there were significant differences (p < 0.01) between the control groups untreated with H₂O₂ and the experimental groups for the mean comet tail length and percentage of tailed DNA in K562 and MCF-7 cells. And we found that the damage induced by H₂O₂ was aggrav...