Mechnism Of DNA Damage Responses In Stem Cells Induced By Chromium(VI) Compounds

Induced pluripotent stem(i PS) cells are derived from somatic cells through genetic reprogramming, capable of self renewal and differentiation into a variety of cell types. Recent studies have shown that human i PS cells also offer a valuable alternative to human embryonic stem cells for drug development, as well as for in vitro expansion and differentiation into cells of the hematopoietic lineage. It is well known that cells undergoing in vitro expansion are constantly exposed to a variety of environmental insults including genotoxic agents and oxidative stress. Given the great potential of i PS cells, it is imperative to understand the characteristics of these cells, especially regarding their genomic stability after exposure to environmental genotoxic agents.SSCs are drived from PGCs(primordial germ cells),capable of differentiated into sperm and also has the capacity of self renewal and differentiation. It can be maintained for the purpose of cryopreservation and transplantation. SSCs thus represent a valuable resources in medical,biology and animal science. Although, SSCs can transfer genetic information to its descendants, this transformation was still determined by the surrounding environmental conditions. When the situation was bad, SSCs will stopped to differentiate into sperm, thus disturbed spermatogenesis and caused male reproductive dysfunction.Chromium(VI) compounds are well established environmental carcinogens that produce genotoxic effects leading to human cancers. Although some studies have been carried out with an emphasis on toxic and carcinogenic effects of Cr(VI) compounds on terminal differentiation cells, its effect on human i PS cells remains largely unknown. Given this, the present study was designed to conducted DNA damage responses caused by genotoxic agents Cr(VI) in i PS cells and using two other genotoxic agents of H2O2 and Doxorubicin(DOX)as parallel control. Extensive research in the past has identified DNA damage response entails a series of signaling events including auto-phosphorylation of ATM S1981 and phosphorylation of histone H2 AX s139 and p53 ser15, p53 ser20, p53 ser392, et al. results responses The results indicate that i PS cells possess a rather distinct pattern of DNA damage response after Cr(VI)induction. Detail conclusions are as follows:(1)The p-ATM level in i PS cells exhibited a biphasic pattern of induction after Cr(VI) treatment. It was slightly induced at about 1 h after treatment and then declined below the basal level until it increased again after cells were treated for more than 24 h.(2)The mechanism of Chromium(VI) induced DNA damaged response is believed to generate reactive oxygen species(ROS), which will trigger oxidative stress to damage cell. We next examined the effect of H2O2 and Doxorubicin(DOX) on the activation of key components of DNA damage responses in i PS. We observed that different cell types responded to H2O2 in a manner similar to that of Cr(VI) with regard to p53 induction and activation. The p-ATM level in i PS cells treated with H2O2 displayed a biophasic pattern similar to that of Cr(VI) treatment which means at least partly, one of the main mechanisms for Cr(VI)-induced DNA damage responses is the generation of reactive oxygen species(ROS) caused genome instability.(3) p53 was situated at the crossroads of a network of DNA damage signalling pathways that are essential for cell growth regulation and apoptosis induced by genotoxic and non-genotoxic stresses. In normal unstressed cells, the level of p53 protein is down regulated. After genotoxic or non-genotoxic stresses, p53 was activated and play its tran sactivation functions. Downstream signaling includes a large series of genes that are activated by the transactivation properties of p53. We found p53 was phosphorylated at the site of ser15 and ser392, but we failed to detect p53 ser20 which indicates p53 ser20 doesn't involved in p53 activation during Cr(VI)induced DNA damage in i PS cells.(4) The striking difference in p53 S392 phosphorylation between three cell types in response to different stimuli prompted us to further explore on the potential kinases that phosphorylate this residue in these cells. Previous studies have shown that the p38 MAP kinase and casein kinase 2(CK2) are the main kinases that phosphorylate p53 S392. Our study with specific chemical inhibitors p38(SB203580), CK2(TBBz) examined whether these two kinases are responsible for the observed differential phosphorylation of this residue. Intriguingly, contrast to the previous studies, we observed that inhibition of CK2, but not p38, promotes phosphorylation of p53 ser392 in i PS cells.(5) Cr(VI)induced oxidative stress in SSCs and caused p53 activation by increasing p53 expression, decreasing apoptosis inhibition protein Bcl-2 expression and promoting apoptosis promoting protein Bax levels. Cr(VI) also lead to lower mitochondrial membrane potential, down regulate Caspase3 protein kinase family, which cleaced PARP-1, and eventually cause the effect of SSCs apoptosis.(6) In vivo study confirmed Cr(VI) exposure caused male germ cell dysfunction leading to impairment of spermatogenesis in mice. Cr(VI) causes reduction of diameter of seminiferous tubules, thinning of the basement membrane, disintegration of peritubular membrane, disorganization of germ cells, reduce in spermatogenic cell population, early arrest of spermatogenesis, vacuolization of seminiferous epithelium, Leydig cell atrophy, prominent Sertoli cells and collapsed blood vessels in mice.(7) Since oxidative stress is suggested to be the main factor that causes apoptosis in male germ cells. Melatonin(Melatonin) as the most potent endogenous radical scavenger against oxidative stress can attenuate Cr(VI) induced SSCs apoptosis and abnormal testicular structure in mice model.As above all, the current study indicated that i PS cells possess a rather distinct pattern of responses compared with established cell lines: ATM sensors DSB in Cr(VI)induced DNA damage and become activation in the form of p-ATM, p-ATM phosphorylated his tone H2 AX and transcription factor p53 at the site of ser392, CK2 regulates p53 phosphorylation at the site of ser392,phosphorylation of p53 ser392 stabilize total p53 protein. Environmental stresses are easily caused DNA damage by DNA structure changes. The current studies may help to unravel mechanism of diseases caused by environmental carcinogens. For early detection and prevention human diseases susceptibility to environmental causes, to be better explored for regenerative medicine and potential drug development.