| 1BackgroundCells are constantly exposed to environmental and metabolic insults such as radiation, chemical agents and oxidative stress. Such exposure may generate DNA lesions that lead to mutations and DNA strand breaks and cause genomic instability. To overcome these attacks and maintain the integrity of the genome, eukaryotic cells have evolved a complex network to detect, signal the presence of and repair DNA damage, which is referred as DNA damage response(DDR) pathway. DDR is composed of numerous checkpoint and repair proteins that coordinate a complex signaling cascade to assess the damage, then either arrest cell cycle to provide time for DNA repair or DNA damage tolerance or trigger apoptosis.Recent evidences have suggested that human DNA polymerase eta (Polη) has been implicated in DDR. Polη is the product of the xeroderma pigmentosum variant (XPV) gene, which is mutated in the cancer-prone genetic disorder, xeroderma pigmentosum variant. Polη is a Y-family DNA polymerase with the ability to perform translesion synthesis across cys-syn pyrimidine dimmers (CPDs), the primary lesion induced by UV radiation, which blocks replicative DNA polymerases. In addition to CPDs, Polη has been shown to replicate across other DNA lesions, including cisplatin adducts, butadiene-derived2’-deoxyuridine adducts, and the nitric oxide-derived adduct2’-Deoxyinosine. Moreover, Polη is also involved in somatic hypermutation of immunoglobulin genes, homologous recombination, and replication of DNA fragile sites. Recent studies have found that Polη plays a suppressive role in the mutagenesis by8-hydroxyguanine in human cells and plays an important role in preventing genome instability and avoiding y-H2AX which is associated with double-strand breaks and single-stranded DNA in human cells. These studies suggest that in addition to its roles in DNA damage tolerance by translesion synthesis, Polη has other biological roles in response to DNA damage caused by a variety of endogenous and exogenous genotoxic agents.Hydroquinone (HQ) occurs naturally in bacteria, plants and some animals and is also manufactured for commercial use. It is used in cosmetics as a skin lightening agent, in photography as a black and white developer, in the production of antioxidants for rubber and as a polymerization inhibitor for vinyl acetate and acrylic monomers. HQ also occurs naturally in cigarette smoke, the leaves and bark of several plant species as a component of glucopyranoside, arbutin, and plant-derived foods such as coffee, wheat-based products, and red wine. Human exposure to HQ can occur following environmental, occupational, dietary and cigarette smoke exposure, and from exposure to benzene, which can be metabolized to HQ. HQ is absorbed dermally, orally or by inhalation, and is mainly metabolized in the liver.Due to the widespread use of HQ, numerous toxicity studies have been conducted. In chronic toxicity studies with rodents some evidence of carcinogenicity was demonstrated, including benign tumors of the kidney and liver and mononuclear cell leukemia. Many studies have indicated that HQ can trigger an oxidative stress process, giving rise to reactive oxygen species, and induce significant chromosome abnormities and DNA damage via a different mechanism. Thus, DNA lesions are usually used as toxic end points. In addition, HQ can induce apoptosis and interfere with cell cycle progression in human cells. Despite its well-established role in translesion synthesis, the role of Polη in DDR has not been well explored.In order to better understand the functional roles and importance of Polη in human hepatic cells, we used RNA interference (RNAi) to selectively knockdown Polη in hepatocytes and then determined how the loss of Polη affected the cytotoxicity and genotoxicity of HQ by measuring its effects on cell proliferation, apoptosis, cell cycle progression, DNA strand breaks, and the activation of the DNA damage response pathways in hepatocytes. The goal of this work was to characterize preliminarily the mechanism of polymerase Polη in DNA damage response induced by HQ in hepatocytes and provide scientific theoretical basis to establish the body defense system to the damage caused by hydroquinone.2Methods2.1Construction and identification of Polη-deficient hepatic cell lineAccording to the targeting sequences of POLH, three pairs of oligonucleotide encoding shRNAs and one pair of oligonucleotide encoding non-target shRNA were designed using siRNA design software, and synthesized by Sangon. The oligonucleotides were annealed and cloned into the AgeI and EcoRI sites of the pLKO.1vector to generate RNAi vectors, and these recombination vectors were then transformed into competent JM109cells. Bacterial colonies were pooled and used for plasmid preparation. The positive clones were confirmed by sequencing. The resulting plasmids were designated as pLKO.1-POLH1, pLKO.1-POLH2, pLKO.1-POLH3and pLKO.1-C.293FT cells were then transfected with pLKO.1-POLHs or pLKO.1-C using Lipofectamine2000according to the manufacturer’s instructions. The lentivirus expression vectors were established and replication incompetent lentiviruses carrying POLH shRNA were produced by transfected293FT cells. L-02cells were stably infected with lentivirus carrying POLH shRNA or negative control shRNA. After infection, stable cell lines were generated by selection with puromycin. The puromycin-resistant clones selected were considered to be stably infected cells and designated as L02-POLH-sh1, L02-POLH-sh2, L02-POLH-sh3and L02-POLH-nsc (non-specific siRNA control) cells, respectively. POLH mRNA and protein expression levels were confirmed by Real-time PCR and Western blot. In addition, the biological characteristics(cell morphology, cell proliferation and cell cycle) between the above cell lines were compared.2.2Exploration on effects of inhibition of Polη expression on cell biological characteristics and DNA damage in hepatocytes exposed by HQ(1) Exponentially growing hepatic cells were treated with HQ of different final concentrations(0,10,20,40,80,160, and320μM) for24h, then morphological features of hepatic cells were observed by inverted microscope and cellular viability was determined by the MTT assay.(2) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, apoptosis was determined by flow cytometry, and the nuclei changes induced by HQ were observed under fluorescence microscope after DAPI staining.(3) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, and cell cycle distribution was determined using flow cytometry. At least10,000cells per sample were collected and the data were then analyzed by a FACSAriaTM flow cytometer using CellQuest software. The percentage of cell cycle phases was quantified using ModFit LT software.(4) L02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, DNA damage was determined by the Comet assay and the parameters of each comet was calculated using CASP analysis software; y-H2AX foci were visualized by confocal laser scanning fluorescence microscopy.2.3Exploration on effects of inhibition of Polη expression on the expression, phosphorylation and subcellular localization of DNA checkpoint proteins in hepatocytes exposed by HQL02-POLH-nsc and L02-POLH-sh Cells were treated with different concentration of HQ (0,10,20, and40μM) for24h, the mRNA expression levels of DNA damage checkpoint genes(ATM, ATR, Chk1, Chk2, p53, H2AX and RPA2) were determined by real-time PCR and the data were analyzed by RQ Manager1.2software; the protein expression levels of DNA damage checkpoint proteins(ATM, ATR, Chk1, Chk2, p53, H2AX and RPA2) and its phosphorylation(p-Chkl, p-Chk2, p-p53, y-H2AX and p-RPA2) were determined by Western blot, the relative amounts of target proteins were calculated from the scanning profiles and analyzed by ImageJ software; subcellular localization of DNA damage checkpoint proteins were visualized by confocal laser scanning fluorescence microscopy.2.4Statistical analysisAll data are presented as mean±SD. Statistical evaluation of data analysis was carried out using SPSS14.0for Windows. Differences between the mean values of two groups were analyzed by t test. Differences between the mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. P<0.05was considered statistically significant.3Results3.1Polη-deficient hepatic cell line was constructed by RNAiThe results of real-time PCR analysis showed that the expression levels of Polη mRNA were down-regulated by61%in L02-POLH-sh1cells, by60%in L02-POLH-sh2cells and by82%in L02-POLH-sh3cells compared with normal L-02cells. Different degrees of POLH knockdown were also observed at the protein level in these cells, which were down-regulated by58%in L02-POLH-shl cells, by72%in L02-POLH-sh2cells and by86%in L02-POLH-sh3cells compared with normal L-02cells. The expression levels of both Polη, mRNA and protein were not obviously changed in L02-POLH-nsc and L02-POLH-nvc cells. In addition, there were no significant differences in cell biology characteristics between L02-POLH-sh1, L02-POLH-sh2, L02-POLH-sh3, L02-POLH-nsc, L02-POLH-nvc and L-02cells, however, the levels of Polη mRNA and protein were significantly reduced in L02-POLH-sh1, L02-POLH-sh2and L02-POLH-sh3cells, compared to L-02cells(P<0.05). Because Polη was down-regulated most effectively in L02-POLH-sh3cells, this clone was used in all subsequent studies, and is hereafter referred to as L02-POLH-sh.3.2Effects of inhibition of Polη expression on cell biological characteristics and DNA damage in hepatocytes exposed by HQ(1) Cell viability of L-02and L02-POLH-nsc cells in the treated groups (160and320μM) was significantly less than that in the untreated group (0μM)(P<0.01). However, cell viability in the treated groups (40,80,160and320μM) of L02-POLH-sh cells was significantly less than that in the untreated group (P<0.01). In particular, the viability of L02-POLH-sh cells was significantly lower than that of L-02and L02-POLH-nsc cells treated with40,80,160or320μM HQ, respectively (P<0.01). Whereas the cytotoxicity of HQ in parental L-02cells was similar to that observed in L02-POLH-nsc cells (P>0.05). The results also showed that L02-POLH-sh cells were2-fold more sensitive to HQ compared with L-02and L02-POLH-nsc cells. In addition, the results of morphological observations were consistent with the MTT assay.(2) The results showed that HQ induced apoptosis in L02-POLH-nsc and L02-POLH-sh cells in a dose-dependent manner, and suppression of Polη expression had little effect on the percentage of apoptotic cells in untreated controls. However, treatment with increasing concentrations of HQ resulted in significant dose-dependent increases in the levels of apoptosis in both cell lines. The rate of apoptosis increased from2.73%to3.77%, to5.27%, to6.63%in L02-POLH-nsc cells and from3.24%to6.78%, to10.70%, to14.03%in L02-POLH-sh cells at0,10,20, and40μM HQ treatment, respectively, and a significantly higher percentage of apoptosis in all treated L02-POLH-sh cells was observed compared with L02-POLH-nsc cells(P<0.01). In addition, the results of DAPI staining assay were consistent with above data.(3) Exposure of cells to HQ induced a significant alteration in cell cycle distribution with a decrease in the fraction of cells in G1phase and a corresponding increase in the fraction of cells in S and G2phase in both cell lines. No significant alteration was found in S phase cell populations after incubation with10and20μM HQ as compared with untreated cells (P>0.05), whereas treatment with40μM HQ significantly increased the percentage of L02-POLH-nsc cells in S phase (P<0.05), the proportion of cells in S phase increased from25.93%in the control to27.54,27.93and35.02%, respectively. In contrast, treatment with10,20and40μM HQ significantly increased the percentage of L02-POLH-sh cells in S phase (P<0.05):27.80,36.42,40.72and44.42%, respectively. In addition, our data revealed that the fraction of L02-POLH-sh cells in S phase was significantly higher than that in L02-POLH-nsc cells treated with10,20, and40μM HQ (P<0.05).(4) Comet assay showed that HQ induced a marked dose-dependent increase in DNA damage in L02-POLH-nsc and L02-POLH-sh cells, as measured by comet parameters. However, DNA damage due to HQ treatment was significantly greater in L02-POLH-sh cells as compared with L02-POLH-nsc cells after24h of exposure(P<0.01). Further more, immunofluorescence assay showed that treatment of L02-POLH-nsc and L02-POLH-sh cells with HQ could induce the accumulation of y-H2AX foci in the nucleus in a dose-dependent manner, y-H2AX foci were more abundant in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment(P<0.05).3.3Effects of inhibition of Polη expression on the expression, phosphorylation and subcellular localization of DNA checkpoint proteins in hepatocytes exposed by HQ(1) Results of real-time PCR showed that HQ induced a dose-dependent increase in mRNA expression levels of ATM, ATR, CHK1, CHK2, P53, and RPA2in L02-POLH-nsc and L02-POLH-sh cells, and the mRNA expression levels of CHK1, P53and RPA2were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment.(2) Results of Western blot analysis showed that HQ induced an increase in protein expression levels of ATM, ATR, Chk2, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX in L02-POLH-nsc and L02-POLH-sh cells; the protein expression levels of Chkl was increased in L02-POLH-sh, but not in L02-POLH-nsc cells following HQ treatment. In addition, the protein expression levels of Chk1, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment.(3) Results of immunofluorescence assay showed that HQ induced an increase in the fluorescence intensity of ATM, ATR, Chk1, Chk2, p53, RPA2, p-chkl, p-chk2, p-p53, p-RPA2and y-H2AX in L02-POLH-nsc and L02-POLH-sh cells, and the fluorescence intensities of Chkl, p53, RPA2, p-chk1, p-chk2, p-p53, p-RPA2and y-H2AX were higher in L02-POLH-sh cells than in L02-POLH-nsc cells following the same HQ treatment. Subcellular localization analysis showed that ATM and Chk2translocated from the nucleus to the cytoplasm in L02-POLH-nsc and L02-POLH-sh cells following HQ treatment. Polη deficiency resulted in translocation of Chkl and p53from the cytoplasm to the nucleus. In addition, the majority of RPA2colocalized with γ-H2AX in the nucleus of L02-POLH-nsc and L02-POLH-sh cells, and this co-localization could be enhanced after HQ treatment in dose-dependent manner; furthermore, inhibition of Polη enhanced co-localication of RPA2and γ-H2AX.4Conclusion4.1Polη-deficient L02-POLH-sh cell line was successfully constructed by RNAi technology mediated by lentiviral vector, which provided basis for later studies.4.2Inhibition of Polη expression led to a decrease in cell proliferation and an enhanced susceptibility to HQ cytotoxicity in hepatocytes.4.3Polη deficiency significantly increased the formation or accumulation of DSBs induced by HQ in hepatocytes.4.4Inhibition of Polη expression resulted in an enhanced apoptosis and more pronounced S phase arrest upon HQ treatment in hepatocytes.4.5Polη deficiency significantly increased the expression levels and nuclear localization of Chkl, P53and RPA2in hepatocytes induced by HQ.4.6Phosphorylation of Chkl, Chk2, p53, H2AX and RPA2was strongly activated by HQ, and this activation is enhanced in hepatic cells lacking Polη.4.7The results demonstrate that Polη may play an important role in the DNA damage response induced by HQ in human hepatic cells, through regulating cell proliferation, apoptosis, cell cycle progression, DNA double-strand break repair and activation of DNA damage checkpoint proteins. |
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