title: Down-regulation of Polη expression leads to increased DNA damage, apoptosis and enhanced S phase arrest in L-02 cells exposed to hydroquinone.
abstract: DNA polymerase eta (Polη), the product of the xeroderma pigmentosum variant gene, is required for translesion DNA synthesis, and plays a pivotal role in preventing genome instability after DNA damage induced by genotoxic agents. Studies have previously suggested a link between Polη and susceptibility to hydroquinone (HQ)-induced toxicity. To further address the role of Polη in the response of L-02 cells to HQ, we employed RNA interference to silence Polη expression in L-02 cells and examined the susceptibility of these Polη-deficient cells to the toxic effects of HQ. In this study, cell survival rate was determined using the MTT assay, DNA damage was determined by the Comet assay, apoptosis and cell cycle distribution were determined using flow cytometry, the mRNA expression levels of Polη were determined by real-time PCR, and the protein expression levels of Polη and γ-H2AX were determined by Western blot, γ-H2AX foci were visualized by confocal laser scanning fluorescence microscopy after cells were exposed to HQ at various concentrations for 24h in vitro. The results showed that stable Polη-knockdown cells were successfully constructed and more than 80% inhibition of Polη expression was confirmed. The results also showed that down-regulation of Polη led to a decrease in cell proliferation and an enhanced susceptibility to HQ-induced cytotoxicity. Polη-deficient cells were 2-fold more sensitive to HQ when compared with nonspecific siRNA control cells. Moreover, Polη-silenced L-02 cells treated with HQ displayed an increased level of DNA double-strand breaks as measured by olive tail moment, and an elevated DNA damage response as indicated by the induction of γ-H2AX. In addition, knockdown of Polη resulted in more enhanced apoptosis and more pronounced S phase arrest following HQ treatment. Together, these results show that Polη plays an important role in the response of L-02 cells to HQ-induced DNA damage.
authors.affiliation: Department of Toxicology, School of Public Health, Central South University, 110 Xiangya Road, Changsha 410078, Hunan, China.
authors.forename: Gonghua
authors.initials: G
authors.lastname: Hu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.forename: Lingqing
authors.initials: L
authors.lastname: Yang
authors.forename: Caigao
authors.initials: C
authors.lastname: Zhong
authors.forename: Bo
authors.initials: B
authors.lastname: Xia
authors.forename: Yuan
authors.initials: Y
authors.lastname: Yang
authors.forename: Jianjun
authors.initials: J
authors.lastname: Liu
authors.forename: Desheng
authors.initials: D
authors.lastname: Wu
authors.forename: Qingchen
authors.initials: Q
authors.lastname: Liu
authors.forename: Zhixiong
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D059447:Cell Cycle Checkpoints / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D020552:Comet Assay; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D006873:Hydroquinones / Q000633:toxicity*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction; D016196:S Phase / Q000187:drug effects* / Q000502:physiology; D000099270:Y-Family DNA Polymerases
chemical_list: D004259:DNA-Directed DNA Polymerase; D006657:Histones; D006873:Hydroquinones; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C468783:H2AX protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C031927:hydroquinone