[DNA Repair Function and Mutation of an H2B Monoubiquitination Factor WDR70 in Ovarian Cancer].

OBJECTIVE: To investigate the roles of enzyme DCAF proteinDNA damagebinding protein 1 (DDB1)/cullin4 (CRL4) complex family members CRL4WD40 repeat domain protein 70 (WDR70) in DNA repair process and its mutation in ovarian cancer. METHODS: Immunofluorescent assay was employed to measure H2AX (γH2AX) and phosphorylated replication protein A2 (RPA32) formed in siDDB1 or siWDR70 ovarian cancer cells after the treatments of chemical medicine and radioactive threapy. 5Brdu immunohistochemical staining was used to explore the function of WDR70 in DNA replication. The expressions of WDR70 and histone protein H2B monoubiquitination (uH2B) was measured by immunohistochemistry,the function of DNA repair,expression and mutations of CRL4 in ovarian cancer were detected by semiquantitative PCR and DNA sequencing. RESULTS: Immunofluorescent assay indicated that distinct subunits of CRL4 played different roles in checkpoint activation and H2Bmonoubiquitinationdepedendent homologous recombination,while the scaffold subunit DDB1 participated in both processes,WDR70 was only required for DNA end resection,chromatin loading of RPA32 and HR. The dose of WDR70 was not effect on DNA replication. Ovarian cancer had different expression of WDR70 and uH2B compared with normal tissue,transcripts of WDR70 was diminished or truncated in 50% of ovarian cancer,which corresponded to multiple mutations. CONCLUSION: CRL4 ubiquitin ligase plays multiple roles in DNA repair and is critical for genome stability. It may be an potential anticancer barrier against ovarian malignancies.

[HPV E7 Function in DNA Damage Response of Cervical Intraepithelial Neoplasia.]

OBJECTIVES: To determine the function of human papillomavirus (HPV) E7 in DNA damage response of cervical intraepithelial neoplasia (CIN) 3 cells. METHODS: Samples of CIN 3 and child foreskin tissues were collected,with pathologically confirmed HPV positive and negative,respectively.Collagenase A was used for digesting tissues prior to primary culture.The HPV negative cells were infected with lentivirus E7 and pLV.Proteins(53BP1,NBS1,BRCA1 and RPA32) responsive to DNA double break damages were detected by indirect immunofluorescent staining after 0-8 h treatment with X-ray (2 or 5 Gy). RESULTS: After treatment with 2 or 5 Gy X-ray,53BP1,NBS1,BRCA1 and RPA32 foci in HPV+ cells increased compared with HPV- cells (P<0.05).Less 53BP1,RPA32,BRCA1 and NBS1 foci positive cells (foci>5) were found in E7 infected cells than in pLV infected cells(P<0.05).Both of them reached the peak at 6 h (2 Gy) or 4 h (5 Gy). CONCLUSIONS: We have successfully established a model to detect the function of HPV E7 in DNA damage response using primary culture of CIN fibroblasts.With the progression of CIN,HPV E7 can inhibit DNA double break repair.

[Employing DNA Damage Response Inhibitors to Enhance Chemosensitivity of Ovarian Carcinoma Cells].

OBJECTIVE: To assess the sensitisation effects of DDR inhibitors combined with conventional chemotherapeutics agents (cisplatin et al) in a drug-resistant ovarian cancer cell line(OVCAR-8). METHODS: Inhibitors of DDR regulators with cisplatin were applied to challenge OVCAR-8, and evaluated the DNA damage response (DDR) and cytotoxic effects of different combination of chemicals. Inhibition of proliferation to OVCAR-8 of different drugs was evaluated by MTT assay. The activation of phosphorylation of histone family 2A variant (yH2AX) and p53 binding protein 1 (53BP1) in OVCAR-8 were evaluated by immunofluorescence to observe their ability of recruitment and forming foci at DNA damage site. RESULTS: We observed that combined treatment of ataxia-telangiectasia mutated (ATM)/ATM and Rad 3-related (ATR) inhibitor and cisplatin can suppress the activation of damage repair mechanisms and weakened the proliferative activity of OVCAR-8 cells (P<0. 01) ; ATR pathway was suppressed and the signal of γH2AX weakened and cell survival rate significantly reduced when combination therapy of HU and Wortmannin (P < 0.05); poly ADP-ribose polymerase (PARP) inhibitor could not enhance chemosensitivity in OVCAR-8 cells when combined with cisplatin (P > 0.05). CONCLUSION: We substantiated that appropriate inhibitors of DNA damage response may have a potential to improve the anti-tumor effect of conventional chemotherapy drugs and prevent drug resistances.

[Functional Analysis of DNA Damage Repair Factor WDR70 and Its Mutation in Ovarian Cancer].

OBJECTIVES: To analyze the cellular function of the newly discovered DNA damage repair factor WDR70, and investigate the mutation in ovarian cancer to verify if function loss of the WDR70gene was associated with ovarian cancer. METHODS: The WDR70 gene was silenced by using siRNA technique or overexpressed its wild and mutation type by with lentivirus and plasmid in hunman cells. The subcellular localization and biochemical function of WDR70 was analyzes by indirect immunofluorescence and immunoblotting. The expression level of WDR70 and the mutations of its cDNA was checked with RT-PCR sequencing for 1 normal ovarian tissue and 16 ovarian cancer specimen. RESULTS: We found gene silencing of WDR70 or overexpression of WDR70 mutation type disrupts the phosphorylation level of homologous recombination functional protein RPA32 and the ability of recruitment at DNA damage site of recombinase RAD51, the loss of function of WDR70 also causes the elevation of the chromosome breakage in metaphase. Meanwhile, we also noticed that the existence of multiple mutations in genomic WDR70 in ovarian cancer specimen. CONCLUSIONS: Our results defined that in vitro system, WDR70 is a DNA damage repair gene, silencing of WDR70 or overexpression of WDR70 mutation type disrupts homologous recombination and chromosomal instability; the frequent mutations of WDR70 gene in genome of ovarian cancer specimens could also lead to DNA repair defeat and gene instability. Consequently WDR70 gene could represent an anti-cancer mechanism for ovarian cancer.