[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.

[DNA damage response of epithelial ovarian cancer cells (primary culture) to chemo-radiotherapy].

OBJECTIVE: To detect protein dynamic changes of cellular localization and the DNA damage response of epithelial ovarian cancer cells to chemo-radiotherapy. METHODS: 28 specimens of epithelial ovarian cancer were collected, with 6 cases diagnosed as borderline serous cystadenoma, 5 as highly differentiated, 6 as medium differentiated and 11 as poorly differentiated cystadenocarcinoma. Collagenase A was used for digesting tissues before primary culture. We compared the characteristics of cells cultured in different mediums (MCDB/M199 medium, primary culture medium, and DMEM medium) supplemented with multiple growth-promoting factors. The characteristics of cells were examined in terms of the maintenance of normal cell morphology, proliferation potential, and cell fibrosis proteins (53BP1 and gamma-H2AX) responsive to DNA damage [those in the ATM checkpoint pathway determined by indirect immunofluorescent staining after treatment with camptothecin (CPT) and X-ray]. RESULTS: Normal morphology was maintained relatively well in the cells cultured in MCDB/M199 medium and its cell fibrosis was slow compared with the cells cultured in other media. Gradually increased endogenous damage was demonstrated by the expression of 53BP1 and gamma-H2AX foci (P < 0.05) in all of the ovarian primary cells. After treatment with CPT and ionizing radiation, increased levels of DNA double-strand breaks were observed indicating a classic DNA damage response. CONCLUSION: We have successfully established a protocol for the primary culture of epithelial ovarian cancer cells, which provides an important platform for characterizing DNA damage responses of the cells. With the progression of epithelial ovarian cancers, the ATM checkpoint pathway is activated by endogenous DNA lesions. This signaling pathway can be further activated by CPT or X-ray irradiation, hampering the growth of tumor cells and further progression of cancers.

Decreased histone H2B monoubiquitination in malignant gastric carcinoma.

AIM: To investigate H2B monoubiquitination (uH2B) and H3K4 di- and tri-methylation (H3K4-2me, H3K4-3me) levels and their clinical significance in gastric cancer (GC). METHODS: Immunohistochemistry (IGC) was used to detect the differential levels of uH2B, H3K4-2me and H3K4-3me modifications in GC specimens from chemo/radiotherapy-naïve patients who underwent potentially curative surgical resection (n = 159) and in a random sampling of non-tumor gastric epithelium specimens (normal controls, n = 20). The immunohistochemistry (IHC)-detected modifications were classified as negative, low-level, or high-level using a dual-rated (staining intensity and percentage of positively-stained cells) semi-quantitative method. The relationships between uH2B modification levels and clinicopathological parameters of GC were assessed by a Wilcoxon rank sum test (pairwise comparisons) and the Kruskal-Wallis H test (multiple comparisons). The correlation between uH2B modification and survival was estimated by Kaplan-Meier analysis, and the role of uH2B as an independent prognostic factor for survival was assessed by multivariate Cox regression analysis. RESULTS: The presence and level of H3K4-2me and H3K4-3me IHC staining was similar between the normal controls and GC specimens. In contrast, the level of uH2B was significantly lower in the malignant gastric tissues (vs normal control tissues) and decreased along with increases in dedifferentiation (well differentiated > moderately differentiated > poorly differentiated). The level of uH2B correlated with tumor differentiation (P < 0.001), Lauren's diffuse- and intestinal-type classification (P < 0.001), lymph node metastasis (P = 0.049) and tumor-node-metastasis stage (P = 0.005). Patients with uH2B+ staining had higher 5-year survival rates than patients with uH2B-staining (52.692 ± 2.452 vs 23.739 ± 5.207, P < 0.001). The uH2B level was an independent prognostic factor for cancer-specific survival (95%CI: 0.237-0.677, P = 0.001). CONCLUSION: uH2B displays differential IHC staining patterns corresponding to progressive stages of GC. uH2B may contribute to tumorigenesis and could be a potential therapeutic target.

[DNA damage response in ovarian clear cell adenocarcinoma].

OBJECTIVE: To study the relationship between ovarian clear cell adenocarcinoma and DNA damage. METHODS: 14 samples were selected from clinical ovarian cases including 3 cases with normal ovarian tissue, 6 cases with poorly differentiated ovarian tumor, 5 cases with ovarian clear cell adenocarcinoma, treated by X-ray irradiation and frozen sections respectively. DNA damage response was analyzed by immunofluorescence and Western blot. RESULTS: Before X-ray irradiation, compared to normal ovarian tissue, a large number of endogenous damage existed in ovarian clear cell adenocarcinoma, and phosphorylation of histone family 2A variant (H2AX) was abnormally enhanced 1 hour after irradiation treatment, however, DNA repair was normal in ovarian clear cell adenocarcinoma. Phosphorylation of H2AX was dispensable for p53 binding protein 1 (53BP1) activation and couldn't be colocalized in clear-type ovarian cancer tissues. CONCLUSION: The abnormal DNA damage activation implies that the network of DNA damage signaling pathway may be defective.