RESUMO
Malignancies including ovarian cancer (OvCa) are genetically unstable. Genomic integrity is maintained by tumor suppressor p53 and DNA damage response network, which crosstalk to each other via not well characterized mechanisms. In this work, we characterize features of damage-related signals in cultured epithelial OvCa cells and tumor biopsies. We found that endogenous burden of DNA damage in OvCa tissues were ubiquitously accumulated in high-grade malignancies than lower grade of cancer that cannot be obviously explained by disturbed function of in DNA damage response (DDR). In contrast, CHK1 phosphorylation (CHK1-pS345) marking the checkpoint activation in nucleolar compartments are prevalent in high-grade OvCa, coincident to the elevated DNA damage in nucleoplasm. Generation of CHK1-pS345 requires the presence of p53 protein in addition to the well-known activities of ATM/ATR kinases. Apparently, mutant forms of p53 possess higher activity in triggering CHK1 phosphorylation than wild type, implying a potential role of p53 in maintaining rDNA integrity. Loss of p53 function would cause replication stress in nucleoli. Altogether, our study reveals endogenous nucleoli stress in OvCa that is coupled to perturbed function of p53 in DNA repair.
Assuntos
Neoplasias Ovarianas , Proteína Supressora de Tumor p53 , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma Epitelial do Ovário/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA/genética , Feminino , Humanos , Neoplasias Ovarianas/genética , Fosforilação , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Quebras de DNA de Cadeia Dupla , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Camptotecina/farmacologia , Carcinoma Epitelial do Ovário , Proteínas de Ciclo Celular/metabolismo , Meios de Cultura , Feminino , Histonas/metabolismo , Humanos , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/radioterapia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/radioterapia , Transdução de Sinais , Raios XRESUMO
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.
