Microarray Analysis Of DNA Damage Repair Gene Expression Profiles In Radioresistant Cervical Cancer Cells

Cervical cancer is one of the most common malignancies in China, with a high incidence and mortality and more than 130,000 new cases reported and 50,000 women dying of the disease per year. Radiotherapy is particularly effective for patients with cervical cancers at an advanced stage or that cannot be cured surgically. In 1998, we first treated cervical cancer with 252Cf rays combined with X-rays in China. It obtained satisfactory clinical effect, tumor local control rate was 85.2% and 5-year survival rate was 88.7% respectively. But we observed the existence of radioresistance phenomenon in some primarily cervical cancer patients and secondary radioresistance in course of treatment. Therefore, it was very important that we investigated influencing factors of radiosensitivity in cervical cancer.The activity of the DNA damage repair pathway is one of the most important factors leading to radioresistance in tumors, including cervical cancer. In many human tumor lines, radiosensitivity correlated with DNA damage induction and repair, so the research of DNA damage repair gene expression profiles is helpful to elucidate the mechanism of radioresistance in cervical cancer and supports to look for the targets of improving radiosensitivity. But the mechanism of DNA damage repair so complex that it couldn't be explained by a little of genes and need much information of DNA repair genes. DNA microarray technology is a powerful technique to detect the biological response of thousands of genes and can be used to study the mechanism that DNA damage repair gene leads to radioresistance in cervical cancer. Hence, we first investigate the expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer. Then, long-term 252Cf neutron ray and X-ray irradiation of Hela cells was used to generate two radioresistant cell sub-lines, HelaNR and HelaXR, which provided a model system for studying the radioresistance mechanisms of cervical cancer cells. Whereafter, microarray analysis was used to identify the gene expression patterns of two radioresistant sub-lines derived from Hela cells and the differentially expressed genes were selected, for example GADD45αand BTG2. Finally, we constructed the eukaryotic expression vector of GADD45αgene and investigated its role in radiosensitivity to radiotherapy in cervical cancer.Objective1. To explore the relationship between APE1 and radioresistance in cervical cancer.2. To investigate the mechanism that DNA damage repair gene leads to radioresistance in cervical cancer.3. To investigate primarily the function that eukaryotic expression vector of GADD45αgene enhance radiosensitivity in cervical cancer.Materials and Methods1. The expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer: The expression of APE1 was detected by immunohistochemistry technique in 89 cases of cervical cancer (treated by 252Cf), 15 cases cervical intraepithelial neoplasia(CIN) and 10 cases of normal cervical tissue, and its association with clinicopathological data as well as prognosis was analyzed.2. The generation of two radioresistant cell sub-lines: Hela cells were treated with fractionated 252Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HelaNR and HelaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay.3. The screening of differentially expressed genes relate to DNA damage repair genes in cervical cancer: Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by western blotting and real-time PCR.4. The investigation of eukaryotic expression vector of GADD45αgene enhancing radiosensitivity in cervical cancer: we constructed the eukaryotic expression vector of GADD45αgene. Then, The expression of GADD45αmRNA was detected after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains by real-time PCR and the apoptosis was detected after eukaryotic expression vector of GADD45αgene was transfected into Hela cells by FCM. Results1. The expression feature of APE1, a DNA damage repair gene, and its correlation with clinicopathology and prognostic significance after 252Cf radiotherapy in cervical cancer: The expression of APE1 in cervical cancer is higher significantly than that in normal cervical tissue and CIN(P<0.01). In normal cervical tissue and CIN, the APE1 express was located in the nucleus. In cervical cancer, the APE1 express was located in the nucleus (59), cytoplasm (8) or nucleus and cytoplasm (22), the location of APE1 was related with FIGO stage and pathological grade (P<0.01), and not related with lymph node metastasis. The level of APE1 express related with FIGO stage, pathological grade and lymph node metastasis (P<0.05), and not related with age and pathological type. The Kaplan-Meier survival analysis showed that the survival time of the group of APE1 nucleus expression (median survival time is 70.9 months) and the group of APE1 low expression (median survival time is 75.8 months) is longer significantly than that of the group of APE1 cytoplasm expression (median survival time is 57.8 months) and the group of APE1 high expression (median survival time is 56.5 months) (P=0.025, 0.001).2. The generation of two radioresistant cell sub-lines: (1) Under equal doses of 252Cf neutron ray and X-ray irradiation, the D0, Dq, and SF2 values of the HelaNR and HelaXR cells were higher than those of the Hela cells, indicating that the sub-lines were more radioresistant than the parent cells. (2) In Hela cells, microvilli were present on the surface and the cytoplasm contained abundant mitochondria and ribosome. In the radioresistant sub-lines, swelling of mitochondria, vacuolization, dilatation of the endoplasmic reticulum, and myelin figures were observed. Thus, in the HelaNR and HelaXR sub-lines, the ultrastructural changes induced by long-term irradiation persisted even > 2 months after the last radiation treatment. (3) The cell doubling times of the HelaNR and HelaXR cells (33.12±3.67 h, and 36.94±3.16 h, respectively) were longer than those of the parent Hela cells (28.62±2.77 h). (4) In Hela cells, exposure to a radiation dose of 4 Gy significantly increased the proportion of cells in G2 and decreased the proportion of cells in G1. The proportion of G2-arrested Hela cells was even greater following 16-Gy irradiation. In the HelaNR and HelaXR sub-lines, however, the proportion of cells in G2 did not increase after irradiation with 4 Gy. At 16 Gy, cells of both sub-lines became arrested in G2 but the proportion was much lower than in Hela cells. Thus, cells of the radioresistant sub-lines probably arrested in the G1 phase of the cell cycle. (5) At 0 Gy, the apoptosis rate was 0.93–2.71% for all three cell lines. At 4 and 16 Gy, the apoptosis rate of Hela cells was much higher than that of the radioresisitant sub-lines (4Gy: 9.32 vs 3.84, 7.94 vs 5.43; 16Gy: 22.47 vs 7.28, 20.03 vs 11.1, p < 0.05). Thus, the sub-lines HelaXR and HelaNR were more radioresistant than the parental Hela cell line.3. The screening of differentially expressed genes relate to DNA damage repair genes in cervical cancer: Using stringent criteria for array analysis (≥2-fold change in expression), we identified 113 genes related to DNA damage signaling pathways that were differentially expressed in the parental Hela cells compared to radioresistant HelaXR and HelaNR cells. Of the 24 genes significantly altered by at least 2-fold in HelaNR cells, 19 were up-regulated and 5 down-regulated. Likewise, of the 41 genes significantly altered by at least 2-fold in HelaXR cells, 38 were up-regulated and 3 down-regulated. For the two radioresistant sub-lines, the similar overall trend in gene-expression changes indicated that long-term exposure to 252Cf neutron and X-rays had resulted in a similar induction of genes involved in DNA damage signaling pathways. The number of genes that underwent homologous recombination and nonhomologous end-joining, processes that result in altered gene expression, was higher in HelaXR cells (8 and 3, respectively) than in HelaNR cells (2 and 1, respectively). Furthermore, the number of cell cycle arrest and mismatch repair genes expressed was higher in HelaXR cells (7 and 3, respectively) than in HelaNR cells (3 and 1, respectively) whereas the number of base excision repair genes expressed in the two sub-lines was almost the same (4 in HelaXR cells and 5 in HelaNR cells). Thus, genes encoding double-strand break (DSB) repair, mismatch repair, and cell cycle arrest functions were more highly expressed in cells resistant to X-rays than in cells resistant to 252Cf neutron rays. Two genes, BTG2 and GADD45α, were chosen for further analysis and validation of the microarray data by Western blotting and real-time PCR. As shown by Western blotting, BTG2 protein expression was up-regulated in the resistant sub-lines, especially in HelaNR cells, while the expression of GADD45αprotein was down-regulated in both resistant sub-lines. PCR analysis showed that BTG2 and GADD45αmRNA expression paralleled that of the respective proteins.4. The investigation of eukaryotic expression vector of GADD45αgene enhancing radiosensitivity in cervical cancer: Through gene sequencing and BLASTn in PUBMED, we determined that eukaryotic expression vector of GADD45αgene was constructed successfully. The expression of GADD45αmRNA increased significantly after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains (P<0.05). Apoptosis rate for almost 0.81–1.91% was found at 0Gy dose point in three groups,which was increased with higher radiation doses. A significant difference was observed between the transfected group and control group, lipofectin group (p < 0.05). There was no significant difference between control group and lipofectin group (p> 0.05).Conclusion1. The dystopic express of APE1 might play a pivotal role in carcinogenesis and progression of cervical cancer, and the express of APE1 might estimate the prognosis after 252Cf radiotherapy. It showed that the DNA damage repair function of APE1 might correlate with radioresistance in cervical cancer.2. Hela cells were irradiated with fractionated 252Cf neutron and X-rays, yielding two radioresistant cell sub-lines HelaNR and HelaXR. The radioresistant sub-lines were more radioresisitant to parental Hela cells by detecting their radioresistant characteristics.3. Microarray analysis was used to identify the gene expression patterns of two radioresistant sub-lines derived from Hela cells and the differentially expressed genes relate to DNA damage repair genes in cervical cancer were selected. It supported to look for the targets of improving radiosensitivity in cervical cancer.4. The eukaryotic expression vector of GADD45αgene was constructed. The expression of GADD45αmRNA and radiation-induced apoptosis increased significantly after eukaryotic expression vector of GADD45αgene was transfected into cervical cancer cell strains. It showed that eukaryotic expression vector of GADD45αgene might enhance radiosensitivity in cervical cancer.