Adenovirus type 5 E1A gene therapy for ovarian clear cell carcinoma. Free

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Clear cell carcinoma (CCC) of the ovary, a distinct histologic entity in the World Health Organization classification of ovarian tumors, behaves differently than other epithelial ovarian cancers. CCC constitutes only 4% to 12% of epithelial ovarian cancers but has a very poor prognosis (median survival, 12.7 months in advanced disease), in part because of its resistance to conventional platinum- or taxane-based chemotherapy. Molecular analyses of various types of ovarian tumors recently showed that HER-2/neu (HER2) is upregulated more often in CCC than in other histologic types of epithelial ovarian cancer. We, therefore, explored two molecules thought to target HER2: trastuzumab and adenovirus type 5 E1A. HER2 protein was detected at various levels in all 10 cell lines by western blotting and in five CCC cell lines by immunohistochemical staining; HER2 gene amplification was detected in only one cell line (RMG-I) by fluorescence in situ hybridization. In breast cancer, Her-2 gene amplification is the best predictor of response to trastuzumab. Thus, we examined the effects of trastuzumab on the proliferation of four CCC cell lines (RMG-I, SMOV-2, OVTOKO, and OVSAYO) that expressed a range of HER2 protein levels. Trastuzumab did not inhibit proliferation in any of the four CCC cell lines and did not downregulate the expression of phosphorylated Akt protein, which is regulated by HER2 signaling. We next examined the cytotoxicity of E1A in CCC cell lines. Transfection with E1A reduced colony formation by 47% to 86% relative to control vectors in all 10 CCC cell lines regardless of HER2 expression level. Infection of RMG-I and SMOV-2 cells with an adenoviral vector encoding E1A led to significant (P < 0.05) suppression of proliferation and enhancement of cell death. Because CCC rarely expresses mutant p53, and E1A can stabilize the p53 protein, we transfected siRNA against p53 (si-p53) and examined E1A-induced cell death in RMG-I and SMOV-2 cells. The combination of E1A and si-p53 reduced the cytotoxic effect of E1A in RMG-I and SMOV-2 cells compared with the combination of Ad.E1A(+) and a scrambled siRNA sequence (P < 0.05). P53 and Bax protein levels were upregulated and cleaved caspase 9 was detected in the Ad.E1A(+)-treated cells. However, cells in which p53 was knocked down showed reduced p53 expression and no upregulation of Bax or cleavage of caspase 9. Further, we examined p73, another member of the p53 protein family that can be upregulated by E1A and induce p53-independent apoptosis. We found that Ad.E1A(+) infection did not affect p73 protein expression, nor was E1A-induced cytotoxicity affected by knocking down p73 in RMG-I and SMOV-2 cells, suggesting that p73 is not involved in E1A-induced apoptosis of CCC. These results indicate that E1A gene therapy, because of its ability to stabilize wild type p53, is worth exploring as a treatment modality for women with ovarian CCC, which is commonly known to have wild type p53.