'IN VITRO SCREENING OF SELECTED CHEMOPREVENTIVE AGENTS U

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Survivin is a protein uniquely expressed in cells which are resistant to apoptosis. The project's objective is to determine survivin expression in neoplastic and preneoplastic tissue samples from common cancer types. Survivin may serve as a efficacy biomarker, especially for modulation in the preinvasive stages. The study identifies which cancer types in which survivin can be used as a intervention target, and provides proof of principle that survivin overexpression in normal cells decreases the apoptotic index. Tissue samples are being acquired from at least 10 frozen tumor specimens and/or paraffin blocks representing various degrees of malignancy (especially premalignant lesions, and the surrounding normal tissues) from each of the following common cancers: breast, lung, colon, brain, cervical, ovarian, prostate, and melanoma. The lesions are carefully histopathologically characterized according to standard criteria. Expression of survivin is being measured quantitatively by immunohistochemistry as well as by in situ hybridization and a possible correlations with the degree of malignancy are being determined. The project uses various expression constructs and/or antisense nucleotides for expressing/introducing antisense survivin in at least three human cancer cell lines. These human cancer cell lines and/or cell lines derived from premalignant tissues found to be positive in #1, especially in precancerous lesions, are being used for blocking survivin expression. Apoptosis is being monitored by internucleosomal DNA fragmentation (TUNEL) assay or by caspase 3-dependent hydrolysis of the fluorogenic substrate, N-acetyl-Asp-Glu-Asp-aldehyde. Four chemopreventive compounds belonging to the class of antiproliferative agents, 4 HPR, DFMO, Sulindac, and Perillyl Alcohol are being used at five doses in a nontoxic range. These experiments are being carried out in the lung carcinoma cell line, A427. The cells line is transfected to either overexpress the survivin gene or to block its expression. The expression of survivin in normal human cells: Survivin is thought to contribute to cancer by blocking apoptosis. It is not expressed in normal cells. Inhibition of apoptosis in normal cells by ectopic expression of survivin will provide proof of principle for the antiapoptotic activity of survivin. The study involves transfecting at least three normal human cell types, endothelial, epithelial, and fibroblast, with the cDNA expression vectors of the survivin gene. The expression of the survivin gene shall be measured both at the RNA and protein level and the apoptotic index shall be measured and compared with the untransfected and vector-transfected controls.