'IN VITRO SCREENING OF SELECTED CHEMOPREVENTIVE AGENTS U

Survivin是一种在细胞中特异表达的抗凋亡蛋白。 该项目的目的是确定生存素在常见癌症类型的肿瘤和癌前组织样本中的表达。生存素可以作为一种疗效生物标志物，特别是对于侵袭前阶段的调节。 该研究确定了哪些癌症类型中生存素可以用作干预靶点，并提供了生存素在正常细胞中过表达降低凋亡指数的原则证据。 从至少10个冷冻肿瘤标本和/或石蜡块中采集组织样本，这些标本和/或石蜡块代表了以下常见癌症的不同恶性程度（尤其是癌前病变和周围正常组织）：乳腺癌、肺癌、结肠癌、脑癌、宫颈癌、卵巢癌、前列腺癌和黑色素瘤。根据标准标准，对病变进行仔细的组织病理学表征。 生存素的表达正在通过免疫组织化学和原位杂交进行定量测定，并正在确定与恶性程度的可能相关性。 该项目使用各种表达构建体和/或反义核苷酸在至少三种人类癌细胞系中表达/引入反义生存素。 这些人癌细胞系和/或来源于在#1中发现阳性的癌前组织的细胞系，特别是在癌前病变中，被用于阻断存活素表达。 通过核小体间DNA片段化（TUNEL）测定或通过荧光底物N-乙酰基-Asp-Glu-Asp-aldehyde的半胱天冬酶3依赖性水解来监测细胞凋亡。 属于抗增殖剂类别的四种化学预防化合物，4 HPR、DFMO、舒林酸和紫苏醇以无毒范围内的五种剂量使用。 这些实验在肺癌细胞系A427中进行。转染细胞系以过表达存活素基因或阻断其表达。 生存素在正常人体细胞中的表达：生存素被认为通过阻断细胞凋亡而导致癌症。 它在正常细胞中不表达。 抑制凋亡在正常细胞的异位表达生存素将提供证据的原则，生存素的抗凋亡活性。 该研究涉及用存活素基因的cDNA表达载体转染至少三种正常人类细胞类型，内皮细胞、上皮细胞和成纤维细胞。 应在RNA和蛋白质水平上测量生存素基因的表达，并测量凋亡指数，并与未转染和载体转染的对照进行比较。

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.