'IN VITRO SCREENING OF SELECTED CHEMOPREVENTIVE AGENTS U

Survivin是一种在抗凋亡细胞中独特表达的蛋白。该项目的目的是确定来自常见癌症类型的肿瘤和肿瘤前组织样本中的survivin表达。Survivin可以作为一种疗效生物标志物，特别是在侵袭前阶段的调节。本研究确定了survivin在哪些癌症类型中可以作为干预靶点，并提供了survivin在正常细胞中过表达降低凋亡指数的原理证明。组织样本取自至少10个冷冻肿瘤标本和/或石蜡块，代表不同程度的恶性肿瘤（特别是癌前病变和周围的正常组织），这些肿瘤来自以下常见癌症：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。通过免疫组织化学和原位杂交定量测量survivin的表达，并确定其与恶性程度的可能相关性。该项目使用各种表达结构和/或反义核苷酸在至少三种人类癌细胞系中表达/引入反义survivin。这些人类癌细胞系和/或来源于癌前组织的细胞系在#1中呈阳性，特别是在癌前病变中，被用于阻断survivin的表达。凋亡是通过核小体间DNA片段（TUNEL）测定或半胱天冬酶3依赖性水解荧光底物n -乙酰- asp - glu - asp -醛来监测的。四种属于抗增殖剂类的化学预防化合物，4 HPR， DFMO， Sulindac和紫苏醇在无毒范围内以五种剂量使用。这些实验是在肺癌细胞系A427中进行的。转染细胞系后，要么过度表达survivin基因，要么阻断其表达。正常人类细胞中survivin的表达：survivin被认为通过阻断细胞凋亡而促进癌症的发生。它在正常细胞中不表达。survivin异位表达对正常细胞凋亡的抑制作用为survivin抗凋亡活性提供了理论依据。该研究涉及用survivin基因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。检测survivin基因在RNA和蛋白水平上的表达，检测细胞凋亡指数，并与未转染和载体转染的对照组进行比较。

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.