'IN VITRO SCREENING OF SELECTED CHEMOPREVENTIVE AGENTS U

选定化学预防剂的体外筛选 U

• 批准号: 6314984
• 负责人: DAVID L. MCCORMICK
• 金额: $ 24.72万
• 依托单位: IIT RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6314984
• 关键词: antineoplastics; apoptosis; biomarker; brain neoplasms; breast neoplasms; cancer prevention; cell line; cervix neoplasms; chemoprevention; colon neoplasms; drug screening /evaluation; gene expression; histopathology; immunocytochemistry; in situ hybridization; lung neoplasms; melanoma; neoplastic cell; ovary neoplasms; prostate neoplasms; terminal nick end labeling; transfection

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.

生存素是一种在抗凋亡细胞中独特表达的蛋白质。 该项目的目标是确定常见癌症类型的肿瘤和肿瘤前组织样本中生存素的表达。生存素可以作为一种功效生物标志物，特别是对于侵袭前阶段的调节。 该研究确定了生存素可作为干预靶点的癌症类型，并提供了正常细胞中生存素过度表达可降低细胞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，代表以下每种常见癌症的不同程度的恶性肿瘤（特别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。根据标准对病变进行仔细的组织病理学表征。 通过免疫组织化学以及原位杂交定量测量存活蛋白的表达，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。 这些人类癌细胞系和/或源自癌前组织的细胞系在#1中被发现呈阳性，特别是在癌前病变中，被用于阻断存活蛋白的表达。 通过核小体间 DNA 片段化 (TUNEL) 测定或荧光底物 N-乙酰基-天冬氨酸-谷氨酸-天冬氨酸-醛的半胱天冬酶 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、舒林酸和紫苏醇正在以无毒范围内的五种剂量使用。 这些实验是在肺癌细胞系 A427 中进行的。转染细胞系以过度表达生存素基因或阻断其表达。 生存素在正常人类细胞中的表达：生存素被认为通过阻止细胞凋亡而导致癌症。 它在正常细胞中不表达。 生存素异位表达对正常细胞凋亡的抑制将为生存素的抗凋亡活性提供原理证明。 该研究涉及用生存素基因的 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，并测量细胞凋亡指数并与未转染和载体转染的对照进行比较。