METAL TRANSFORMATION IN 10TL/2 AND HUMAN FIBROBLASTS

10TL/2 和人类成纤维细胞中的金属转化

• 批准号: 3250548
• 负责人: JOSEPH R LANDOLPH
• 金额: $ 12.34万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-09-28 至 1989-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3250548
• 关键词: Retroviridae; Retroviridae disease; arsenic; cellular oncology; chemical carcinogenesis; chromium; environment related neoplasm /cancer; metal poisoning; molecular cloning; molecular oncology; neoplasm /cancer genetics; nickel; oncogenes

The goals of this proposal are to continue our studies into the mechanisms and molecular biology of metal-salt transformation of C3H/10T1/2 cells and diploid human foreskin fibroblasts. We will finalize inducing nickel subsulfide and hexavalent chromium salt transformed 10T1/2 cell lines and characterizing them for anchorage independence and tumorigenicity. Then, we will study the molecular biology of transformation in arsenic, nickel, and chromium salt transformed 10T1/2 cell lines. Poly A+ mRNA extracted from arsenic, nickel, and chromium transformed 10T1/2 cell lines will be probed with cloned murine and avian RNA tumor virus oncogene probes in RNA dot and Northern blotting analyses to determine whether proto-oncogenes are expressed at higher steady-state levels in metal-transformed 10T1/2 cell lines. DNA from metal salt transformed 10T1/2 cell lines will be probed with cloned RNA tumor virus oncogenes in restriction enzyme-Southern blotting analyses to determine whether proto-oncogenes are amplified, rearranged, or under-methylated in metal-transformed 10T1/2 cells. DNA from metal-transformed 10T1/2 cell lines will be transfected into NIH3T3 cells to determine whether metal-induced morphological transformation is encoded on DNA. Patterns of restriction endonuclease sensitivity of ability of DNA's from metal-transformed cell lines to transfect transformed phenotypes will be studied. Cotransfection of DNA from metal transformed cell lines and PBr322 and construction of total genomic libraries from DNA of secondary transfectants will be used to clone metal-induced transforming genes. These genes will be probed with RNA tumor virus oncogenes in restriction endonuclease-Southern blotting analyses to identify them. These genes will be used to isolate their normal homologs from a total genomic mouse DNA library. We will also determine whether there are perturbations in oncogene expression in arsenic, nickel, and chromium induced anchorage-independent human cell strains we previously derived and whether DNA from these cell strains can transfect anchorage-independence to NIH3T3 cells. We will also attempt to provoke full transformation of these anchorage-independent human cell strains to focus formation, immortality, and tumorigenicity by a) transfecting cloned, mutated, transforming myc genes into these cell strains or by b) treating these cell strains with tumor promoters or with colcemid to induce chromosomal aneuploidy.

该提案的目标是继续我们对机制的研究 C3H/10T1/2 细胞金属盐转化和分子生物学 二倍体人包皮成纤维细胞。 我们将最终确定诱导低硫化镍和六价铬盐 转化 10T1/2 细胞系并表征它们的贴壁 独立性和致瘤性。 然后，我们将学习分子生物学 砷、镍、铬盐转化10T1/2 细胞系。 从砷、镍和铬中提取的 Poly A+ mRNA 转化的 10T1/2 细胞系将​​用克隆的鼠类和禽类进行探测 RNA 点和 Northern 印迹分析中的 RNA 肿瘤病毒癌基因探针 确定原癌基因是否在较高稳态下表达 金属转化的 10T1/2 细胞系中的水平。 金属盐 DNA 转化的 10T1/2 细胞系将​​用克隆的 RNA 肿瘤病毒进行探测 限制性内切酶-Southern印迹分析中的癌基因，以确定 原癌基因是否被扩增、重排或甲基化不足 金属转化的 10T1/2 细胞。 来自金属转化 10T1/2 细胞的 DNA 将细胞系转染至 NIH3T3 细胞以确定是否 金属诱导的形态转变是在 DNA 上编码的。 的图案 DNA 的限制性内切酶敏感性 转染转化表型的金属转化细胞系将 研究过。 来自金属转化细胞系的 DNA 的共转染和 PBr322 和从二级 DNA 构建总基因组文库 转染子将用于克隆金属诱导的转化基因。 这些基因将在限制性条件下用RNA肿瘤病毒癌基因进行探测 核酸内切酶-Southern印迹分析来识别它们。 这些基因将 用于从小鼠总基因组 DNA 中分离其正常同源物 图书馆。 我们还将确定癌基因是否存在扰动 砷、镍和铬诱导的锚定独立表达 我们之前衍生的人类细胞株以及这些细胞的 DNA 是否来自 菌株可以不依赖贴壁地转染 NIH3T3 细胞。 我们还将 试图激发这些不依赖锚定的人类的全面转变 细胞株通过 a) 集中形成、永生和致瘤性 将克隆、突变、转化的 myc 基因转染到这些细胞中 细胞株或通过b)用肿瘤促进剂或用 秋水酰胺诱导染色体非整倍体。