METAL TRANSFORMATION IN 10TL/2 AND HUMAN FIBROBLASTS

这项建议的目标是继续我们对这些机制的研究 和C3H/10T1/2细胞金属盐转化的分子生物学 二倍体人包皮成纤维细胞。 我们将最终确定诱导亚硫化镍和六价铬盐 转化10T1/2细胞系及其贴壁特性的研究 独立性和致瘤性。然后，我们将学习分子生物学 转变成10T1/2的砷、镍和铬盐中的转变 细胞系。从砷、镍和铬中提取的Poly A+mRNA 转化的10T1/2细胞系将与克隆的小鼠和禽类进行探测 RNA斑点杂交和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的稳定状态下表达 金属转化的10T1/2细胞株中的表达水平。从金属盐中提取DNA 转化的10T1/2细胞将用克隆的RNA肿瘤病毒进行探测 用限制性内切酶-Southern印迹分析确定癌基因 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。金属转化10T1/2细胞的DNA 将这些细胞系导入NIH3T3细胞，以确定 金属诱导的形态转化编码在DNA上。模式： DNA来源能力的限制性内切酶敏感性 金属转化细胞系将转化表型 学习。金属转化细胞系DNA与DNA共转染的研究 PBr322及其DNA全基因组文库的构建 转染体将被用于克隆金属诱导的转化基因。 这些基因将与rna肿瘤病毒癌基因进行限制性检测。 核酸内切酶-Southern印迹分析以鉴定它们。这些基因将 用于从小鼠总基因组DNA中分离它们的正常同源物 图书馆。 我们还将确定癌基因是否存在扰动 砷、镍和铬诱导的锚定非依赖性表达 我们之前获得的人类细胞株以及这些细胞中的DNA 菌株可将不依赖于锚定的基因导入NIH3T3细胞。我们还将 试图激起这些独立于锚地的人类的完全转变 细胞株通过a)集中形成、不朽和致瘤性) 将克隆的、突变的、转化的myc基因导入这些细胞 菌株或通过b)用肿瘤促进剂或 诱导染色体非整倍体。

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.