METAL TRANSFORMATION IN 10TL/2 AND HUMAN FIBROBLASTS

本提案的目的是继续研究 和金属盐转化C3 H/10 T1/2细胞的分子生物学， 二倍体人包皮成纤维细胞 我们将最终诱导低硫化镍和六价铬盐 转化的10 T1/2细胞系，并表征它们的锚定 独立性和致瘤性。 然后，我们将研究分子生物学 在砷、镍和铬盐中转化10 T1/2 细胞系 从砷、镍和铬中提取的Poly A+ mRNA 转化的10 T1/2细胞系将用克隆的鼠和禽进行探测 RNA肿瘤病毒癌基因探针的RNA斑点和北方印迹分析 以确定原癌基因是否在较高的稳态下表达 在金属转化的10 T1/2细胞系中的水平。 金属盐DNA 将用克隆的RNA肿瘤病毒探测转化的10 T1/2细胞系 限制性内切酶-Southern印迹法分析癌基因，以确定 原癌基因是否扩增、重排或甲基化不足， 金属转化的10 T1/2细胞。 金属转化10 T1/2细胞DNA 将细胞系转染入NIH 3 T3细胞，以确定是否 金属诱导的形态学转化在DNA上编码。 模式 限制性内切酶敏感性 金属转化的细胞系，以抑制转化的表型， 研究了 来自金属转化细胞系的DNA的共转染和 PBr 322和从次级植物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。 这些基因将与限制性RNA肿瘤病毒癌基因进行探测 核酸内切酶-Southern印迹分析以鉴定它们。 这些基因将 用于从总基因组小鼠DNA中分离其正常同源物 图书馆 我们还将确定是否有癌基因的扰动， 砷、镍和铬诱导的锚定非依赖性表达 我们以前获得的人类细胞株以及这些细胞的DNA是否 菌株对NIH 3 T3细胞的贴壁不依赖性。 我们还将 试图激发这些锚定独立的人类的全面转变， 通过a）将细胞株聚焦于形成、永生和致瘤性 将克隆的、突变的、转化的myc基因导入这些细胞 或通过B）用肿瘤促进剂或用 秋水仙胺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.