METAL INDUCED TRANSFORMATION IN C3H10T1/2 CELLS

C3H10T1/2 细胞中金属诱导的转化

• 批准号: 3250547
• 负责人: JOSEPH R LANDOLPH
• 金额: $ 13.09万
• 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 1986-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3250547
• 关键词: Retroviridae; arsenic; cellular oncology; chemical carcinogenesis; chromium; environment related neoplasm /cancer; metal poisoning; molecular cloning; molecular oncology; neoplasm /cancer genetics; nickel; oncogenes

The goal of this proposed research is to use carcinogenic metal salts to induce neoplastic transformation in C3H/10T1/2 CI 8 mouse fibroblasts and to study the molecular biology of neoplastic transformation induced by carcinogenic metal salts. Arsenic, chromium, and nickel salts, metal compounds that are strongly suspected to be human carcinogens, will be used to induce transformation in C3H/10T1/2 cells in this proposal. I will isolate a number of transformed C3H/10T1/2 cell lines derived from induction of Type II and Type III foci by carcinogenic metal salts. These cell lines will be seeded into soft agorose to determine whether they are anchorage independent and injected into nude mice to determine whether they are tumorigenic. Poly A containing mRNA will be extracted from metal-transformed cell lines and a battery of cloned murine and avian RNA tumor virus oncogene probes will be used in dot blotting and Northern blotting analyses to determine whether any particular proto-oncogenes are expressed at higher steady-state levels in metal-transformed C3H/10T1/2 cell lines. Where this is the case, I will use known cloned RNA tumor virus oncogene probes in restriction enzyme-Southern blotting analyses to determine whether any of the analogous proto-oncogenes are transposed in metal-transformed C3H/10T1/2 cells of whether they are differentially under-methylated in metal-transformed compared to in nontransformed C3H/10T1/2 cells. To study genes involved in transformation by carcinogenic metal salts, I will extract DNA from metal-transformed C3H/10T1/2 cell lines and determine whether the metal-transformed phenotypes of morphological transformation and anchorage independence can be transfected on DNA to nontransformed NIH3T3 and C3H/10T1/2 cells. The pattern of restriction endonuclease sensitivity of the ability of DNA's extracted from various metal-transformed cell lines to transfect transformed phenotypes will be studied. Contransfection of DNA from metal transformed cell lines and PBr322 and the construction of total genomic libraries from the DNA of secondary transfectants will be used to clone metal-transformed genes, and these gene probes will be used to isolate their normal homologs from a library of total genomic mouse DNA. Further restriction and Southern blotting analyses utilizing these genes and RNA tumor virus oncogenes will be done to provide information on the identify of these genes.

这项拟议研究的目标是使用致癌金属盐， 在C3 H/10 T1/2 C18小鼠成纤维细胞中诱导肿瘤转化， 目的：研究肿瘤转化的分子生物学机制， 致癌金属盐。 金属砷、铬和镍盐 强烈怀疑是人类致癌物的化合物，将被使用 诱导C3 H/10 T1/2细胞转化。 我将分离一些转化的C3 H/10 T1/2细胞系，这些细胞系来源于 致癌金属盐诱发II型和III型病灶。 这些 将细胞系接种到软琼脂中，以确定它们是否 锚定独立的，并注射到裸鼠，以确定它们是否 是致癌的 将从金属转化细胞系中提取含Poly A的mRNA 以及一组克隆的鼠和禽RNA肿瘤病毒癌基因探针 将用于斑点印迹和北方印迹分析以确定 是否有任何特定的原癌基因在较高的稳态下表达 在金属转化的C3 H/10 T1/2细胞系中的水平。 在这种情况下， 我将使用已知的克隆RNA肿瘤病毒癌基因探针在限制性内切酶 酶-Southern印迹分析，以确定是否有任何类似物 原癌基因在金属转化的C3 H/10 T1/2细胞中转座， 无论它们在金属转化的 与未转化的C3 H/10 T1/2细胞相比。 为了研究致癌金属盐转化相关基因，我 将从金属转化的C3 H/10 T1/2细胞系中提取DNA，并确定 形态转化的金属转化表型 并且锚定独立性可以在DNA上转染到非转化的 NIH 3 T3和C3 H/10 T1/2细胞。 限制性内切酶谱 从不同的组织中提取DNA的能力的灵敏度 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DNA构建总基因组文库 二次转染子将用于克隆金属转化的基因， 这些基因探针将被用于从一种 小鼠总基因组DNA文库。 进一步限制和南方 利用这些基因和RNA肿瘤病毒癌基因的印迹分析将 为这些基因的鉴定提供信息。