CARCINOGEN-TRANSFORMED HUMAN CELLS: GENETIC TRAITS

目标是确定使二倍体人类 成纤维细胞获得肿瘤相关表型，主要是 锚地--独立增长。我们计划做一些实验，涉及到 将激活的癌基因转移到人成纤维细胞以及 BP反义寡核苷酸的分子表征实验 二醇-环氧化物诱导的不依赖锚定的人成纤维细胞。这些 将进行实验，以确定相关的人类转化 基因。这些离散遗传事件的互补性将在 了解肿瘤发生的遗传学基础的尝试 人成纤维细胞的转化。几种技术方法将是 用于这些实验，包括：转染法或电穿孔法 将克隆的癌基因导入人成纤维细胞；诱变剂诱导 人成纤维细胞的非锚定生长和基因特异性DNA及 RNA杂交以识别基因扩增/过度表达事件； 从锚定非依赖性人成纤维细胞中筛选DNA 将NIH/3T3细胞转化为肿瘤细胞并随后克隆 用粘粒抢救技术转化基因；从 锚定非依赖性人成纤维细胞诱导分化能力的研究 受体人成纤维细胞的贴壁非依赖性生长及其克隆 这个基因位于一个可选择的粘粒中，该粘粒在染色体外复制 人类成纤维细胞。克隆的NIH/3T3和人转化基因AS 以及它们来自人类DNA文库的野生型同源物，将是 进行DNA序列分析。 我们的目标是将诱变剂诱导的癌症相关的获得联系起来 表型，如人类细胞中的锚定独立性，到分子 在特定基因座上的变化以了解转化 人类成纤维细胞。

The goal is to identify genetic events which enable diploid human fibroblasts to acquire neoplasia-associated phenotypes, principally anchorage-independent growth. We propose to do experiments involving the transfer of activated oncogenes into human fibroblasts as well as experiments involving the molecular characterization of BP anti diol-epoxide-induced, anchorage-independent human fibroblasts. These experiments will be done in order to identify relevant human transforming genes. Complementation of these discrete genetic events will be studied in an attempt to understand the genetic basis of the tumorigenic transformation of human fibroblasts. Several technical approaches will be used in these experiments, these include: transfection or electroporation of cloned oncogenes into human fibroblasts; mutagen induction of anchorage-independent growth in human fibroblasts and gene-specific DNA and RNA hybridization to identify gene amplification/over-expression events; screening DNA from anchorage-independent human fibroblasts for its ability to tumorigenically transform NIH/3T3 cells and subsequent cloning of the transforming gene by a cosmid rescue technique; screening DNA from anchorage-independent human fibroblasts for its ability to induce anchorage-independent growth in recipient human fibroblasts and cloning of this gene within a selectable cosmid which replicates extrachromosomally in human fibroblasts. The cloned NIH/3T3 and human transforming genes, as well as their wild-type homologues from a human DNA library, will be subjected to DNA sequence analysis. Our goal is to relate mutagen-induced acquisition of cancer-related phenotypes, such as anchorage-independence in human cells, to molecular changes at specific gene loci in order to understand transformation of human fibroblasts.