CARCINOGEN-TRANSFORMED HUMAN CELLS: GENETIC TRAITS

目的是确定使二倍体人类能够 成纤维细胞获得肿瘤相关表型，主要是 非锚定生长 我们建议做一些实验， 将活化的癌基因转移到人成纤维细胞中，以及 实验涉及BP抗 二醇-环氧化物诱导的锚定非依赖性人成纤维细胞。 这些 实验将进行，以确定相关的人类转化 基因. 这些离散的遗传事件的互补将在 试图了解肿瘤发生的遗传基础， 人成纤维细胞的转化。 将采取若干技术措施， 在这些实验中使用的方法包括：转染或电穿孔 将克隆癌基因导入人成纤维细胞;致突变剂诱导 人成纤维细胞和基因特异性DNA的锚定非依赖性生长， RNA杂交以鉴定基因扩增/过表达事件; 从锚定非依赖性人成纤维细胞中筛选DNA， 以致瘤性转化NIH/3 T3细胞，并随后克隆 通过粘粒拯救技术转化基因;从 锚定非依赖性人成纤维细胞的诱导能力 受体人成纤维细胞的锚定非依赖性生长和克隆 该基因位于可选择的粘粒内， 人成纤维细胞。 克隆的NIH/3 T3和人转化基因，如 以及它们来自人类DNA文库的野生型同源物， 进行DNA序列分析。 我们的目标是将诱变剂诱导的癌症相关基因的获得 表型，如人类细胞中的锚定独立性，以分子 在特定基因位点的变化，以了解转化 人成纤维细胞。

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.