GENETIC ANALYSIS OF TRANSFORMATION OF HUMAN FIBROBLASTS

我们最近开发了一种独特的无限寿命的人类成纤维细胞 菌株MSU-1.0，其为二倍体，表型正常，和 非致瘤性的 从这个细胞株中，我们分离出一种自发的 变异株，命名为（MSU-1.1），具有稳定的核型（45 染色体），其包括两个独特的染色体。 来自MSU-1.1 细胞株，我们分离出一个自发的变异，具有相同的稳定 核型，但在无外源生长的培养基中增殖迅速 因素 该菌株被命名为MSU-1.2。 这些菌株都不是 致瘤的将fes癌基因转染MSU-1.2细胞， 分离出几种克隆转染细胞株。 所有这些 菌株高度恶性，潜伏期短。 转染 fes癌基因导入MSSU-1.1细胞后，未产生肿瘤细胞 直接. 然而，表达转染的H-ras癌基因的MSU-1.1细胞， 或v-sis癌基因通过转染转化为恶性细胞 fes基因。 MSU-1.1细胞也转化为恶性细胞 通过转染的H-ra或N-ras癌基因的高表达。 MSU-1.1和 MSU-1.2细胞也可被致癌物转化为肿瘤细胞 治疗和适当的选择。 MSU-1.0细胞从未被 通过任何程序肿瘤性转化。 这些结果清楚地 这表明，当肿瘤细胞生长到一定程度时， 从MSU-1.0细胞产生MSU-1.1细胞。 而且他们 表明致癌物处理导致MSU-1.1和MSU-1.2细胞 经历一些关键的变化将它们转化为肿瘤细胞。 这 一项新的研究试图确定这两个基因发生了什么变化。 转化这些人类成纤维细胞的步骤。 细胞融合物 将进行，以确定是否之间的关键区别 细胞株是抑制基因活性的丧失或一种 癌基因 如果有证据表明 信息作为因果事件，我们将返回丢失的染色体 碎片到细胞。 我们将研究抑制基因的状态 当证据表明这些基因已经失去表达时。 在 如果出现显性癌基因被激活的情况，我们将 检查最明显的候选基因（例如，ras和sis）。 如果一个 或者另一种努力没有成功，我们将克隆基因， 通过在EBV载体中制备合适的cDNA文库来参与， 将这些cDNA转移到谱系中合适的受体细胞， 鉴定表型修饰的细胞。 将从以下物质中分离质粒： 这样的细胞，富集，重新测试，看看他们是否可重复地导致 正在测试的表型变化。 如果是这样，那么所涉及的cDNA将是 测序并与已知序列进行比较。

We recently developed a unique, infinite life span human fibroblast cell strain, MSU-1.0, that is diploid, phenotypically normal, and nontumorigenic. From this cell strain, we isolated a spontaneously variant strain, designated (MSU-1.1), that has a stable karyotype (45 chromosomes), which includes two unique chromosomes. From the MSU-1.1 cell strain we isolated a spontaneous variant that has the same stable karyotype, but proliferates rapidly in medium without exogenous growth factors. This strain is designated MSU-1.2. None of these strains are tumorigenic. The fes oncogene was transfected into MSU-1.2 cells, and several clonal transfectant cell strains were isolated. All of these strains were highly malignant with a short latency. Transfection of the fes oncogene into MSSU-1.1 cells did not give rise to tumor cells directly. However, MSU-1.1 cells expressing a transfected H-ras oncogene or the v-sis oncogene were converted to malignant cells by transfection of the fes gene. MSU-1.1 cells were also converted to malignant cells by high expression of transfected H-ra or N-ras oncogenes. MSU-1.1 and MSU-1.2 cells can also be converted to tumor cells by carcinogen treatment and appropriate selection. MSU-1.0 cells have never been neoplastically transformed by any procedure. These results clearly indicate that some critical change for carcinogenesis occurred when the MSU-1.1 cells were generated from the MSU-1.0 cells. Furthermore, they indicate that carcinogen treatment causes MSU-1.1 and MSU-1.2 cells to undergo some change critical to convert them to tumor cells. This proposal seeks to determine what genetic change occurred at these two steps in transforming these human fibroblast-derived cells. Cell fusions will be carried out to determine whether the critical difference between cell strains is the loss of suppressor gene activity or activation of an oncogene. In the case where evidence points to the loss of genetic information as the causal event, we will return the lost chromosome fragment to the cells. We will examine the status of suppressor genes when the evidence suggests that such genes have lost expression. In cases where it appears a dominant oncogene has been activated, we will examine the most obvious candidate genes (e.g., ras, and sis). If one or the other of these efforts does not succeed, we will clone the gene(s) involved by making an appropriate cDNA library in an EBV vector and transfer these cDNA's to suitable recipient cells in the lineage to identify phenotypically modified cells. Plasmids will be isolated from such cells, enriched, retested to see if they reproducibly cause the phenotypic change being tested. If so, the cDNA involved will be sequenced and compared with known sequences.