GENETIC ANALYSIS OF TRANSFORMATION OF HUMAN FIBROBLASTS

我们最近开发了一种独特的、无限寿命的人成纤维细胞 MSU-1.0，为二倍体，表型正常， 不会致癌的。从这个细胞株中，我们自发地分离出一种 变异株，命名为(MSU-1.1)，具有稳定的核型(45 染色体)，它包括两条独特的染色体。从MSU-1.1 我们分离出一种自发变异的细胞株，它具有相同的稳定性 核型，但在没有外源生长的培养基中快速增殖 各种因素。该菌株被命名为MSU-1.2。这些菌株都不是 致癌的。将fes癌基因导入MSU-1.2细胞，并 分离得到了几株克隆的转基因细胞株。所有这些都是 菌株恶性程度高，潜伏期短。转染法的研究 FeS癌基因导入MSSU-1.1细胞不产生肿瘤细胞 直接去吧。然而，表达H-ras癌基因的MSU-1.1细胞 或通过转染法将v-sis癌基因转化为恶性细胞。 FES基因的基因。MSU-1.1细胞也转化为恶性细胞 高表达H-ra或N-ras癌基因。MSU-1.1和 MSU-1.2细胞也可以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 通过任何手术而肿瘤性转化的。这些结果清楚地表明 表明癌症发生的一些关键变化发生在 MSU-1.1细胞由MSU-1.0细胞分化而来。此外，他们还 表明致癌物处理可使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 一项提案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 将进行的是确定是否存在临界差异 细胞株是指抑制基因活性的丧失或一种 致癌基因。在有证据表明基因缺失的情况下 信息为因果事件，我们将找回丢失的染色体 碎片传给细胞。我们将检查抑制基因的状态 当证据表明这些基因已经失去表达时。在……里面 在显性癌基因被激活的情况下，我们将 检查最明显的候选基因(例如，ras和sis)。如果有 或者这些努力中的另一个不成功，我们将克隆基因(S) 通过在EBV载体中制作适当的cDNA文库和 将这些cDNA转移到谱系中合适的受体细胞，以 识别表型修饰的细胞。我们将从 这些细胞，经过浓缩，重新测试，看看它们是否可重复地导致 正在测试表型变化。如果是这样的话，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.