Notario V, Castro R, Flessate D M, Doniger J, DiPaolo J A
Department of Radiation Medicine, Vincent T. Lombardi Cancer Research Center, Georgetown University School of Medicine, Washington, DC 20007.
Oncogene. 1990 Sep;5(9):1425-30.
Carcinogen-caused transformation of Syrian hamster embryo cells has been widely used as a model for experimental carcinogenesis. However, analysis of the molecular mechanisms of hamster cell transformation has been limited. To expand the understanding of the molecular basis of this system, 22 independently derived Syrian hamster neoplastic cell lines initiated with chemical carcinogens were screened for the presence of dominant transforming sequences by DNA transfection into mouse NIH3T3 cells. High molecular weight DNAs from 12 (55%) of these cell lines transformed NIH3T3 cells through serial transfection cycles. NIH3T3 transformants contained hamster-specific repetitive sequences, which co-segregated with the transformed phenotype in successive transfection rounds. Results from Southern hybridization analyses and p21ras mobility assays indicated the presence of N-ras oncogenes, presumably activated by point mutations at codon 61, in 3 of the 12 (25%) transfection positive lines, all initiated with sodium bisulfite; non-ras transforming sequences were apparently activated in the remaining 9 (75%) lines. DNA prepared from NIH3T3 transformants derived from cell line 81C39 was analysed by Southern hybridization with a battery of 38 probes including non-ras oncogenes known to score as positive in the NIH3T3 assay as well as other retroviral and mammalian oncogenes. Each probe hybridized to DNA fragments showing the mobility characteristic of NIH3T3 protooncogenes, but failed to detect homolog sequences of hamster origin, even under hybridization conditions which allowed their detection in hamster DNA. Results show that ras activation occurs at a low frequency in hamster neoplastic transformation and strongly suggest that novel transforming sequences are activated, thus validating the use of this system for investigating the role of non-ras transforming sequences in neoplasia.
致癌物诱导的叙利亚仓鼠胚胎细胞转化已被广泛用作实验性致癌作用的模型。然而，对仓鼠细胞转化分子机制的分析一直有限。为了扩大对该系统分子基础的理解，通过将22个独立衍生的、由化学致癌物引发的叙利亚仓鼠肿瘤细胞系的DNA转染到小鼠NIH3T3细胞中，筛选显性转化序列的存在。这些细胞系中有12个（55%）的高分子量DNA通过连续转染周期转化了NIH3T3细胞。NIH3T3转化体含有仓鼠特异性重复序列，其在连续转染轮次中与转化表型共分离。Southern杂交分析和p21ras迁移率测定结果表明，在12个（25%）转染阳性细胞系中的3个中存在N-ras癌基因，推测是由第61位密码子的点突变激活的，所有这些细胞系均由亚硫酸氢钠引发；其余9个（75%）细胞系中显然激活了非ras转化序列。用包括在NIH3T3检测中已知为阳性的非ras癌基因以及其他逆转录病毒和哺乳动物癌基因在内的38种探针，通过Southern杂交分析从细胞系81C39衍生的NIH3T3转化体中制备的DNA。每个探针都与显示NIH3T3原癌基因迁移特征的DNA片段杂交，但即使在允许在仓鼠DNA中检测到它们的杂交条件下，也未能检测到仓鼠来源的同源序列。结果表明，ras激活在仓鼠肿瘤转化中发生频率较低，并强烈表明新的转化序列被激活，从而验证了该系统用于研究非ras转化序列在肿瘤形成中作用的用途。
