Chiodino C, Jones R F, Ethier S P
Department of Pathology, Wayne State University, Detroit, Michigan.
Mol Carcinog. 1991;4(4):286-96. doi: 10.1002/mc.2940040407.
To determine if activation of the c-Ha-ras-1 gene is involved in the acquisition of growth factor independence in 7,12-dimethylbenz[a]anthracene (DMBA)--and N-nitrosomethylurea (NMU)--induced rat mammary carcinomas, three strategies were used. First, Ha-ras DNA from growth factor-independent DMBA-induced rat mammary tumor cells was amplified using the polymerase chain reaction and examined for the presence of mutations in the first and second exons of Ha-ras-1 by restriction fragment length polymorphism analysis, allele-specific oligonucleotide hybridization, and direct sequencing. No mutations were found in the codon 12/13 or codon 61 regions of the Ha-ras-1 gene. Second, a similar analysis of an NMU-induced mammary carcinoma showed that it harbored an activating mutation in codon 12 of Ha-ras-1. When analyzed for growth factor requirements, these cells were found to express limited growth potential in all media tested, in contrast to growth factor-independent cells, which proliferated extensively in the presence or absence of exogenous growth factors. Third, growth factor-dependent rat mammary tumor cells and spontaneously immortalized rat normal mammary epithelial cells were transfected with an activated form of the Ha-ras-1 (T24) gene, and the growth factor requirements of the transfected cells were examined. The ras-transfected cells retained the growth factor requirements of the normal cells. In addition, ras-transfected cells were transplanted into syngeneic rats and nude mice, and no tumors developed after 6 mo in vivo. These results indicate that, in rat mammary tumor cells, neither growth factor independence in vitro nor transplantability are directly mediated by Ha-ras oncogenes. The results also suggest that ras activation and growth factor independence may be associated with independent pathways to malignancy in rat mammary tumorigenesis.
为了确定c-Ha-ras-1基因的激活是否参与7,12-二甲基苯并[a]蒽（DMBA）和N-亚硝基甲基脲（NMU）诱导的大鼠乳腺癌中生长因子非依赖性的获得，采用了三种策略。首先，使用聚合酶链反应扩增来自生长因子非依赖性DMBA诱导的大鼠乳腺肿瘤细胞的Ha-ras DNA，并通过限制性片段长度多态性分析、等位基因特异性寡核苷酸杂交和直接测序检查Ha-ras-1第一和第二外显子中是否存在突变。在Ha-ras-1基因的密码子12/13或密码子61区域未发现突变。其次，对NMU诱导的乳腺癌进行的类似分析表明，它在Ha-ras-1的密码子12处存在激活突变。当分析生长因子需求时，发现这些细胞在所有测试培养基中表达的生长潜力有限，这与生长因子非依赖性细胞形成对比，后者在有无外源性生长因子的情况下都能广泛增殖。第三，用激活形式的Ha-ras-1（T24）基因转染生长因子依赖性大鼠乳腺肿瘤细胞和自发永生化的大鼠正常乳腺上皮细胞，并检查转染细胞的生长因子需求。ras转染细胞保留了正常细胞的生长因子需求。此外，将ras转染细胞移植到同基因大鼠和裸鼠体内，6个月后体内未形成肿瘤。这些结果表明，在大鼠乳腺肿瘤细胞中，体外生长因子非依赖性和可移植性均不是由Ha-ras癌基因直接介导的。结果还表明，ras激活和生长因子非依赖性可能与大鼠乳腺肿瘤发生中恶性转化的独立途径相关。
