High-level expression of c-H-ras1 fails to fully transform rat-1 cells
High-level expression of c-H-ras1 fails to fully transform rat-1 cells
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c-H-ras1的高水平表达未能完全转化rat-1细胞
DOI：
10.1128/mcb.8.4.1460-1468.1988
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发表时间：
1988
影响因子：
5.3
通讯作者：
A. Levinson
中科院分区：
文献类型：
--
作者：
M. Ricketts;A. Levinson
将编码正常(Gly-12)、非激活(Pro-12)和激活(Val-12和Ile-12)p21H-ras的p21H-ras的质粒在可扩增的二氢叶酸还原酶标记物存在的情况下导入RAT-1细胞。用甲氨蝶呤筛选法扩增p21H-ras基因，以确定p21H-ras的表达与细胞转化的各种标志之间的关系。与细胞活力一致的p21H-ras(Gly-12)的最大水平约为细胞总蛋白的0.13%(每个细胞约60,000个分子)；这是内源蛋白水平的44倍。第二种非转化形式的p21H-ras(Pro-12)的最大耐受水平约为该水平的一半。在大鼠1细胞中，编码高致瘤性的Val-12和Ile-12形式的p21H-ras的H-ras基因的扩增不能通过甲氨蝶呤的选择而实现，这提供了强有力的证据，表明在大鼠1细胞中合成的活化的p21H-ras超过一定的阈值(约占总蛋白的0.02%)与细胞活力不相容。分离单个细胞系，研究它们的形态、非贴壁生长、致瘤性以及对生长因子的反应和产生。我们报告说，表达接近最大耐受水平的正常或亲12形式的p21H-ras的细胞系没有表达更适度水平的高度致癌(Val-12)形式的细胞那么转化，这表明ras对转化表型的完整阐述至少部分取决于区分细胞蛋白和病毒蛋白的突变。我们发现，表达正常p21(H-ras)水平升高的细胞可以被激活的(val-12)形式完全转化，并且这些细胞继续过表达p21(H-ras)(Gly-12)，这与正常ras基因在抑制突变激活的对应物的致癌潜力中的作用相反。
Rat-1 cells were transfected with plasmids encoding normal (Gly-12), nonactivated (Pro-12), and activated (Val-12 and Ile-12) p21H-ras in the presence of an amplifiable dihydrofolate reductase marker. The introduced DNA was amplified by selection in methotrexate to establish the relationship between p21H-ras expression and various hallmarks of cellular transformation. The maximum level of p21H-ras (Gly-12) consistent with cell viability was approximately 0.13% of total cell protein (approximately 60,000 molecules per cell); this is 44-fold greater than the level of the endogenous protein. The maximum tolerated level of a second nontransforming form of p21H-ras (pro-12) was about half of this. Amplification in Rat-1 cells of H-ras genes encoding the highly oncogenic Val-12 and Ile-12 forms of p21H-ras could not be achieved by methotrexate selection, providing strong evidence that synthesis of activated p21H-ras above a certain threshold (about 0.02% of total protein) in Rat-1 cells is incompatible with cell viability. Individual cell lines were isolated and their morphology, anchorage-independent growth, tumorigenicity, and response to and production of growth factors were studied. We report that cell lines expressing near-maximum tolerated levels of either the normal or pro-12 form of p21H-ras were not as transformed as cells expressing much more modest levels of the highly oncogenic (Val-12) form, suggesting that the complete elaboration of the transformed phenotype by ras depends, at least in part, on mutations that distinguish the cellular and viral proteins. We found that cells expressing elevated levels of the normal p21(H-ras) could be fully transformed by the activated (Val-12) form and that such cells continued to overexpress p21(H-ras) (Gly-12), arguing against a role for normal ras genes in suppression of the oncogenic potential of their mutationally activated counterparts.