Growth suppression of renal cell carcinoma cell lines by a dominant negative H-ras mutant

我们研究了N116Y是否可以抑制肾细胞癌（RCC）细胞系的生长，N116Y是通过酪氨酸取代天冬酰胺-116而从v-H-ras癌基因中衍生出来的。为了研究显性H-ras阴性突变体N116Y的高表达是否会影响肿瘤细胞的增殖，我们构建了一个高效的N116Y表达载体_pZIP-N116Y，并通过脂质转染程序转染了3个RCC细胞系（ACHN,NT-2, SMKT-R3）。转染_pZIP-N116Y完全抑制ACHN、NT-2、SMKT-R3的集落形成，G418选择后没有细胞存活。尽管_pZIP-N116Y可能是一种有效的RCC细胞抑制因子，但_pZIP-N116Y的抑制活性可能取决于neo基因的失活。为了排除这种可能性，我们以10:1和0:1的比例共转染含有_pZIP-N116Y和_pSV2_<neo>的质粒，检测了6株RCC细胞系（ACHN、NT-2、OSRC2、SMKTR-2、SMKTR-3、SMKTR-4）的集落形成能力。与对照转染（0∶1）相比，g418耐药菌落数分别为4% （ACHN）、6% （NT-2）、10% （OSRC2）、13%（SMKTR-2）、20% （SMKTR-3）和9% （SMKTR-4）。为了阐明N116Y对RCC细胞株生长抑制的作用机制，我们分析了介导Ras蛋白激活的so蛋白的表达。AII5 RCC细胞株（ACHN、NT-2、SMKT-R-3、SMKT-R-4、OS-RC-2）中Sos蛋白表达水平较高，而正常肾组织中Sos蛋白表达量极少。这些结果表明，显性负H-ras突变体N116Y可以抑制RCC细胞系的增殖。

We examined whether N116Y,which derived from the v-H-ras oncogene by substituting the asparagine-116 with tyrosine, can inhibit the growth of renal cell carcinoma (RCC) cell lines.In order to examine whether high expression of a dominant negative H-ras mutant, N116Y,affects tumor cell proliferation, we constructed an efficient N116Y expression vector, _pZIP-N116Y,and transfected three RCC cell lines (ACHN,NT-2, SMKT-R3) by the lipofection procedure. Transfection of _pZIP-N116Y completely inhibited the colony formation of ACHN,NT-2, SMKT-R3 and no cell survived after G418 selection. Although _pZIP-N116Y may be a potent suppressor of RCC cells, it is possible that the suppressor activity of _pZIP-N116Y depends on neo gene inactivation. To exclude this possibility, we examined the colony forming ability of 6 RCC cell lines (ACHN,NT-2, OSRC2, SMKTR-2, SMKTR-3, SMKTR-4) containing both _pZIP-N116Y and _pSV2_<neo> by cotransfection of these plasmids at the rates of 10 : 1 and 0 : 1. The numbers of G418-resistant colonies were 4% (ACHN), 6% (NT-2) , 10% (OSRC2) , 13%(SMKTR-2) , 20% (SMKTR-3) , and 9% (SMKTR-4) in comparison with the control transfection (0 : 1).To clarify the mechanism of growth suppression in RCC cell lines by N116Y,we analyzed the expression of son of sevenless (Sos) protein which mediates the the activation of Ras protein . AII5 RCC cell lines (ACHN,NT-2, SMKT-R-3, SMKT-R-4, OS-RC-2) expressed compartively high levels of Sos protein, while the amount of Sos protein was very small in normal kidney tissues.These results suggest that a dominant negative H-ras mutant, N116Y,can suppress the proliferation of RCC cell lines.