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

为了检测显性阴性H-ras突变体N116Y的高表达对肿瘤细胞增殖的影响，构建了高效的N116Y表达载体pZIP-N116Y，并通过脂质体转染法将N116Y导入肾癌细胞株ACHN、NT-2、SMKT-R3。_pZIP-N116Y可完全抑制ACHN、NT-2、SMKT-R3的集落形成，G418筛选后无细胞存活。虽然_pZIP-N116Y可能是肾癌细胞的有效抑制因子，但它的抑制活性可能依赖于neo基因的失活。为了排除这种可能性，我们检测了6株同时含有_pZIP-N116Y和_pSV2_&lt；neo&gt；的肾癌细胞系(ACHN、NT-2、OSRC2、SMKTR-2、SMKTR-3、SMKTR-4)的集落形成能力。G418抗性克隆数分别为4%(ACHN)、6%(NT-2)、10%(OSRC2)、13%(SMKTR-2)、20%(SMKTR3)和9%(SMKTR4)。为了阐明N116Y抑制肾癌细胞生长的机制，我们分析了介导RAS蛋白活化的SOS蛋白的表达。AII5肾癌细胞株(ACHN、NT-2、SMKT-R-3、SMKT-R-4、OS-RC-2)分别高表达SOS蛋白，而正常肾组织中SOS蛋白含量很低，提示显性阴性H-ras突变体N116Y可抑制肾癌细胞的增殖。

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.