Analysis of the RAS oncogene function by regulating the cellular RAS activity

我们已经发现，H-ras突变体N116Y对正常ras功能表现出明显的负活性，并且能够抑制ras介导的信号通路。在这项研究中，我们证明了N116Y抑制了ras p21s活性gtp结合形式的产生。提示N116Y可能消耗鸟嘌呤核苷酸交换因子，使细胞ras功能失活。接下来，我们检测了N116Y突变体是否可以抑制人类肿瘤细胞的生长。N116Y极显著抑制A431（外阴）、PC3（前列腺）、T24（膀胱）、MCF7（乳腺）、NKPS和TMK1（胃）癌细胞的增殖。A431和PC3细胞对N116Y易感。为了测试N116Y对肿瘤表型的影响，我们将效率较低的N116Y表达载体转染到A431细胞中。经过G418的选择，几乎所有克隆都存活了下来。然而，他们没有保留N116Y基因，只有一个克隆微弱表达N116Y。该表达N116Y的克隆在体内无致瘤性，并表现出形态变形和DNA断裂，提示N116Y可能诱导了凋亡细胞死亡。因此，N116Y可能适用于广泛的人类肿瘤的基因治疗。

We have already found that an H-ras mutant, N116Y,exhibited a dominant negative activity toward normal ras function and was capable of inhibiting the ras-mediated signaling pathways. In this study, we demonstrated that N116Y inhibited the production of the active GTP-bound form of ras p21s. This suggests that N116Y may consume the guanine nucleotide exchange factors and inactivate the cellular ras function.Next, we examined whether the N116Y mutant could suppress the growth of human tumor cells. N116Y extremely inhibited the proliferation of A431 (vulva), PC3 (prostate), T24 (bladder), MCF7 (breast), NKPS and TMK1 (stomach) cancer cell lines. A431 and PC3 cells were particularly susceptible to N116Y.In order to test the effects of N116Y on the neoplastic phenotypes, we transfected a less efficient N116Y expression vector into A431 cells. Almost all clo nes survived after G418 selection. However, they did not retain the N116Y gene and only one clone faintly expressed N116Y.This N116Y expressing clone had no tumorigenecity in vivo, and revealed deformed morphology and DNA fragmentation, suggesting that N116Y might have induced apoptotic cell death. Thus, N116Y may be applicable for gene therapy of a wide spectrum of human tumors.