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

显性失活 H-ras 突变体对肾细胞癌细胞系生长的抑制作用

• 批准号: 08671786
• 负责人: SHINOHARA Nobuo
• 金额: $ 1.34万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08671786/
• 关键词: renal cell carcinoma cell line; Activation of Ras protein; Gene Transfer; Growth suppression; Son of Sevenless (Sos) protein; 腎癌; ras癌遺伝子; 遺伝子治療; GDP; GTP交換因子

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.

本研究通过将v-H-ras癌基因的天冬酰胺-116替换为酪氨酸而获得的N116 Y基因，构建了N116 Y基因的高效表达载体pZIP-N116 Y，用脂质体法转染ACHN、NT-2、SMKT-R3三种肾癌细胞株。转染pZIP-N116 Y后，ACHN、NT-2、SMKT-R3的殖民地形成完全被抑制，经G418筛选无细胞存活。pZIP-N116 Y可能是肾细胞癌的有效抑制基因，但其抑制活性可能依赖于neo基因的失活。为了排除这种可能性，我们通过以10：1和0：1的比率共转染这些质粒，检测了含有_pZIP-N116 Y和_pSV 2_的6种RCC细胞系（ACHN、NT-2、OSRC 2、SMKTR-2、SMKTR-3、SMKTR-4）的殖民地形成能力<neo>。与对照转染（0：1）.探讨N116 Y对肾癌细胞生长抑制的机制。我们分析了介导Ras蛋白激活的Sos蛋白的表达。结果显示，肾癌细胞株ACHN、NT-2、SMKT-R-3、SMKT-R-4、OS-RC-2均表达较高水平的Sos蛋白，而正常肾组织中Sos蛋白的表达量很低，提示H-ras基因显性失活突变体N116 Y可抑制肾癌细胞株的增殖。

项目成果

Shinohara, N.et al.: "Growth suppression of renal cell carcinoma cell lines by a dominant negative H-ras mutant." Journal of Urology. 155(5). 407A (1996)

Shinohara, N.等人：“显性失活 H-ras 突变体对肾细胞癌细胞系的生长抑制。”

Shinohara,N.et.al.: "Growth suppression of renal cell carcinoma cell lines by a dominant negative H-ras mutant." Journal of Urology. 155(5). 407A- (1996)

Shinohara,N.et.al.：“显性失活 H-ras 突变体对肾细胞癌细胞系的生长抑制。”

Shinohara, N.et al: "The significance of ras guanine nucleotide exchange factor, son of sevenless protein, in renal cell carcinoma cell lines." Journal of Urology. 158(3). 908-911 (1997)

Shinohara, N. 等人：“七少蛋白之子 ras 鸟嘌呤核苷酸交换因子在肾细胞癌细胞系中的意义。”

Shinohara,N.et.al.: "The significance of ras guanine nucleotide exchange factor,son of sevenless protein,in renal cell carcinoma cell lines." Journal of Urology. 158(3). 908-911 (1997)

Shinohara, N.et.al.：“七少蛋白之子 ras 鸟嘌呤核苷酸交换因子在肾细胞癌细胞系中的意义。”

Shinohara, N.et al.: "The significance of ras guanine nucleotide exchange factor,son of sevenless protein,in renal cell carcinoma cell lines." Journal of Urology. 158(3). 908-911 (1997)

Shinohara, N. 等人：“七少蛋白之子 ras 鸟嘌呤核苷酸交换因子在肾细胞癌细胞系中的意义。”