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

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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.