Expression of activated N-ras in a primary melanoma cell line counteracts growth inhibition by transforming growth factor-beta

One critical factor in melanoma progression is the change from radial growt h phase to vertical growth phase. We previously showed a high incidence of ras mutations in progressing but not early human melanomas. We also found t hat stable expression of activated Ras in a primary human melanoma cell lin e (WM35) led to enhanced proliferation, anchorage-independent survival, mig ration and invasion in vitro and enhanced subcutaneous tumor formation in v ivo, transforming the melanoma phenotype from the radial growth phase to th e vertical growth phase. Inhibitory cytokines, especially transforming grow th factor-beta, are important in homeostasis of normal human melanocytes. P roliferation of early melanoma cells can be inhibited by transforming growt h factor-beta, whereas more aggressive stages lose this response. Using a t ransforming growth factor-beta activated luciferase reporter transiently tr ansfected into WM35, WM35N-ras, and WM35H-ras (WM35 transfected with mutant N-ras or H-ras genes), we demonstrated significant decreases (p < 0.04) in transforming growth factor-beta induced reporter expression in both ras tr ansfected cell lines. Transforming growth factor-beta also induced signific ant decreases (p < 0.002) in the proportion of WM35 cells in S-phase of the cell cycle; this effect was not observed in WM35N-ras cells. Furthermore, we demonstrated that an important controlling factor in transforming growth factor-beta inhibition of cell cycle progression, the phosphorylation of t he Rb protein, was altered in WM35N-ras; transforming growth factor-beta ca used a marked relative increase in hypophosphorylated pRb in WM35 cells, bu t not in WM35N-ras. These data suggest that activated Ras plays an importan t part in melanoma progression from the radial growth phase to the vertical growth phase by counteracting inhibition by cytokines such as transforming growth factor-beta, thus providing a growth advantage.