MECHANISMS OF AUTONOMOUS PROLIFERATION OF HUMAN RENAL CANCER CELLS

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Since a human renal cancer cell line, SMKT-R3, is able to grow in serum-free media, it is supposed to proliferate autonomously. On the other hand, glycolipid sulfotransferase (GST) is highly expressed in human renal cancer cells. In order to elucidate the mechanisms of autonomous proliferation and GST expression, we performed the following experiments. Genistein, a tyrosine kinase inhibitor, not only cancelled the enhancement of the cell growth by EGF but also completely inhibited the proliferation of non-treated cells and their GST activity. These observations suggested that endogenous tyrosine kinase (s) are involved in the autonomous proliferation and the expression of GST in human renal cancer cells. The fact that 120 kDa tyrosine-phosphorylated protein was detected without EGF treatment indicates that endogenous tyrosine kinase (s) other than the EGF receptor are activated in SMKT-R3 cells. Although the 120 kDa tyrosine-phosphorylated protein was observed on a western blotting, it … More was not able to be precipitated with anti-phosphotyrosine antibody. Therefore, we could not identify the tyrosine kinase that phosphorylates the 120 kDa protein. Then we examined whether Ras is involved. We introduced v-H-ras gene into SMKT-R3 cells and obtained cell lines that stably express activated Ras. The v-Ras expressing cells showed higher GST activity than control cells but their growth rate was not enhanced significantly. The v-Ras expressing cells did not respond to additional EGF because the signal was saturated downstream of the EGF receptor. On the other hand, When genistein was added to the v-Ras expressing cells, the reduction of GST activity was not observed, indicating that the tyrosine kinases including the EGF receptor act upstream of Ras to increase GST activity. However, the growth of v-Ras expressing cells was suppressed by genistein. This suggests that the tyrosine kinases involved in the proliferation act on other points. To inhibit endogenous Ras activity, we introduced a dominant-negative ras gene into SMKT-R3 cells. The dominant-negative Ras expressing cells came to be impossible to grow, suggesting that the endogenous Ras is essential for the autonomous proliferation of renal cancer cells. Less