MECHANISMS OF AUTONOMOUS PROLIFERATION OF HUMAN RENAL CANCER CELLS

由于人肾癌细胞系SMKT-R3能够在无血清培养基中生长，因此它应该自主增殖。另一方面，糖脂磺基转移酶（GST）在人肾癌细胞中高度表达。为了阐明自主增殖和GST表达的机制，我们进行了以下实验。酪氨酸激酶抑制剂Genistein不仅能消除EGF对细胞生长的促进作用，而且能完全抑制未处理细胞的增殖和GST活性。这些结果表明，内源性酪氨酸激酶参与了人肾癌细胞的自主增殖和GST表达。在没有EGF处理的情况下检测到120 kDa酪氨酸磷酸化蛋白质的事实表明，在SMKT-R3细胞中除了EGF受体之外的内源性酪氨酸激酶被激活。尽管在蛋白质印迹上观察到120 kDa酪氨酸磷酸化蛋白， ...更多信息 不能用抗磷酸酪氨酸抗体沉淀。因此，我们无法鉴定使120 kDa蛋白磷酸化的酪氨酸激酶。然后我们检查了Ras是否参与其中。我们将v-H-ras基因导入SMKT-R3细胞，获得了稳定表达活化Ras的细胞株。表达v-Ras的细胞表现出比对照细胞更高的GST活性，但它们的生长速率没有显著提高。表达v-Ras的细胞对额外的EGF没有反应，因为EGF受体下游的信号饱和。另一方面，当向表达v-Ras的细胞中加入染料木黄酮时，没有观察到GST活性的降低，表明包括EGF受体在内的酪氨酸激酶在Ras上游起作用以增加GST活性。然而，v-Ras表达细胞的生长被染料木黄酮抑制。这表明参与增殖的酪氨酸激酶作用于其他点。为了抑制内源性Ras活性，我们将显性负性ras基因导入SMKT-R3细胞。显性阴性表达Ras的细胞不能生长，表明内源性Ras对肾癌细胞的自主增殖是必需的。少

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