MECHANISMS OF AUTONOMOUS PROLIFERATION OF HUMAN RENAL CANCER CELLS

人肾癌细胞自主增殖机制

基本信息

批准号：
07671700
负责人：
HONKE Koichi
金额：
$ 1.47万
依托单位：
RESEARCH INSTITUTE,OSAKA MEDICAL CENTER FOR MATERNAL AND CHILD HEALTH
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671700/
关键词：
RENAL CELL CARCINOMA TYROSINE KINASE EGF TYROSINE KINASE INHIBITOR RAS

项目摘要

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

由于人类肾脏癌细胞系SMKT-R3能够在无血清培养基中生长，因此预计它将自动增殖。另一方面，在人肾脏癌细胞中高度表达糖脂磺胺硫代磺胺硫代磺胺硫代化酶（GST）。为了阐明自主增殖和GST表达的机制，我们进行了以下实验。 Genastein是一种酪氨酸激酶抑制剂，不仅取消了EGF的细胞生长增强，而且完全抑制了未处理细胞的增殖及其GST活性。这些观察结果表明，内源性酪氨酸激酶（S）参与了人类肾脏癌细胞中GST的自主扩散和表达。未经EGF处理的未检测到120 kDa酪氨酸磷酸化蛋白的事实表明，除EGF受体以外的其他酪氨酸激酶（S）在SMKT-R3细胞中被激活。尽管在蛋白质印迹上观察到了120 kDa酪氨酸磷酸化的蛋白质，但它……更多无法用抗磷酸酪氨酸抗体沉淀。因此，我们无法鉴定磷酸化120 kDa蛋白的酪氨酸激酶。然后我们检查了RAS是否参与其中。我们将V-H-RAS基因引入SMKT-R3细胞中，并获得了稳定表达活化Ras的细胞系。 V-RAS表达细胞的GST活性高于对照细胞，但其生长速率并未显着提高。表达V-RAS的细胞对EGF没有响应，因为该信号已饱和，EGF受体的下游饱和。另一方面，当将染料木黄酮添加到表达V-RAS的细胞中时，未观察到GST活性的降低，表明包括RAS上游的酪氨酸激酶在RAS上游，以增加GST活性。然而，染料木黄酮抑制了V-RAS表达细胞的生长。这表明酪氨酸激酶涉及其他要点。为了抑制内源性RAS活性，我们将显性阴性的RAS基因引入了SMKT-R3细胞。表达的主要阴性RAS不可能生长，这表明内源性RAS对于肾脏癌细胞的自主扩散至关重要。较少的