Investigation of transforming activity of cyctatin which has a similarity to Ras

与Ras相似的cyctatin转化活性的研究

• 批准号: 07808084
• 负责人: HIWASA Takaki
• 金额: $ 1.34万
• 依托单位: Chiba Cancer Center Research Institute
• 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-07808084/
• 关键词: ras; cysteine proteinase; cystatin; transformation; プロテアーゼ; 形質転換

We have found a potent cysteine proteinase inhibitory activity of ras gene product (Ras). To elucidate whether the protease-inhibitory activity of Ras is involved in carcinogenesis, the present study was carried out using the following probes ; (gene 1) cyctatin which hasa similar cysteine proteinase-inhibitory activity but no GTP-binding activity, (gene 2) cystatin which has a CAAX motif of Ras at the carboxy-terminus, (gene 3) RasDELTA42-49 which has a deletion between amino acide 42 and 49 and retains the cysteine proteinase-inhibitory activity but has completely lost GTP-binding activity. These genes were transfected into NIH3T3 cells and the transfected cells were isolated. The expression of the introduced genes were effectively induced by treatment with dexamethasone. Then, the colony-forming ability in soft agar medium was investigated in the presence of dexamethasone. The results showed that cells were not transformed by expression of these genes. However, high colony-forming efficiencies were observed after induction of genes 2 or 3 in the presence of a tumor promoter, TPA,suggesting that the cells were anchorage-independently transformed.Induction of gene 1 resulted in a moderate increase in the colony-forming abilty. These results suggest that suppression of cycteine proteinases induces an initial chage in carcinogenesis.

我们发现ras基因产物（Ras）具有很强的半胱氨酸蛋白酶抑制活性。为了阐明Ras的蛋白酶抑制活性是否参与致癌作用，本研究使用以下探针进行：（基因1）具有类似半胱氨酸蛋白酶抑制活性但无GTP结合活性的半胱氨酸蛋白酶抑制剂，（基因2）在羧基末端具有Ras的CAAX基序的半胱氨酸蛋白酶抑制剂，（基因3）RasDELTA 42 -49，其在氨基酸42和49之间具有缺失，并保留半胱氨酸蛋白酶抑制活性，但完全丧失了GTP结合活性。将这些基因转染到NIH 3 T3细胞中，并分离转染的细胞。地塞米松处理可有效诱导导入基因的表达。然后，在地塞米松的存在下，在软琼脂培养基中的集落形成能力进行了研究。结果表明，这些基因的表达没有转化细胞。然而，在存在肿瘤启动子TPA的情况下，在基因2或3的诱导下观察到高的集落形成效率，这表明细胞是锚定非依赖性转化的。这些结果表明，抑制半胱氨酸蛋白酶诱导癌发生的初始变化。

项目成果

Hiwasa,T.et al.: "Enhancement of catalytic activities of serine proteases by tripeptide compounds." FEBS Lett.,. 386. 47-50 (1996)

Hiwasa,T.et al.：“三肽化合物增强丝氨酸蛋白酶的催化活性。”

Hiwasa, T.: Biological effects of Ras and cystatin alpha in mouse fibroblasts.Proteolysis and Protein Turnover. V.K.Hopsu-Havu, (ed.), IOS Press, Amsterdam,

Hiwasa, T.：Ras 和胱抑素 α 在小鼠成纤维细胞中的生物效应。蛋白水解和蛋白质周转。

Hiwasa,T.and Kominami,E.: "Physical association of Ras and cathepsins B and L in the conditioned medium of v-Ha-ras-transformed NIH3T3 cells." Biochem.Biophys.Res.Commun.216. 828-834 (1995)

Hiwasa,T. 和 Kominami,E.：“v-Ha-ras 转化 NIH3T3 细胞的条件培养基中 Ras 和组织蛋白酶 B 和 L 的物理关联。”

Takaki Hiwasa et al.: "Increase of cyclin B by cverexpression fo cystatin α." Cell Biochem. Func.13. 293-296 (1995)

Takaki Hiwasa 等人：“通过表达胱抑素 α 来增加细胞周期蛋白 B”。Cell Biochem.13（1995）。

Takaki Hiwasa et al.: "Loss of Raf-1binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151." Signal trunsduction. (in press). (1996)

Takaki Hiwasa 等人：“通过删除氨基酸残基 64-72 和 143-151，导致 v-Ha-Ras 的 Raf-1 结合活性丧失。”