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

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。

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.