Melanovytic tumor development in transgenic mice carrying the ret oncogene

我们通过引入融合到小鼠金属硫蛋白- 1启动子增强子（MT/ret）上的ret癌基因，获得了4个表现出全身严重黑素病的转基因小鼠系。黑素细胞瘤经常发生在四条细胞系中的三条。一个系的小鼠在面部和颈部的真皮、腿部肌肉、纵隔和腹膜后腔发生肿瘤，而另外两个系的小鼠主要在面部的真皮发生肿瘤。Northern杂交和原位杂交分析表明，肿瘤细胞和非肿瘤黑色素产生细胞高水平表达该基因。为了分析ret致癌基因产物的信号转导途径，我们从MT/ret转基因小鼠的黑色素细胞肿瘤中建立了一个细胞系（Mel-ret）。与原发黑色素细胞肿瘤不表现出恶性特征不同，Mel-ret细胞移植到裸鼠体内后具有转移能力。我们在黑色素细胞肿瘤和Mel-ret细胞的细胞裂解物中检测到100kd、125kd和135kd酪氨酸磷酸化蛋白。此外，在Mel-ret细胞中特异性存在一条85kd酪氨酸磷酸化带。Mel-ret细胞中酪氨酸磷酸化的总体水平远高于原发肿瘤，提示酪氨酸磷酸化的增加可能与恶性转化有关。免疫荧光和细胞分离研究表明，Mel-ret细胞中的ret蛋白和大部分酪氨酸磷酸化蛋白定位于膜组分。

We generated four transgenic mouse lines which showed severe melanosis of the whole body by introducing the ret oncogene fused to the mouse metallothionein-I promoter-enhancer (MT/ret). Melanocytic tumors frequently developed in three of the four lines. Mice of one line developed tumors in the dermis of the face and neck, the leg muscle, the mediastinum and the retroperitoneal cavity while mice of the other two lines developed them predominantly in the dermis of the face. Northern hybridization and in situ hybridization analyses showed that tumors cells and nontumorous melanin-producing cells expressed the transgene at high levels. To analyze the signal transduction pathway of the ret oncogene product, we established a cell line (Mel-ret) from a melanocytic tumor developed in a MT/ret transgenic mouse. Unlike primary melanocytic tumors which did not showed malignant features, the Mel-ret cells had the metastatic ability when they were transplanted into nude mice. We detected 100kd, 125kd and 135kd tyrosine phosphorylated proteins in both cell lysates of melanocytic tumors and Mel-ret cells. In addition, an 85kd tyrosine phosphorylated band was present specifically in the Mel-ret cells. The overall level of tyrosine phosphorylation in the Mel-ret cells was much higher than that in the primary tumors, suggesting that the increase of tyrosine phosphorylation may be responsible for malignant transformation. Immunofluorescence and cell fractionation studies showed that the ret protein and most of tyrosine phosphorylated proteins in the Mel-ret cells localized in the membrane fraction.