Melanovytic tumor development in transgenic mice carrying the ret oncogene

我们产生了四个转基因小鼠品系，表现出严重的全身黑变病，通过引入ret癌基因融合到小鼠金属硫蛋白-I启动子-增强子（MT/ret）。黑素细胞肿瘤经常在三个四行。一个系的小鼠在面部和颈部、腿部肌肉、纵隔和腹膜后腔的真皮中形成肿瘤，而另外两个系的小鼠主要在面部的真皮中形成肿瘤。北方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表达高水平的转基因。为了分析ret癌基因产物的信号转导途径，我们从MT/ret转基因小鼠中产生的黑素细胞肿瘤建立了细胞系（Mel-ret）。与原发性黑素细胞瘤不具有恶性特征不同，Mel-ret细胞移植到裸鼠体内后具有转移能力。我们在黑素细胞瘤和Mel-ret细胞的细胞裂解物中检测到100 kd、125 kd和135 kd酪氨酸磷酸化蛋白。此外，在Mel-ret细胞中特异性地存在一条85 kd的酪氨酸磷酸化条带。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是恶性转化的原因。免疫荧光和细胞分级分离研究表明，ret蛋白和大多数酪氨酸磷酸化蛋白在Mel-ret细胞定位于膜组分。

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.