Avvedimento V E, Musti A M, Ueffing M, Obici S, Gallo A, Sanchez M, DeBrasi D, Gottesman M E
Institute of Cancer Research, Columbia University, New York, New York 10032.
Genes Dev. 1991 Jan;5(1):22-8. doi: 10.1101/gad.5.1.22.
Exposure of rat thyroid cells for 1 week to a temperature-sensitive variant of Kirsten murine sarcoma virus (KiMSV) Ras inactivated the thyroglobulin promoter (pTg). Cellular dedifferentiation was paralleled by the loss of the thyroid-specific trans-acting factor, TgTF1, which binds to pTg. When Ras was denatured by shifting cells to 39 degrees C, TgTF1 binding and pTg function recovered rapidly without the synthesis of new protein. TgTF1 could be reactivated in vitro by treating nuclear extracts with protein kinase A. After 4 weeks of exposure to the oncogene, denaturation of Ras no longer restored TgTF1 binding or reactivated pTg. Incubation of nuclear extracts with protein kinase A likewise did not reactivate TgTF1. Cells chronically exposed to Ras did, however, yield differentiated clones after treatment with 5-azacytidine. We suggest that Ras induces dedifferentiation in two sequential steps: (1) Ras reduces PKA activity; TgTF1 (or an auxiliary protein) becomes dephosphorylated, and binding to pTg is abolished. (2) The effects of Ras become imprinted by methylation, possibly of the TgTF1 gene.
将大鼠甲状腺细胞暴露于柯斯顿鼠肉瘤病毒（KiMSV）Ras的温度敏感变体1周，可使甲状腺球蛋白启动子（pTg）失活。细胞去分化与甲状腺特异性反式作用因子TgTF1的丧失同时发生，TgTF1可与pTg结合。当通过将细胞转移至39℃使Ras变性时，TgTF1结合和pTg功能迅速恢复，且无需合成新蛋白。通过用蛋白激酶A处理核提取物，可在体外重新激活TgTF1。在暴露于癌基因4周后，Ras的变性不再恢复TgTF1结合或重新激活pTg。用蛋白激酶A孵育核提取物同样不会重新激活TgTF1。然而，长期暴露于Ras的细胞在用5-氮杂胞苷处理后确实产生了分化克隆。我们认为Ras通过两个连续步骤诱导去分化：（1）Ras降低PKA活性；TgTF1（或一种辅助蛋白）去磷酸化，与pTg的结合被消除。（2）Ras的作用可能通过甲基化（可能是TgTF1基因的甲基化）被印记下来。
