RAS and Thyroid Cell Survival

描述（由申请人提供）：Ras突变是人类肿瘤的标志，包括甲状腺肿瘤。Ras突变在滤泡性腺瘤和癌中发现频率很高。虽然Ras突变在乳头状癌中不常见，但ret突变在这些肿瘤中发生的频率很高，ret已被证明通过Ras发出信号。尽管多年的积极研究，Ras对肿瘤转化的贡献尚未得到很好的理解。Ras信号通过复杂的信号网络以细胞类型依赖的方式被利用。Ras激活后的表型结果取决于Ras活性的水平和持续时间、被Ras激活的效应物以及重要的细胞环境。活化的Ras在原代甲状腺细胞中的作用是不寻常的。与原代成纤维细胞不同，在原代成纤维细胞中，激活的Ras诱导生长停滞，而Ras刺激人甲状腺细胞的持续增殖。我们已经证明，激活Ras的急性表达刺激大鼠甲状腺细胞凋亡。在这些细胞中，如人甲状腺细胞，细胞凋亡发生在细胞增殖之前。然而，细胞周期进程对急性Ras表达的反应是高度异常的。表达ras的细胞进入G1期，在S期被延迟，并因凋亡而死亡。此外，Ras对细胞周期机制的影响与甲状腺细胞有丝分裂原明显不同。急性表达后，Ras降低cyclin D1和p27蛋白水平，升高p21蛋白表达。有趣的是，Ras诱导细胞周期蛋白依赖性激酶-2 （cdk-2）活性显著增加，主要发生在凋亡细胞中。这伴随着细胞周期蛋白A和p27在凋亡细胞中的选择性切割。在甲状腺肿瘤中观察到，cyclin D1在被激活的Ras组成表达存活的甲状腺细胞中上调。我们的假设是，Ras诱导的细胞凋亡是由Ras对周期蛋白依赖性激酶抑制剂p27和p21的作用引发的cdk-2活性不受限制的直接结果。本应用程序的具体目标是确定cdk-2活性对于Ras刺激的细胞凋亡是否是必要的和充分的，阐明激活Ras急性表达后cdk-2活性无限制和细胞凋亡的分子机制，并确定导致含有Ras的甲状腺细胞存活的继发性变化。

DESCRIPTION (provided by applicant): Ras mutations are a hallmark of human tumors, including those of the thyroid gland. Ras mutations are found at high frequency in follicular adenomas and carcinomas. Although Ras mutations are infrequent in papillary carcinomas, ret mutations occur at high frequency in these tumors and RET has been shown to signal through Ras. Despite years of active investigation, the contribution of Ras to neoplastic transformation is not well understood. Ras signals through complex signaling networks that are utilized in a cell type-dependent manner. The phenotypic consequences that follow Ras activation depend upon the level and duration of Ras activity, the effectors activated by Ras and importantly, cell context. The effects of activated Ras in primary thyroid cells are unusual. Unlike primary fibroblasts where activated Ras induces growth arrest, Ras stimulates sustained proliferation in primary human thyroid cells. We have shown that acute expression of activated Ras stimulates apoptosis in rat thyroid cells. In these cells as in human thyrocytes, apoptosis is preceded by cell proliferation. However, cell cycle progression in response to acute Ras expression is highly aberrant. Ras-expressing cells progress through G1, are delayed in S phase and perish by apoptosis. Moreover, the effects of Ras on the cell cycle machinery are strikingly different from those of thyroid cell mitogens. Following its acute expression, Ras decreased cyclin D1 and p27 protein levels, and increased p21 expression. Intriguingly, Ras elicited a marked increase in cyclin-dependent kinase-2 (cdk-2) activity predominantly in apoptotic cells. This was accompanied by the cleavage of cyclin A and p27 selectively in apoptotic cells. As observed in thyroid tumors, cyclin D1 was upregulated in thyroid cells selected to survive constitutive expression of activated Ras. It is our hypothesis that apoptosis induced by Ras is a direct consequence of unrestrained cdk-2 activity initiated by Ras effects on the cyclin-dependent kinase inhibitors, p27 and p21. The specific goals of this application are to determine whether cdk-2 activity is both necessary and sufficient for apoptosis stimulated by Ras, to elucidate the molecular mechanisms that contribute to unrestrained cdk-2 activity and apoptosis following acute expression of activated Ras, and to identify the secondary changes that transpire to allow for the survival of thyroid cells harboring Ras.