RAS and Thyroid Cell Survival

描述(申请人提供)：RAS突变是人类肿瘤的标志，包括甲状腺肿瘤。RAS基因突变在滤泡性腺瘤和癌中出现频率较高。虽然Ras突变在乳头状癌中很少见，但Ret突变在这些肿瘤中发生的频率很高，并且Ret已被证明通过RAS发出信号。尽管多年来的积极研究，RAS在肿瘤转化中的作用还不是很清楚。RAS信号通过复杂的信令网络，这些信令网络以依赖于细胞类型的方式使用。RAS激活后的表型结果取决于RAS活性的水平和持续时间、RAS激活的效应器以及重要的细胞环境。激活的RAS对原代甲状腺细胞的影响是不寻常的。与原代成纤维细胞激活RAS诱导生长停滞不同，RAS刺激原代人甲状腺细胞持续增殖。我们已经证明，激活的RAS的急性表达刺激了大鼠甲状腺细胞的凋亡。在这些细胞中，就像在人类甲状腺细胞中一样，细胞增殖在细胞凋亡之前。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的细胞通过G1期进入G1期，延迟于S期，并因细胞凋亡而死亡。此外，RAS对细胞周期机制的影响与甲状腺细胞有丝分裂原显著不同。在其急性表达后，RAS降低了细胞周期蛋白D1和p27的蛋白水平，增加了p21的表达。耐人寻味的是，RAS诱导细胞周期蛋白依赖性激酶-2(CDK-2)活性显著增加，主要是在凋亡细胞中。伴随而来的是细胞周期蛋白A和p27在凋亡细胞中的选择性切割。正如在甲状腺肿瘤中观察到的那样，细胞周期蛋白D1在甲状腺细胞中上调，以存活激活的RAS的组成性表达。我们的假设是，RAS诱导的细胞凋亡是由于RAS对细胞周期蛋白依赖性激酶抑制剂p27和p21的作用而引起的cdk-2活性不受抑制的直接结果。该应用的具体目标是确定CDK-2活性对于RAS刺激的细胞凋亡是否既是必要的也是充分的，阐明导致CDK-2活性不受抑制和RAS急性表达后细胞凋亡的分子机制，以及识别允许携带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.