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的频率很高。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频率很高，并且RET已被证明通过Ras发出信号。尽管多年的积极调查，Ras的肿瘤转化的贡献是没有得到很好的理解。Ras通过复杂的信号网络发送信号，这些信号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激活的效应子以及重要的细胞环境。激活的Ras在原代甲状腺细胞中的作用是不寻常的。与激活的Ras诱导生长停滞的原代成纤维细胞不同，Ras刺激原代人甲状腺细胞的持续增殖。我们已经表明，急性表达激活Ras刺激大鼠甲状腺细胞凋亡。在这些细胞中，如在人甲状腺细胞中，细胞凋亡之前是细胞增殖。然而，响应于急性Ras表达的细胞周期进展是高度异常的。表达Ras的细胞进展通过G1期，在S期延迟并通过凋亡而死亡。此外，Ras对细胞周期机制的影响与甲状腺细胞有丝分裂原的影响截然不同。Ras急性表达后，细胞周期蛋白D1和p27蛋白水平降低，p21表达增加。有趣的是，Ras引起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。这是伴随着细胞周期蛋白A和p27选择性地在凋亡细胞中裂解。正如在甲状腺肿瘤中所观察到的那样，在选择的甲状腺细胞中，细胞周期蛋白D1在活化Ras的组成型表达中存活。我们假设Ras诱导的细胞凋亡是Ras对细胞周期蛋白依赖性激酶抑制剂p27和p21的作用引发的不受限制的cdk-2活性的直接结果。本申请的具体目标是确定cdk-2活性是否是Ras刺激的细胞凋亡所必需和充分的，以阐明导致活化Ras急性表达后不受限制的cdk-2活性和细胞凋亡的分子机制，并鉴定允许携带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.