HORMONAL CONTROL OF PROLIFERATION

糖皮质激素可杀死正常和恶性淋巴细胞。因此，这类类固醇仍然是恶性和非恶性淋巴增生性疾病化疗的主要工具。糖皮质激素还调节正常的免疫发育和分化，是免疫反应的重要药理和生理调节因子。在美国，几乎所有人在一生中都会出于某种目的而使用糖皮质激素。然而，我们还不清楚这类类固醇影响正常或转化的淋巴样细胞增殖和死亡的机制。这项建议提出了一个令人信服的假说来解释糖皮质激素如何杀死恶性T淋巴样细胞，这是该领域20多年工作的继续。我们已经确定糖皮质激素抑制细胞周期蛋白D3的表达。这是一个转录后过程，加入地塞米松后，细胞周期蛋白D3mRNA的降解迅速增加。失稳涉及至少两种蛋白质，它们与细胞周期蛋白D3mRNA的3‘非翻译区内的一个确定的反应元件结合。大约一半的项目(特定目标1)涉及这两种蛋白质的分离和鉴定，分析它们的作用机制，以及鉴定可能参与糖皮质激素介导的细胞周期蛋白D3mRNA失稳的其他蛋白质。我们已经确定，抑制细胞周期蛋白D3的表达导致细胞周期蛋白依赖的激酶抑制剂p27Kip1的重新分布，当细胞周期蛋白D3的丰度下降时，p27Kip1从含有CDK4的复合体中解离出来，并与细胞周期蛋白E/CDK2复合体结合并抑制。我们最近发现抗凋亡蛋白BC12是CDK2的底物。糖皮质激素和其他药物对CDK2的抑制会导致Bcl2的净去磷酸化。我们认为，BC12的去磷酸化导致细胞死亡，可能是通过降低BC12与Bax的亲和力，有利于Bax同源二聚体的形成和细胞死亡。这项提议的剩余一半(具体目标2)涉及检验这一假设。我们将分析表达显性阴性CDKs和p27的细胞的表型，特别关注BC12的磷酸化和细胞死亡。我们还将分析表达不能磷酸化的BC12突变体以及模拟结构性磷酸化的突变体的细胞的表型。在完成这些具体目标后。我们希望第一次有一个机制，可以解释糖皮质激素抑制CDK2活性、细胞增殖和细胞死亡之间的关系。

Glucocorticoids kill both normal and malignant lymphoid cells. Consequently, steroids of this class remain a principle tool in chemotherapy of both malignant and non-malignant lymphoproliferative diseases. Glucocorticoids also mediate normal immune development and differentiation, and are very important pharmacological and physiological modulators of the immune response. Virtually all individuals in the United States will use glucocorticoids for some purpose during their life. Nevertheless, we do not understand the mechanisms whereby such steroids affect proliferation and death of either normal or transformed lymphoid cells. This proposal presents a cogent hypothesis to explain how glucocorticoids kill malignant T lymphoid cells, and is a continuation of more than 20 years of work in this area. We have determined that glucocorticoids inhibit the expression of cyclin D3. This is a post- transcriptional process in which the degradation of cyclin D3 mRNA increases rapidly upon addition of dexamethasone. Destabilization involves at least two proteins that bind to a defined response element within the 3' untranslated region of cyclin D3 mRNA. Approximately half of the project (Specific Aim 1) deals with isolation and characterization of these two proteins, analysis of their mechanism of action, and identification of other proteins that may participate in glucocorticoid-mediated destabilization of cyclin D3 mRNA. We have determined that inhibition of cyclin D3 expression causes a redistribution of the cyclin-dependent kinase inhibitor, p27Kip1, which dissociates from Cdk4-containing complexes as the abundance of cyclin D3 falls and binds to and inhibits cyclin E/Cdk2 complexes. We have recently discovered that the antiapoptotic protein Bc12 is a substrate for Cdk2. Inhibition of Cdk2 by glucocorticoids and other agents causes net dephosphorylation of Bcl2. We propose that dephosphorylation of Bc12 leads to cell death, perhaps by decreasing the affinity of Bc12 for Bax, favoring the formation of Bax homodimers and cell death. The remaining half of this proposal (Specific Aim 2) deals with testing this hypothesis. We will analyze the phenotype of cells that express dominant negative Cdks and p27, with particular attention to Bc12 phosphorylation and cell death. We will also analyze the phenotype of cells that express Bc12 mutants that cannot be phosphorylated as well as mutants that mimic constitutive phosphorylation. Upon completion of these specific aims. we hope to have, for the first time, a mechanism that accounts for the relationship between glucocorticoid inhibition of Cdk2 activity, cell proliferation, and cell death.