HORMONAL CONTROL OF PROLIFERATION

糖皮质激素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组织增生性疾病化疗的主要工具。 糖皮质激素还介导正常的免疫发育和分化，并且是免疫应答的非常重要的药理学和生理学调节剂。 在美国，几乎所有的人都会在他们的生活中使用糖皮质激素。然而，我们不了解这些类固醇影响正常或转化淋巴细胞增殖和死亡的机制。 该提案提出了一个令人信服的假设来解释糖皮质激素如何杀死恶性T淋巴细胞，并且是该领域20多年工作的延续。 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，其中加入地塞米松后细胞周期蛋白D3 mRNA的降解迅速增加。去稳定化涉及至少两种蛋白质，其与细胞周期蛋白D3 mRNA的3'非翻译区内的确定的反应元件结合。 大约一半的项目（具体目标1）涉及这两种蛋白质的分离和表征，分析其作用机制，并鉴定可能参与糖皮质激素介导的细胞周期蛋白D3 mRNA不稳定的其他蛋白质。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性激酶抑制剂p27 Kip 1的重新分配，p27 Kip 1从含Cdk 4的复合物中解离，因为细胞周期蛋白D3的丰度福尔斯，并结合和抑制细胞周期蛋白E/Cdk 2复合物。 我们最近发现，抗凋亡蛋白Bc 12是Cdk 2的底物。 糖皮质激素和其他药物对Cdk 2的抑制导致Bcl 2的净去磷酸化。 我们认为Bc 12的去磷酸化可能通过降低Bc 12对Bax的亲和力，促进Bax同源二聚体的形成和细胞死亡而导致细胞死亡。 本提案的其余部分（具体目标2）涉及检验这一假设。 我们将分析表达显性负性Cdks和p27的细胞的表型，特别关注Bc 12磷酸化和细胞死亡。我们还将分析表达不能磷酸化的Bc 12突变体以及模拟组成性磷酸化的突变体的细胞的表型。 完成这些具体目标后。我们希望第一次有一种机制来解释糖皮质激素对Cdk 2活性的抑制、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.