Cell cycle regulation in acinar cell death

腺泡细胞死亡中的细胞周期调控

• 批准号: 6298776
• 负责人: MARY VERA RAYNOLDS
• 金额: $ 19万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-15 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6298776
• 关键词: DNA damage; acinar cell; apoptosis; cell cycle; cell line; cyclin dependent kinase; cysteine endopeptidases; cytoprotection; enzyme activity; laboratory mouse; laboratory rat; oncoprotein p21; p53 gene /protein; salivary glands; tissue /cell culture

Salivary gland hypofunction following chemotherapeutic regimens or head/neck irradiation presents a major oral health problem. Loss of salivary gland acinar cells under these conditions appears to be through apoptosis, a genetically programmed, energy requiring multi-step process. One of the mechanisms through which apoptotic stimuli might mediate cell death is through altered cell cycle regulation. Cell death in response to diverse stimuli including growth factor withdrawal, activation of 'death' receptors or DNA damage may be accompanied by ectopic activation of cyclin dependent kinases (cdks) which may lead to inappropriate cell cycle re-entry or progression in diverse types of cells, including secretory epithelial cells. Over-expression of cyclin dependent kinase inhibitors (CKIs), proteins that inhibit cdks and therefore cell cycleprogression, can protect cells from apoptosis associated with different stresses. It is our hypothesis that inappropriate activation of cyclin dependent kinases is an integral part of the apoptotic pathway in acinar cells, and that cyclin dependent kinase inhibitors can attenuate apoptosis through modulating cdk activity. Our preliminary data suggest that suggest that DNA increases the kinase activity of specific cyclin/cdk complexes in immortalized and in primary acinar cells. 'Ectopic' activation of cdks is an early event in acinar cell death, occurring at the same time as caspase-3 activation. Increased cyclin/cdk kinase activity also correlates with loss of expression of the CKI p27/kip1. In this proposal, we will examine the role of DNA damage-induced cdk activation in animal cell apoptosis. We will determine whether 'ectopic' cdk activation precedes or requires caspase--3 activation in primary cultures of acinar cells. We will resolve whether cdk activation is necessary for acinar cell death, and we will ascertain whether cdks phosphorylate normal cell cycle substrates or perhaps act on novel substrates during apoptosis. We will explore the ability of the CKIs/p27/kip1 and p21/cip1 to modulate DNA-damage induced apoptosis through cdk activity. In addition, we will verify the protective functions of either p21/cip1 or p27/kip1 in CKI null cells. Activation of the tumor suppressor 53 mediates DNA damage induced apoptosis through induction of p21/cip1 gene transcription in many types of cell. Therefore, we will examine the role of the tumor suppressor in acinar cell death by testing the effects of DNA damage and CKI expression in p53 null cells.

化疗方案或头/颈部照射后唾液腺功能减退是一个主要的口腔健康问题。在这些条件下唾液腺腺泡细胞的损失似乎是通过细胞凋亡造成的，这是一种基因编程的、需要多步骤能量的过程。细胞凋亡刺激可能介导细胞死亡的机制之一是通过改变细胞周期调节。响应多种刺激（包括生长因子撤回、“死亡”受体激活或 DNA 损伤）的细胞死亡可能伴随着细胞周期蛋白依赖性激酶 (cdks) 的异位激活，这可能导致不同类型细胞（包括分泌性上皮细胞）不适当的细胞周期重新进入或进展。细胞周期蛋白依赖性激酶抑制剂 (CKI) 的过度表达（CKI 是抑制 cdks 从而抑制细胞周期进展的蛋白质）可以保护细胞免受与不同应激相关的细胞凋亡。我们的假设是，细胞周期蛋白依赖性激酶的不当激活是腺泡细胞凋亡途径的一个组成部分，并且细胞周期蛋白依赖性激酶抑制剂可以通过调节 cdk 活性来减弱细胞凋亡。我们的初步数据表明，DNA 增加了永生化细胞和原代腺泡细胞中特定细胞周期蛋白/cdk 复合物的激酶活性。 cdks 的“异位”激活是腺泡细胞死亡的早期事件，与 caspase-3 激活同时发生。细胞周期蛋白/cdk 激酶活性的增加也与 CKI p27/kip1 表达的丧失相关。在本提案中，我们将研究 DNA 损伤诱导的 cdk 激活在动物细胞凋亡中的作用。我们将确定在腺泡细胞原代培养物中“异位”cdk 激活是否先于或需要 caspase--3 激活。我们将解决 cdk 激活是否是腺泡细胞死亡所必需的问题，并且我们将确定 cdk 是否磷酸化正常细胞周期底物或可能在细胞凋亡过程中作用于新底物。我们将探索 CKIs/p27/kip1 和 p21/cip1 通过 cdk 活性调节 DNA 损伤诱导的细胞凋亡的能力。此外，我们将验证p21/cip1或p27/kip1在CKI无效细胞中的保护功能。在许多类型的细胞中，肿瘤抑制因子 53 的激活通过诱导 p21/cip1 基因转录来介导 DNA 损伤诱导的细胞凋亡。因此，我们将通过测试 p53 缺失细胞中 DNA 损伤和 CKI 表达的影响来研究肿瘤抑制因子在腺泡细胞死亡中的作用。