ROLE OF DNA STRAND BREAKS AND PARP ON IONIZING RADIATION INDUCED APOPTOSIS

DNA 链断裂和 PARP 对电离辐射诱导细胞凋亡的作用

• 批准号: 6651742
• 负责人: MARK SMULSON
• 金额: $ 34.88万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6651742
• 关键词: DNA binding protein; DNA damage; apoptosis; cell line; cellular pathology; enzyme activity; ionizing radiation; laboratory mouse; molecular cloning; molecular pathology; pentosyltransferase; transfection; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): We propose to continue to molecularly clarify specific roles of PARP in radiation (IR)-induced apoptosis. It is based on the overall hypothesis that the covalent p(ADP-ribose)n of proteins by long, negatively charged chains of polymer renders target DNA-binding proteins "DNA phobic" and prevents their binding to DNA. Specific Aim 1 focuses on the regulation of human Ca2+/Mg2+ DNASIL3 during apoptosis, which causes late nucleosomal degradation of genomic DNA. DNASIL3 is inhibited and hence regulated in vitro when in the p(ADP-ribose)n state. Having successfully cloned the human DNASIL3, we wish to clarify its role in vivo via PARP regulation in IR-induced cell death. Cells transfected with a caspase-resistant PARP will be used to assess the consequences during IR-induced apoptosis of a now stable, uncleaved polymer, covalently bound to DNASIL3. Approaches in Specific Aim 2 will establish cell lines, stably transfected with inducible antisense and sense cDNA for PARP glycohydrolase (PARG). This enzyme specifically cleaves PAR from proteins such as DNAS1L3 and p53. Accordingly, PARG will be either eliminated or over expressed in cells. In the initial case we will test how the lack of ability to cleave PAR from DNAS1L3 and p53 will influence the regulation imposed by p(ADP-R)n on nuclear proteins. Complimentary PARG overexpression experiments will be performed to test the effects of a stable p(ADP-R)n state of the DNASIL3 and p53. Specific Aim 3 will focus on DFF45, an inhibitor of the nuclease, DFF40. This nuclease plays a key role in chromatin restructuring by 50 Kb DNA cleavage early in apoptosis. Cells derived from DFF45-/- and +/+ mice will be used to establish the mechanism underlying the protection of DFF45-/- cells against strand breaks-induced cell death. This involves key apoptotic events and a PARP-1-associated amplification loop. We will test whether IR causes a biphasic activation of PARP-1 during radiation-induced apoptosis immediately after direct DNA damage and secondly after DFF activation and the generation of 50 kb DNA breaks.

描述（由申请人提供）：我们建议继续从分子水平上阐明 PARP 在辐射 (IR) 诱导的细胞凋亡中的具体作用。它基于这样的总体假设：通过带负电荷的聚合物长链形成的蛋白质共价 p(ADP-核糖)n 使目标 DNA 结合蛋白具有“DNA 恐惧性”，并阻止其与 DNA 结合。具体目标 1 重点关注细胞凋亡过程中人 Ca2+/Mg2+ DNASIL3 的调节，这会导致基因组 DNA 的晚期核小体降解。当 DNASIL3 处于 p(ADP-核糖)n 状态时，DNASIL3 会受到抑制，因此在体外受到调节。 成功克隆人类 DNASIL3 后，我们希望通过 PARP 调节在 IR 诱导的细胞死亡中阐明其在体内的作用。用抗 caspase 的 PARP 转染的细胞将用于评估 IR 诱导的与 DNASIL3 共价结合的稳定的、未切割的聚合物凋亡过程中的后果。具体目标 2 中的方法将建立细胞系，并用 PARP 糖水解酶 (PARG) 的诱导型反义和有义 cDNA 稳定转染。该酶专门从 DNAS1L3 和 p53 等蛋白质中裂解 PAR。因此，PARG将在细胞中被消除或过度表达。在最初的情况下，我们将测试缺乏从 DNAS1L3 和 p53 切割 PAR 的能力将如何影响 p(ADP-R)n 对核蛋白的调节。将进行免费的 PARG 过表达实验，以测试稳定的 p(ADP-R)n 状态的效果 DNASIL3 和 p53。具体目标 3 将重点关注 DFF45，它是核酸酶 DFF40 的抑制剂。这种核酸酶在细胞凋亡早期通过 50 Kb DNA 切割进行染色质重组中发挥着关键作用。来自 DFF45-/- 和 +/+ 小鼠的细胞将用于建立保护 DFF45-/- 细胞免受链断裂诱导的细胞死亡的机制。这涉及关键的细胞凋亡事件和 PARP-1 相关的放大环路。我们将测试 IR 是否会在直接 DNA 损伤后立即在辐射诱导的细胞凋亡过程中引起 PARP-1 的双相激活，然后在 DFF 激活和产生 50 kb DNA 断裂后立即进行。