Zinc Protoporphyrin and the Cell Cycle in Neonatal Mice

锌原卟啉和新生小鼠的细胞周期

• 批准号: 7057832
• 负责人: PHYLLIS A. DENNERY
• 金额: $ 40.52万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7057832
• 关键词: DNA footprinting; apoptosis; bilirubin; cell cycle; cell cycle proteins; cell growth regulation; cell proliferation; chemical binding; cyclins; enzyme activity; genetic promoter element; genetic transcription; genetically modified animals; heme oxygenase; immunoprecipitation; laboratory mouse; metalloporphyrins; newborn animals; p53 gene /protein; porphyrin biosynthesis; spectrometry; tissue /cell culture; transcription factor; western blottings; zinc

DESCRIPTION (provided by applicant): Zinc protoporphyin (ZnPP) is a naturally occurring metalloporphyrin (Mp) that is formed endogenously during heme biosynthesis. This compound is synthesized at higher levels in conditions of iron deficiency anemia and lead intoxication. Under these conditions, red blood cell turnover allows for deposition of ZnPP in tissues such as the liver, spleen. Animal experiments suggest that ZnPP is an effective inhibitor of bilirubin production. However, other studies demonstrate that ZnPP is an inhibitor of bone marrow cell proliferation and that it results in apoptosis in hamster fibroblasts ceils in culture. Therefore, it may be that ZnPP plays a role in cell cycle regulation. The Specific Aims of this proposal are to: 1) describe the effect of ZnPP on cell cycle regulation in vitro, 2) describe the effect of ZnPP on cell cycle regulation in vitro and 3) elucidate the mechanisms by which ZnPP mediates cell cycle regulation in vitro. Using primary hepatocytes in culture, we will evaluate the effect of exogenous ZnPP on cell cycle progression as well as the expression of cyclins and other proteins that regulate the cell cycle. Neonatal mice will be injected with ZnPP at various concentrations. Tissues will be evaluated for ZnPP incorporation. Tissues demonstrating ZnPP deposition will be further examined for markers of cell proliferation and apoptosis and for cell cycle protein expression. The amount of ZnPP binding to HO-1 and HO-2 proteins will be determined by immunoprecipitation techniques and Western analysis. To document ZnPP-DNA binding, we will detect radiolabeling of nuclear DNA using DNAse footprinting after incubation with radiolabeled 65ZnPP. This will allow us to determine whether there are direct or indirect effects of ZnPP on gene transcription. We will also evaluate how incubation with ZnPP specifically alters the cell cycle. We hypothesize that ZnPP mediates its effects, in part, through induction of early growth response transcription factor (Egr-1) and through Egr-l-mediated gene transcription. Using hepatocytes derived from Egr-1 and p53 null mutants, we will specifically address whether ZnPP-mediated changes in cell cycle occur through Egr-1 and/or p53 dependent pathways. These experiments will allow us to determine specifically how ZnPP serves as a signaling molecule to suppress cell proliferation. A better understanding of the role of ZnPP in cell cycle regulation will determine whether ZnPP functions as a modifier of the cell cycle. This may also lead to therapeutic strategies to modify ZnPP so as to prevent abnormal proliferation in cells.

描述（由申请人提供）： 锌原卟啉（ZnPP）是一种天然存在的金属卟啉（Mp），在血红素生物合成过程中内源形成。在缺铁性贫血和铅中毒的情况下，该化合物的合成水平较高。在这些条件下，红细胞更新允许 ZnPP 在肝脏、脾脏等组织中沉积。动物实验表明，ZnPP 是胆红素产生的有效抑制剂。然而，其他研究表明 ZnPP 是骨髓细胞增殖的抑制剂，并导致培养的仓鼠成纤维细胞凋亡。因此，ZnPP可能在细胞周期调节中发挥作用。该提案的具体目标是：1）描述 ZnPP 对体外细胞周期调节的作用，2）描述 ZnPP 对体外细胞周期调节的作用，3）阐明 ZnPP 介导细胞周期调节的机制。体外。使用培养的原代肝细胞，我们将评估外源 ZnPP 对细胞周期进程以及细胞周期蛋白和其他调节细胞周期的蛋白质表达的影响。新生小鼠将被注射不同浓度的 ZnPP。将评估组织中 ZnPP 的掺入情况。将进一步检查显示 ZnPP 沉积的组织的细胞增殖和凋亡标记以及细胞周期蛋白表达。与 HO-1 和 HO-2 蛋白结合的 ZnPP 量将通过免疫沉淀技术和 Western 分析来确定。为了记录 ZnPP-DNA 结合，我们将在与放射性标记的 65ZnPP 一起孵育后，使用 DNAse 足迹法检测核 DNA 的放射性标记。这将使我们能够确定 ZnPP 对基因转录是否有直接或间接影响。我们还将评估 ZnPP 孵育如何具体改变细胞周期。我们假设 ZnPP 部分通过诱导早期生长反应转录因子 (Egr-1) 和 Egr-1 介导的基因转录来介导其作用。使用源自 Egr-1 和 p53 无效突变体的肝细胞，我们将专门解决 ZnPP 介导的细胞周期变化是否通过 Egr-1 和/或 p53 依赖性途径发生。这些实验将使我们能够具体确定 ZnPP 如何作为信号分子来抑制细胞增殖。更好地了解 ZnPP 在细胞周期调节中的作用将确定 ZnPP 是否起到细胞周期调节剂的作用。这也可能导致修饰 ZnPP 的治疗策略，以防止细胞异常增殖。