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）：锌原生态（ZNPP）是一种天然存在的金属卟啉（MP），在血红素生物合成期间内源形成。在铁缺乏贫血和铅中毒条件下，该化合物在较高水平上合成。在这些条件下，红细胞的转换允许在肝脏，脾脏等组织中沉积ZNPP。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，它导致仓鼠成纤维细胞的凋亡是培养中天花板的凋亡。因此，可能是ZNPP在细胞周期调节中起作用。该提案的具体目的是：1）描述ZNPP对体外细胞周期调节的影响，2）描述ZNPP对细胞周期调节体外的影响，3）阐明了ZNPP在体外介导细胞周期调节的机制。在培养中使用原代肝细胞，我们将评估外源ZNPP对细胞周期进展的影响以及细胞周期蛋白和其他调节细胞周期的蛋白质的表达。新生小鼠将以各种浓度注入ZNPP。将评估ZNPP掺入的组织。证明ZNPP沉积的组织将进一步检查细胞增殖和凋亡的标志物以及细胞周期蛋白表达。 ZNPP与HO-1和HO-2蛋白的结合量将由免疫沉淀技术和西方分析确定。为了记录ZNPP-DNA结合，我们将使用DNase足迹与放射性标记的65ZNPP孵育后检测核DNA的放射性标记。这将使我们能够确定ZNPP对基因转录的直接或间接影响。我们还将评估与ZNPP孵育如何特异性改变细胞周期。我们假设ZNPP通过诱导早期生长反应转录因子（EGR-1）和EGR-L介导的基因转录来部分介导其效应。使用源自EGR-1和p53空突变体的肝细胞，我们将特别解决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特别确定ZNPP如何用作信号分子以抑制细胞增殖。更好地理解ZNPP在细胞周期调节中的作用将确定ZNPP是否作为细胞周期的修饰符。这也可能导致修饰ZNPP的治疗策略，以防止细胞中异常增殖。