Novel Effects of DNA Intercalators: Inhibition of Epigenetic Modifications

DNA 嵌入剂的新作用：抑制表观遗传修饰

• 批准号: 7483522
• 负责人: Beth O Van Emburgh
• 金额: $ 4.18万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483522
• 关键词: American; Anthracycline Antibiotics; Anthracyclines; Apoptotic; Biological Assay; Breast Cancer Treatment; Cancer Etiology; Cancer Patient; Cells; Cessation of life; Class; Clinic; DNA; DNA Binding; DNA Double Strand Break; DNA Methylation; DNA Methyltransferase; DNA Modification Methylases; DNA methyltransferase inhibition; DNA-Directed RNA Polymerase; Data; Development; Doxorubicin; Drug usage; Electrophoretic Mobility Shift Assay; Enzymes; Epigenetic Process; Gene Expression; Gene Silencing; Generations; Genes; Genetic Transcription; Genome; Genomics; Goals; High Pressure Liquid Chromatography; Histone Acetylation; Histones; Hypermethylation; In Vitro; Intercalating Agents; Knowledge; Laboratories; Malignant Neoplasms; Measurement; Measures; Mediating; Methylation; Methyltransferase; Mitoxantrone; Modification; Molecular; Monitor; Outcome; Patients; Pattern; Pharmaceutical Preparations; Polymerase Chain Reaction; Post-Translational Protein Processing; Promoter Regions; Public Health; Reactive Oxygen Species; Recombinants; Reporting; Research; Reverse Transcriptase Polymerase Chain Reaction; System; Testing; Time; Topoisomerase II; Topoisomerase II inhibition; Toxic effect; Tumor Suppressor Genes; Woman; Work; base; bisulfite; cancer cell; cancer type; chemotherapeutic agent; chromatin immunoprecipitation; cytotoxic; cytotoxicity; enzyme activity; immune function; improved; in vitro Assay; in vivo; killings; magnetic beads; malignant breast neoplasm; novel

DESCRIPTION (provided by applicant): The DNA intercalator doxorubicin is a highly effective chemotherapeutic agent used to treat several cancer types including breast cancer The mechanism of toxicity of DNA intercalators is generally attributed to their ability to inhibit topoisomerase II which results in the generation of double strand breaks in DNA. Emerging studies report that DNA intercalators are capable of modulating epigenetic modifications, specifically inhibiting DNA methylation, resulting in reexpression of silenced genes. These studies were supported in this laboratory with both doxorubicin and mitoxantrone. Previous studies in this laboratory have discovered a novel function for doxorubicin as it is capable of inhibiting the activity of the DNA methyltransferase DNMT1 in vitro. These studies also determined that DNMT1 is an important contributor to the apoptotic activity of doxorubicin. The objectives of this proposal are to characterize the mechanism of intercalator mediated epigenetic alterations and how DNA methyltransferase inhibition contributes to the cytotoxic effects of these drugs. Improved understanding of the the molecular basis of DNA intercalator mediated cytotoxicity will allow for more effective use of the drugs clinically. The specific aims are (1) to characterize the effects of DNA intercalators on DNA methyltransferase activity and interaction with DNA in vitro and (2) to characterize the effects of DNA intercalators on DNA methylation patterns and histone modifications in vivo. The DNMT-magnetic Beads Assay, which was previously developed in this laboratory, will be used to analyze the effects of DNA intercalators on methyltransferase activity using recombinant DNMT1, DnmtSa, and DnmtSb. Gel shift assays will be used to monitor the effects on enzyme and DNA binding interactions in vitro. In order to monitor the effects of DNA intercalators on DNA methylation in vivo, the methylation status of silenced genes in DNA intercalator treated cells will be measured both qualitatively using methylation specific PCR and quantitatively with bisulfite genomic sequencing. The effects, on gene expression will be determined using real-time RT-PCR. Global methylation will be measured using HPLC and changes in histone modifications will be studied using chromatin immunoprecipitation. Public Health Relevance: The results of this study will improve the understanding of how an important and commonly used class of chemotherapeutic drugs like doxorubicin is capable of killing cancer cells. This knowledge will enable them to be used more effectively in the clinic, which is expected to improve the outcome for cancer patients.

描述（由申请人提供）：DNA介导剂阿霉素是一种非常有效的化学治疗剂，用于治疗几种癌症类型，包括乳腺癌，DNA插量的毒性机制通常归因于其抑制拓扑异构酶II的能力，从而在DNA中产生双链断裂。新兴研究报告说，DNA介导剂能够调节表观遗传修饰，特别抑制DNA甲基化，从而导致沉默基因的重新表达。这些研究在该实验室中都得到了阿霉素和米托酮的支持。该实验室的先前研究发现了阿霉素的新功能，因为它能够在体外抑制DNA甲基转移酶DNMT1的活性。这些研究还确定DNMT1是阿霉素凋亡活性的重要因素。该提案的目标是表征嵌入剂介导的表观遗传改变的机制，以及DNA甲基转移酶抑制作用如何有助于这些药物的细胞毒性作用。对DNA互化器介导的细胞毒性的分子基础的理解得以提高，可以在临床上更有效地使用这些药物。 （1）的具体目的是表征DNA介导剂对DNA甲基转移酶活性的影响，并在体外与DNA与DNA相互作用，（2）表征DNA介导剂对体内DNA甲基化模式和组蛋白修饰的影响。先前在该实验室中开发的DNMT磁珠测定法，将使用重组DNMT1，DNMTSA和DNMTSB分析DNA插量对甲基转移酶活性的影响。凝胶转移测定将用于监测体外对酶和DNA结合相互作用的影响。为了监测DNA介导剂对体内DNA甲基化的影响，将使用特异性PCR进行定性测量DNA互化细胞中沉默基因的甲基化状态，并用Bisulfite基因组测序进行定量。对基因表达的影响将使用实时RT-PCR确定。将使用HPLC测量全局甲基化，并将使用染色质免疫沉淀研究组蛋白修饰的变化。 公共卫生相关性：这项研究的结果将提高人们对诸如阿霉素等重要和常用类化学治疗药物的理解，能够杀死癌细胞。这些知识将使它们能够在诊所中更有效地使用，这有望改善癌症患者的结果。