Imbalancing DNA BER to enhance Ovarian Tumor Sensitivity

不平衡 DNA BER 可增强卵巢肿瘤敏感性

• 批准号: 7227896
• 负责人: Mark R. Kelley
• 金额: $ 28.63万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7227896
• 关键词: Alkylating Agents; Alkylation; Animal Model; Antineoplastic Protocols; Base Excision Repairs; Binding; Cancer cell line; Cell Death; Cell Line; Cell Nucleus; Cells; Cisplatin; Clinic; DNA Repair; DNA-3-methyladenine glycosidase II; Data; Dominant-Negative Mutation; Dose; Effectiveness; Elements; Enzymes; Folic Acid; Gene Transfer; Genes; Goals; Human; In Vitro; Internal Ribosome Entry Site; Intervention; Ionizing radiation; Light; Liposomes; Malignant Neoplasms; Malignant neoplasm of ovary; Mitochondria; Modeling; Monitor; Mus; Nuclear; Nucleotide Excision Repair; Numbers; Outcome; Pathway interactions; Plasmids; Protein Overexpression; Proteins; RNA; Radiation Tolerance; Reagent; Research Personnel; Role; SCID Mice; Site; Small Interfering RNA; Standards of Weights and Measures; Structure; System; Telomerase; Therapeutic; Tissues; Transcriptase; Xenograft procedure; adduct; antitumor agent; base; cancer cell; cancer therapy; cell killing; chemotherapeutic agent; chemotherapy; endonuclease; expression vector; gene therapy; human APEX1 protein; in vivo; killings; knock-down; mutant; neoplastic cell; ovarian neoplasm; prevent; programs; promoter; repaired; response; therapeutic gene; treatment effect; tumor

DESCRIPTION (provided by applicant): The overall significance of this project relates to the ability to imbalance the DNA base excision repair (BER) pathway in ovarian tumor ceils, increasing their sensitivity to chemotherapeutic and ionizing radiation (IR) agents. We will attempt to accomplish this goal using mutants of the human apurinic/apyrimidinic endonuclease (APE1) enzyme, overexpression of N-methylpurine DNA glycosylase (MPG), both targeted to the nucleus and mitochondria, as well as small (short) interfering RNA (siRNA) for APE1. We will also utilize folic acid-derivatized liposomes and adenoviral targeting along with tumor specific promoter expression using the human telomerase reverste transcriptase (hTERT) promoter in both cell lines and an NOD/SCID animal model to develop the usefulness of this approach. Hypothesis: Overexpression of MPG in the nucleus and/or mitochondrial compartments, altered human APE1 proteins (dominant-negative), or siRNA for APE1 either independently, or in various combinations will enhance ovarian cancer cells to standard or decreased levels of commonly used chemotherapeutic agents (e.g. alkylators) and/or IR. The Specific Aims are: Specific Aim 1: This first aim includes determining the effectiveness of overexpressing MPG or dominant-negative APE1 in multiple ovarian cancer lines and evaluating tumor cell response to chemotherapeutic and IR treatment. This includes both nuclear and mitochondrial targeting of the MPG enzyme and overexpression and the knockdown of APE1 with siRNA. Specific Aim 2: Determine the effects of co-overexpression of nucMPG, mitoMPG and nuclMPG+mitoMPG, nucMPG+APE1 mutant, mitoMPG+APE1 mutant and nucMPG or mitoMPG and APEI-siRNA. We will monitor whether combined expression enhances the tumor cell killing effect of chemotherapeutic agents or IR. Specific Aim 3: Constructs using the hTERT promoter will be used in ovarian cancer cell lines in both plasmid (folic acid-derivatized liposome) and adenoviral based delivery systems for tumor specific expression studies using best candidate APE1 mutants, nuc- or mitoMPG, or siRNA as determined by the results in Aims 1-2. Specific Aim 4: Determine in vivo chemo- and radiosensitivity due to the expression of the various constructs of APE1 mutants, or nuc-/mitoMPG as well as APEI-siRNA in NOD/SCID mice. Adenoviral constructs with the hTERT promoter as well as folic acid-derivatized liposomes containing selected genes from the first three aims will be used with xenograft NOD/SCID mice. If successful, we feel these studies will create very effective reagents in a therapeutic gene transfer/therapy setting in the clinic, as well as shed light on the role of both nuclear and mitochondrial BER in cancer cells.

描述（由申请人提供）：该项目的总体意义涉及平衡卵巢肿瘤细胞中 DNA 碱基切除修复 (BER) 途径的能力，从而提高其对化疗和电离辐射 (IR) 药物的敏感性。我们将尝试使用人类无嘌呤/无嘧啶核酸内切酶 (APE1) 突变体、N-甲基嘌呤 DNA 糖基化酶 (MPG) 的过表达（均靶向细胞核和线粒体）以及小（短）干扰 RNA 来实现这一目标。 APE1 的 siRNA）。我们还将在细胞系和 NOD/SCID 动物模型中利用叶酸衍生脂质体和腺病毒靶向以及肿瘤特异性启动子表达，使用人端粒酶逆转录酶 (hTERT) 启动子来开发该方法的实用性。 假设：细胞核和/或线粒体区室中 MPG 的过度表达、改变的人 APE1 蛋白（显性阴性）或 APE1 的 siRNA 单独或以各种组合形式将增强卵巢癌细胞至标准水平或降低常用化疗药物的水平（例如烷基化剂）和/或 IR。具体目标是： 具体目标 1：第一个目标包括确定在多种卵巢癌细胞系中过表达 MPG 或显性失活 APE1 的有效性，并评估肿瘤细胞对化疗和 IR 治疗的反应。这包括 MPG 酶的核和线粒体靶向以及通过 siRNA 过度表达和敲低 APE1。具体目标 2：确定 nucMPG、mitoMPG 和 nuclMPG+mitoMPG、nucMPG+APE1 突变体、mitoMPG+APE1 突变体和 nucMPG 或 mitoMPG 和 APEI-siRNA 共过表达的效果。我们将监测联合表达是否增强化疗药物或IR的肿瘤细胞杀伤作用。具体目标 3：使用 hTERT 启动子的构建体将用于卵巢癌细胞系中的质粒（叶酸衍生化脂质体）和基于腺病毒的递送系统，以使用最佳候选 APE1 突变体、nuc-或 mitoMPG 或 siRNA 进行肿瘤特异性表达研究由目标 1-2 中的结果确定。具体目标 4：确定 NOD/SCID 小鼠中由于 APE1 突变体或 nuc-/mitoMPG 以及 APEI-siRNA 的各种构建体的表达而产生的体内化学和放射敏感性。具有 hTERT 启动子的腺病毒构建体以及含有来自前三个目标的选定基因的叶酸衍生脂质体将用于异种移植 NOD/SCID 小鼠。如果成功，我们认为这些研究将在临床治疗基因转移/治疗环境中创造出非常有效的试剂，并揭示核和线粒体 BER 在癌细胞中的作用。