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: 7050537
• 负责人: Mark R. Kelley
• 金额: $ 29.5万
• 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/7050537
• 关键词: Adenoviridae; DNA damage; DNA repair; N glycosidase; NOD mouse; SCID mouse; carbon oxygen lyase; cell line; enzyme activity; folate; gene expression; gene mutation; ionizing radiation; liposomes; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplastic cell; ovary neoplasms; radiation sensitivity; small interfering RNA; telomerase; terminal nick end labeling; transfection /expression vector; vitamin receptor; xenotransplantation

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）：该项目的总体意义与卵巢肿瘤天花板中DNA碱基切除修复（BER）途径失衡的能力有关，从而提高了它们对化学治疗和电离辐射（IR）药物的敏感性。我们将尝试使用人磷灰岩/磷灰蛋白核酸内切酶（APE1）酶，N-甲基嘌呤DNA糖基酶（MPG）的过表达来实现这一目标，均针对核和小线粒体，以及小（短）（短）（短）（短）（短）RNA（短）RNA（短）。我们还将在细胞系和点头/ScID动物模型中使用人端粒酶Reverste转录酶（HTERT）启动子利用叶酸衍生化的脂质体和腺病毒靶向以及肿瘤特异性启动子表达。 假设：在细胞核和/或线粒体室中MPG的过表达，改变了人APE1蛋白（主导性）或APE1的siRNA或siRNA独立或各种组合将增强卵巢癌细胞，以提高卵巢癌细胞对标准或降低常用的使用化学治疗剂的标准或降低水平（例如，使用的化学治疗剂水平）（例如Alkyls）和/或IR/或IR/或IR/或IR/或IR/或IR/或IR。具体目的是：具体目标1：第一个目标包括确定在多个卵巢癌系中过表达MPG或显性阴性APE1的有效性，并评估肿瘤细胞对化学治疗和IR治疗的反应。这包括MPG酶和过表达的核和线粒体靶向，以及用siRNA敲除APE1。具体目标2：确定Nucmpg，mitompg和nuclmpg+mitompg，nucmpg+ape1突变体，mitompg+ape1突变体以及nucmpg或mitompg和mitompg和apei-siRNA的共同表达的影响。我们将监控联合表达是否增强化学治疗剂或IR的肿瘤细胞杀死作用。具体目标3：使用HTERT启动子的构建体将用于质粒（叶酸衍生化脂质体）和基于腺病毒的递送系统的卵巢癌细胞系中，用于肿瘤特异性表达研究，使用最佳候选APE1突变体，NUC-或MITOMPG或mITOMPG或SIRNA作为由AIMS 1-2中的结果确定的。具体目标4：由于APE1突变体的各种构建体的表达，NUC-/Mitompg以及NOD/SCID小鼠中的APEI-SIRNA，确定体内化学和放射敏感性。带有HTERT启动子的腺病毒构建体以及含有前三个目标中选定基因的叶酸衍生脂质体的腺病毒构建体将与异种移植点NOD/SCID小鼠一起使用。如果成功，我们认为这些研究将在诊所的治疗基因转移/治疗环境中产生非常有效的试剂，并阐明核和线粒体在癌细胞中的作用。