Imbalancing DNA BER to enhance Ovarian Tumor Sensitivity

描述（由申请人提供）：该项目的总体意义涉及卵巢肿瘤细胞中DNA碱基切除修复（BER）途径失衡的能力，增加其对化疗和电离辐射（IR）试剂的敏感性。我们将尝试使用人脱嘌呤/脱嘧啶核酸内切酶（APE 1）的突变体，N-甲基嘌呤DNA糖基化酶（MPG）的过表达，靶向细胞核和线粒体，以及APE 1的小（短）干扰RNA（siRNA）来实现这一目标。我们还将利用叶酸衍生的脂质体和腺病毒靶向沿着肿瘤特异性启动子表达，在两种细胞系和NOD/SCID动物模型中使用人端粒酶逆转录酶（hTERT）启动子来开发这种方法的有用性。 假设：MPG在细胞核和/或线粒体区室中的过表达，改变了人APE 1蛋白（显性阴性）或针对APE 1的siRNA独立地或以各种组合将卵巢癌细胞增强至标准水平或降低的常用化疗剂水平具体目的是：具体目的1：第一个目标包括确定多个卵巢癌细胞系中过表达MPG或显性阴性APE 1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过表达和用siRNA敲低APE 1。具体目标二：确定nucMPG、mitoMPG和nuclMPG+mitoMPG、nucMPG+ APE 1突变体、mitoMPG+ APE 1突变体和nucMPG或mitoMPG和APEI-siRNA的共过表达的影响。我们将监测组合表达是否增强化疗剂或IR的肿瘤细胞杀伤作用。使用hTERT启动子的构建体将在基于质粒（叶酸衍生的脂质体）和腺病毒的递送系统中用于卵巢癌细胞系中，用于使用最佳候选APE 1突变体、nuc-或mitoMPG或siRNA的肿瘤特异性表达研究，如通过目的1-2中的结果所确定的。具体目标4：确定NOD/SCID小鼠中由于APE 1突变体或nuc-/mitoMPG以及APEI-siRNA的各种构建体的表达而导致的体内化疗和放射敏感性。具有hTERT启动子的腺病毒构建体以及含有来自前三个目的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/治疗环境中创造非常有效的试剂，并阐明核和线粒体BER在癌细胞中的作用。

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.