Imbalancing DNA BER to enhance Ovarian Tumor Sensitivity

描述(申请人提供)：该项目的总体意义在于能够平衡卵巢肿瘤细胞的DNA碱基切除修复(BER)途径，增加其对化疗和电离辐射(IR)药物的敏感性。为了实现这一目标，我们将试图通过人类脱嘌呤/脱嘧啶核酸内切酶(APE1)的突变体，针对细胞核和线粒体的N-甲基嘌呤DNA糖基酶(MPG)的过表达，以及APE1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，以及在两种细胞系和NOD/SCID动物模型中使用人类端粒酶逆转录酶(HTERT)启动子表达肿瘤特异性启动子来发展这种方法的有效性。 假设：MPG在细胞核和/或线粒体中的过度表达，改变的人类APE1蛋白(显性-阴性)，或APE1的siRNA单独或在各种组合中的过度表达将使卵巢癌细胞达到常用化疗药物(如烷化剂)和/或IR的标准或降低水平。具体目标是：特定目标1：第一个目标包括确定MPG或显性阴性APE1在多个卵巢癌细胞系中过表达的有效性，并评估肿瘤细胞对化疗和IR治疗的反应。这包括核靶向和线粒体靶向的MPG酶和过表达，以及用siRNA击倒APE1。特异性目的2：检测nucMPG、mitoMPG和nuclMPG+mitoMPG、nucMPG+APE1突变体、mitoMPG+APE1突变体和nucMPG或mitoMPG与APEI-siRNA共表达的效应。我们将监测联合表达是否增强化疗药物或IR对肿瘤细胞的杀伤作用。具体目标3：使用hTERT启动子的构建物将用于卵巢癌细胞系的质粒(叶酸衍生脂质体)和腺病毒递送系统，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siRNA，如AIMS 1-2中的结果所确定的那样。具体目标4：确定在NOD/SCID小鼠体内由于APE1突变体或nuc-/mitoMPG以及APEI-siRNA表达的不同结构导致的化疗和放射敏感性。带有hTERT启动子的腺病毒载体以及含有前三个目的基因的叶酸衍生脂质体将用于异种移植的NOD/SCID小鼠。如果成功，我们认为这些研究将在临床的治疗性基因转移/治疗环境中创建非常有效的试剂，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.