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）药物的敏感性。我们将尝试使用人类无尿嘧啶/无嘧啶核酸内切酶（APE1）酶的突变体，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脂质体和腺病毒靶向以及肿瘤特异性启动子表达，在两种细胞系和NOD/SCID动物模型中使用人类端粒酶逆转录酶（hTERT）启动子来开发这种方法的实用性。

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.