Role of MnSOD in Acquired Resistance to Cancer Therapy

描述（由申请人提供）：尽管活性氧中间体（ROI）长期以来一直被怀疑在放疗和化疗药物的细胞杀伤中起核心作用，但负责ROI介导的细胞死亡或存活的特定靶基因尚未被确定。阐明ROI介导的信号可能为增强肿瘤细胞对抗癌治疗的敏感性提供新的基因靶点。最近的研究结果表明，线粒体抗氧化酶，含锰超氧化物歧化酶（MnSOD），是由暴露于电离辐射转化的人细胞诱导的。其他证据表明，编码MnSOD的基因可能通过应激反应转录因子NF-kappaB表达。通过对辐照细胞和过表达MnSOD的细胞的基因表达谱分析，发现了两个关键的促生存基因，cyclin B1和14-3-3 - zeta，它们可以通过阻断MnSOD的表达来抑制，而在so2敲除（-/-）细胞中不受辐射的诱导。此外，利用突变体IkappaB抑制NF-kappaB，可以抑制MnSOD、cyclin b1和14-3-3 -3zeta的表达。因此，涉及NF-kappaB、MnSOD和一组促生存效应基因的信号网络似乎被激活，并且在信号细胞对ROI介导的细胞毒性的敏感性中是必需的。本研究将验证由MnSOD引起的稳态H2O2水平的变化在调节促生存基因cyclin B1和14-3-3zeta中起关键作用，以及阻断cyclin B1和14-3-3zeta基因表达是否会增加细胞对辐射诱导的细胞毒性的敏感性。四个特定目标将验证这一假设：a)确定在so2 -/-和MCF-7细胞的线粒体中MnSOD功能的重建是否恢复了细胞周期蛋白B1和14-3-3zeta的表达，氧化还原敏感的转录因子是否负责MnSOD介导的细胞周期蛋白B1和14-3-3zeta转录基因启动子的激活；b)利用靶向过氧化氢酶（CAT）或谷胱甘肽过氧化物酶（GPx）的无复制能力腺病毒载体，确定mnsod诱导的线粒体H2O2稳态水平的变化是否会导致细胞周期蛋白B1和14-3-3zeta表达的改变；c)通过分析cJun和STAT的磷酸化速率以及p300与cJun和STAT的相互作用，确定氧化还原敏感亚基（AP-1、STAT和p300）是否控制mnsod诱导的细胞周期蛋白B1和14-3-3zeta的表达；d)确定细胞周期蛋白B1和/或14-3-3zeta过表达是否是辐射抗性表型所需的关键信号通路之一。选择稳定的过表达cyclin b1和14-3-3 -3zeta的MCF-7和HK18细胞系进行辐射毒性试验。然后将辐射处理的MCF-7和HK18细胞的抗性克隆以及MCF+SOD和HK18+SOD细胞暴露于细胞周期蛋白B1或14-3-3zeta的siRNA中，以确定是否可以逆转辐射抗性表型。

DESCRIPTION (provided by applicant): Although reactive oxygen intermediates (ROI) has long been suspected to play a central role in cell killing by radiation and chemotherapeutic agents, specific target genes responsible for ROI mediated cell death or survival have not been identified. Elucidation of ROI mediated signaling may provide new gene targets for enhancing tumor cell sensitivity to anticancer therapy. Recent results suggest that the mitochondrial antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), is induced by exposure of transformed human cells to ionizing radiation. Additional evidence demonstrates that expression of the gene that encodes MnSOD may be via the stress responsive transcription factor NF-kappaB. Analysis of gene expression profiles in irradiated cells and cells over expressing MnSOD has identified two key pro-survival genes, cyclin B1 and 14-3-3zeta, that can be inhibited by blocking MnSOD expression and were not induced by radiation in Sod2 knockout (-/-) cells. Moreover, inhibiting NF-kappaB using mutant IkappaB, inhibited expression of MnSOD as well as cyclin B 1 and 14-3-3zeta. Therefore, a signaling network involved NF-kappaB, MnSOD, and a group of pro-survival effector genes appears to be activated and required in signaling cell sensitivity to ROI mediated cytotoxicity. This proposal will test the hypothesis that changes in steady state levels of H2O2 caused by MnSOD plays a key role in regulating pro-survival genes cyclin B1 and 14-3-3zeta and if blocking cyclin B1 and 14-3-3zeta gene expression increases cell sensitivity to radiation-induced cytotoxicity. Four Specific Aims will test this hypothesis: a) Determine if reconstitution of MnSOD function in mitochondria of Sod2-/- and MCF-7 cells restores cyclin B1 and 14-3-3zeta expression and if redox-sensitive transcription factors are responsible for MnSOD mediated activation of gene promoter for cyclin B 1 and 14-3-3zeta transcription; b) Determine if MnSOD-induced changes in steady state levels of mitochondrial H2O2 contribute to alterations in the expression of cyclin B1 and 14-3-3zeta using replication incompetent adenoviral vectors targeting the H2O2 scavenging enzymes, catalase (CAT) or glutathione peroxidase (GPx), to mitochondria; c) Determine if redox sensitive subunits of transcription factors (AP-1, STAT and p300) control MnSOD-induced cyclin B1 and 14-3-3zeta expression by analysis of the rate of cJun and STAT phosphorylation and p300 interaction with cJun and STAT; and d) Determine if cyclin B1 and/or 14-3-3zeta overexpression is one of the key signaling pathways required for the radioresistance phenotype. Stable MCF-7 and HK18 cells lines overexpressing cyclin B 1 and 14-3-3zeta will be selected and resistance to radiation-induced toxicity tested. Then resistant clones from radiation treated MCF-7 and HK18 cells as well as MCF+SOD and HK18+SOD cells will be exposed to siRNA to cyclin B1 or 14-3-3zeta to determine if the radioresistant phenotype can be reversed.