Role of MnSOD in Acqired Resistance to Cancer Therapy

描述（由申请人提供）：尽管长期以来一直怀疑活性氧中间体（ROI）在放射和化学治疗剂的细胞杀伤中起核心作用，但尚未鉴定出负责ROI介导的细胞死亡或存活的特异性靶基因。阐明ROI介导的信号传导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的转化人类细胞诱导。另外的证据表明，编码MnSOD的基因的表达可能是通过应激反应转录因子NF-κ B。在辐射细胞和细胞过表达MnSOD的基因表达谱的分析已经确定了两个关键的促生存基因，细胞周期蛋白B1和14-3- 3 zeta，可以通过阻断MnSOD表达而被抑制，并且在Sod 2敲除（-/-）细胞中不被辐射诱导。此外，使用突变体Ikappa B抑制NF-κ B，抑制MnSOD以及细胞周期蛋白B 1和14-3- 3 ζ的表达。因此，涉及NF-κ B、MnSOD和一组促存活效应基因的信号传导网络似乎被激活，并且在信号传导细胞对ROI介导的细胞毒性的敏感性中是必需的。该提案将测试MnSOD引起的H2 O2稳态水平的变化在调节促生存基因细胞周期蛋白B1和14-3- 3 zeta中起关键作用的假设，以及阻断细胞周期蛋白B1和14-3- 3 zeta基因表达是否增加细胞对辐射诱导的细胞毒性的敏感性。a）确定MnSOD功能在Sod 2-/-和MCF-7细胞的线粒体中的重建是否恢复细胞周期蛋白B1和14-3- 3 ζ的表达，以及氧化还原敏感性转录因子是否负责MnSOD介导的细胞周期蛋白B 1和14-3- 3 ζ转录的基因启动子的激活; B）使用靶向H2 O2清除酶的复制缺陷型腺病毒载体，确定MnSOD诱导的线粒体H2 O2稳态水平的变化是否有助于细胞周期蛋白B1和14-3- 3 ζ表达的改变，过氧化氢酶（CAT）或谷胱甘肽过氧化物酶（GPx），线粒体; c）确定是否氧化还原敏感亚基的转录因子（AP-1、STAT和p300）通过分析cJun和STAT磷酸化速率和p300与cJun和STAT的相互作用来控制MnSOD诱导的细胞周期蛋白B1和14-3- 3 zeta表达;和d）确定细胞周期蛋白B1和/或14-3- 3 ζ过表达是否是辐射抗性表型所需的关键信号传导途径之一。将选择过表达细胞周期蛋白B 1和14-3- 3 ζ的稳定MCF-7和HK 18细胞系，并测试对辐射诱导的毒性的抗性。然后将来自辐射处理的MCF-7和HK 18细胞以及MCF+SOD和HK 18 +SOD细胞的抗性克隆暴露于针对细胞周期蛋白B1或14-3- 3 ζ的siRNA，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.