Role of MnSOD in Acqired Resistance to Cancer Therapy

描述(由申请人提供)：尽管长期以来人们一直怀疑活性氧中间产物(ROI)在放射和化疗药物对细胞的杀伤中起核心作用，但负责ROI介导的细胞死亡或存活的特定靶基因尚未确定。阐明ROI介导的信号转导可能为提高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)是由转化的人类细胞暴露在电离辐射中而诱导的。更多证据表明，编码MnSOD的基因可能是通过应激反应转录因子NF-kappaB表达的。对照射细胞和高表达MnSOD细胞的基因表达谱分析发现，在Sod2基因敲除(-/-)细胞中，两个关键的促生存基因Cyclin B1和14-3-3zeta可以通过阻断MnSOD的表达而被抑制，而辐射不能诱导它们的表达。此外，用突变体IkappaB抑制NF-kappaB，可抑制MnSOD、Cyclin B1和14-3-3zeta的表达。因此，一个包括核因子-kappaB、MnSOD和一组促生存效应基因在内的信号网络似乎被激活，并在信号转导细胞对ROI介导的细胞毒作用方面是必需的。这项提议将检验这样一种假设，即MnSOD引起的H_2O_2稳态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基因的表达是否会增加细胞对辐射诱导的细胞毒性的敏感性。四个特定的目标将验证这一假说：a)确定Sod2-/-和MCF-7细胞线粒体中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，以及氧化还原敏感的转录因子是否参与了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)利用针对过氧化氢清除酶过氧化氢酶(CAT)或谷胱甘肽过氧化物酶(GPX)的复制失效腺病毒载体，确定MnSOD诱导的线粒体H_2O_2稳态水平的变化是否有助于细胞周期蛋白B1和14-3-3_zeta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，确定氧化还原敏感的转录因子亚单位(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；以及d)确定细胞周期蛋白B1和/或14-3-3zeta的过度表达是否是辐射抗性表型所需的关键信号通路之一。将选择稳定表达细胞周期蛋白B1和14-3-3zeta的MCF-7和HK18细胞株，并测试其对辐射毒性的耐受性。然后，将经辐射处理的MCF-7和HK18细胞以及MCF+SOD和HK18+SOD细胞的抗性克隆暴露于Cyclin 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.