SUPEROXIDE REGULATION OF FIBROSARCOMA CELL GROWTH

描述：(申请人描述) 这个应用程序的长期目标是通过以下方式描述这些机制 哪种线粒体超氧化物歧化调控肿瘤的恶性表型 纤维肉瘤细胞的体外和体内实验。进行的初步研究 申请人证明了锰超氧化物歧化酶(MnSOD) 过量表达，一种催化从细胞中清除超氧化物的酶 线粒体与细胞生长抑制呈负相关。在……里面 此外，IL-1-α水平对MnSOD的变化具有特异性反应 活动。当MnSOD水平增加两到四倍时，或者细胞 低氧培养(3%)，基底细胞和肿瘤细胞均减少 观察肿瘤坏死因子诱导的IL-L-α的变化。然而，作为MnSOD 活性进一步增强，IL-L-α相应增加 观察到纤维肉瘤的生长减少。 细胞。细胞生长抑制和IL-L-α水平升高 高表达MnSOD的细胞(10-15倍)可被生长抑制 在3%的氧气中。后一项发现表明， 锰超氧化物歧化酶过表达对细胞生长和IL-L-α水平的影响 线粒体超氧化物歧化。核启动、启动子与NFkB-DNA结合 活度分析表明，稳态时的变化 MnSOD过表达和低生长对IL-1-αmRNA水平的影响 氧气并不伴随IL-1-α转录的变化。这个 候选人还证明了MnSOD的过度表达或氧气改变 IL-L基因表达水平通过调节其稳定性来实现。在低氧或微妙的情况下 纤维肉瘤细胞MnSOD活性、IL-1mRNA转换率升高 是增加的。相反，在21%的氧气或高MnSOD下 过表达IL-L-α基因后趋于稳定。他打算把自己的特点 人IL-L-α基因的表达及其与超氧化物歧化的关系 信使核糖核酸的响应区。详细的缺失和突变分析 该信使核糖核酸将利用5‘和3’非翻译区进行 将IL-L-α基因连接到报告基因构建体中 瞬时转染人纤维肉瘤细胞株HT-1080。这些研究将 建立IL-L-α基因的特定区域，这些区域是 氧化剂的调节。他将进一步谈到参与 一旦他定义了超氧化物敏感RNA结合蛋白的区域 反应超氧化物的核糖核酸。他还建议将 线粒体氧化剂的产生和完整性的变化导致 锰超氧化物歧化酶对IL-1-α水平和细胞生长的影响 过度表达。IL-1-α是一种主要的炎性细胞因子，一直以来 显示通过增强宿主抗肿瘤防御来介导肿瘤消退 机械装置。因此，他将调查肿瘤消退是否与 体内MnSOD的过度表达部分是由IL-1-α介导的。这些 研究不仅应该阐明调节 MnSOD逆转肿瘤生长，但也定义了这一新发现的模式 对IL-1-α的调节。

DESCRIPTION: (Applicant's Description) The long-term goal of this application is to characterize the mechanisms by which mitochondrial superoxide regulates the malignant phenotype of fibrosarcoma cells in vitro and in vivo. Preliminary studies conducted by the applicant demonstrate that manganese superoxide dismutase (MnSOD) overexpression, an enzyme which catalyzes the removal of superoxide from the mitochondria, is inversely correlated with cellular growth inhibition. In addition, the levels of IL-1-alpha specifically respond to changes in MnSOD activity. When MnSOD levels are increased two- to four-fold or cells are cultured in low oxygen (3 percent), a decrease in both basal and tumor necrosis factor (TNF)-induced IL-l-alpha is observed. However, as MnSOD activity increases further, a corresponding increase in IL-l-alpha is observed with an associated decrease in the growth of the fibrosarcoma cells. Both the growth inhibition and the increase in IL-l-alpha levels in cells which highly overexpress MnSOD (10-15-fold) can be blocked by growth in three percent oxygen. This latter finding implies that the effects of MnSOD overexpression on cell growth and IL-l-alpha levels are mediated by mitochondrial superoxide. Nuclear run-on, promoter, and NFkB-DNA binding activity analysis demonstrate that the alterations in the steady state levels of IL-1-alpha mRNA by either MnSOD overexpression or growth in low oxygen are not accompanied by changes in IL-1-alpha transcription. The candidate has also demonstrated that MnSOD overexpression or oxygen alter IL-l mRNA levels by modulating its stability. Under low oxygen or a subtle increase in the MnSOD activity of the fibrosarcoma cells, IL-1 mRNA turnover is increased. Conversely, under 21 percent oxygen or high MnSOD overexpression IL-l-alpha mRNA becomes stable. He proposes to characterize the human IL-l-alpha mRNA and hence define the putative superoxide responsive region in the mRNA. A detailed deletion and mutation analysis of the mRNA will be conducted utilizing both the 5' and 3'-untranslated regions of the IL-l-alpha mRNA ligated into a reporter gene construct and transiently transfected into HT-1080 fibrosarcoma cells. These studies will establish the specific regions of the IL-l-alpha mRNA that are necessary for regulation by oxidants. He will further address the involvement of superoxide sensitive RNA-binding proteins once he has defined the regions of the RNA which respond superoxide. He also proposes to characterize the changes in the mitochondrial oxidant production and integrity which lead to alterations in IL-1-alpha levels and cell growth in response to MnSOD overexpression. IL-1-alpha is a major inflammatory cytokine which has been shown to mediate tumor regression by enhancing host antitumor defense mechanisms. Thus, he will investigate if tumor regression associated with MnSOD overexpression in vivo is mediated in part by IL-1-alpha. These studies should not only shed light on the mechanisms which mediate the reversal of tumor growth by MnSOD but also define this newly discovered mode of regulation of IL-1-alpha.