SUPEROXIDE REGULATION OF FIBROSARCOMA CELL GROWTH

描述：（申请人的描述） 本申请的长期目标是通过以下方式表征机制： 线粒体超氧化物调节恶性表型 纤维肉瘤细胞在体外和体内。 进行的初步研究 申请人证明锰超氧化物歧化酶（MnSOD） 过表达，一种酶，催化从超氧化物中去除超氧化物， 线粒体，与细胞生长抑制呈负相关。 在 此外，IL-1-α的水平特异性地响应于MnSOD的变化， 活动 当MnSOD水平增加两到四倍或细胞 低氧培养（3%），基底细胞和肿瘤细胞减少， 观察到坏死因子（TNF）诱导的IL-1-α。 然而，作为MnSOD 活性进一步增加，IL-1-α的相应增加是 观察到纤维肉瘤生长的相关减少 细胞 生长抑制和IL-1-α水平的增加都可以抑制肿瘤细胞的生长。 高度过表达MnSOD（10-15倍）的细胞可被生长阻断 3%的氧气 后一项发现意味着， MnSOD过表达对细胞生长和IL-1-α水平的影响是由 线粒体超氧化物 核运行、启动子和NFkB-DNA结合 活性分析表明，在稳态的变化， 通过MnSOD过表达或低浓度生长， 氧不伴随IL-1-α转录的变化。 的 候选人还证明MnSOD过表达或氧改变了 IL-1 mRNA水平通过调节其稳定性。 在低氧或微妙的 纤维肉瘤细胞MnSOD活性增加，IL-1 mRNA周转增加 提高了 相反，在21%的氧气或高MnSOD下， 过表达IL-1-α mRNA变得稳定。 他建议将 人IL-1-α mRNA，因此确定了假定的超氧化物 mRNA中的响应区域。 详细的缺失和突变分析， mRNA将利用5 ′和3 ′-非翻译区进行 将IL-1-α mRNA连接到报告基因构建体中， 瞬时转染HT-1080纤维肉瘤细胞。 这些研究将 建立IL-1-α mRNA的特定区域， 氧化剂的调节。 他将进一步讨论参与 一旦他确定了超氧化物敏感的RNA结合蛋白的区域， 对超氧化物应答RNA。 他还建议， 线粒体氧化剂产生和完整性的变化，导致 MnSOD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， 显示通过增强宿主抗肿瘤防御介导肿瘤消退 机制等 因此，他将研究肿瘤消退是否与 体内MnSOD过表达部分由IL-1-α介导。 这些 研究不仅应该阐明介导这种疾病的机制， MnSOD逆转肿瘤生长，但也定义了这种新发现的模式 IL-1-alpha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.