Cytokine modulated novel growth inhibitory mechanisms

细胞因子调节的新型生长抑制机制

• 批准号: 7033005
• 负责人: DHAN V. KALVAKOLANU
• 金额: $ 26.75万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-07 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7033005
• 关键词: CD95 molecule; RNA interference; apoptosis; athymic mouse; chromatin immunoprecipitation; gene induction /repression; gene mutation; genetic transcription; interferon beta; nuclear proteins; oncogenes; protein protein interaction; protein structure function; retinoate; retinoid binding proteins; site directed mutagenesis; tissue /cell culture; transcription factor; tumor suppressor proteins

DESCRIPTION (provided by applicant): Cytokines play a central role in regulating neoplastic cell growth/n v/vo. They exert such effects either by a direct induction of growth suppressor genes in the tumor or by mounting an immune response against it. In particular, the interferon family of cytokines regulates several physiologic responses such as antiviral, antitumor, and immune functions. By interacting with other cytokines, IFNs form a large network of intercellular signaling molecules that control neoplastic cell growth and host defenses against pathogens. Among their various actions, the regulation of growth suppression by IFNs is not fully understood. In an earlier study, we demonstrated that the IFN-induced anti-tumor actions, especially apoptosis, could be synergistically augmented upon combination with retinoic acid. This vitamin-A metabolite by itself acts as a growth inhibitor in many experimental models. Some clinical studies also demonstrated the potent tumor inhibitory effects of IFN/RA. In the light of these data, it is important to understand the mechanisms of action of the molecular regulators that cause tumor growth suppression. We hypothesized that IFN/RA employs either novel gene products for inducing cell death. To identify the critical molecules involved in IFN/RA-induced apoptosis, we have taken a genetic approach that identifies genes on the basis of their functions. Here, we have characterized a novel gene product, (gene associated with Retinoid-lnterferon induced Mortality-19 (GRIM-19), which induces apoptosis. The anti-oncogenic effects of GRIM-19 were further highlighted in our recent studies, where its apoptotic functions were suppressed by DNA viral oncoproteins. GRIM-19-expression appears to be inhibited in some human tumors. These characteristics indicate its potent anti-oncogenic characteristics. To gain a better insight into the mechanisms of action, we have used a yeast-2-hybrid screen and identified Signal Transducing Activator of Transcription-3 (STAT3) as a GRIM-19 binding protein. Several recent studies have shown that STAT3, an otherwise dormant signal-regulated transcription factor, is constitutively activated in human tumors. A number of viral and cellular oncoproteins constitutively stimulate its activity to promote cell survival. Based on our preliminary observations, we have hypothesized that GRIM-19 inhibits STAT3-regulated transcription. In this proposal we will investigate the mechanisms of GRIM-19 induced inhibitory effects on tumor cell growth. In particular, we will study the anti-oncogenic effects of GRIM-19 on a constitutively active STAT3 variant, and an oncogenic Src, which uses STAT3 for promoting cell growth. We will also define the critical domains that mediate GRIM-19:STAT3 interactions. Using chromatin immunoprecipitation (CHIP) assays, STAT3-regulated reporter genes and mutant cell lines lacking STAT3, we will investigate the mechanisms of the anti-STAT3 effects of GRIM-19. GRIM-19 may be: 1) a novel tumor suppressor; 2) serve as a marker for the tumor response to therapeutic agents 3) inhibit several other cancer cells by participating in other growth suppressive pathways; 4) be useful in gene therapy once its mode of action has been defined. Thus, an understanding of GRIM-19 action will further help in defining the tumor cell-specific dysfunctions that ablate this new growth regulatory pathway. In summary, the studies proposed in this application will uncover a novel mechanism of cell growth regulation employed by a newly discovered anti-oncogenic protein.

描述（由申请人提供）：细胞因子在调节肿瘤细胞生长中起核心作用。它们要么通过直接诱导肿瘤中的生长抑制基因，要么通过对其进行免疫反应来发挥这种作用。特别是，细胞因子干扰素家族调节多种生理反应，如抗病毒、抗肿瘤和免疫功能。通过与其他细胞因子的相互作用，ifn形成了一个大的细胞间信号分子网络，控制肿瘤细胞的生长和宿主对病原体的防御。在它们的各种作用中，ifn对生长抑制的调控尚不完全清楚。在早期的研究中，我们证明了ifn诱导的抗肿瘤作用，特别是细胞凋亡，可以在与维甲酸联合使用时协同增强。这种维生素a代谢物本身在许多实验模型中起生长抑制剂的作用。一些临床研究也证实了IFN/RA的有效肿瘤抑制作用。根据这些数据，了解导致肿瘤生长抑制的分子调节因子的作用机制是很重要的。我们假设IFN/RA利用其中一种新的基因产物诱导细胞死亡。为了确定参与IFN/ ra诱导的细胞凋亡的关键分子，我们采用了一种基于功能识别基因的遗传方法。在这里，我们描述了一个新的基因产物，与类视黄酮干扰素诱导死亡-19 （GRIM-19）相关的基因，它可以诱导细胞凋亡。我们最近的研究进一步强调了GRIM-19的抗肿瘤作用，其凋亡功能被DNA病毒癌蛋白抑制。在一些人类肿瘤中，grim -19的表达似乎受到抑制。这些特征表明其具有强大的抗肿瘤特性。为了更好地了解其作用机制，我们使用酵母-2杂交筛选并鉴定了信号转导激活因子转录-3 （STAT3）作为GRIM-19结合蛋白。最近的几项研究表明，STAT3是一种休眠的信号调节转录因子，在人类肿瘤中被组成性激活。许多病毒和细胞癌蛋白组成性地刺激其活性以促进细胞存活。基于我们的初步观察，我们假设GRIM-19抑制stat3调控的转录。在本研究中，我们将探讨GRIM-19诱导肿瘤细胞生长抑制作用的机制。特别是，我们将研究GRIM-19对组成型活性STAT3变体和致癌Src的抑癌作用，后者使用STAT3促进细胞生长。我们还将定义介导GRIM-19:STAT3相互作用的关键结构域。利用染色质免疫沉淀（CHIP）实验、STAT3调控的报告基因和缺乏STAT3的突变细胞系，我们将研究GRIM-19抗STAT3作用的机制。GRIM-19可能是：1)一种新的肿瘤抑制因子；2)作为肿瘤对治疗剂反应的标志；3)通过参与其他生长抑制途径抑制其他几种癌细胞；4)一旦确定了其作用方式，就可用于基因治疗。因此，对GRIM-19作用的理解将进一步有助于定义破坏这一新的生长调节途径的肿瘤细胞特异性功能障碍。总之，本研究将揭示一种新发现的抗肿瘤蛋白调控细胞生长的新机制。