Cytokine modulated novel growth inhibitory mechanisms

描述（由申请方提供）：细胞因子在调节肿瘤细胞生长中发挥核心作用/n v/vo。它们通过直接诱导肿瘤中的生长抑制基因或通过建立针对肿瘤的免疫应答来发挥这种作用。特别是，干扰素家族的细胞因子调节几种生理应答，如抗病毒、抗肿瘤和免疫功能。通过与其他细胞因子相互作用，IFN形成控制肿瘤细胞生长和宿主对病原体防御的细胞间信号分子的大网络。在它们的各种作用中，IFN对生长抑制的调节作用尚未完全了解。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，可以协同增强后，与维甲酸组合。这种维生素A代谢物本身在许多实验模型中作为生长抑制剂。一些临床研究也证明了IFN/RA的有效的肿瘤抑制作用。根据这些数据，了解导致肿瘤生长抑制的分子调节剂的作用机制是很重要的。我们假设IFN/RA采用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，我们采取了遗传学方法，根据基因的功能来确定基因。在这里，我们已经表征了一种新的基因产物，（与类视黄醇干扰素诱导的死亡率-19（GRIM-19）相关的基因，其诱导细胞凋亡。GRIM-19的抗肿瘤作用在我们最近的研究中得到进一步强调，其中其凋亡功能被DNA病毒癌蛋白抑制。GRIM-19的表达似乎在一些人类肿瘤中受到抑制。这些特征表明其有效的抗癌特性。为了更好地了解作用机制，我们使用酵母双杂交筛选并将信号转导转录激活因子3（STAT 3）鉴定为GRIM-19结合蛋白。最近的几项研究表明，STAT 3，否则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STAT 3调节的转录。在这个提议中，我们将研究GRIM-19诱导的对肿瘤细胞生长的抑制作用的机制。特别是，我们将研究GRIM-19对组成型活性STAT 3变体和致癌Src的抗癌作用，Src使用STAT 3促进细胞生长。我们还将定义介导GRIM-19：STAT 3相互作用的关键结构域。使用染色质免疫沉淀（CHIP）分析，STAT 3调节的报告基因和缺乏STAT 3的突变细胞系，我们将研究GRIM-19的抗STAT 3作用的机制。GRIM-19可能是：1）一种新的肿瘤抑制剂; 2）作为肿瘤对治疗剂反应的标志物; 3）通过参与其他生长抑制途径抑制几种其他癌细胞; 4）一旦确定其作用模式，可用于基因治疗。因此，对GRIM-19作用的理解将进一步有助于确定消除这种新的生长调节途径的肿瘤细胞特异性功能障碍。总之，本申请中提出的研究将揭示一种新发现的抗癌蛋白所采用的细胞生长调节的新机制。

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.