Cytokine modulated novel growth inhibitory mechanisms

描述(申请人提供)：细胞因子在调节肿瘤细胞生长中起中心作用/n v/vo。它们通过直接诱导肿瘤中的生长抑制基因或通过启动针对肿瘤的免疫反应来发挥这种作用。特别是，干扰素家族的细胞因子调节多种生理反应，如抗病毒、抗肿瘤和免疫功能。通过与其他细胞因子相互作用，IFN形成一个巨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。在他们的各种行动中，对IFN抑制增长的监管还没有完全被理解。在早期的一项研究中，我们证明了干扰素诱导的抗肿瘤作用，特别是凋亡，与维甲酸联合使用可以协同增强。在许多实验模型中，这种维生素A代谢物本身就起到了生长抑制作用。一些临床研究也证明了干扰素/维甲酸具有很强的抑瘤作用。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。我们假设干扰素/类风湿因子使用两种新的基因产物中的任何一种来诱导细胞死亡。为了确定与干扰素/维甲酸诱导的细胞凋亡有关的关键分子，我们采取了一种遗传学方法，即根据基因的功能识别基因。在这里，我们鉴定了一种新的基因产物，与维甲酸干扰素诱导的死亡率-19(GRIM-19)相关的基因，它可以诱导细胞凋亡。GRIM-19的抗肿瘤作用在我们最近的研究中得到了进一步的强调，其中DNA病毒癌蛋白抑制了GRIM-19的凋亡功能。在某些人类肿瘤中，GRIM-19的表达似乎被抑制。这些特征表明其具有很强的抗肿瘤作用。为了更好地了解作用机制，我们使用了酵母2-杂交筛选，并鉴定了信号转导转录激活因子-3(STAT3)作为GRIM-19结合蛋白。最近的一些研究表明，STAT3是一种休眠的信号调节转录因子，在人类肿瘤中被结构性激活。许多病毒和细胞癌蛋白结构性地刺激其活性，促进细胞存活。根据我们的初步观察，我们假设GRIM-19抑制STAT3调控的转录。在这项建议中，我们将探讨GRIM-19诱导抑制肿瘤细胞生长的机制。特别是，我们将研究GRIM-19对一个结构性活性的STAT3变异体和一个致癌的Src的抗肿瘤作用，该变异体使用STAT3促进细胞生长。我们还将定义调解GRIM-19：STAT3相互作用的关键域。利用染色质免疫沉淀(ChIP)分析、STAT3调控的报告基因和缺失STAT3的突变细胞株，我们将探讨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)作为肿瘤对治疗药物反应的标志物；3)通过参与其他生长抑制途径抑制其他几种癌细胞；4)一旦其作用模式确定，将在基因治疗中发挥作用。因此，了解GRIM-19的作用将进一步有助于确定肿瘤细胞特异性功能障碍，从而消除这一新的生长调节途径。综上所述，本申请中提出的研究将揭示一种新发现的抗癌蛋白调节细胞生长的新机制。

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.