Biological Function of the Retinoblastoma Gene Product

描述(申请人提供)：视网膜母细胞瘤蛋白，Rb，是细胞增殖和凋亡的负调节因子。Rb的抗增殖作用是其抑瘤活性的基础。Rb的抗细胞凋亡作用应该会促进肿瘤的发展，然而，Rb在这方面的活性还没有被研究。我们已经使用我们培育的一种小鼠品系获得了RB促进肿瘤潜能的体内证据。在这个小鼠品系中，我们突变了Rb基因中的两个密码子，以使Rb C末端的caspase裂解位点失活。由此产生的Rb-MI等位基因编码一种抗caspase的Rb-MI蛋白，该蛋白可以保护细胞免受肿瘤坏死因子(TNF)诱导的凋亡。肿瘤坏死因子激活caspase-8裂解Bid，导致线粒体渗漏和细胞死亡。我们已经证明，在RB-MI细胞中，肿瘤坏死因子诱导的BID裂解受到损害，导致死亡反应减少。在体内，RB-MI小鼠肠上皮细胞对内毒素诱导的肿瘤坏死因子依赖性细胞凋亡具有抵抗作用。此外，RB-MI在p53缺失的遗传背景下促进自发性结肠肿瘤。这些结果建立了Rb-m1介导的对肿瘤坏死因子诱导的细胞凋亡的保护作用与肠道肿瘤的发展之间的强烈相关性。肿瘤坏死因子是一种炎性细胞因子，协调全身对感染和损伤的反应。作为一种可以杀死肿瘤细胞的生理因子，肿瘤坏死因子被证明也能促进肿瘤的生长，特别是那些与肝脏和肠道的慢性炎症相关的肿瘤。肿瘤坏死因子在促进肿瘤和抑制肿瘤之间的转换机制尚不完全清楚。我们认为，在RB-MI细胞中被结构性激活的RB依赖的抗凋亡机制可以在炎症条件下促进肿瘤生长依赖于肿瘤坏死因子。为了验证这一假说，我们将追求四个特定的目标：目的1-研究Rb-MI对炎症相关肿瘤小鼠模型发展的影响；目的2-研究caspase-8缺陷对炎症相关结肠癌的影响；目的3-表征Rb和Rb-MI的蛋白结合功能；目的4-研究Rb对caspase-8的调节。这项拟议的研究将产生新的结肠癌小鼠模型，并阐明Rb依赖的抗凋亡途径。拟议的研究结果将有助于开发新的策略来降低炎症相关癌症的风险。

DESCRIPTION (provided by applicant): The retinoblastoma protein, RB, is a negative regulator of cell proliferation and apoptosis. The anti- proliferation function of RB underlies its tumor suppression activity. The anti-apoptotic function of RB should promote tumor development, however, this aspect of the RB activity has not been investigated. We have obtained in vivo evidence for the tumor promoting potential of RB using a mouse strain we have generated. In this mouse strain, we mutated two codons in the Rb gene to inactivate a caspase cleavage site at the C- terminus of RB. The resulting Rb-MI allele encodes a caspase-resistant RB-MI protein that protects cells from tumor necrosis factor (TNF)-induced apoptosis. TNF activates caspase-8 to cleave Bid and thus causing mitochondria leakage and cell death. We have shown that TNF-induced Bid cleavage is impaired in Rb-MI cells, resulting in a reduced death response. In vivo, the intestinal epithelial cells of Rb-MI mice are resistant to TNF-dependent apoptosis induced by endotoxin. Furthermore, Rb-MI promotes spontaneous colon tumors in the p53-null genetic background. These results establish a strong correlation between Rb- Ml-mediated protection from TNF-induced apoptosis and the development of intestinal tumors. TNF is an inflammatory cytokine that orchestrates the systemic response to infections and injuries. Discovered as a physiological factor that can kill tumor cells, TNF has been shown to also promote tumor growth, particularly those associated with chronic inflammation in the liver and the gut. The mechanisms underlying the switch between tumor-promotion versus tumor-suppression by TNF are not fully understood. We propose that the RB-dependent anti-apoptotic mechanism, which is constitutively activated in Rb-MI cells, can promote TNF- dependent tumor development under conditions of inflammation. To test this hypothesis, we will pursue four specific aims: Aim 1-To investigate the effect of Rb-MI on inflammation-associated tumor development in mouse models; Aim 2- To investigate the effect of caspase-8 deficiency on inflammation-associated colon cancer; Aim 3- To characterize the protein binding functions of RB and RB-MI; Aim 4-To investigate the regulation of caspase-8 by RB. The proposed study will generate new mouse models for colon cancer and elucidate the RB-dependent anti-apoptotic pathway. Results from the proposed research will facilitate the development of new strategies to reduce the risk of inflammation-associated cancer.