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的抗增殖功能是其抑瘤活性的基础。RB的抗凋亡功能可能促进肿瘤的发展，然而，这方面的RB活性尚未被研究。我们已经利用我们所产生的小鼠品系获得了RB促进肿瘤潜力的体内证据。在这个小鼠品系中，我们突变了Rb基因的两个密码子，使Rb的C端caspase切割位点失活。由此产生的Rb-MI等位基因编码抗caspase的Rb-MI蛋白，保护细胞免受肿瘤坏死因子（TNF）诱导的细胞凋亡。TNF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tnf诱导的Rb-MI细胞的Bid切割受损，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tnf依赖性凋亡具有抗性。此外，Rb-MI在p53-null遗传背景下促进自发性结肠肿瘤。这些结果表明，Rb- ml介导的对tnf诱导的细胞凋亡的保护作用与肠道肿瘤的发生之间存在很强的相关性。TNF是一种炎性细胞因子，协调对感染和损伤的全身反应。作为一种可以杀死肿瘤细胞的生理因子，TNF已被证明也能促进肿瘤生长，特别是那些与肝脏和肠道慢性炎症相关的肿瘤生长。TNF在肿瘤促进与肿瘤抑制之间转换的机制尚不完全清楚。我们提出rb依赖性抗凋亡机制，在Rb-MI细胞中组成性激活，可以促进炎症条件下TNF依赖性肿瘤的发展。为了验证这一假设，我们将追求四个具体目标：目的1：在小鼠模型中研究Rb-MI对炎症相关肿瘤发展的影响；目的2-探讨caspase-8缺乏对炎症相关性结肠癌的影响；目的3-表征RB和RB- mi的蛋白结合功能；目的：探讨RB对caspase-8的调控作用。该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。拟议研究的结果将有助于制定新的策略来降低与炎症相关的癌症的风险。

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.