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.
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