Retinoblastom protein, lacking in any catalytic domain, is a scaffolding protein. It functions through bringing protein complexes together and has been shown to regulate cells processes such as cell cycle progression and programmed cell death, apoptosis. Regulation of RB and its binding partners occurs in a variety of ways. In the E2F transcription factor case, RB is hyperphosphorylated to induce intra-domain interaction thus releasing E2F. Other avenues to regulating RB includes the process of TNF induced apoptosis which proceeds with a specific caspase cleavage of the last 42 amino acids of RB. By applying mass spectrometry and a number of in vitro binding assays, we have identified several proteins that interact with the C-terminal region of RB. Our results demonstrate that survivin and [Beta]-catenin interact with the C-terminal portion of RB. Nonmuscle Myosin Heavy Chain II A has been shown to specifically interact with the terminal 42 amino acids of RB. Our results demonstrate multiple binding partners of RB C-terminal region; a region that is relevant to apoptosis regulation

Abstract Background The intestinal crypt homeostasis is maintained by a combination of growth factors including Wnt, R-Spondin1, Noggin and the epidermal growth factor (EGF). In human colorectal cancer, the Wnt pathway is constitutively activated through genetic and epigenetic alterations in as many as 11 genes encoding components of this crypt stem-cell maintenance mechanism. Although the proliferation of colon cancer cells does not require Wnt, it is possible that colon cancer cells can still respond to the crypt growth factors in the colonic microenvironment. A number of studies have shown that epithelial cells behave differently in 3-D versus 2-D cultures. Because the 3-D conditions more closely mimic the in vivo environment, we examined the effects of Wnt and other crypt growth factors on colon cancer cell growth in 3-D culture. Methods Colon cancer cells were grown in 3-D matrigel supplemented with different combinations of crypt growth factors and colonies were examined for morphology and pathways. Results When colon cancer cells were cultured in 3-D with EGF, they grew as round spheroid colonies. However, colon cancer cells also grew as flat, disc-like colonies when cultured with EGF plus Wnt, R-Spondin1 and Noggin. Disc colonies were found to have comparable levels of E-cadherin as the spheroid colonies, but showed decreased E-cadherin at the cell-matrix contact sites. Disc colonies also elaborated F-actin rich protrusions (FRP) at the cell-matrix edge, reminiscent of an invasive phenotype but without the expression of vimentin. These E-cadherin and F-actin alterations were not induced by the four growth factors in 2-D culture. Formation of the disc colonies was inhibited by the knockdown of β-catenin and by protein kinase inhibitors such as gefitinib, imatinib and MK-2206. Furthermore, withdrawal of the crypt growth factors was able to revert the disc colonies to spheroid growth, showing that the invasive phenotype was reversible dependent on the availability of growth factors. Conclusions These findings show that colon cancer cells remain responsive to the growth factors in the crypt microenvironment and can be induced to undergo morphological transformation in the more physiologically relevant 3-D culture.