| IBP (IRF-4 binding protein, also know as: Def-6, SLAT) was identified during searching out the potential partner of the lymphoid-restricted transcription factor IRF-4. IBP is expressed in myeloid progenitors but down-regulated after induction of differentiation into macrophages, granulocytes, and erythrocytes. Murine IBP, which is expressed at high levels in lymphocyte, plays an important role in the development and maturation of Th2 cells and is recruited to the immunological synapse upon TCR stimulation. IBP-trapped mice display an obvious defect at the earliest stages of thymocyte development. It also has been found that IBP can regulate cell morphology in cooperation with activated Rac1 and influence cell differentiation in cooperation with integrin. Previous studies suggested a significant physiological role for IBP; however, the biological function of IBP in mammalian cells remains largely unexplored. IBP is postulated to be directly associated with cell survival. T cells from IBP-deficient mice display a defective ability to undergo apoptosis via a cell autonomous pathway. Abnormal cell survival and death have been tightly linked with oncogenesis. As yet, most of these studies were limited to the immune system. IBP function in tumorigenesis remains unclear, little is known about the expression or the regulation of IBP in human cancer in vivo. Therefore, we were interested in detecting the expression of IBP in colorectal cancer, which is the third most prevalent cancer in the world and it is an important cause of cancer-related death.Methods and results1. Preparation and characteration of anti-IBP polyclonal antibody.1)According to the bioinformatics analysis, we cloned, expressed and purified the most idio-fragment of IBP(aa 410-631). Polyclonal antiserum was raised in rabbits according to the modified method.2)The IgG fractions of Polyclonal antiserum were purified using Protein A-Sepharose。3)To remove antibodies reacting to the Trx tag and residual bacterial protein, the antibodies eluted from the Protein A column were passed sequentially over CNBr-activated Sepharose 4 Fast Flow to which a bacterial protein, expressed from a empty pET-32a vector, had been coupled according to the instructions provided by the manufacturer.4)These antibodies show high specificity and sensitivity against IBP in ELISA, western blot and ICC.2. Expressed of IBP in colon cancer1)We examined IBP expression in four human colon cancer cell lines by RT-PCR . High expression of IBP can be detected at the mRNA level in SW480 and SW620 cells, lower expression can be detected in HT29 cells, but not in LoVo cells.2)We examined IBP expression in four human colon cancer cell lines by western blot. Consist with the RT-PCR result, high expression of IBP can be detected at the protein level in SW480 and SW620 cells, lower expression can be detected in HT29 cells, but not in LoVo cells.3)Confocal analysis was employed to locate IBP using anti-IBP polyclonal antibodies. IBP is predominantly localized in the cytoplasm of SW480 cells. Immunostaining of IBP in nuclear was not observed4)Colorectal cancer tissues and normal colorectal tissues were collected from 63 patients undergoing surgical resections of primary colorectal cancer, as well as adenoma tissues were collected form 15 patients at the Southwest Hospital affiliated to Third Military Medical University between January 2005 and 2008. IBP expression was detected by IHC with anti-IBP polyclonal antibodies.5)Pearson'sχ2 test was performed to evaluate the IBP expression in different colorectal specimens. All the analyses were performed with SPSS software. A value of p < 0.05 was regarded as statistically significant.In this study, first, we developed anti-IBP antibodies with the most unique portion of IBP, and purified them by affinity chromatography. These antibodies show high specificity and sensitivity against IBP in ELISA, western blot and ICC. Consistent with previous reports, our result confirmed the high expression of IBP in immune cells. Second, IBP expression was evaluated in four human colon cancer cell lines. We demonstrated that expression of IBP can be detected at both mRNA and protein levels in three human colon cancer cell lines detected. Confocal analysis indicated that IBP is predominantly located in cytoplasm of cancer cells. These data will be useful in the future study of the relevance of IBP function in colorectal cancer progression and tumor behavior. Third, the expression of IBP was evaluated in the cancer tissue compared with paired normal tissue of the same patient, as well as 15 cases of adenoma tissues. Overexpression of IBP was detected in colorectal cancer tissues and correlated strongly with the grades of differentiation of the tumors. But the expression of IBP was undetectable in neither non-cancerous surrounding tissues nor 15 adenoma tissues. In summary, we propose that expression of IBP may correlate to the occurrence and progress of human colorectal cancer. The biological role of IBP may not be merely limited to the immune system. IBP might be a novel promising tumor marker of human colorectal cancer. The mechanisms of the overexpression of IBP in human colorectal cancer, and the biological function of IBP in human colorectal cancer or a larger variety of other human tumors need further explanation. Moreover, the novel antibodies we produced provide useful tool for both basic research and clinical studies.
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