Expression of the 40 kD protein in DLD-1 colon cancer cells and the effect of cytokines

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Abstract
We recently reported the presence of an organ-specific 40 kD colonic protein which acts as an autoantigcn(s) in patients with ulcerative colitis. Using a specific monoclonal antibody directed against 40 kD protein (7E12H12, IgM isotype), in conjunction with immunocytochemistry and flow cytometry. we examined the presence of the 40 kD protein on human colon cancer cells. DLD-1, and also characterized the ability of cytokines. IFN-y and tumour necrosis factor, to modulate the expression of this protein on these tumour cells. The presence of the 40 kD protein was localized to the plasma membrane; less was present within the cytoplasm. Following exposure to IFN-γ (10–1000 Lty ml). DLD-1 colon tumour cells showed a dose- and time-dependent increase in 7E12H12 antibody associated immunofluorescence. with the maximum 7H12H12 antibody binding observed with 100 U/ml IFN-γ at 48 h. In contrast, tumour necrosis factor did not alter the levels of anti-40 kD antibody binding over that of control cells. Since IFN-γ is also known to induce class II major histocompatibility antigens, we examined the possibility of cross-reactivity of HLA class II antigens and Mr40 kD epitope. Neither pre-incubation of DLD-1 colon tumour cells with anti-HLA class II antibodies followed by 7E12H12 nor co-incubation of both antibodies altered the amount of 7E12H12 antibody binding. Using a direct ELISA, a highly enriched preparation of M, 40 kD protein reactive to anti-40 kD antibody did not react with HLA class II antibodies. The present results suggest that 40 kD protein is present on DLD-1 human colon tumour cells and that although the 40 kD protein epitope expression is increased by the lymphocyte-derived cylokine, IFN-y. the epitope is separate and distinct from the class II HLA antigens. Further studies on the 40 KD protein may elucidate its autoantigenic role in the pathogencsis of inflammatory bowel disease.