Abstract
Introduction: Secretion of carcinoembryonic antigen (CEA) is an indicator of poor prognosis for colorectal cancer. Circulating CEA is removed by receptor mediated endocytosis by Kupffer cells and this results in an inflammatory response characterized by a cytokine cascade (IL-1, IL-6, IL-10 and TNF-α) that increases the metastatic potential of the cancer cells. The transcription factor c-jun has been shown to be a binding partner with the CEA receptor hnRNP M. There is also activation of the AP-1 transcription factor in Kupffer cells exposed to CEA. We have used differentiated THP-1 macrophages as a model for CEA induced macrophage activation and have demonstrated hnRNP M on the surface of these terminally differentiated cells. Exposure to CEA results in production of cytokines and these influence the ability of CEA producing cancers to implant and grow in the liver. Methods: THP-1 a human monocyte cell line was differentiated overnight to produce a macrophage phenotype with phorbol myristate acetate (PMA) (200ng/ml). Differentiated THP-1 cells were treated with CEA (2μg?ml) and the cells harvested at a number of time points between 0 and 4 hours. Cells were extracted following treatment at these various times, with RIPA buffer containing a cocktail of protease and phosphatase inhibitors. These extracts were run on one or 2-D SDS-PAGE and transferred to membranes for Western blotting. Membranes were probed with antibodies against c-jun, phospho c-jun, JNK 1,2 and 3, p38, phospho-tyrosine and phospho serine/threonine. Results: We showed a significant down regulation of c-jun expression on treatment of the differentiated cells with CEA while an increase in c-jun phosphorylation was observed. JNK-1, and 2 expressions also increase with CEA treatment that is consistent with phosphorylation of c-jun. JNK-3 levels however, decrease following CEA binding. There was also a down regulation of the JNK pathway protein p38. These data point to role for the JNK/c-jun/AP-1 system in signaling by hnRNP-M4. Because hnRNP M4 has a tyrosine phosphorylation motif in its N-terminal domain we have examined the effect of CEA binding on phosphorylation at this locus. Use of western blotting showed increased tyrosine phosphorylation of both isoforms of hnRNP M (M3 and M4). Using 2D electrophoresis we have shown changes in the isoelectric point of the receptor isoforms that would be consistent with phosphorylation. Use of phospho-serine/threonine antibodies showed phosphorylation of 3 proteins distinct from hnRNP M in differentiated THP-1 cells. CEA treatment causes a decline in one band and an increase in phosphorylation of the other two. We are presently attempting to identify these proteins. Conclusions: There is involvement of the c-jun pathway in the initiation of macrophages by CEA to produce cytokines and involves up regulation of JNK-1 and 2. Activation of macrophages by CEA probably involves the transcription factor AP-1. Both isoforms of the receptor appear to be phosphorylated on the consensus tyrosine located in the N-terminal domain. These pathways may be potential targets for intervention to prevent implantation of CEA producing colorectal cancer cells in the liver.
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