Abnormal Expression Of Cytokeratins In Hepatocellular Carcinoma Cell Lines And Induction Of Cytoderatin 19 Expression By Laminin

In normal adult liver, the cytokeratin (CK) expression pattern in hepatocytes is different from that in ductal biliary cells. Intermidiate filament (IF) cytoskeleton of hepatocytes consists of only CK8 and CK18 ("hepatocyte type" CKs), while the bile ductal and ductular cells contain CK7 and CK19 ("bile duct type" CKs) hi addition to CKS and CK18. It was believed that the difference between these two types of liver cells in their CK patterns could be maintained under all pathological conditions, even after neoplastic transformation. Therefore, it appears that CKs can serve as important "lineage markers" in differential diagnosis of liver neoplasms. However, accumulating data indicates that CK pattern in hepatocytes can change under some pathological conditions. Expression of CK19, with or without CK7, can be observed in hepatocyte in diseased human livers, which is called abnormal CK expression. According to the data from our and other laboratories, abnormal deposition of extracellular matrix components, particularly laminin (LN) may play an important role in this process.In the present study, five approaches were used to clarify relationship between expression of CK19 and LN in six hepatocellular cell lines growing in vitro. (l)An immunocytochemical assay conducted demonstrate CKS, CK18, CK7, CK19, LN and LN receptor in hepatocellular carcinoma cell lines; (2) expression of CK19 and LN in hepatocellular carcinoma cell lines were cojocalized using a confocal laser scanning microscopy technique; (3) expression of CK18, CK7 and CK19 were also detected via Western blotting in heaptocellular carcinomacell lines; (4) expression of CK19 was induced by administration of purified LN protein into the culture medium in two hepatocelular carcinoma cell lines formerly negative for CK19; (5) polyclonal antibodies against LN can block the induction effect of LN on the expression of CK19 in hepatocellular carcinoma cell lines.It was found that CK8 and CK18 were expressed in all of the six cell lines examined. CK7 was also observed in all of them, but its level being different. CK19 was positive in four, and negative in two cell lines, which was correlative well to LN-immunoreactivity. The positive products of both molecules were demonstrated to be present in the same cells as demonstrated via laser confocal scanning microscopy. LN receptor was expressed in all of the six cell lines. With the LN protein added in culture medium, two hepatocellular carcinoma cell lines, formerly negative for CK19, began to express CK19. The reaction is dose-dependent. Polyclonal antibodies against LN that added in the cell culture medium could block the induction effect of LN.Our data have demonstrated that abnormal expression of CK7 and CK19 occurred frequently in HCC cell lines, providing cell models for further understanding the mechanism of the abnormal CK19 expression in hepatocytic cells. Our previous reports have suggested that CK19 expression in liver parenchyma cells is associated with abnormal LN deposition. The concept has been verified in this study by the following findings: (1) the correlative expression and colocalization of CK19 and LN in HCC cell lines expressing LN receptors; (2) the induction of CK19 in two cell lines formerly negative for both CK19 and LN by adding a certain amount of LN directly into the culture medium; and (3) the successful blockage of CK19 expression in two cells lines by a medium containing LN antibodies. These data are helpful for the further understanding of hepatocyte differentiation and hepatocarcinogenesis, and are important for differential diagnosis of liver neoplasms.