N-glycosylation is required for the surface localization of MUC17 mucin.

The nucleic acid sequence of the human gene, MUC17, indicates that this mucin contains an SEA domain, a transmembrane domain, and putative N-glycosylation sites in the carboxyl terminus. Mucins that possess an SEA domain are usually proteolytically cleaved within that domain to yield two subunits, the smaller of which is associated with the surface membrane. Homogenates of ASPC-1 pancreatic cancer cells showed three main bands of immunoreactivity with alpha-SEA (a polyclonal antibody directed against a site downstream of the postulated cleavage site) after SDS-PAGE and Western blotting (38, 45, and 49 kDa). Experiments utilizing N-glycan specific hydrolases showed that the 38 kDa band contained high mannose glycans whereas the 45 and 49 kDa bands contained complex-type glycans. Only two smaller alpha-SEA reactive bands (30 and 32 kDa) were present after cells had been treated with the N-glycosylation inhibitor tunicamycin. Surface biotinylation studies showed that only the forms possessing complex-type N-glycans were localized to the cell surface. Both tunicamycin and brefeldin A, an inhibitor of protein transport, reduced surface localization. In summary, our results indicate that the surface localization of the smaller subunit of MUC17 is dependent on its N-glycosylation status.

Methylation status of promoters and expression of MUC2 and MUC5AC mucins in pancreatic cancer cells.

Anomalous expression of certain types of mucins occurs in pancreatic tumors, but little is known about the causes. MUC2 and MUC5AC are not expressed in normal pancreas. In the present study an immunohistochemical screening showed that MUC2 antigen was expressed in 6% of invasive tumors. Of the pancreatic cell lines, 2.4% expressed MUC2 message and antigen. In contrast, MUC5AC antigen was expressed in 86-100% of invasive adenocarcinoma (depending on the antibody). MUC5AC message and antigen were expressed in 66.7% of the cell lines tested. A polymerase chain reaction based assay was used to determine if methylation of CpG sites immediately 5' of the transcription initiation sites of the MUC2 and MUC5AC genes could be related to mucin expression in the cell lines. Digestibility by the methylation sensitive restriction enzyme HpaII correlated with the presence or absence of mRNA in 100% and 77.8% of the cell lines for MUC2 and MUC5AC, respectively. Treatment of a cell line that did not express MUC2 or MUC5AC gene products with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in an increase in MUC2 message but no change in MUC5AC message. In summary, the expression of MUC2 gene products correlated well with methylation of the proximal region of the promoter whereas expression of MUC5AC may involve additional regions or other mechanisms.

Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas.

BACKGROUND & AIMS: It has recently been suggested that infiltrating adenocarcinoma of the pancreas arises from histologically well-defined precursor ductal lesions called pancreatic intraepithelial neoplasia (PanIN-1A, -1B, -2, and -3). This study examined alterations in the pattern and the level of expression of several mucin genes (MUC1, MUC2, MUC5AC, and MUC6) and mucin-associated tumor antigens (Nd2 and sialyl Tn) in these precursor lesions. METHODS: We examined 139 PanINs and 68 infiltrating ductal adenocarcinomas of the pancreas by using immunohistochemistry and in situ hybridization methods. RESULTS: Overexpression of MUC1, a pan-epithelial mucin, and MUC6, a pyloric-gland mucin, and de novo expression of MUC5AC, a gastric foveolar mucin, was observed in all stages of PanINs and invasive ductal adenocarcinoma. In contrast, the expression of mucin-associated carbohydrate antigen, sialyl Tn, was markedly increased only in PanlN-3 and invasive ductal adenocarcinoma. In addition, a decrease in the expression of these mucin-associated peptide and carbohydrate antigens was correlated with the degree of differentiation of the tumor. CONCLUSIONS: Expression of both gastric-foveolar and pyloric-gland mucin in PanINs is an early event, whereas sialyl Tn expression is a late event in the recently defined progression model of pancreatic carcinogenesis. This altered mucin gene expression provides new insight into the role of cell lineage-associated metaplasia in pancreatic carcinogenesis.

Granulocyte-colony stimulating factor enhances chimeric antibody Nd2 dependent cytotoxicity against pancreatic cancer mediated by polymorphonuclear neutrophils.

Nd2 is a monoclonal antibody against pancreatic cancer. We have previously reported that human/mouse chimeric antibody Nd2 (c-Nd2) can induce antibody-dependent cell-mediated cytotoxicity (ADCC) with peripheral blood mononuclear cells (PBMs) as effectors. In this study, we investigated whether c-Nd2 can induce ADCC with poly-morphonuclear neutrophils (PMNs) as effector cells and the effects of granulocyte-colony stimulating factor (G-CSF) in enhancing this cytotoxicity. Cytotoxicities for pancreatic cancer cell line, SW1990 were dose-dependently increased by c-Nd2 during co-culture with PMNs and these cytotoxicities were significantly suppressed by the addition of neutralizing antibodies against CD16, which is Fcgamma receptor expressed on PMN membranes. PMNs treated with G-CSF significantly enhanced in vitro ADCC activity against SW1990 induced by c-Nd2. The in vivo growth of subcutaneously transplanted SW1990 tumor in nude mouse was significantly inhibited by i.p. administration of c-Nd2 compared to control (non-specific IgG1). In addition, this inhibitory effect was enhanced by the combination of c-Nd2 and G-CSF. Immunohistochemical study with anti-mouse neutrophil elastase antibody demonstrated strong infiltrations of PMNs into and around the transplanted tumor, treated with c-Nd2 and G-CSF. These results suggest that PMNs play an important role in c-Nd2 inducing ADCC and that combination immunotherapy of c-Nd2 with G-CSF may have clinical applications in the treatment of patients with pancreatic cancer by enhancing ADCC.

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP.

MUC5AC mucin is not expressed in normal pancreas but is expressed in tumors. Little is known about the mechanisms that lead to this atypical expression. In this study, we demonstrate that stimulation of adenylyl cyclase and the protein kinase A (PKA) pathway by forskolin and vasoactive intestinal peptide (VIP) increased MUC5AC antigen expression and release from pancreatic cancer cells. Stimulation of the PKA pathway also increased MUC5AC mRNA. When SW1990 pancreatic cancer cells were grown on porous membranes they released MUC5AC mucins apically in response to VIP (10(-7) M) applied to their basolateral surfaces. SW1990 cells, as have been reported for other pancreatic cancer cells, have high affinity (<10(-7) M) VIP receptors and low affinity (>10(-6) M) secretin receptors. We also showed that four antibodies (CLH2, 21M1, 45M1, and Nd2) react with MUC5AC antigen in different cellular compartments of both tissues and cultured cells. In conclusion, the PKA pathway may contribute to the up-regulation of MUC5AC expression seen in pancreatic tumors.