ABSTRACT
Nd2 antibody recognizes an antigen that is tumor specific in pancreas. It has been used successfully in clinical radioimmunodetection studies to identify exocrine pancreatic tumors. In the present study we show that the uptake of radiolabeled Nd2 antibody by SW1990 pancreatic carcinoma cells was increased by the adenyl cyclase activator, forskolin. Dibutyryl cyclic AMP and forskolin were both effective in increasing the level of Nd2 antigen in SW1990 cells. Immunoprecipitation studies showed that the Nd2 epitope is associated with MUC1 mucin. Forskolin increased Nd2/MUC1 antigen in both a membrane fraction and a high buoyant density mucin-like fraction. Nd2 immunoreactivity was reduced by treatment of mucins with proteases and beta-mercaptoethanol. Immunohistochemical studies showed that periodate catalyzed beta-elimination greatly reduced Nd2 immunoreactivity. These results suggest that the Nd2 epitope is unusual in having characteristics of both a peptide and a carbohydrate, protease and conformation sensitivities and involvement of O-linked oligosaccharides. Nd2 antibody does not react with several known pancreatic cancer antigens. In summary, activation of the cyclic AMP pathway increased cellular uptake of Nd2 antibody and the cellular expression of the tumor-specific, mucin-associated Nd2 antigen. These results suggest a means of improving the effectiveness of monoclonal antibodies in targeting tumor antigens for the diagnosis and treatment of malignancy.
Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/biosynthesis , Carcinoma, Pancreatic Ductal/metabolism , Colforsin/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Mucin-1/biosynthesis , Pancreatic Neoplasms/metabolism , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Carbohydrate Conformation , Carcinoma, Pancreatic Ductal/immunology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Endopeptidases/pharmacology , Enzyme Inhibitors/pharmacology , Epitopes/biosynthesis , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Glycosylation , Humans , Immunoenzyme Techniques , Mercaptoethanol/pharmacology , Molecular Sequence Data , Mucin-1/chemistry , Mucin-1/genetics , Mucin-1/immunology , Oxidation-Reduction , Pancreatic Neoplasms/immunology , Periodic Acid/pharmacology , Protein Conformation , Protein Processing, Post-Translational , Tumor Cells, CulturedABSTRACT
The carbohydrate antigen sialyl-Lewis(a) is important to pancreatic tumour biology because the circulating antigen is used in serological tests for malignancy and because cell surface antigen is involved in tumour cell binding to the endothelial adhesion molecule, E-selectin, in extravasation. In this study, we examined the effects of the adenylyl cyclase activator, forskolin, and the diacylglycerol analogue, phorbol 12-myristate 13-acetate (PMA), on the expression and release of sialyl-Lewis(a) in human pancreatic cancer cells. Increases in the release of sialyl-Lewis(a) from SW1990 cells produced by forskolin and PMA were associated with increases in the activities of protein kinases A and C, respectively, and could be blocked by inhibitors specific for these enzymes. Immunoprecipitation experiments showed that sialyl-Lewis(a) was associated with MUC1 mucin. Forskolin also increased the cellular content of antigen and MUC1 mRNA. Actinomycin D and a protein kinase A inhibitor, H8, blocked these effects. In contrast, PMA reduced cellular antigen and MUC1 mRNA levels, although it produced a temporary increase in release of the antigen. The effects of PMA were blocked by the protein kinase C inhibitor, H7. PMA also reduced cell binding to the adhesion molecule E-selectin. In summary, PKA and PKC alter cell MUC1-associated sialyl-Lewis(a) in opposite directions. These changes may have clinical utility in the diagnosis of pancreatic cancer and the prevention of metastases.
Subject(s)
Colforsin/pharmacology , Mucin-1/metabolism , Oligosaccharides/metabolism , Pancreatic Neoplasms/metabolism , Phorbol Esters/pharmacology , Blotting, Western , Cyclic AMP-Dependent Protein Kinases/metabolism , Dactinomycin/pharmacology , Humans , Protein Kinase C/metabolism , RNA, Messenger/metabolism , Sialyl Lewis X Antigen , Tumor Cells, CulturedABSTRACT
In this study we report that phorbol 12-myristate 13-acetate (PMA) transiently reduced the level of EGF receptor tyrosine phosphorylation in three pancreatic cancer cell lines (HPAC, SW1990, and UCVA-1) in response to EGF. The effect was maximal at 40-90 min. Pretreatment with the protein kinase C inhibitor GF 109203X reduced the PMA effect. Flow cytometry experiments showed that PMA produced only a slight reduction in the surface expression of EGF-R. The phosphotyrosine phosphatase inhibitor bpV(phen) returned phosphorylation to almost control levels. Moreover, homogenates of PMA treated pancreatic cells reduced the phosphorylation of activated receptor that was immunoprecipitated from A431 epidermoid cells. A combination of orthovanadate and NaF or bpV(phen) inhibited the effect of the homogenates. These results suggest that PMA activates a phosphotyrosine phosphatase activity that reduces the steady-state level of tyrosine phosphorylation of the receptor that is induced by EGF.
