sodium-dodecyl-sulfate and Pancreatic-Neoplasms

Although the hamster pancreas does not express A, B or H blood group antigens, all hamster pancreatic ductal adenocarcinomas induced by treatment with N-nitrosobis(2-oxopropyl)amine express blood group-A antigen. Thus, the acquisition of blood group-A antigen expression in this system is a cancer-associated alteration. We have purified three major blood group-A antigen bearing glycoproteins (gp120, gp135 and gp150) from hamster pancreatic cancer cell membrane preparations using affinity chromatography on DBA (Dolichos biflorus) agglutinin-agarose. When assayed by immunoblotting, gp120 and gp135 showed strong blood group-A reactivity, which was removed by treating membrane samples with peptide-N-glycosidase F. Blood group-A reactivity was unchanged by treatment of the membrane fractions with endoglycosidases F and H. In addition, these two glycoproteins bearing blood group-A antigen also bound L-PHA (Phaseolus vulgaris leucoagglutinin). These results demonstrate that gp120 and gp135 express blood group-A antigen on Asn-linked multi-antennary complex type glycan structures. The gp150 showed weak blood group-A expression. This is the first demonstration of the neoexpression of cancer-associated blood group-A determinants which reside on Asn-linked glycan structures.

Topics: ABO Blood-Group System; Animals; Antigens, Neoplasm; Carcinogens; Carcinoma, Intraductal, Noninfiltrating; Cricetinae; Electrophoresis, Polyacrylamide Gel; Epitopes; Glycoside Hydrolases; Immunoblotting; Lectins; Membrane Glycoproteins; Nitrosamines; Oligosaccharides; Pancreas; Pancreatic Neoplasms; Plant Lectins; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

The subcellular localization and biochemical characteristics of blood group A antigen were studied by immunogold methods and by SDS-PAGE and Western blotting procedures in N-nitrosobis)2-oxopropyl)amine (BOP)-induced pancreatic cancer (PC) in Syrian hamsters, in the pancreatic cancer cell line (PC-1) derived from a primary induced pancreatic cancer, and in intrapancreatic and subcutaneous transplants of PC-1 cells. Normal hamster duodenal epithelial cells expressing A antigen were compared with the normal hamster pancreas (lacking A antigen), human PC tissues from patients with blood group A and human PC cell lines. Blood group A antigen was present on the membrane of hamster duodenal cells, but was absent in the normal pancreatic cells. A antigen was localized mainly on the cell membrane of the hamster cancer cells both in vivo and in vitro. Glycoproteins with blood group A specificity were observed by SDS-PAGE and Western blotting procedures in the membrane fraction of PC-1 cells, with a major component of molecular mass of approximately 120 kd. Similar migration patterns were observed in the primary induced PC and in subcutaneous and intrapancreatic transplants of PC-1 cells. Membrane preparations from cell lines derived from two primary pancreatic cancers from patients of blood group A and from human pancreatic cell lines, CD11 and CD18, showed a major A reactive component with a molecular mass similar to that found in the hamster PC cells. These findings suggest that: (i) both the hamster and human PC cells in vitro produce glycoproteins with blood group A specificity of similar molecular masses; (ii) differences exist in the structure of the glycoprotein immunoreactive with the anti-A antigen between the normal and cancerous cells; and (iii) differences exist in the molecular mass of the anti-A reactive substance between hamsters and human PC cells and between tissues in vivo and in vitro.

Topics: ABO Blood-Group System; Adenocarcinoma; Animals; Antibodies, Monoclonal; Antigens, Differentiation; Blotting, Western; CD11 Antigens; CD18 Antigens; Cell Membrane; Cricetinae; Duodenum; Electrophoresis, Polyacrylamide Gel; Humans; Immunohistochemistry; Injections, Subcutaneous; Microscopy, Electron; Neoplasm Transplantation; Nitrosamines; Pancreas; Pancreatic Neoplasms; Receptors, Leukocyte-Adhesion; Sodium Dodecyl Sulfate; Subcellular Fractions; Tumor Cells, Cultured

The combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peanut agglutinin lectin blotting has been used to identify a pancreatic cancer-related glycoprotein in serum. Its high molecular weight and its lectin-binding characteristics suggest that it probably is a mucin. Comparison with immunoblotting with Ca 19-9 antibody suggests that the epitopes for Ca 19-9 and peanut agglutinin may be variably expressed on different sites of the same mucin. The peanut agglutinin-binding glycoprotein was demonstrated in 12 of 34 pancreatic cancer sera, including four with normal Ca 19-9 assay, and none of 96 control sera, including sera from patients with obstructive jaundice and other cancers. These preliminary results suggest that it may become a useful addition to the current serum markers for pancreatic cancer. The technique of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and lectin blotting may well prove valuable in the search for other cancer-related glycoprotein markers.

Topics: Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Humans; Immunoassay; Lectins; Molecular Weight; Neoplasm Proteins; Pancreatic Neoplasms; Peanut Agglutinin; Sodium Dodecyl Sulfate

To clarify the difference in the protein composition of pancreatic juice between patients with pancreatic cancer and normal controls, the proteins of pure pancreatic juice from three cases of cancer of the head of the pancreas and six apparently healthy adults were analyzed by two-dimensional gel electrophoresis (two-DE) followed by silver staining. Two minor proteins of Mr 59,000 and Mr 78,000 present in all the three patients were not detected in the six controls. We have identified the minor proteins with Mr 59,000 and Mr 78,000 as alpha 1-antitrypsin and transferrin, respectively, using Western blotting with anti-alpha 1-antitrypsin and anti-transferrin antibodies. In addition, serum albumin also identified by antibody binding was abundantly present in patients compared to controls. The increase in the amount of serum albumin in the patient was also confirmed by a quantitative analysis using SDS-PAGE and gel densitometry. The data indicate that not only serum albumin but also alpha 1-antitrypsin and transferrin are increased in the pancreatic juice of the patients with pancreatic cancer, as compared with apparently healthy adults. The data also suggests that the analysis of pancreatic juice proteins by two-DE with silver staining is useful for the diagnosis of pancreatic diseases.

Topics: Adenocarcinoma; alpha 1-Antitrypsin; Amylases; Carboxypeptidase B; Carboxypeptidases; Carboxypeptidases A; Densitometry; Electrophoresis; Electrophoresis, Polyacrylamide Gel; Enzyme Precursors; Humans; Lipase; Pancreatic Juice; Pancreatic Neoplasms; Proteins; Serum Albumin; Sodium Dodecyl Sulfate; Staining and Labeling; Transferrin

Topics: Electrophoresis, Polyacrylamide Gel; Enzymes; Humans; Isoelectric Focusing; Neoplasm Proteins; Pancreatic Juice; Pancreatic Neoplasms; Proteins; Sodium Dodecyl Sulfate