intrinsic-factor and Colonic-Neoplasms

A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 x 10(9) 1/mol at pH 7.4 and 4 degrees C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4 degrees C and at 37 degrees C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.

Topics: Chloroquine; Chromatography, Gel; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Endocytosis; Humans; Intrinsic Factor; Kinetics; Receptors, Cell Surface; Tumor Cells, Cultured; Vitamin B 12

Human colon adenocarcinoma (Caco-2) cells express both intrinsic factor-cobalamin receptor and transcobalamin II (TC II). The expression of these activities began to rise by day 6 and reached peak levels between 10 and 15 days in culture. The postconfluent Caco-2 cell membranes bound approximately 30-35 fmol of intrinsic factor (IF) [57Co]Cbl/mg protein. The size of the mature receptor expressed in the apical brush border had a relative molecular mass of 230 kDa. The intracellular form of TC II had a Mr of 43, 5 higher than the secreted form of TC II. TC II was secreted unidirectionally via the basolateral direction when Caco-2 cells were grown on culture inserts. When grown on culture inserts, the Caco-2 cells were polarized (electrical resistance greater than 200 omega/cm2) and transcytosed [57Co]Cbl bound to IF from apical-to-basal but not from basal-to-apical direction. Under these conditions, [57Co]Cbl complexed to haptocorrin was not transported. These cells also transcytosed free [57Co]Cbl, although less efficiently. The [57Co]Cbl transcytosed using either IF[57Co]Cbl or free [57Co]Cbl as ligands was bound exclusively to TC II. Intracellular [57Co]Cbl decreased during transcytosis with a slow (t1/2 = 4 h) transfer of [57Co]Cbl from IF to TC II. These results show that the transport of Cbl in Caco-2 cells is very similar to the human enterocyte system.

Topics: Adenocarcinoma; Biological Transport; Cell Line; Colonic Neoplasms; Electrophoresis, Polyacrylamide Gel; Humans; Intrinsic Factor; Kinetics; Methionine; Molecular Weight; Sulfur Radioisotopes; Transcobalamins; Vitamin B 12

Caco-2 cells grown on 0.45-micron filters, in Millicell chambers, form intact monolayers with many of the properties of polarized intestinal epithelial cells. It is reported here that these cells bind and internalize intrinsic factor-cobalamin complexes and that after 14-28 days in culture this specific binding is exclusively located on the apical membrane. Caco-2 cells also synthesize and secrete a protein with properties similar to transcobalamin II. This protein is secreted from the basolateral side of the cells after 20 days in culture. Specific apical-to-basolateral transcellular transport of [57Co]cobalamin also occurs between 20 and 28 days in culture. Thus, Caco-2 cells provide the first polarized human cell system for studying the transepithelial transport of cobalamin.

Topics: Biological Transport; Carcinoma; Cell Line, Transformed; Chromatography, Ion Exchange; Colon; Colonic Neoplasms; Electric Conductivity; Epithelium; Humans; Intrinsic Factor; Microscopy, Electron; Protein Binding; Time Factors; Tumor Cells, Cultured; Vitamin B 12

Topics: Anemia; Anemia, Hypochromic; Anemia, Macrocytic; Carbachol; Celiac Disease; Cobalt Isotopes; Colonic Neoplasms; Duodenal Ulcer; Folic Acid; Folic Acid Deficiency; Gastric Acidity Determination; Gastric Juice; Histamine; Humans; Insulin; Intrinsic Factor; Male; Pharmacology; Physiology; Sprue, Tropical; Vitamin B 12