Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.5 (5'-nucleotidase)
 3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human pancreatic exocrine adenocarcinoma cells established in tissue culture expressed both HLA and beta 2-microglobulin (beta 2M). Plasma-membrane components of this pancreatic cancer cell line were purified from plasma membrane fractions enriched by sucrose density-gradient centrifugation, using immunoaffinity chromatography on immobilized anti-human beta 2M antibody. Both rabbit and mouse monoclonal anti-beta 2M IgG were used, with a 20--25-fold overall purification of 5'-nucleotidase. The method was applicable to 5 x 10(7) cells and permitted the solubilization of membranes retained on the column, with the selective desorption of components not associated with beta 2M before the subsequent elution at pH 3 of beta 2M-associated macromolecules. The acid eluate contained one major and two minor bands in the 40--45,000 mol.-wt range with two additional enriched components of 18,000 and 22,000 dalton. A major carbohydrate-containing component of high mol. wt was also found to be associated with the pancreatic cancer-cell plasma membrane.
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PMID:Isolation of plasma-membrane components from cultured human pancreatic cancer cells by immuno-affinity chromatography of anti-beta 2M sepharose 6MB. 9 30

Differences in cell morphology, concanavalin A-induced receptor redistributions, and the cooperativity of the inhibition of 5'-nucleotidase (AMPase) by concanavalin A (Con A) have been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma cells treated with microfilament- and microtubule-perturbing drugs. By scanning electron microscopy MAT-C1 cells exhibit a highly irregular surface, covered with microvilli extending as branched structures from the cell body. MAT-A, MAT-B, and MAT-B1 cells have a more normal appearance, with unbranched microvilli, ruffles, ridges, and blebs associated closely with the cell body. MAT-C cells have an intermediate morphology. Treatment of MAT-A, MAT-B, or MAT-B1 cells with Con A causes rapid redistribution of Con A receptors. Both cytochalasins and colchicine cause alternations in the receptor redistributions. Receptors on MAT-C1 cells are highly resistant to redistribution, even in the presence of cytoskeletal perturbant drugs. The cooperativity of the inhibition of AMPase by Con A was investigated in MAT-A and MAT-C1 cells. Untreated cells exhibit no cooperativity. If either subline is treated with colchicine, cytochalasin B or D, or dibucaine, cooperativity is observed. Lumicolchicine has no effect. Theophylline or dibutyryl cyclic AMP prevents the effects of either colchicine or cytochalasin. The concentration required for half-maximal induction of cooperativity is 0.3--0.4 microM for both colchicine and cytochalasin D, which is in the appropriate range for specific microtubule and microfilament disruptions. The effectiveness of the cytochalasins (E greater than D greater than B) is consistent with their known effects on microfilaments. No direct correlation was observed between the induction of cooperativity and drug-induced changes in Con A receptor redistribution or cell morphology. The morphology of MAT-A cells is grossly altered by cytochalasins or dibucaine and somewhat less by colchicine. MAT-C1 cells exhibit more minor alterations in morphology as a result of these drug treatments. The results of this study indicate that the inhibition of AMPase, which is a Con A receptor, is a different process from the redistribution of the bulk of the Con A receptors, possibly short range membrane interactions rather than global effects on the cell.
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PMID:Effects of cytoskeletal perturbant drugs on ecto 5'-nucleotidase, a concanavalin A receptor. 23 Jan 91

A subcellular fractionation method to isolate simultaneously apical and basolateral plasma membrane fractions from the human adenocarcinoma cell line Caco-2, grown on filter supports, is described. The method employs sucrose-density-gradient centrifugation and differential precipitation. The apical membrane fraction was enriched 14-fold in sucrase-isomaltase and 21-fold in 5'-nucleotidase compared with the homogenate. The basolateral membrane fraction was enriched 20-fold relative to the homogenate in K(+)-stimulated p-nitrophenylphosphatase. Alkaline phosphatase was enriched 15-fold in the apical membrane fraction and 3-fold in the basolateral membrane fraction. Analytical density-gradient centrifugation showed that this enzyme was a true constituent of both fractions, and experiments measuring alkaline phosphatase release following treatment with phosphatidylinositol-specific phospholipase C showed that in both membrane fractions the enzyme was glycosyl-phosphatidylinositol-linked. There was very little contamination of either membrane fraction by marker enzymes of the Golgi complex, mitochondria or lysosomes. Both membrane fractions were greater than 10-fold purified with respect to the endoplasmic reticulum marker enzyme alpha-glucosidase. Protein composition analysis of purified plasma membrane fractions together with domain-specific cell surface biotinylation experiments revealed the presence of both common and unique integral membrane proteins in each plasma membrane domain. The post-synthetic transport of endogenous integral plasma membrane proteins was examined using the devised subcellular fractionation procedure in conjunction with pulse-chase labelling experiments and immunoprecipitation. Five common integral membrane proteins immunoprecipitated by an antiserum raised against a detergent extract of the apical plasma membrane fraction were delivered with the same time course to each cell-surface domain.
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PMID:The post-synthetic sorting of endogenous membrane proteins examined by the simultaneous purification of apical and basolateral plasma membrane fractions from Caco-2 cells. 131 18

Four mouse monoclonal antibodies (PTN63, PTN108, PTN124, PTN514) against the ecto-5'-nucleotidase purified from a human pancreatic adenocarcinoma cell line (PaTu II) have been raised and characterized. All four monoclonal antibodies recognize the protein moiety of the glycosylated ecto-5'-nucleotidase. In competition assays it was demonstrated that three of the antibodies (PTN63, PTN108, PTN514) recognize different epitopes within the protein moiety. Furthermore, PTN108, PTN124, and PTN514 reduced the 5'-nucleotidase AMPase activity in contrast to PTN63 having no inhibitory effect. The antibodies show no cross-reactivity with ecto-5'-nucleotidases from rat liver, bull seminal plasma, chicken gizzard and human peripheral blood cells. When assayed by indirect immunofluorescence the antibodies react with the plasma membrane of human pancreatic tumor cells with varying staining intensity. Immunocytochemistry on paraffin sections of normal human pancreas revealed a prominent staining of the pancreatic duct cells. No staining of the acinar and islet cells could be detected. Thus, 5'-nucleotidase is a marker enzyme for pancreatic duct cells and can be used to determine the origin of pancreatic tumor cells. PTN63 reduced the attachment to fibronectin substratum of a human pancreatic adenocarcinoma tumor cell line possessing a high amount of plasma membrane bound ecto-5'-nucleotidase, but had no effect on a cell line lacking the membrane bound AMPase. In contrast, PTN108 and PTN514, which inhibit the AMPase activity, exhibited no influence on the adhesion of human pancreatic tumor cells to fibronectin substratum.
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PMID:Monoclonal antibodies against 5'-nucleotidase from a human pancreatic tumor cell line: their characterization and inhibitory capacity on tumor cell adhesion to fibronectin substratum. 164 65

We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.
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PMID:5'-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol-glycan. 255 91

DU-PAN-2 is a high-molecular-weight glycoprotein defined by a murine monoclonal antibody (MAb) elicited against a human pancreatic adenocarcinoma cell line. This MAb recognizes an oncofetal antigen present on the surface of normal pancreatic and bile-duct epithelium, normal bronchus epithelium, and some adenocarcinomas. Elevated levels of the antigen (greater than 400 U/ml) have been detected in the serum of 79% of patients with adenocarcinoma of the pancreas and in a small percentage of patients with other adenocarcinomas, by means of a competition radioimmunoassay. Here, we have studied DU-PAN-2 antigen levels in sera of patients with a spectrum of hepatobiliary diseases and controls. Serum DU-PAN-2 antigen was elevated in 59% of 112 patients with non-malignant hepatobiliary diseases and in 50% of hepatoma patients. None of 50 healthy controls had elevated serum DU-PAN-2 levels. Patients in every category of hepatobiliary disease studied had elevated median serum DU-PAN-2 levels; the highest median levels were seen in patients with primary biliary cirrhosis (1,296 U/ml) and the lowest in stable cirrhosis (300 U/ml). Elevated serum DU-PAN-2 levels in one patient with primary biliary cirrhosis and in one patient with hepatoma returned to normal following liver transplantation. Serum DU-PAN-2 levels did not correlate well with alkaline phosphatase, 5'-nucleotidase, bilirubin, or alpha-fetoprotein. Using an immunoperoxidase technique on formalin-fixed, deparaffinized liver sections, we showed that DU-PAN-2 MAb reacted heterogeneously with bile-duct epithelium but never stained hepatocytes or hepatoma cells. While serum DU-PAN-2 levels may be useful in detecting and monitoring pancreatic adenocarcinoma, they are not specific for this disease.
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PMID:Detection of an oncofetal antigen (DU-PAN-2) in sera of patients with non-malignant hepatobiliary diseases and hepatomas. 283 18

The inhibition of the cell surface enzyme 5'-nucleotidase by concanavalin A is being studied as a model for understanding transmembrane modulation of cell surface functions. Nucleotidase of 13762 MAT-C1 ascites rat mammary adenocarcinoma cells is inhibited by concanavalin A in a noncooperative process. When cells are treated with the cytoplasmic effectors cytochalasins, colchicine, energy poisons, calcium plus ionophore or hypotonic buffers, the concanavalin A inhibition of the enzyme becomes cooperative. 5'-Nucleotidase of isolated MAT-C1 microvilli is also inhibited by concanavalin A in a noncooperative process; however, treatment of the microvilli with the same cytoplasmic effectors does not induce cooperativity. Since previous studies in several systems have suggested an association of nucleotidase with actin-containing microfilaments or the cell cytoskeleton, one explanation for the cooperativity changes is that they result from a change in the association of the enzyme with the cytoskeleton. However, Triton X-100 extractability of nucleotidase is the same for MAT-C1 cells exhibiting cooperative or noncooperative concanavalin A inhibition. Moreover, enzyme from cells exhibiting cooperative inhibition can be extracted into the zwitterionic detergent Zwittergent in a cooperative form, while enzyme exhibiting noncooperative behavior can be extracted into Zwittergent in a noncooperative form. Gel filtration and rate-zonal sucrose density gradient centrifugation showed little discernible size or sedimentation difference between enzyme samples exhibiting noncooperative and cooperative inhibition. These results indicate that changes in the cooperativity of the concanavalin A inhibition of nucleotidase are not a result of changes in the association of the enzyme with the cytoskeleton. These studies emphasize the caution which must be exercised in interpreting the effects of cytoskeletal perturbants on cell surface functions.
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PMID:Transmembrane modulation of the concanavalin A inhibition of 5'-nucleotidase is not due to a direct association of the enzyme with the cytoskeleton. 298 74

The cell-surface enzyme 5'-nucleotidase in microvilli from 13762 rat mammary adenocarcinoma cells remains largely associated with microfilament-containing high-speed pellets from Triton X-100 extracts of the microvilli. The fraction remaining with the insoluble portion is higher under ionic conditions which enhance microfilament stability. To minimize trapping and cosedimentation we have analyzed the distribution of microfilaments and 5'-nucleotidase activity on velocity sedimentation sucrose gradients of the microvillar extracts. A large fraction of the total enzyme activity is found in the filament fractions in the middle of the gradient. When phalloidin is included in the extraction buffer to stabilize the microfilaments, both the microfilaments and the bulk of the nucleotidase activity are shifted further into the gradients. Both the position of the filament fraction and the percentage of the total nucleotidase activity remaining with the filament fraction varies with extraction buffer composition and conditions. Nonetheless, under all conditions tested, a large percentage of the activity was shifted, along with the microfilaments, in the presence of phalloidin. These results are consistent with a specific association of 5'-nucleotidase with microfilaments in the ascites tumor cell microvilli.
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PMID:Demonstration of the association of the cell-surface enzyme, 5'-nucleotidase, with microvillar microfilaments by phalloidin shift on velocity sedimentation gradients. 300 89

The interaction of tumor cells with extracellular-matrix components is suspected to play an important role in tumorigenesis induction. The tumorigenicity of a poorly tumorigenic human colon-adenocarcinoma cell line (BCS-TC2) was induced by co-injection with Matrigel. A new cell sub-line, BCS-TC2.1, was isolated and established from these tumors. Implantation of these cells in nude mice in the absence of Matrigel-generated tumors which allowed the establishment of another tumorigenic cell sub-line, BCS-TC2.2. Matrigel and laminin, but not collagens, promote the tumorigenicity of BCS-TC2 cells, probably due to specific interactions of a pre-existing minor cell sub-population with laminin, which facilitate the initial growth of these cells in vivo. Cytogenetic analysis reveals that both sub-lines originate from the parental one, but a new marker in chromosome 9 is observed. These sub-lines present a lower degree of differentiation, as deduced from the lower CEA content, 5'-nucleotidase and alkaline-phosphatase activities. No variation is observed in the mRNA and protein expression of the 67-kDa laminin-binding protein. However, an increase in beta1 integrins and a parallel decrease in beta4 integrin were detected. Thus, the new sub-lines, compared to the parental cells, present karyotypic and phenotypic differences such as the expression of a distinctive integrin pattern. This system represents a useful model for understanding the development and progression of tumorigenicity in cancer cells.
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PMID:Characterization of tumorigenic sub-lines from a poorly tumorigenic human colon-adenocarcinoma cell line. 878 56

We have analysed the major effects of sodium butyrate on the morphology, protein content and induction of epithelial differentiation markers in human colon adenocarcinoma BCS-TC2 cells. Sodium butyrate alters the cell morphology, inducing a larger cellular size, flattening and vacuolization. The protein content per cell increases, whereas the proliferation rate is reduced. Moreover, cell death by apoptosis is also observed. Butyrate-treated cells show higher levels of alkaline phosphatase activity and carcinoembryonic antigen, suggesting that this agent induces the in vitro differentiation of BCS-TC2 cells. These effects are reversible and time and dose dependent. In addition, we have observed that the ectoenzyme 5'-nucleotidase activity also increases during this treatment, suggesting that it could be considered as a new differentiation marker for this type of carcinoma cells. These results contribute to the understanding of the action of sodium butyrate as a potential cancer chemotherapeutic agent.
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PMID:Differentiation of BCS-TC2 human colon adenocarcinoma cells by sodium butyrate: increase in 5'-nucleotidase activity. 926 51


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