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1.
Cancer Res ; 57(12): 2529-37, 1997 Jun 15.
Article in English | MEDLINE | ID: mdl-9192836

ABSTRACT

Many cell types transport vitamin C solely in its oxidized form, dehydroascorbic acid, through facilitative glucose transporters. These cells accumulate large intracellular concentrations of vitamin C by reducing dehydroascorbic acid to ascorbate, a form that is trapped intracellularly. Certain specialized cells can transport vitamin C in its reduced form, ascorbate, through a sodium-dependent cotransporter. We found that normal human melanocytes and human malignant melanoma cells are able to transport vitamin C using both mechanisms. Melanoma cell lines transported dehydroascorbic acid at a rate that was at least 10 times greater than the rate of transport by melanocytes, whereas both melanoma cells and melanocytes transported ascorbate with similar efficiency. Dehydroascorbic acid transport was inhibited by deoxyglucose and cytochalasin B, indicating the direct participation of facilitative glucose transporters in the transport of oxidized vitamin C. Melanoma cells accumulated intracellular vitamin C concentrations that were up to 100 times greater than the corresponding extracellular dehydroascorbic acid concentrations, whereas intracellular accumulation of vitamin C by melanocytes never exceeded the extracellular level of dehydroascorbic acid. Melanoma cells transported dehydroascorbic acid through at least two different transporters, each with a distinct K(m), a finding that agreed well with the presence of several glucose transporter isoforms in these cells. Only one kinetic component of ascorbate uptake was identified in both melanocytes and melanoma cells, and ascorbate transport was sodium dependent and inhibited by ouabain. Both cell types were able to accumulate intracellular concentrations of vitamin C that were greater than the extracellular ascorbate concentrations. The data indicate that melanoma cells and normal melanocytes transport vitamin C using two different transport systems. The transport of dehydroascorbic acid is mediated by a facilitated mechanism via glucose transporters, whereas transport of ascorbic acid involves a sodium-ascorbate cotransporter. The differential capacity of melanoma cells to transport the oxidized form of vitamin C reflects the increased expression of facilitative transporters associated with the malignant phenotype.


Subject(s)
Ascorbic Acid/pharmacokinetics , Dehydroascorbic Acid/pharmacokinetics , Melanocytes/metabolism , Melanoma/metabolism , Ascorbic Acid/analysis , Biological Transport, Active/drug effects , Biological Transport, Active/physiology , Cells, Cultured , Cytochalasin D/pharmacology , Deoxyglucose/pharmacology , Dose-Response Relationship, Drug , Humans , Immunohistochemistry , Lithium Chloride/pharmacology , Melanocytes/chemistry , Melanoma/chemistry , Models, Biological , Monosaccharide Transport Proteins/drug effects , Monosaccharide Transport Proteins/physiology , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Sucrose/pharmacology , Time Factors , Tumor Cells, Cultured
2.
J Clin Invest ; 100(11): 2842-8, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9389750

ABSTRACT

Vitamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.


Subject(s)
Ascorbic Acid/pharmacokinetics , Blood-Brain Barrier , Brain/metabolism , Dehydroascorbic Acid/pharmacokinetics , Monosaccharide Transport Proteins/metabolism , Animals , Autoradiography , Brain/diagnostic imaging , Capillary Permeability , Deoxyglucose/pharmacology , Glucose Transporter Type 1 , Image Processing, Computer-Assisted , Kinetics , Mice , Mice, Inbred BALB C , Oxidation-Reduction , Radiography , Rats , Rats, Inbred F344 , Time Factors
3.
Blood ; 85(4): 973-80, 1995 Feb 15.
Article in English | MEDLINE | ID: mdl-7849318

ABSTRACT

While the primary targets for granulocyte-macrophage colony-stimulating factor (GM-CSF) are hematopoietic precursors and mature myeloid cells, GM-CSF receptors (GMR) are also found on normal tissues including placenta, endothelium, and oligodendrocytes as well as certain malignant cells. The function of GMR in these nonhematopoietic cells is unknown. We studied the function of GMR in human melanoma cell lines. Six of seven cell lines tested (clones 1-5 and 3.44 of SK-MEL-131, SK-MEL-188, SK-MEL-23, SK-MEL-22, and SK-MEL-22A) expressed mRNA encoding the membrane-bound and soluble isoforms of the alpha subunit of the GMR. Melanoma cell lines in early stages of differentiation expressed the largest quantities of alpha-subunit mRNA. Although five of these lines expressed trace levels of mRNA encoding the beta subunit of the GMR, Scatchard analysis of equilibrium binding data derived from three of the cell lines showed that they expressed only low-affinity GMR. Clones 3.44 and 1-5 of SK-MEL-131, and SK-MEL-188 cells expressed receptors with a dissociation constant (kd) for GM-CSF in the following ranges: 0.7 to 0.8, 1.2 to 1.8, and 0.4 to 0.8 nmol/L, respectively. GM-CSF stimulated glucose uptake in four of the melanoma cell lines expressing the alpha subunit, presumably through facilitative glucose transporters, as uptake was blocked by cytochalasin B but not cytochalasin E. Stimulation of glucose uptake was transient, with maximum stimulation occurring at approximately 30 minutes in the presence of 1 nmol/L GM-CSF. GM-CSF stimulated glucose uptake 1.4- to 2.0-fold but did not stimulate cell proliferation. These results suggest a metabolic role for the low-affinity GMR in melanoma cell lines and indicate that the alpha subunit of the GMR can signal for increased glucose uptake in nonhematopoietic tumor cells.


Subject(s)
Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Melanoma/metabolism , Monosaccharide Transport Proteins/metabolism , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Signal Transduction/physiology , Base Sequence , Biological Transport, Active , Cell Line , Cytochalasin B/pharmacology , Cytochalasins/pharmacology , DNA Primers , Deoxyglucose/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Kinetics , Leukemia, Myeloid , Macromolecular Substances , Molecular Sequence Data , Monosaccharide Transport Proteins/drug effects , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Signal Transduction/drug effects , Tumor Cells, Cultured
4.
J Biol Chem ; 269(15): 11663-71, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-8157699

ABSTRACT

We recently cloned murine and human cDNAs that encode CP2, a cellular transcription factor that interacts with the alpha-globin promoter as well as with additional cellular and viral promoter elements. We have now characterized the genomic structure, chromosome location, promoter, and expression pattern of the factor. Genes for the murine and human mRNAs contained 16 and 15 exons, respectively. Both genes spanned approximately 30 kilobases of chromosomal DNA, and among coding exons, all exon/intron boundaries were conserved. The human gene for CP2 was found to reside on chromosome 12 while the murine gene mapped to the distal end of chromosome 15, near Gdc-1, Wnt-1, and Rarg, a region syntenic with human chromosome 12. The murine and human promoters initiated mRNAs at multiple start sites in a conserved region that spanned more than 450 nucleotides. Lastly, a study of the pattern of CP2 gene expression showed that the factor was expressed in all adult and fetal murine tissues examined from at least day 9.5 of development.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 12 , DNA-Binding Proteins/genetics , Gene Expression Regulation , Globins/genetics , Promoter Regions, Genetic , Transcription Factors/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Exons , Humans , Introns , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Organ Specificity , Polymerase Chain Reaction , RNA Splicing , RNA, Messenger/biosynthesis , RNA, Messenger/metabolism , RNA-Binding Proteins , Restriction Mapping
5.
Mol Endocrinol ; 2(4): 344-9, 1988 Apr.
Article in English | MEDLINE | ID: mdl-2454399

ABSTRACT

Cyclic AMP has been shown to stimulate synthesis of tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) by increasing the amount of its mRNA through an increase in initiation of transcription. However, cAMP also has posttranscriptional effects on the enzyme's synthesis, as evidenced by the 4- to 5-fold enhanced decline seen when cultured hepatoma cells are exposed to cAMP and transcription is inhibited. As a direct test of the possibility that cAMP exerts this effect by destabilizing the mRNA for tyrosine aminotransferase, we analyzed the rate of decay of the mRNA using the transcriptional inhibitor 5,6-dichlororibofuranosylbenzimidazole, Northern blot analysis, and an internal standard consisting of prelabeled rRNA. It was found that the half-life of the mRNA (2.0 +/- 0.2 h) was not changed by treatment of cultured hepatoma cells under conditions which increase intracellular cAMP levels. These mRNA half-life values were not significantly different from the decline in the rate of synthesis of the enzyme after induction in dexamethasone-treated cells. We conclude that cAMP does not affect the stability of the mRNA for tyrosine aminotransferase and discuss other possible explanations for the paradoxical effect of cAMP on deinduction of this enzyme.


Subject(s)
Cyclic AMP/pharmacology , Liver Neoplasms, Experimental/enzymology , RNA, Messenger/metabolism , Tyrosine Transaminase/genetics , 1-Methyl-3-isobutylxanthine/pharmacology , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , DNA/genetics , Dichlororibofuranosylbenzimidazole/pharmacology , Enzyme Induction/drug effects , Half-Life , Kinetics , Nucleic Acid Hybridization , Plasmids , Tumor Cells, Cultured , Tyrosine Transaminase/biosynthesis
6.
Article in English | MEDLINE | ID: mdl-2887599

ABSTRACT

Undegraded tyrosine aminotransferase was purified to near homogeneity from rat liver and was confirmed to be a substrate for the beef heart cyclic AMP dependent protein kinase catalytic subunit. Specific antibody was used to quantitate the amount of phosphate incorporated into the enzyme. Phosphate incorporation was maximal at a catalytic subunit to tyrosine aminotransferase molar ratio of 7:1 using 200 microM ATP for 30 to 60 min at 30 degrees C. Phospho-peptide maps of tyrosine aminotransferase phosphorylated in vitro by the catalytic subunit were compared with those of amino-transferase immunoprecipitated from 32P labeled cells treated with and without 8-Br cAMP. Whereas the phospho-peptide maps of tyrosine aminotransferase isolated from cells treated with and without 8-Br cAMP were identical, differences were observed in the peptide map of tyrosine aminotransferase phosphorylated in vitro and in vivo. These results were taken to indicate that the catalytic subunit is not responsible for tyrosine aminotransferase phosphorylation in vivo.


Subject(s)
Cyclic AMP/pharmacology , Protein Kinases/metabolism , Tyrosine Transaminase/metabolism , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Adenosine Triphosphate/pharmacology , Animals , Cattle , Chymotrypsin/metabolism , Immunosorbent Techniques , Liver/enzymology , Male , Myocardium/enzymology , Peptide Fragments/metabolism , Phosphates/metabolism , Phosphorylation , Rats , Rats, Inbred Strains
7.
Article in English | MEDLINE | ID: mdl-3700775

ABSTRACT

Highly purified 4-aminobutyrate aminotransferase from pig brain is susceptible to phosphorylation by the purified cAMP-dependent protein kinase catalytic subunit. Up to 0.7 moles of phosphate from ATP-(gamma)-32P can be incorporated per mole of dimeric holoenzyme. Maximum phosphorylation was observed within about 90 minutes at 30 degrees C. Despite the extensive degree of phosphorylation observed, no kinetic property of the enzyme was perceptibly altered. Removal of cofactor had no detectable impact on the extent of phosphorylation but thermal inactivation of the enzyme increased and mild reduction with sodium borohydride decreased the phosphorylatability of the aminotransferase. It was possible to separate the enzyme into phospho and dephospho forms by the use of DEAE chromatography. Validation that the two fractions represented genuine aminotransferase was obtained by proteolytic peptide mapping. The phospho form of the enzyme was found to possess little or no aminotransferase activity while that of the dephospho form exhibited higher specific activity than the purified enzyme prior to phosphorylation. Furthermore, the dephospho form of the enzyme could not be detectably phosphorylated by reincubation with the kinase following DEAE chromatography unless it was subjected to thermal inactivation. The stoichiometry of phosphorylation of the fraction containing 32P from DEAE chromatography was approximately 1 mole/mole of dimer. These results suggest that the substrate for phosphorylation by the kinase is a form of the aminotransferase which is somehow inactivated during routine purification even when extensive precautions are taken to maximally preserve catalytic activity.


Subject(s)
4-Aminobutyrate Transaminase/metabolism , Protein Kinases/metabolism , Animals , Borohydrides/metabolism , Chromatography, DEAE-Cellulose , Hot Temperature , In Vitro Techniques , Oxidation-Reduction , Phosphorylation , Swine , Time Factors
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