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The Journal of Biological Chemistry Apr 1985The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino...
The nitric oxide (N = O) free radical exhibits potent cytocidal, mutagenic and vasodilatory properties. We have examined the hypothesis that the hydroxynitrosamino functionality (see sequence in text), which occurs naturally in antineoplastic and antihypertensive agents, will directly generate N = O following peroxidatic 1-electron oxidation. Cupferron (see sequence in text) is indeed an excellent (k greater than 10(7) m-1 s-1) substrate for horseradish peroxidase. The products are N = O and nitrosobenzene (phi - N = O) which are generated and consumed as follows. First, cupferron is oxidized by the classical peroxidatic mechanism to form an unstable nitroxide free radical (see sequence in text) which then forms N = O and phi - N = O spontaneously (see sequence in text). The N = O then reacts with phi - N = O to reform cupferron (see sequence in text) or with the enzyme to generate the characteristic peroxidase--N = O chromophore. Simultaneously, in a competitive reaction with O2, the N = O is converted to NO-2 (4N = O + O2 + 2H2O------------4NO-2 + 4H+). The reactivity of hydroxynitrosamino compounds with horseradish peroxidase is in the order cupferron greater than hydroxynitrosaminomethane greater than alanosine. These model reactions, involving direct oxidation of the hydroxynitrosamino moiety, comprise a novel pathway for the biological production of N = O.
Topics: Chemical Phenomena; Chemistry; Horseradish Peroxidase; Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxylamines; Mathematics; Nitric Oxide; Nitrosamines; Nitroso Compounds; Oxygen Consumption
PubMed: 3980468
DOI: No ID Found -
Blood Oct 1996Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute... (Comparative Study)
Comparative Study
Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute lymphoblastic leukemia (ALL). Because the MTAP gene is located adjacent to the tumor-suppressor gene p16 on chromosome 9p21 and more than 60% of T-cell ALL (T-ALL) patients have deletion in the p16 gene, we examined the status of the MTAP gene in T-ALL patients. Quantitative polymerase chain reaction amplification of exon 8 of MTAP showed a deletion in 16 of 48 (33.3%) patients at diagnosis and in 13 of 33 (39.4%) patients at relapse. Southern blot analysis showed that, in addition to deletion of the entire MTAP gene, a common break point was between exons 4 and 5, resulting in deletion of exons 5 through 8. The finding of frequent deficiency of MTAP in T-ALL offers the possibility of an enzyme targeted therapy for T-ALL. MTAP(-) T-ALL-derived cell line, CEM cells were very sensitive to methionine deprivation, with cell viability at 50% of control as early as 48 hours after methionine deprivation. In contrast, methionine deprivation had little effect on the viability of normal lymphocytes or on their proliferative response to phytohemagglutinin. Alanosine, an inhibitor of AMP synthesis, inhibited the growth of both MTAP(+) (Molt-4 and Molt-16) and MTAP(-) (CEM and HSB2) cell lines. However, the addition of methylthioadenosine, the substrate of MTAP, protected the MTAP(+) cells but not the MTAP(-) cells from alanosine toxicity. These findings suggest the possibility of targeting MTAP for selective therapy of T-ALL.
Topics: Adenosine Monophosphate; Alanine; Antimetabolites, Antineoplastic; Chromosomes, Human, Pair 9; DNA Mutational Analysis; DNA, Neoplasm; Exons; Genes; Genes, Tumor Suppressor; Humans; Leukemia-Lymphoma, Adult T-Cell; Methionine; Neoplasm Proteins; Polymerase Chain Reaction; Purine-Nucleoside Phosphorylase; Sequence Deletion; T-Lymphocytes; Tumor Cells, Cultured
PubMed: 8874207
DOI: No ID Found -
The Journal of Biological Chemistry Jul 1983The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the...
The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.
Topics: Adenosine Deaminase; Animals; Cell Line; Coformycin; Electrophoresis, Polyacrylamide Gel; Electrophoresis, Starch Gel; Gene Amplification; Genes; Karyotyping; Mice; Molecular Weight; Nucleoside Deaminases; Pentostatin; Thymidine Kinase
PubMed: 6602803
DOI: No ID Found -
Molecular and Cellular Biology Oct 1985Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet...
Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) pseudorevertant cell lines were isolated under selective conditions requiring adenine salvage for survival; yet they were found to be deficient in measurable APRT activity and resistant to the purine analog 2'6'-diaminopurine (DAP) (M.S. Turker, J. A. Tischfield, P. Rabinovitch, P.J. Stambrook, J.J. Trill, A.C. Smith, C.E. Ogburn, and G.M. Martin, manuscript in preparation). Adenine salvage was examined in two APRT pseudorevertant cell lines, their two APRT homozygous deficient parental cell lines, and a genotypic APRT revertant cell line (i.e., one with measurable APRT activity and DAP sensitivity). Adenine accumulation was observed in both revertant phenotypes and was demonstrated by high-performance liquid chromatography to be linked with adenine metabolism. The ability to salvage adenine declined substantially in the pseudorevertant cell lines when they were removed from selective media containing inhibitors of de novo 5'-AMP synthesis (alanosine and azaserine); for one pseudorevertant cell line this decline was accelerated by the addition of DAP to the medium. The readdition of alanosine or azaserine to the growth medium of the pseudorevertant lines induced adenine salvage to its previous levels. An APRT-like cross-reacting material was found in the pseudorevertant cell lines, although its relationship to adenine salvage is unknown. A low level of constitutive adenine salvage was found in the parental APRT-deficient lines, and it was also possible to induce adenine salvage in these cell lines. These findings suggest a novel regulatory mechanism for adenine salvage.
Topics: 2-Aminopurine; Adenine; Adenine Phosphoribosyltransferase; Alanine; Animals; Azaserine; Cell Line; Cross Reactions; Drug Resistance; Gene Expression Regulation; Karyotyping; Mice; Mycoplasma; Pentosyltransferases
PubMed: 3837181
DOI: 10.1128/mcb.5.10.2662-2668.1985 -
Biochimica Et Biophysica Acta Apr 1995GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The...
GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [14C]glycine (to label the de novo pathway) or [3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or L-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73-81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, L-alanosine decreased ATP mass by 26-33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.
Topics: Adenosine Triphosphate; Alanine; Animals; Cytosol; Guanosine Triphosphate; Insulin; Islets of Langerhans; Male; Mercaptopurine; Mycophenolic Acid; Rats; Rats, Sprague-Dawley
PubMed: 7718617
DOI: 10.1016/0167-4889(94)00235-7 -
The Journal of Biological Chemistry Nov 1993Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines, including dopamine. This...
Wild type PC12 pheochromocytoma cells express a Na(+)-dependent norepinephrine transporter that operates in the uptake of catecholamines, including dopamine. This transporter is not expressed in two spontaneously occurring flat cell variants of PC12 or in two other flat cell variants whose phenotype was induced by expression of the Wnt-1 oncogene. However, each of the flat cell variants, including those that express Wnt-1, exhibit a Na(+)-dependent, Cl(-)-independent glutamate/aspartate transporter activity that is not present in wild type PC12 cells. The flat cell variants took up glycine by a Na(+)-dependent process as well as did wild type cells. All of the flat cell variants have decreased levels of norepinephrine transporter mRNA but normal levels of glycine transporter mRNA. Glutamate/aspartate transporter mRNA was detected only in the variants that exhibited glutamate/aspartate transporter activity, and the nucleotide sequence of a partial glutamate/aspartate transporter cDNA from these cells demonstrated that it was the glial form of the transporter that was expressed. These variants were more sensitive than was wild type PC12 to alanosine, a toxic aspartate analog that enters cells by a transporter-mediated system such as the glutamate/aspartate transporter; however, these variants were as sensitive as wild type cells to another toxic aspartate analog, N-(phosphonacetyl)-L-aspartic acid, which is believed to enter cells by endocytosis. We suggest that the Wnt-1 gene product, or a homolog, may be involved in glial differentiation and that the mechanisms that alter the expression of the norepinephrine and glutamate/aspartate transporters in wild type and variant PC12 cells may also operate to regulate neurotransmitter transporter expression in vivo.
Topics: Alanine; Amino Acid Sequence; Amino Acid Transport System X-AG; Animals; Aspartic Acid; Biological Transport; Carrier Proteins; DNA, Complementary; Gene Expression; Glycine; Glycoproteins; Molecular Sequence Data; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Oncogene Proteins; PC12 Cells; Phosphonoacetic Acid; RNA, Messenger; Symporters
PubMed: 8226929
DOI: No ID Found -
Proceedings of the National Academy of... Jan 1974A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made...
A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16.
Topics: Adenine; Animals; Autoradiography; Carbon Radioisotopes; Chromosome Mapping; Chromosomes, Human, 16-18; Clone Cells; Electrophoresis, Polyacrylamide Gel; Fluorescence; Genes; Humans; Hybrid Cells; Karyotyping; Lung; Mice; Pentosyltransferases; Quinacrine; Staining and Labeling
PubMed: 4129802
DOI: 10.1073/pnas.71.1.45 -
The Journal of Biological Chemistry Oct 1988The pathways of 2',3'-dideoxyadenosine (ddAdo) metabolism, a selective inhibitor of the replication of human immunodeficiency virus, were investigated with use of the...
The pathways of 2',3'-dideoxyadenosine (ddAdo) metabolism, a selective inhibitor of the replication of human immunodeficiency virus, were investigated with use of the human T-lymphoid cell line CCRF-CEM which is deficient in either deoxycytidine kinase or adenosine kinase activity, or both. At an extracellular concentration of 10 microM, which blocks the cytopathic effect of human immunodeficiency virus in vitro, ddAdo was found to be metabolized to its mono-, di-, and triphosphates and to dideoxyinosine monophosphate (ddIMP). The metabolism of ddAdo in the kinase-deficient mutants was found to be unchanged by comparison with that in parental cells; however, the inhibition of ddAdo deamination to 2',3'-dideoxyinosine (ddIno) by the adenosine deaminase inhibitor, 2'-deoxycoformycin, reduced ddAdo nucleotide formation in deoxycytidine kinase-deficient, adenosine kinase-deficient, and doubly kinase-deficient mutants by 42, 54, and 80%, respectively. Incubation of the CCRF-CEM cells with 20 microM L-alanosine, an amino acid antagonist that inhibits purine biosynthesis at the level of adenylosuccinate/lyase synthetase, resulted in 80% inhibition in the accumulation of ddAdo nucleotides in both wild-type and kinase-deficient mutants and also increased ddIMP accumulation 2- to 3-fold. These findings indicate that ddAdo activation in human T-lymphoblasts can occur by three metabolic pathways: directly, by phosphorylation to ddAMP by the action of either deoxycytidine kinase or adenosine kinase and, indirectly, through deamination to ddIno with consequent phosphorylation of ddIno to ddIMP, and reamination to ddAMP in a reaction catalyzed by adenylosuccinate synthetase/lyase. However, in the absence of 2'-deoxycoformycin, the activation of ddAdo to ddATP in T-lymphoid cells is primarily a function of the indirect route.
Topics: Adenosine Kinase; Antiviral Agents; Cell Line; Chromatography, High Pressure Liquid; Deoxycytidine Kinase; Dideoxyadenosine; Dideoxynucleosides; HIV; Humans; Kinetics; Virus Replication
PubMed: 3262616
DOI: No ID Found -
Biochemical and Biophysical Research... May 1996Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation)....
Nucleophosmin/B23 (NPM) is a nucleolar phosphoprotein which shifts from nucleoli to the nucleoplasm in cells treated with certain cytotoxic agents (NPM-translocation). NPM requires GTP for localization into nucleoli (J. Biol. Chem. 268, 5823-5827, 1993). To understand more about NPM's dynamic localization, the effects of lowering ATP on NPM-translocation and rRNA synthesis were studied. When the ATP level in HeLa cells was reduced by sodium azide, NPM-translocation was blocked. Similar results were obtained when ATP was depleted by other agents, suggesting that ATP depletion was responsible for the blocking of NPM-translocation. It was found that newly synthesized rRNA accumulated in the nuclei during ATP-depletion. Significantly larger than normal nucleoli were also observed. These results indicate that NPM may be involved in the transportation of newly synthesized ribosomes.
Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Alanine; Antibiotics, Antineoplastic; Azides; Cell Nucleus; Dinitrophenols; HeLa Cells; Humans; Kinetics; Mycophenolic Acid; Nuclear Proteins; Nucleolus Organizer Region; Nucleophosmin; RNA, Ribosomal; Ribosomes; Sodium Azide
PubMed: 8670243
DOI: 10.1006/bbrc.1996.0782 -
Antimicrobial Agents and Chemotherapy Jun 1973A sensitive, precise microbiological assay was developed for the determination of tissue distribution of dl-alanosine, a new antitumor agent.
A sensitive, precise microbiological assay was developed for the determination of tissue distribution of dl-alanosine, a new antitumor agent.
Topics: Animals; Antibiotics, Antineoplastic; Biological Assay; Escherichia coli; Hydroxylamines; Mice; Mice, Inbred Strains; Nitroso Compounds; Propionates; Time Factors
PubMed: 4597740
DOI: 10.1128/AAC.3.6.739