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Proceedings of the National Academy of... Dec 197113 clonal cell lines were isolated from a hybrid cell population established by cell fusion between cloned BALB/c myeloma cells that were resistant to 8-azaguanine and...
13 clonal cell lines were isolated from a hybrid cell population established by cell fusion between cloned BALB/c myeloma cells that were resistant to 8-azaguanine and produced immunoglobulin G and free kappa chain and C(67)BL/6n lymphoma cells that were resistant to bromodeoxyuridine and did not produce immunoglobulins. Some of the histocompatibility-2 antigens of both parental cell lines could be demonstrated on all of the hybrid clones. 11 clones synthesized only free kappa chain. Two clones synthesized IgG of BALB/c type, as well as free kappa chain. These clones had higher chromosome number than did the clones that synthesized only kappa chains.
Topics: Animals; Antigens; Azaguanine; Bromodeoxyuridine; Cell Fusion; Cell Line; Clone Cells; Complement System Proteins; Culture Media; Histocompatibility; Hybrid Cells; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin G; Immunoglobulins; Karyotyping; Lymphoma; Mice; Multiple Myeloma; Rabbits; Species Specificity
PubMed: 5002282
DOI: 10.1073/pnas.68.12.3045 -
Pathophysiology : the Official Journal... May 2007Dipeptidyl peptidase IV, a cell membrane surface protease also known as CD26 (CD26/DPPIV), is known to play multiple functions in human organism, where it is largely...
Dipeptidyl peptidase IV, a cell membrane surface protease also known as CD26 (CD26/DPPIV), is known to play multiple functions in human organism, where it is largely expressed, for instance, in the development of human cancer and metastasis as well as in chemotherapy response. The objective of this work was to study the CD26 membrane expression and DPPIV activity in T-acute leukaemia cell lines (CEM and MOLT3) in culture, in order to observe the modification of its expression under the 8-azaguanine treatment. Cell line samples were incubated, some without different azaguanine concentration and others with, ranging from 10 to 100muM. Cell surface CD26 expression has been identified by flow cytometry and DPPIV activity, in cultured medium, was fluorimetrically measured. Results we have observed showed that 8-azaguanine induced a decrease in cell viability in a dose, time and cell type dependent manner with MOLT3 cells being the most sensitive to 8-azaguanine citotoxic effects (24h IC50: +/-10muM) when compared with CEM cells (24h IC50: +/-100muM). In the same experimental conditions, MOLT3 cell treated with 8-azaguanine shows an increase in CD26 expression (MIF) compared with that of CEM cell submitted to the same conditions (65.4+/-1.3 versus 18.7+/-1.7). DPPIV activity in culture medium supernatant of CEM versus MOLT3 controls cells (1.91+/-0.43 versus 2.06+/-0.50) and of CEM versus MOLT3 treated cells (2.10+/-0.16 versus 1.89+/-0.04) did not show a significant difference. These preliminary results suggest that 8-azaguanine stimulates CD26 expression which may be related to cellular sensitivity to 8-azaguanine.
PubMed: 17055708
DOI: 10.1016/j.pathophys.2006.09.003 -
Molecular Cancer Therapeutics Jan 2016Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and...
Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and identify potential anticancer compounds selective for the high-ploidy state. Among 354 human breast cancers, 10% have mean chromosome copy number exceeding 3, and this is most common in triple-negative and HER2-positive types. Women with high-ploidy breast cancers have higher risk of recurrence and death in two patient cohorts, demonstrating that it represents an important group for improved treatment. Because high-ploidy cancers are aneuploid, rather than triploid or tetraploid, we devised a two-step screen to identify selective compounds. The screen was designed to assure both external validity on diverse karyotypic backgrounds and specificity for high-ploidy cell types. This screen identified novel therapies specific to high-ploidy cells. First, we discovered 8-azaguanine, an antimetabolite that is activated by hypoxanthine phosphoribosyltransferase 1 (HPRT1), suggesting an elevated gene-dosage of HPRT1 in high-ploidy tumors can control sensitivity to this drug. Second, we discovered a novel compound, 2,3-diphenylbenzo[g]quinoxaline-5,10-dione (DPBQ). DPBQ activates p53 and triggers apoptosis in a polyploid-specific manner, but does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. Structure-function analysis defines the core benzo[g]quinoxaline-5,10 dione as being necessary for the polyploid-specific effects of DPBQ. We conclude that polyploid breast cancers represent a high-risk subgroup and that DPBQ provides a functional core to develop polyploid-selective therapy. Mol Cancer Ther; 15(1); 48-59. ©2015 AACR.
Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Discovery; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Karyotype; Polyploidy; Prognosis; Proline; Signal Transduction; Tumor Suppressor Protein p53
PubMed: 26586723
DOI: 10.1158/1535-7163.MCT-15-0527 -
Journal of Biomolecular Screening Jun 2012Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are...
Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that affects multiple organs. Treatment is mainly surgical, and effective systemic therapies are needed. We developed a cell-based screening tool to identify compounds that stabilize or upregulate full-length, point-mutated VHL protein. The 786-0 cell line was infected with full-length W117A-mutated VHL linked to a C-terminal Venus fluorescent protein. This VHL-W117A-Venus line was used to screen the Prestwick drug library and was tested against proteasome inhibitors MG132 and bortezomib. Western blot validation and evaluation of functional readouts, including hypoxia-inducible factor 2α (HIF2α) and glucose transporter 1 (Glut1) levels, were performed. We found that bortezomib, MG132, and the Prestwick compounds 8-azaguanine, thiostrepton, and thioguanosine upregulated VHL-W117A-Venus in 786-0 cells. 8-Azaguanine downregulated HIF2α levels and was augmented by the presence of VHL W117A. VHL p30 band intensities varied as a function of compound used, suggesting alternate posttranslational processing. Nuclear-cytoplasmic localization of VHL-W117A-Venus varied among the different compounds. In conclusion, a 786-0 cell line containing VHL-W117A-Venus was successfully used to identify compounds that upregulate VHL levels, with differential effect on VHL intracellular localization and posttranslational processing. Further screening efforts will broaden the number of pharmacophores available to develop therapeutic agents that will upregulate and refunctionalize mutated VHL.
Topics: Cell Line; Cysteine Proteinase Inhibitors; Drug Discovery; High-Throughput Screening Assays; Humans; Leupeptins; Mutant Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Reproducibility of Results; Small Molecule Libraries; Von Hippel-Lindau Tumor Suppressor Protein
PubMed: 22357874
DOI: 10.1177/1087057112436557 -
The Biochemical Journal Oct 19681. The ribosomal components in the postmitochondrial supernatant of a rat hepatoma (hepatoma 7800) and the corresponding host liver were examined for diversity and...
1. The ribosomal components in the postmitochondrial supernatant of a rat hepatoma (hepatoma 7800) and the corresponding host liver were examined for diversity and functional competence. 2. The ;free' and ;membrane-bound' polyribosomes of both tissues were equally active in vivo and had equilibrated with newly synthesized ribosomes 4hr. after administration of [6-(14)C]orotic acid. 3. The inactive monomer-dimer pool in hepatoma 7800 was unattached to membranes and a larger fraction of the polyribosomes was free in hepatoma than in liver. 4. By using sensitivity to puromycin as a criterion, evidence was obtained that most of the polyribosomes in hepatoma 7800 were active in vivo. 5. Actinomycin, azaguanine and carbon tetrachloride caused marked conversion of polyribosomes into inactive monomers and dimers in the host liver and moderate conversion in the hepatoma. 6. Significant accumulation of ferritin and shifts in the mean polyribosome size to the lighter species occurred in the host liver of rats bearing large hepatomas.
Topics: Animals; Azaguanine; Carbon Isotopes; Carbon Tetrachloride; Carcinoma, Hepatocellular; Dactinomycin; Ferritins; Liver; Liver Neoplasms; Neoplasms, Experimental; Orotic Acid; Puromycin; Rats; Ribosomes
PubMed: 4300830
DOI: 10.1042/bj1090617 -
Proceedings of the National Academy of... Jun 1963
Topics: Azaguanine; Cyanides; Learning; RNA; Ristocetin
PubMed: 14019945
DOI: 10.1073/pnas.49.6.918 -
The Biochemical Journal Mar 19671. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose...
1. In kidney, but not in rat whole brain and liver, guanine-deaminase activity was localized almost exclusively in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, as in brain and liver, the enzymic activity recovered in the supernatant was higher than that in the whole homogenate. The particulate fractions of kidney, especially the heavy mitochondria, brought about powerful inhibition of the supernatant guanine-deaminase activity. 2. In spleen, as in kidney, guanine-deaminase activity was localized in the 15000g supernatant fraction of iso-osmotic sucrose homogenates. However, the particulate fractions did not inhibit the activity of the supernatant. 3. Guanine-deaminase activity in rat brain was absent from the cerebellum and present only in the cerebral hemispheres. The inhibitor of guanine deaminase was located exclusively in the cerebellum, where it was associated with the particles sedimenting at 5000g from sucrose homogenates. 4. Homogenates of cerebral hemispheres, the separated cortex or the remaining portion of the hemispheres had significantly higher guanine-deaminase activity than homogenates of whole brain. The enzymic activity of the subcellular particulate fractions was nearly the same. 5. Guanine deaminase was purified from the 15000g supernatant of sucrose homogenates of whole brain. The enzyme separated as two distinct fractions, A and B, on DEAE-cellulose columns. 6. The guanine-deaminase activity of the light-mitochondrial fraction of whole brain was fully exposed and solubilized by treatment with Triton X-100, and partially purified. 7. Tested in the form of crude preparations, the inhibitor from kidney did not act on the brain and liver supernatant enzymes and the inhibitor from cerebellum did not act on kidney enzyme, but the inhibitor from liver acted on both brain and kidney enzyme. 8. The inhibitor of guanine deaminase was purified from the heavy mitochondria of whole brain and liver and the 5000g residue of cerebellum, isolated from iso-osmotic homogenates. The inhibitor appeared to be protein in nature and was heat-labile. The inhibition of the enzyme was non-competitive. 9. Kinetic, immunochemical and electrophoretic studies with the preparations purified from brain revealed that the enzyme from light mitochondria was distinct from enzyme B from the supernatant. A distinction between the two forms of supernatant enzyme was less certain. 10. Guanine deaminase isolated from light mitochondria of brain did not react with 8-azaguanine or with the inhibitor isolated from heavy mitochondria.
PubMed: 16742482
DOI: 10.1042/bj1020691 -
Journal of Bacteriology Jan 1962Mangalo, R. (University of Illinois, Urbana) and J. T. Wachsman. Effect of 8-azaguanine on growth and viability of Bacillus megaterium. J. Bacteriol. 83:27-34. 1962.-The...
Mangalo, R. (University of Illinois, Urbana) and J. T. Wachsman. Effect of 8-azaguanine on growth and viability of Bacillus megaterium. J. Bacteriol. 83:27-34. 1962.-The addition of 8-azaguanine to exponentially growing cells of Bacillus megaterium results in an inhibition of growth after a lag of approximately 30 min. However, 8-azaguanine-2-C(14) is incorporated into the nucleic acids in a linear fashion without a detectable lag. The inhibitory action is reversed by purines and their derivatives, but not by uridine, thymidine, or cytidine. 8-Azaguanine is bactericidal, especially under conditions where growth (ribonucleic acid synthesis) is possible. Growth in the presence of a complete amino acid mixture, either before or during exposure to 8-azaguanine, increases the rate of killing. Chloramphenicol has little or no effect on the bactericidal action of the analogue.
Topics: Amino Acids; Antimetabolites; Azaguanine; Bacillus; Bacillus megaterium; Nucleic Acids; Purines; RNA; Thymidine; Uridine
PubMed: 14469259
DOI: 10.1128/jb.83.1.27-34.1962 -
Journal of Bacteriology Mar 1982Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more...
Growth of Streptomyces viridochromogenes on a solid glycerol-NH4NO3 salts medium was accompanied by the formation of aerial mycelia and spores. Adding 0.5% or more casein hydrolysate to the medium stimulated growth while completely repressing the formation of aerial mycelia and spores. This repression was temporary, as evidenced by the fact that transfer of the organisms to media not containing casein hydrolysate resulted in the appearance of aerial mycelia and spores. The effects of individual amino acids were tested. Glycine retarded growth and repressed formation of both aerial mycelia and spores. L-Aspartic acid, L-glutamic acid, and L-histidine stimulated or had little effect on growth and repressed formation of spores but not aerial mycelia. Repression by casein hydrolysate could not be attributed to the carbon/nitrogen ratio or the pH of the medium. Adding 1.25 to 2.5 mM adenine to the medium caused a reversal of the casein hydrolysate repression of aerial mycelium formation but did not reverse repression of sporulation. Dimethyladenine and 8-azaguanine had an effect similar to that of adenine, but a variety of other purine or pyrimidine derivatives had no effect on casein hydrolysate repression. The repression of aerial mycelium and spore formation by casein hydrolysate occurred only in media containing 15 mM or more phosphate. Aerial mycelia and spores were formed in media containing casein hydrolysate and 3 mM or less phosphate.
Topics: Adenine; Amino Acids; Caseins; Culture Media; Glucose; Phosphates; Potassium; Potassium Compounds; Protein Hydrolysates; Purines; Pyrimidines; Quaternary Ammonium Compounds; Spores, Bacterial; Streptomyces
PubMed: 7061380
DOI: 10.1128/jb.149.3.1102-1111.1982 -
Characterisation of methionine adenosyltransferase from Mycobacterium smegmatis and M. tuberculosis.BMC Microbiology Jun 2003Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One...
BACKGROUND
Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase.
RESULTS
The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM.
CONCLUSION
The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.
Topics: Azaguanine; Azathioprine; Cloning, Molecular; Enzyme Inhibitors; Gene Expression; Methionine Adenosyltransferase; Mycobacterium smegmatis; Mycobacterium tuberculosis; Species Specificity
PubMed: 12809568
DOI: 10.1186/1471-2180-3-12