azaguanine and Lesch-Nyhan-Syndrome

GTP cyclohydrolase I feedback regulatory protein (GFRP) mediates the feedback inhibition of GTP cyclohydrolase I activity by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) through protein complex formation. Since guanine and BH4 have a common pyrimidine ring structure, we examined the inhibitory effect of guanine and its analogs on the enzyme activity. Guanine, 8-hydroxyguanine, 8-methylguanine, and 8-bromoguanine inhibited the enzyme activity in a GFRP-dependent and pH-dependent manner and induced complex formation between GTP cyclohydrolase I and GFRP. The type of inhibition by this group is a mixed type. All these properties were shared with BH4. In striking contrast, inhibition by 8-azaguanine and 8-mercaptoguanine was GFRP-independent and pH-independent. The type of inhibition by 8-azaguanine and 8-mercaptoguanine was a competitive type. The two compounds did not induce complex formation between the enzyme and GFRP. These results demonstrate that guanine compounds of the first group bind to the BH4-binding site of the GTP cyclohydrolase I/GFRP complex, whereas 8-azaguanine and 8-mercaptoguanine bind to the active site of the enzyme. Finally, the possible implications in Lesch-Nyhan syndrome and Parkinson diseases of the inhibition of GTP cyclohydrolase I by guanine and 8-hydroxyguanine are discussed.

Topics: Adjuvants, Immunologic; Animals; Antimetabolites, Antineoplastic; Azaguanine; Binding Sites; Binding, Competitive; Chromatography, Gel; Dose-Response Relationship, Drug; GTP Cyclohydrolase; Guanine; Guanosine; Guanosine Triphosphate; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Kinetics; Lesch-Nyhan Syndrome; Models, Chemical; Parkinson Disease; Rats; Thionucleosides

Our experience with the prenatal detection of the Lesch-Nyhan syndrome (LNS; hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency) in three fetuses at risk is reported. Enzyme activities were measured in cultured amniocytes in two pregnancies, and in tissues and cultures obtained from chorionic villus sampling (CVS) in a third pregnancy. In all tissues the specific activities of HGPRT and adenine phosphoribosyltransferase (APRT) were determined and APRT/HGPRT ratios were calculated. In addition to the enzyme assays, the rate of purine synthesis de novo was assessed in the two amniocyte cultures, and the rate of [14C]hypoxanthine incorporation into nucleotides and sensitivity to azaguanine were measured in one of the amniocyte cultures. We report the diagnosis of normal fetuses by study of amniocytes in two pregnancies and of LNS using CVS in one pregnancy. In all three cases the diagnosis was confirmed.

Topics: Adenine Phosphoribosyltransferase; Amniotic Fluid; Azaguanine; Brain; Chorionic Villi; Chorionic Villi Sampling; Female; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Muscles; Pregnancy; Prenatal Diagnosis; Purines; Skin

The feasibility of using RNA synthesis in freshly isolated, human peripheral blood lymphocytes to detect 6-thioguanine (TG)- and 8-azaguanine (AG)-resistant variants in an autoradiographic assay similar to that of Strauss and Albertini (1979) has been evaluated. In phytohemagglutinin (PHA)-stimulated cultures RNA synthesis and HPRT activity began well in advance of DNA synthesis and increased in parallel during the first 44 h of culture. Introduction of TG or AG with PHA at the beginning of culture completely inhibited DNA synthesis during the first 44 h and reduced RNA synthesis to low levels within 24 h. When TG or AG was added after cells had been in culture for 38 h, DNA synthesis was reduced quickly while RNA synthesis was inhibited more slowly. An autoradiographic assay is described in which freshly isolated lymphocytes are cultured with PHA for 24 h, with or without TG or AG, then labeled with [3H]uridine for 1 h. TG-resistant and AG-resistant variant frequencies for 2 normal individuals and a Lesch-Nyhan individual were determined with this assay. The variant frequencies for the normal individuals ranged from 0.46 to 10.6 X 10(-5) depending upon the selective conditions used. All the Lesch-Nyhan cells were resistant to 0.2 microM-2 mM AG; some were sensitive to 0.2 mM TG and most were sensitive to 2.0 mM TG.

Topics: Autoradiography; Azaguanine; Drug Resistance; Genetic Variation; Humans; Hypoxanthine Phosphoribosyltransferase; In Vitro Techniques; Lesch-Nyhan Syndrome; Lymphocytes; Phytohemagglutinins; RNA; Thioguanine

In the present study, hypoxanthine phosphoribosyltransferase (HPRT) has been investigated in fibroblasts of 19 patients from 16 different families with HPRT deficiency, concerning activity, incorporation of 14C-hypoxanthine, and growth in 8-azaguanine and HAT (hypoxanthine, azaserine, thymidine containing) selection media. According to these data we could classify the patients into 5 groups (patients with classical Lesch-Nyhan syndrome and patients with HPRT variants of types A, B, C, D). In 3 groups (patients with classical Lesch-Nyhan syndrome, HPRT variants C and D), a correlation of residual HPRT activity with the incorporation of 14C-hypoxanthine as well as growth in 8-azaguanine and HAT selection was observed. The variant A, from a patient with the classical Lesch-Nyhan syndrome, exhibited higher HPRT activity than that from all the other patients with the Lesch-Nyhan syndrome. However, the values of hypoxanthine incorporation and growth in selection media were as in the classical syndrome. The cells of variant B were resistant to azaguanine and grew in HAT selection media in the range of control cells, but had HPRT residual activities similar to those of variants A and C. For the characterization of the genetic heterogeneity of HPRT, it seems necessary to study the enzymatic properties in cell extracts as well as the purine uptake and proliferation of cells in different selection media.

Topics: Azaguanine; Cell Line; Cells, Cultured; Culture Media; Fibroblasts; Genetic Variation; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Lesch-Nyhan Syndrome

Topics: Azaguanine; Cell Division; Cells, Cultured; Fibroblasts; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Immunoassay; Kinetics; L Forms; Lesch-Nyhan Syndrome; Mutation

Azaguanine-resistant mutants of Chinese hamster ovary cells were isolated following mutagenesis with ICR-17OG. Of the eight mutant isolates examined, only one, Ag-5 had detectable hypoxanthine(guanine)phosphoribosyltransferase activity. Under normal conditions of growth, de novo purine biosynthesis in the mutants was not significantly different from wild type. However, when the cultures were starved for glutamine over a 2 h period before measuring 5'phosphoribosyl formylglycinamide (a relative measure of de novo purine biosynthesis), elevated levels of 5'-phosphoribosyl formylglycinamide accumulated in some of the mutants, and decreased levels in wild type and Ag-5. The level of purine biosynthesis could be related to the levels of glutamine in the pregrowth medium. The rate of purine biosynthesis correlated with 5-phosphoribosyl pyrophosphate levels, which were enhanced in the mutant (Ag-C) following the starvation period. No alterations were found in levels of 5-phosphoribosyl pyrophosphate synthetase or glutamine synthetase. The extent of feedback inhibition was normal in both mutant and wild type cells. These data suggest that the hypoxanthine (guanine) phosphoribosyltransferase locus is a regulatory gene.

Topics: Azaguanine; Cell Line; Feedback; Glutamine; Glycine; Humans; Hypoxanthine Phosphoribosyltransferase; Lesch-Nyhan Syndrome; Phosphoribosyl Pyrophosphate; Purines; Ribose-Phosphate Pyrophosphokinase

The toxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on cultured diploid human fibroblasts were studied. When 10(5) cells per 60 mm dish were exposed to MNNG for 4 h in Ham's medium F10 containing 0.02 M HEPES buffer at pH 6.8, MNNG concentrations of less than 1 X 10(-6) M resulted in cell survivals near 100%, while the average survival was less than one percent at concentrations greater than 5 X 10(-6) M. After treatment with MNNG, cells were subjected to selection using optimal conditions for the detection of diploid human fibroblasts that are resistant to the guanine-analogs AG and TG because they contain altered or deficient HPRT. The induced mutant frequency was maximized by allowing a 5 to 7 day post-treatment interval for the expression of the mutant phenotype and by replating the cells at the beginning of selection at a population density of less than 450 cells per cm2. Careful attention was given to counting statistically adequate numbers of mutants and to accurately determining cell survival and replating cloning efficiencies. Independent dose-response experiments gave induced mutant frequencies as high as 7.0 X 10(-4) to 8.8 X 10(-4) mutants per viable cell at about 5% survival, compared to a spontaneous mutation rate of 3.7 X 10(-6) to 7.2 X 10(-6) mutants per cell generation. The AGr mutants observed after treatment with MNNG were phenotypically stable and closely resembled prototype AGr cultures derived from humans who have inherited mutant X-chromosomal alleles for HPRT.

Topics: Azaguanine; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance; Fibroblasts; Gout; Humans; Lesch-Nyhan Syndrome; Male; Methylnitronitrosoguanidine; Mutagens; Mutation; Phenotype; Skin

Topics: Azaguanine; Binding Sites; Drug Resistance; Erythrocytes; Fibroblasts; Gout; Guanine; Heterozygote; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Kinetics; Lesch-Nyhan Syndrome; Phosphoribosyl Pyrophosphate

Incorporation of hypoxanthine, resistance to 8-azaguanine and activation by lyophilisation have been studied in cultured human fibroblasts. Cells from one family where there was a boy with Lesch-Nyham syndrome, from two families with variant H-PRT mutations and three cell strains from patients with the Lesch-Nyham syndrome were investigated. Cells from patients with the Lesch-Nyham syndrome showed almost no hypoxanthine incorporation and resistance to concentrations of 8-azaguanine up to 10(-3) M, whereas cells of patients with partial H-PRT deficiency demonstrated variant patterns of hypoxanthine uptake and partial resistance to 8-azaguanine. Lyophilisation of fibroblast sediment from patients with the Lesch-Nyhan syndrome and patients with variant H-PRT mutations showed activation of the deficient or partially deficient H-PRT enzyme. No such activation was observed in healthy controls. Activation of lyophilised fibroblast extract from patients and controls was not obtained. These results suggest that H-PRT could be associated with the cell membranes.

Topics: Azaguanine; Cells, Cultured; Female; Fibroblasts; Freeze Drying; Hot Temperature; Humans; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Lesch-Nyhan Syndrome; Male; Mutation

The cytotoxicity of the "K-region" epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several "K-region" epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ratio of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions. To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by "K-region" epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the "K-region" epoxide of benzo(a)pyrene (BP), 7,12-dimethyl-benz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.

Topics: Azaguanine; Cell Survival; DNA; DNA Repair; Drug Resistance; Ethers, Cyclic; Humans; Lesch-Nyhan Syndrome; Molecular Weight; Mutation; Polycyclic Compounds; Structure-Activity Relationship; Xeroderma Pigmentosum

Topics: Adolescent; Adult; Azaguanine; Evaluation Studies as Topic; Female; Gene Frequency; Hemagglutination Tests; Heterozygote; Humans; Lectins; Lesch-Nyhan Syndrome; Lymphocytes; Male; Methods; Mosaicism; Mutation; Pedigree; Thymidine; Tritium

Topics: Aminopterin; Autoradiography; Azaguanine; Cell Line; Clone Cells; Culture Media; Drug Resistance; Fibroblasts; Guanine; Humans; Hypoxanthines; In Vitro Techniques; Lesch-Nyhan Syndrome; Male; Mutation; Penis; Pentosyltransferases; Probability; Thymidine; Tritium

We have previously described a 14-yr-old boy with hyperuricemia, renal failure, and accelerated purine production resistant in vivo and in vitro to purine analogs. This patient demonstrated normal red cell hypoxanthine-guanine phosphoribosyltransferase (HPRT) heat stability, electrophoresis at high pH, and activity at standard substrate levels. In the present report an abnormal HPRT enzyme was demonstrated by enzyme kinetic study with phosphoribosylpyrophosphate (PRPP) as the variable substrate and inhibitory studies with sodium fluoride. Apparently normal HPRT activity in a patient with hyperuricemia and gout does not exclude a functionally significant HPRT mutation.

Topics: Adolescent; Aminopterin; Azaguanine; Azaserine; Cells, Cultured; Culture Media; Erythrocytes; Fibroblasts; Guanine; Humans; Hydrogen-Ion Concentration; Hypoxanthines; Kinetics; Lesch-Nyhan Syndrome; Male; Mutation; Organophosphorus Compounds; Pentosephosphates; Pentosyltransferases; Phosphoric Acids; Purine-Pyrimidine Metabolism, Inborn Errors; Purines; Temperature; Uric Acid

Topics: Animals; Azaguanine; Bromodeoxyuridine; Cell Line; Cell Transformation, Neoplastic; Chromosome Aberrations; Chromosome Disorders; Chromosomes; Chromosomes, Human, 6-12 and X; Clone Cells; Crosses, Genetic; Drug Resistance; Fibroblasts; Guanine; Humans; Hybrid Cells; Hypoxanthines; Karyotyping; Klinefelter Syndrome; Lesch-Nyhan Syndrome; Male; Mice; Mutation; Pentosyltransferases; Simian virus 40; Skin; Thymidine Kinase

Topics: Azaguanine; Carbon Isotopes; Cell Division; Cells, Cultured; Chlorides; Erythrocytes; Fibroblasts; Guanine; Humans; Hypoxanthines; Lesch-Nyhan Syndrome; Magnesium; Mutation; Pentosyltransferases; Ribonucleotides

Topics: Adenine; Azaguanine; Cells, Cultured; Diploidy; Drug Resistance; Electrophoresis; Fibroblasts; Gels; Genes; Genetics, Medical; Humans; Hypoxanthines; Lesch-Nyhan Syndrome; Male; Molecular Biology; Mutation; Pentosyltransferases; Sex Chromosomes

Topics: Adenine; Adolescent; Aminopterin; Azaguanine; Blood Urea Nitrogen; Carbon Isotopes; Cell Line; Creatinine; Drug Resistance; Electrophoresis, Starch Gel; Erythrocytes; Fibroblasts; Gout; Guanine; Humans; Hypoxanthines; Lesch-Nyhan Syndrome; Male; Mercaptopurine; Pentosyltransferases; Purines; Uric Acid

Topics: Athetosis; Autoradiography; Azaguanine; Biopsy; Compulsive Behavior; Culture Techniques; Female; Fibroblasts; Genetics, Medical; Humans; Huntington Disease; Hypoxanthines; Intellectual Disability; Lesch-Nyhan Syndrome; Mutation; Purine-Pyrimidine Metabolism, Inborn Errors; Purines; Self Mutilation; Skin; Uric Acid