thioguanine-anhydrous and 2-6-diaminopurine

Somatic cells of whole Syrian hamster fetuses (gestation day 13) were isolated and tested by an in vivo/in vitro mutation assay for spontaneous mutation frequencies using independent 6-thioguanine (6-TG), diphtheria toxin (DT), and ouabain mutation selection systems. Optimum conditions were ascertained. For 6-TG mutants, a total of 21 mutants were found in cells from 24 litters on 1993 plates, for an overall mutant frequency of 1.8 x 10(-7) per viable cell with 12 positive litters. In all, 26 litters were tested using DT; 77 mutants were found in 840 plates, yielding an overall mutant frequency of 2.6 x 10(-7), with 20 positive litters. No correlations or familial effects were found among 23 litters tested for both DT and 6-TG. Of 14 litters which were tested for ouabain mutants, 4 were positive, with a total of 5 mutants found on 988 plates, for an overall mutant frequency of 7.6 x 10(-8). For 14 F344 rat fetuses, the overall 6-TG spontaneous mutation frequency was determined to be 1.6 x 10(-7). From the data, estimates of mutation rates were calculated. For mutation to 6-TG resistance the rate was 8.3 x 10(-8), for mutation to DT resistance the rate was 8.1 x 10(-8) and for ouabain, the spontaneous mutation rate was 5.7 x 10(-8). For F344 rat, the spontaneous mutation rate was 1.1 x 10(-7). Induced mutant frequencies after in utero exposure to 1 mmol/kg N-ethyl-N-nitrosourea (ENU) were 311, 135 and 200 times the spontaneous value for 6-TG, DT and ouabain, respectively, for Syrian hamster fetal cells and 125 times the spontaneous 6-TG value for fetal F344 rat cells. Both spontaneous mutation frequencies and underlying spontaneous mutation rates are low, consistent with the view that fetal cells exercise extremely tight control over DNA fidelity.

Topics: 2-Aminopurine; Animals; Brain; Breeding; Cell Survival; Cells, Cultured; Cricetinae; Diphtheria Toxin; Dose-Response Relationship, Drug; Drug Resistance; Female; Fetus; Gene Frequency; Male; Mesocricetus; Mutation; Ouabain; Pregnancy; Rats; Rats, Inbred F344; Thioguanine

We have used the adenine phosphoribosyltransferase gene (APRT; 16q24) to investigate the mechanisms of loss of heterozygosity (LOH) in normal human somatic cells in vivo. APRT-deficient (APRT-/-, APRT-/0) T lymphocytes from the peripheral blood of four obligate APRT heterozygotes (APRT+/-) with characterized germ-line mutations were selected in medium containing 100 microM 2,6-diaminopurine. A total of 80 2,6-diaminopurine-resistant T-cell clones from 2 of the heterozygotes were analyzed for this study. The presence or absence of LOH of proximal linked microsatellite repeat markers was used to divide the clones into two groups: (a) those in which LOH was likely due to localized changes in APRT (e.g., point mutations); and (b) those with LOH at additional loci. A total of 61 clones (76%) exhibited LOH of linked microsatellite repeat markers at different locations on 16q, which extended from the smallest measured region (<5.5 cM) to the entire 16q arm. The remaining 19 clones (24%) had point mutations in APRT or other relatively minor alterations. Ten clones with LOH encompassing different regions of 16q were examined by conventional cytogenetics and by fluorescence in situ hybridization using an APRT cosmid probe. All clones exhibited a normal diploid karyotype, and nine exhibited two copies of APRT. The one clone that was hemizygous for APRT had the smallest observed region of LOH in clones from that individual. These results indicate that mitotic recombination and, to a much lesser extent, deletion may be the primary mechanisms for the relatively high frequency of in vivo LOH observed in normal human T cells. Because LOH leads to the expression of recessive tumor suppressor genes in many cancers, these data have significant implications for the role of LOH in the early stages of tumor development, especially in breast cancer.

Topics: 2-Aminopurine; Adenine Phosphoribosyltransferase; Chromosome Mapping; Clone Cells; Drug Resistance; Female; Gene Deletion; Gene Rearrangement, T-Lymphocyte; Heterozygote; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Microsatellite Repeats; Mitosis; Point Mutation; Recombination, Genetic; T-Lymphocytes; Thioguanine

Co-incubation of human leukemia cell lines with naturally occurring nucleobases (hypoxanthine or adenine) significantly prevented the cytotoxic activity of 6-thiopurines. Extracellular hypoxanthine decreased the transport of 6-mercaptopurine into cells, but adenine had no significant effect. However, intracellular thioinosine monophosphate accumulation in the presence of 10 microM, 6-mercaptopurine was reduced to below 1% or 10% of that of the controls when 50 microM hypoxanthine or adenine was added, respectively. Finally, in adenine phosphoribosyl transferase deficient mutants, adenine provided no protective effect against 6-thiopurines, whereas hypoxanthine retained its modulating activity. These data suggest that the nucleobases compete with 6-thiopurines for the ribose-phosphate donor, 5'-phosphoribosyl-1-pyrophosphate, thus preventing the formation of active metabolites of 6-thiopurines.

Topics: 2-Aminopurine; Adenine; Adenine Phosphoribosyltransferase; Antineoplastic Agents; Biological Transport; Humans; Hypoxanthine; Hypoxanthine Phosphoribosyltransferase; Hypoxanthines; Inosine Monophosphate; Leukemia-Lymphoma, Adult T-Cell; Leukemia, Promyelocytic, Acute; Mercaptopurine; Thioguanine; Thionucleotides; Tumor Cells, Cultured

The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed.

Topics: 2-Aminopurine; Adenine; Adenine Phosphoribosyltransferase; Animals; Azaguanine; Cells, Cultured; Cricetinae; Epithelial Cells; Epithelium; Hypoxanthine Phosphoribosyltransferase; Liver; Mutagenicity Tests; Pentosyltransferases; Thioguanine; Thymine Nucleotides

The ability of excess thymidine (10(-6)-10(-3) M) to enhance the frequency of 6-thioguanine (6-TG) resistant cell mutants and 2,6-diaminopurine (DAP) resistant cell mutants in Friend mouse erythroleukaemia cells, clone 707, was investigated. A significant increase in mutant frequency for both markers was observed at the higher (10(-4) and 10(-3) M) thymidine treatments. Measurements of deoxyribonucleoside triphosphate pool sizes in the cells revealed a dramatic elevation of the deoxythymidine triphosphate and deoxyguanosine triphosphate pools, an increase in the deoxyadenosine triphosphate pool and an almost complete disappearance of the deoxycytidine triphosphate pool at the higher thymidine treatments. This complemented the mutagenesis data. These results support the view that increases in mutant frequency may take place following perturbations in DNA precursor pools through a resultant decrease in the fidelity of DNA synthesis. Measurements of deoxyribonucleoside triphosphate pools were also carried out on clone 707 Friend cells and a thymidine kinase-deficient subclone, 707 BUF. The thymidine kinase-deficient subclone had significantly reduced deoxythymidine triphosphate and deoxyguanosine triphosphate pools relative to those observed in-clone 707 cells. The previously observed mutagen hypersensitivity in thymidine kinase-deficient Friend cells may result through pool imbalance rendering DNA excision repair error prone.

Topics: 2-Aminopurine; Animals; Cell Cycle; Cell Line; Cell Survival; Clone Cells; Deoxyribonucleotides; Drug Resistance; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Mutagens; Mutation; Thioguanine; Thymidine

Mutants of human lymphoblastoid cell lines have been isoalted, which are resistant to 6-thioguanine or to 2,6-diaminopurine. They have been partially characterized and shown to be almost totally deficient in HGPRT and APRT, respectively. Cell lines doubly mutant for HGPRT deficiency and resistance to ouabain have also been isolated. All mutants were indistinguishable from the respective parental lines by criteria other than those leading specifically to their drug resistant phenotypes. The use of these mutants in the production of hybrids between two lines of suspension-growing human lymphoblastoid cells is discussed.

Topics: 2-Aminopurine; Adenine; Adenine Phosphoribosyltransferase; Cell Line; Cell Membrane; Drug Resistance; Humans; Hypoxanthine Phosphoribosyltransferase; Karyotyping; Mutation; Nucleotidases; Ouabain; Thioguanine

Topics: 2-Aminopurine; Amphotericin B; Animals; Antineoplastic Agents; Azaguanine; Bromodeoxyuridine; Carbon Isotopes; Cell Division; Cell Line; Cricetinae; Dactinomycin; Fibroblasts; Floxuridine; Fluorouracil; Hexoses; Hypoxanthines; Mercaptopurine; Mesocricetus; Metabolism; Mitomycin; Mitomycins; Mutation; Nucleosides; Pharmacology; Puromycin; Research; Thioguanine; Tissue Culture Techniques; Tritium; Vinblastine

Topics: 2-Aminopurine; Animals; Fetus; Purines; Rats; Thioguanine