methylnitronitrosoguanidine and thymidine-5--triphosphate

Treatment of Chinese hamster V79 cells with dThd, dCyd, or dThd plus dCyd increased MNNG-induced AGr-, TGr-, and Ouar-mutant frequencies but did not significantly increase background mutant frequencies. All the AGr colonies that were isolated possessed phenotypes characteristic of HGPRT-deficient mutants, and the deoxyribonucleosides did not selectively affect the growth of the mutants, nor the selecting efficiency of AG, and did not significantly enhance background mutagenesis. These data show that both dThd and dCyd facilitated MNNG-induced mutagenesis. This facilitation was maximal when cells were exposed to the deoxyribonucleosides throughout the first doubling time (24 h) after treatment with MNNG and for 4 more doubling times prior to mutant selection with AG. This indicates that one round of DNA replication was sufficient for mispairing of methylated bases in the DNA with the C and T provided by the deoxyribonucleosides, and that 4-6 doublings prior to mutant selection with AG were necessary to deplete pre-existing hypoxanthine: guanine phosphoribosyl transferase in newly mutated cells. The dCyd facilitated mutagenesis by FdUrd, which was not mutagenic without dCyd, indicating that increased dCTP:dTTP ratios were mutagenic. Treatment with FdUrd plus dCyd also induced FdUrdr cells, suggesting that inhibition of dCyd utilization may prevent the development of FdUrd-resistance in cancer chemotherapy. Although dCyd and dThd facilitated mutagenesis in cells treated with monofunctional alkylating agents that methylate DNA oxygens, facilitation of mutagenesis did not occur in cells treated with BCNU, which cross links DNA, nor with benzo(a)pyrene and aflatoxin B1, which are frame shift mutagens, nor with MMS, which produces barely detectable levels of O-methylation in DNA. Virtually non toxic concentrations of dThd potentiated the cytotoxicity of MNNG more than 10-fold but that of MMS was potentiated only about 2-fold showing that O-alkylation of DNA was associated not only with the facilitation of mutagenesis but also with the potentiation of cytotoxicity. The potentiation of MNNG-induced cytotoxicity was maximal in V79 and L1210 cells after only 2 h treatment with dThd, showing that not even one round of DNA replication was necessary for this potentiation. Moreover, dCyd abolished the potentiation, and, at equitoxic concentrations, MNNG induced higher mutant frequencies than did MMS. These data show that the mechanisms by which methylating agents plus d

Topics: Alkylating Agents; Animals; Azaguanine; Cell Line; Cell Survival; Cricetinae; Deoxyadenosines; Deoxycytidine; Deoxycytosine Nucleotides; Deoxyguanosine; Deoxyribonucleosides; DNA; Drug Resistance; Drug Synergism; Floxuridine; Leukemia L1210; Lung; Methylation; Methylnitronitrosoguanidine; Mice; Mutagens; Mutation; Thymidine; Thymidylate Synthase; Thymine Nucleotides

Topics: Alkylating Agents; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Ethyl Methanesulfonate; HL-60 Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Methylnitronitrosoguanidine; Mutagens; Quinazolines; Thiophenes; Thymidylate Synthase; Thymine Nucleotides

The effect of mutational loss of thymidine kinase (TK) on the sensitivity to alkylating agents was investigated in promyelocytic, HL-60, and T-lymphoblastoid, Molt-3, human leukemia cell lines. Although both cell lines exhibited approx. 1% residual TK activity, only HL-60 TK deficient cells had a decreased intracellular TTP pool, i.e., 20% of that of the wild-type. When treated with N-methyl-N'-nitronitrosoguanidine or ethyl methanesulfonate, HL-60 TK deficient cells showed significantly increased killing and mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus relative than did wild-type. Pretreatment of cells with O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, partially abolished those differences. Molt-3 wild-type and TK deficient cells had similar cell survivals and HGPRT mutation frequencies following treatment with alkylating agents. These results indicate that TK deficiency, only when a concomitant decrease of TTP pool is detected, plays a pivotal role in the sensitivity to the cytotoxic and mutagenic effects of alkylating agents.

Topics: Antineoplastic Agents; Antineoplastic Agents, Alkylating; Cell Survival; Ethyl Methanesulfonate; Guanine; HL-60 Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Leukemia, T-Cell; Methylnitronitrosoguanidine; Methyltransferases; Mutagenesis; Mutagens; O(6)-Methylguanine-DNA Methyltransferase; Thymidine Kinase; Thymine Nucleotides; Tumor Cells, Cultured

The effect of an increased intracellular dCTP:dTTP ratio on the specificities of ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis was examined in the yeast Saccharomyces cerevisiae. To do so, we used a dCMP deaminase-deficient (dcd1) strain having a dCTP:dTTP ratio > 77-fold larger than its isogenic wild-type parent under the treatment conditions employed. This DNA precursor imbalance lowered the frequencies of EMS- or MNNG-induced SUP4-o mutations by 75 or 45%, respectively, relative to the corresponding values for the wild-type strain. A total of 405 SUP4-o mutations produced by the alkylating agents in the dcd1 background were characterized by DNA sequencing and the mutational spectra were compared to those for 399 mutations induced in the wild-type parent and 207 mutations that arose spontaneously in the dcd1 strain. Unexpectedly, the frequencies of EMS- and MNNG-induced GC-->AT transitions in the dcd1 strain were found to be reduced by 93 and 68%, respectively, considerably more than the decreases for the overall SUP4-o mutation frequencies. The differences were due mainly to substantial increases in the frequencies of GC-->CG transversions. Although these events were the predominant type of spontaneous substitution in the dcd1 strain, they were more frequent after alkylation treatment and were distributed differently than the spontaneous GC-->CG transversions. Preferences for the EMS- or MNNG-induced GC-->AT transitions to occur at GC sites having the guanine located on the transcribed strand or preceded by a 5' purine, respectively, also were diminished in the dcd1 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkylation; Base Sequence; Deoxycytosine Nucleotides; DNA Mutational Analysis; DNA Repair; DNA Replication; DNA, Fungal; Ethyl Methanesulfonate; Genes, Fungal; Genes, Suppressor; Guanine; Methylnitronitrosoguanidine; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutagens; Nucleic Acid Heteroduplexes; Point Mutation; Saccharomyces cerevisiae; Thymine; Thymine Nucleotides

The cytotoxicity and DNA lesions induced by methotrexate (MTX) were compared in wild-type, hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) and thymidine-kinase-deficient (TK-) HL-60 cells. TK- and HGPRT- cells were approximately 10 and 3 times more sensitive to MTX than wild-type cells, respectively. Following incubation with 2 microM MTX for 16 hr, TK- cells showed a significantly higher number of DNA strand breaks. Concomitantly, DNA fragmentation at the nucleosomal linker region was detected more prominently in TK- cells. Although MTX tended to decrease TTP pools similarly in all 3 cells types, the initial TTP level in TK- cells was only about one-fifth of that found in the wild type. These results indicate that the thymidine salvage pathway has a pivotal role in mediating MTX-induced toxicity and DNA lesions.

Topics: Cell Line; Cell Survival; DNA Damage; DNA, Neoplasm; Humans; Hypoxanthine Phosphoribosyltransferase; Kinetics; Leukemia, Promyelocytic, Acute; Methotrexate; Methylnitronitrosoguanidine; Mutagenesis; Thymidine Kinase; Thymine Nucleotides

Topics: Animals; Aphidicolin; Cell Line; Cell Survival; Cricetinae; Deoxycytosine Nucleotides; Deoxyribonucleotides; Diterpenes; DNA; DNA Polymerase II; Drug Resistance; Female; Methylnitronitrosoguanidine; Mutation; Ovary; Thymine Nucleotides; Ultraviolet Rays