methylnitronitrosoguanidine and Ataxia-Telangiectasia

Radiation-sensitive mutants, SX9 and SX10, were isolated from mouse mammary carcinoma FM3A cells. The mutant SX9 complemented another radiation-sensitive strain M10 of mouse leukemia L5178Y cells, but SX10 did not. SX9 and SX10 cells were more sensitive than the wild-type cells to the lethal effects of bleomycin. The frequency of X-ray-induced mutations to 6-thioguanine resistance was much higher in SX9 cells than in the wild-type cells in the dose range up to 1 Gy, although the induced mutant frequencies were not very different between these two cell strains when compared at equivalent survival. SX9, SX10 and M10 cells were found to be far more sensitive than the respective wild-type cells to hydrogen peroxide inactivation, but the levels of catalase activity were similar among these cell strains. These mutants should be useful for cloning and identifying the human genes responsible for radiation sensitivity.

Topics: Animals; Ataxia Telangiectasia; Bleomycin; Catalase; Cell Survival; Hydrogen Peroxide; Mammary Neoplasms, Experimental; Methylnitronitrosoguanidine; Mice; Mutation; X-Rays

Lymphoblastoid lines from nine Usher's syndrome (recessively inherited retinitis pigmentosa and congenital sensorineural deafness) patients (representing eight kindreds) and from ten Duchenne muscular dystrophy patients (representing seven kindreds) showed a small but statistically significant hypersensitivity to the lethal effects of X-rays, as measured by the cellular ability to exclude the vital dye trypan blue, when compared with lines from 26 normal control subjects. Fibroblast lines from the Usher's syndrome patients, treated with X-rays or the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine, also showed a statistically significant hypersensitivity when compared to normal fibroblast lines. These findings are consistent with the possibility that defective DNA repair mechanisms may be involved in the pathogenesis of these degenerative diseases.

Topics: Adolescent; Adult; Aged; Ataxia Telangiectasia; Cell Line; Cell Survival; Child; Child, Preschool; Deafness; DNA Repair; Female; Fibroblasts; Humans; Infant, Newborn; Lymphocytes; Male; Methylnitronitrosoguanidine; Middle Aged; Muscular Dystrophies; Nerve Degeneration; Retinitis Pigmentosa

The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

Topics: 4-Nitroquinoline-1-oxide; Alkylating Agents; Ataxia Telangiectasia; Bleomycin; Carcinogens; Cell Survival; Cells, Cultured; DNA Repair; DNA Replication; Humans; Methylnitronitrosoguanidine; Mitomycin; Mitomycins; Niacinamide; Oxygen; X-Rays

3 ataxia telangiectasia (AT) fibroblast cell strains, AT4BI, AT5BI and AT2BE (CRL1343) were studied for their colony-forming ability after treatment with various concentrations of 4 different DNA alkylating agents. The results were compared to the response of fibroblast strains from 3 normal individuals. None of the AT strains were abnormally sensitive to N-methyl-N'-nitro-N-nitrosoguanidine. 1 strain (AT5BI) was significantly more sensitive to treatment with methyl methanesulfonate (MMS) based on a survival curve D0 value of 0.29 mM vs. the normal average D0 of 0.38 mM (P less than 0.02) and a D10 value of 0.85 mM vs. the normal average D10 of 1.2 mM (P less than 0.025). Strain AT4BI was also significantly more sensitive to MMS treatment when D10 values were compared (0.73 mM, P less than 0.01). All 3 AT cell strains were significantly more sensitive to treatment with ethyl methanesulfonate when D10 values were the criterion of sensitivity, AT4BI 16 mM, AT5BI 13 mM and AT2BE 15 mM vs. the normal human fibroblast average D10 value of 28 mM (P less than 0.01 for all 3 AT strains). 2 of the 3 AT cell strains (AT4BI and AT2BE) were abnormally sensitive to treatment with 4-nitroquinoline-1-oxide; the D0 values were 0.045 microM and 0.05 microM, respectively, vs. the normal average D0 value of 0.11 microM (P less than 0.01 for both AT strains). The corresponding D10 values were 0.08 microM and 0.11 microM, respectively, vs. the normal average D10 value of 0.27 microM (P less than 0.01 for AT4BI and P less than 0.025 for AT2BE). These results indicate that there is a heterogeneity in the response of AT fibroblast cell strains to treatment with DNA alkylating agents, except possibly in the case of ethylating compounds.

Topics: 4-Nitroquinoline-1-oxide; Alkylating Agents; Ataxia Telangiectasia; Cell Line; Cell Survival; Ethyl Methanesulfonate; Fibroblasts; Humans; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Reference Values; Skin

Topics: Ataxia Telangiectasia; Bleomycin; Cell Line; Dactinomycin; DNA; DNA Replication; Drug Resistance; Humans; Kinetics; Methylnitronitrosoguanidine; Skin

Human lymphoblastoid cell lines from normal individuals and from patients with ataxia telangiectasia were either proficient or deficient in their ability to repair the mutagenic DNA adduct O6-methylguanine that is induced by methylating carcinogens. There was no relationship between the capacity to repair O6-methylguanine and the ataxia telangiectasia phenotype. Time-course studies done following a short pulse (2 min) of alkylation with 0.5 microgram of N-[3H]methyl-N'-nitro-N-nitrosguanidine per ml revealed that the repair of O6-methylguanine in human lymphoblastoid lines proficient in this ability is a rapid process, which proceeds with a half-life of 10 to 15 min. Lymphoblastoid lines with deficient capacity to repair this DNA adduct were hypersensitive to the cytotoxic effect of the methylating carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, and methyl methanesulfonate, and this hypersensitivity was correlated with the relative amount of O6-methylguanine induced by each of the three chemicals. This was taken as an indication of the lethality of unrepaired O6-methylgluanine. The extent of DNA repair synthesis induced by the three carcinogens was the same in cell lines proficient and deficient in O6-methylguanine repair, indicating no major deficiency in an excision repair pathway in the hypersensitive cell lines.

Topics: Adolescent; Adult; Alkylating Agents; Ataxia Telangiectasia; Cell Division; Cells, Cultured; Child; DNA; DNA Repair; Female; Guanine; Humans; Infant, Newborn; Kinetics; Male; Methyl Methanesulfonate; Methylation; Methylnitronitrosoguanidine; Methylnitrosourea

Permanently proliferating lymphoblastoid cell lines (LCLs) and normal unstimulated peripheral blood leukocytes have been used to study the effects of nitrosocimetidine (NC) on cultured human lymphoid cells. The approaches that were used to assess the cells' ability to cope with NC were: (i) determination of cell survival as measured by colony formation in microtiter plates; (ii) quantitation of DNA synthesis and DNA-repair replication by isopyknic sedimentation of DNA density labeled with 5-bromo-2-deoxyuridine (BrdU); (iii) measurement of the induction of alkali labile lesions and strand breaks by NC in 3H-labeled DNA using velocity sedimentation in alkaline sucrose. In summary, treatment with NC was found to inhibit both replicative DNA synthesis and colony formation in LCLs. At the molecular level, NC treatment induced alkali labile lesions in LCL DNA and elicited DNA-repair replication in proliferating LCLs as well as unstimulated lymphocytes. Considered in total, these data indicate that NC is reactive with human DNA in the cellular environment in a manner similar to methylating nitroso compounds which have been shown to be carcinogenic. The significance of these findings will be discussed.

Topics: Ataxia Telangiectasia; Cell Line; Cell Survival; Cimetidine; DNA Replication; Guanidines; Humans; Leukocytes; Methylnitronitrosoguanidine; Nitroso Compounds; X-Rays

Topics: Ataxia Telangiectasia; Cells, Cultured; DNA Repair; Dose-Response Relationship, Drug; Guanine; Humans; Kinetics; Methylnitronitrosoguanidine; Mutation; Xeroderma Pigmentosum

DNA repair synthesis and posttreatment colony-forming ability were measured in six human fibroblast cell strains derived from patients with the autosomal recessive disorder, ataxia telangiectasia (AT), and five cell strains from normal individuals. All 11 cell strains showed approximately equal levels of DNA repair synthesis after methyl methanesulfonate and ultraviolet light treatments assayed by the benzoylated naphthoylated diethylaminoethyl-cellulose method. Four AT cell strains exhibited 27 to 41% of the repair shown by normal cell strains after treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two AT cell strains had normal levels of repair synthesis after MNNG treatment. All the AT strains tested had normal survival after treatment with methyl methanesulfonate and ultraviolet light as assayed by posttreatment colony-forming ability. The six AT cell strains exhibited diminished survival after MNNG treatment. The inhibition of [3H]thymidine uptake as a function of MNNG concentration was similar for all the normal and AT cell strains tested.

Topics: Alkylating Agents; Ataxia Telangiectasia; Cell Division; Clone Cells; DNA Repair; DNA, Neoplasm; Humans; Methylnitronitrosoguanidine; Ultraviolet Rays