methylnitronitrosoguanidine and Lymphoma

To investigate the role of the presumed DNA mismatch repair (MMR) gene Msh2 in genome stability and tumorigenesis, we have generated cells and mice that are deficient for the gene. Msh2-deficient cells have lost mismatch binding and have acquired microsatellite instability, a mutator phenotype, and tolerance to methylating agents. Moreover, in these cells, homologous recombination has lost dependence on complete identity between interacting DNA sequences, suggesting that Msh2 is involved in safeguarding the genome from promiscuous recombination. Msh2-deficient mice display no major abnormalities, but a significant fraction develops lymphomas at an early age. Thus, Msh2 is involved in MMR, controlling several aspects of genome stability; loss of MMR-controlled genome stability predisposes to cancer.

Topics: Animals; Base Composition; Base Sequence; Chimera; DNA; DNA Repair; DNA-Binding Proteins; Fungal Proteins; Genetic Predisposition to Disease; Lymphoma; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Mutant Strains; Molecular Sequence Data; Mutagenesis; MutS Homolog 2 Protein; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Recombination, Genetic; Stem Cells

Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992) J. Biol. Chem. 267, 5272-5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway-defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine-inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan(EthN-P-->)ManGlcN- and EthN-P-6Man(EthN-P-6)Man(EthN- P-->)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D-glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

Topics: Animals; Antigens, Surface; Carbohydrate Sequence; Cell Line; Clone Cells; Flow Cytometry; Glycosylphosphatidylinositols; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Lymphoma; Membrane Glycoproteins; Methylnitronitrosoguanidine; Mice; Molecular Sequence Data; Mutagenesis; Phosphatidylinositols; Thy-1 Antigens; Tumor Cells, Cultured; Uridine Diphosphate Glucose; Uridine Diphosphate N-Acetylglucosamine

We have cloned malignant cells carrying specific antigens associated with ovarian cancer (OVC) and malignant lymphoma (ML) from BS-SHI-4M cells, a line derived from a 1-methyl-3-nitro-1-nitrosoguanidine-treated B-lymphoblastoid cell line isolated from a patient with Bloom syndrome. Since BS-SHI-4M cells react with sera from various individual cancer patients at relatively low frequencies (2-9%), as detected by an indirect immunofluorescence technique, cell clones that specifically react with sera from patients with OVC and ML were separated by the "panning" method in which polystyrene dishes were coated with sera from OVC and ML patients and cells with the corresponding antigens bound to the dishes. Subsequent cloning by limiting dilution provided cell clones highly enriched for OVC- and ML-associated antigens. Karyotype analyses revealed that cell clones with OVC and ML antigens had common marker chromosomes, der(14)t(14;14) (p11;q11),t(6;?)(p25;?) and t(9;?)(q34;?), besides t(17;?) (q25;?) found in the OVC-antigen-positive clones and t(5;?) (p13;?),t(7;?)(q36;?) found in the ML-antigen-positive clones. Interestingly, in cell clones with a strong OVC antigen response, the distal part of the Y chromosome (Yq11) was missing in 100% of the cells. Therefore the cell line BS-SHI-4M appears to be a reservoir of cell clones each of which carries a specific tumor antigen and thus provides a potential tool for rapid serological diagnosis of cancer.

Topics: Antigens, Neoplasm; Bloom Syndrome; Cell Line; Chromobox Protein Homolog 5; Clone Cells; DNA, Neoplasm; Female; Genetic Markers; Humans; Karyotyping; Lymphoma; Methylnitronitrosoguanidine; Ovarian Neoplasms

The reduced tumorigenicity of mutagenized cells appears to be due to the induction or alteration of cellular determinants leading to a more effective immune recognition. We previously reported that clones of the murine T-lymphoma Eb obtained after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment (Eb-MNNG) show a moderate to strong decrease in tumorigenicity and in addition altered expression of several cell surface proteins. We report here that treatment of Eb cells with 5-azacytidine (5-Aza) induces cell surface changes that are very similar to those seen in MNNG clones; likewise, Eb-5-Aza clones were decreased in tumorigenicity and increased in immunogenicity. Regressor mice that had been inoculated with Eb-5-Aza or Eb-MNNG cells could be shown to be protected against a subsequent challenge of Eb cells but not against the syngeneic mastocytoma P 815. Analysis of cytosine methylation of Eb-MNNG and Eb-5-Aza clones by high pressure liquid chromatography revealed decreased levels of methylcytosine content in several clones. Because of these similarities and because both drugs are DNA hypomethylating and gene inducing agents we assume that hypomethylation is involved in causing amplified protein expression. The possible role of amplified proteins in increased immunogenicity is discussed.

Topics: Animals; Azacitidine; Cell Line; DNA, Neoplasm; Epitopes; Lymphoma; Membrane Proteins; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred DBA; Mutagens; Neoplasm Transplantation; T-Lymphocytes, Cytotoxic

From an S49 mouse lymphoma cell subline that carries an electrophoretic marker mutation in one allele for a regulatory (R) subunit of cyclic AMP-dependent protein kinase, 130 cyclic AMP-resistant mutants were isolated and characterized. Of the 77 independent spontaneous and mutagen-induced isolates identified, 74 had kinases with increased apparent activation constants (KaS) for cyclic AMP-dependent activation. The "Ka" phenotype was invariably correlated with an apparent structural lesion in one R subunit allele. "Charge-shift" lesions in 43 independent isolates were mapped to small regions within the R subunit by two-dimensional gel analysis of partial proteolysis peptides. Nine Ka mutations were distinguished by differences in charge or peptide maps of mutant R subunits, and the mutations were clustered in two regions associated with the cyclic AMP-binding sites of the R subunit. The relative frequencies of different mutations differed among spontaneous, ethyl methanesulfonate-induced, and N-methyl-N'-nitro-N-nitrosoguanidine-induced isolates. Mutation frequencies were also markedly different for the two R subunit alleles; this allele preference was strongest for mutagen-induced lesions in the more carboxy terminal cyclic AMP-binding site.

Topics: Animals; Bucladesine; Carrier Proteins; Cell Line; Electrophoresis, Polyacrylamide Gel; Ethyl Methanesulfonate; Intracellular Signaling Peptides and Proteins; Kinetics; Lymphoma; Methylnitronitrosoguanidine; Mice; Mutagens; Mutation; Peptide Fragments; Protein Kinases

The effects of benzene and its metabolites on the rate of DNA synthesis were measured in the mouse lymphoma cell line, L5178YS. The direct toxicity of benzene could be distinguished from that of its metabolites since bioactivation of benzene in L5178YS cells was not observed. Cells were exposed to benzene, phenol, catechol, hydroquinone, p-benzoquinone, or 1,2,4-benzenetriol over the range of 1.0 X 10(-7) to 1.0 X 10(-2) M for 30 min, and the rate of DNA synthesis was measured at various times after chemical washout. Cell viability and protein synthesis were determined by trypan blue dye exclusion and [3H]leucine incorporation, respectively. Effects were designated as "DNA specific" when DNA synthesis was inhibited in the absence of discernible effects on cell membrane integrity and protein synthesis. Concentrations of benzene as high as 1 mM had no effect on DNA synthesis. Comparison of the effects at the maximum nontoxic dose for each compound showed that catechol and hydroquinone were the most effective, inhibiting DNA synthesis by 65%. Phenol, benzoquinone, and benzenetriol inhibited DNA synthesis by approximately 40%. Maximum inhibition was observed 60 min after metabolite washout in each case. Benzoquinone was the most potent inhibitor of DNA synthesis, followed by hydroquinone, benzenetriol, catechol, and phenol with ED50 values of 5 X 10(-6), 1 X 10(-5), 1.8 X 10(-4), 2.5 X 10(-4), and 8.0 X 10(-4), respectively. Cyclic voltammetric experiments were performed on the hydroxylated metabolites of benzene to assess the possible involvement of a redox-type mechanism in their inhibition of DNA synthesis. The ease of oxidation of these metabolites correlated with their ED50 values for inhibition of DNA synthesis (r = 0.997). This suggests that oxidation of phenol or one of its metabolites may be necessary for production of the species involved in inhibition of DNA synthesis.

Topics: Animals; Benzene; Cell Survival; Cells, Cultured; DNA; Dose-Response Relationship, Drug; Lymphoma; Methylnitronitrosoguanidine; Mice; Oxidation-Reduction; Thymidine; Time Factors; Tritium

Hybrids were made between a ouabain-resistant, thioguanine-resistant human lymphoma line able to remove O6-methylguanine from its DNA (Mex+) and human lymphoblastoid lines deficient in this capability (Mex-). The formation of hybrids was confirmed by chromosomal analysis. Hybrid cells had an O6-methylguanine removal capacity per mole of guanine about one third to one half that of the Mex+ parents, i.e., about the same per cell. Cell hybrids removed the same amount of the alkylation adduct 3-methyladenine as did their parents per mole of guanine, i.e., about twice as much per cell. Although the cell hybrids had intermediate resistance to the cytotoxic action of N-methyl-N'-nitro-N-nitrosoguanidine used to induce O6-methylguanine and 3-methyladenine, there is evidence that the ability to remove O6-methylguanine and resistance to the cytotoxic effect of N-methyl-N'-nitro-N-nitrosoguanidine are dissociable characteristics.

Topics: Adenosine; Cell Fusion; Cell Line; Deoxyguanosine; DNA; Drug Resistance; Female; Humans; Hybrid Cells; Karyotyping; Lymphoma; Male; Methylnitronitrosoguanidine; Neoplasms, Experimental

Experimental systems for the detection of somatic cell mutation to hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency in human lymphoma cells were developed. The cell line used was the Raji line established from Burkitt's lymphoma. The cloning efficiency (CE) in soft agar of the cells was enhanced approximately 5-fold by addition of 2 x 10(-4) to 4 x 10(-4)M dithiothreitol (DDT) and 1% human serum, as compared with the CE in control medium. Other thiols such a mercaptoethanol and thioglycerin were also able to increase the CE but not as efficiently as DTT. Under these culture conditions, 10(6) cells per 55-mm dish could be seeded into soft agar medium containing 5 micrograms/ml 6-thioguanine (6-TG) to detect a forward mutation without any metabolic cooperation. The optimum expression time was determined by treating the cells with 125 micrograms/ml ethyl methanesulfonate (EMS) for 20 hr at 37 degrees. A significant increase of mutation frequency was observed approximately 10 days after the 6-TG treatment, by which time the cells had gone through about 10 population doublings. After determining these experimental conditions, a comparison of mutation frequency among three mutagens, EMS, N-methyl-N'-nitro-N-nitrosoguanidine and ICR-170, was carried out. ICr-170 was the most efficient mutagen among these chemicals.

Topics: Aminoacridines; Cell Line; Culture Media; Humans; Lymphoma; Methylnitronitrosoguanidine; Mutagenicity Tests; Mutation; Nitrogen Mustard Compounds; Thioguanine

YAC lymphoma cells were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and then cloned and subcloned. Of 51 clones, 3 were selected for further study. Ten-fold more natural killer (NK) effector cells were required to lyse YAC clone 6 and subclone 6-28 cells compared with clone 19 cells or the YAC parent cell line. The maximum plateau level of cytolysis of the NK-resistant (NKR) variants (20%) never approached that of the NK-sensitive (NKS) variants or YAC parental cells (60%) even after prolonged incubation (20 hr). NKR variants appeared with equal frequency (0.10) on cloning YAC cells that had not been treated with mutagen but these variants were highly unstable with respect to NK sensitivity and were not studied further. Cytolysis of both NKR and NKS lines was mediated by nylon-nonadherent asialo-GM1+ effector cells, and effectors from poly(I) . poly(C)-boosted mice preferentially lysed the NKS lines. The NKR alteration did not appear to change the NK target structure (NK-TS): (i) unlabeled NKR cells competed equally with NKS cells in reciprocal unlabeled-target competition assays; (ii) the frequency of target--effector conjugates was identical with NKR or NKS lines; and (iii) normal rabbit serum, which contains antibodies thought to react with the NK-TS, reacted equally against both NKR and NKS targets. The NKR alteration was selective for NK cells and did not result in a resistance to lysis in general; NKR and NKS variants were equally susceptible to (i) cytolysis mediated by alloimmune or lectin-dependent effector T cells and (ii) antibody- and complement-mediated lysis. These results are compatible with the hypothesis that the NKR variants have an altered acceptor site on the target cell membrane that normally binds the "lytic moiety" delivered by the effector cell.

Topics: Animals; Cell Line; Clone Cells; Genetic Variation; Killer Cells, Natural; Leukemia, Experimental; Lymphoma; Methylnitronitrosoguanidine; Mice; Mutation

Two clones were isolated from mutagenized mouse T-lymphoma cells (S49) which are over 90% deficient in uridine-cytidine kinase. The first clone, AU-200-1, was isolated in two steps by virtue of its resistance to 6-azauridine; whereas the second clone, FU3-70G, was isolated in three steps after exposure to three increasing concentrations of 5-fluorouracil. Extracts of both the AU-200-1 and the FU3-70G cell lines lacked over 90% of the capacity of those from wild type cells to phosphorylate either uridine or cytidine. Furthermore, the uptake of radioactive uridine and cytidine from the medium by intact AU-200-1 and FU3-70G cells was less than 5% of that found for intact wild type cells. By growth rate experiments, these uridine-cytidine kinase-deficient cell lines have altered sensitivities to the toxic pyrimidine analogs, 6-azauridine, 5-fluorouracil, and 5-fluorouridine and thus have been useful in elucidating the biochemical determinants involved in the metabolism of these compounds.

Topics: Animals; Cell Line; Cytidine; Genetic Variation; Lymphoma; Methylnitronitrosoguanidine; Mice; Mutation; Neoplasms, Experimental; Phosphotransferases; Uridine Kinase

The effects of mutagens on three genetic markers--resistance to ouabain, 6-thioguanine, and dibutyryl cyclic AMP (Bt2cAMP), were investigated in a mouse lymphoma cell line, S49. Nitrosoguanidine, ethyl methanesulfonate, ICR 191, and x-rays were used. Mutagen-specific responses were seen. Ouabain resistance was induced by nitrosoguanidine, but not by ICR 191. ICR 191 induced resistance to 6-thioguanine more efficiently than did nitrosoguanidine; the converse was true of resistance to Bt2cAMP. The relative frequency of biochemically distinguishable subtypes of mutants resistant to Bt2cAMP was characteristic of the mutagen used to generate them. The results can be interpreted as follows: nitrosoguanidine and ethyl methanesulfonate frequently, but ICR 191 and x-rays rarely, give rise to DNA base sequence changes that result in structurally altered but functional proteins. This type of change is required for induction of mutants resistant to ouabain and of certain classes of mutants resistant to Bt2cAMP. Resistance to 6-thioguanine and other classes of mutants resistant to Bt2cAMP can result from DNA base sequence changes that lead to extensive alteration of protein structure or expression; these changes are induced by ICR 191 or x-rays.

Topics: Bucladesine; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Drug Resistance; Ethyl Methanesulfonate; Kinetics; Lymphoma; Methylnitronitrosoguanidine; Mutagens; Mutation; Ouabain; Thioguanine

Topics: Animals; Antigens, Neoplasm; Cell Line; Genes; Histocompatibility Antigens; Hybrid Cells; Isoantigens; Leukemia, Experimental; Lymphoma; Methylnitronitrosoguanidine; Mice; Molecular Weight; Neoplasms, Experimental; Rats; T-Lymphocytes

Benzoylated naphthoylated DEAE-cellulose columns can be used to separate DNA growing point regions from the bulk of the DNA. We used the columns to estimate DNA excision repair in both fractions. Repair induced by acetoxy acetyl aminofluorene (AAAF), bromomethyl benz(alpha) anthracene (BMBA), and methyl methanesulfonate (MMS) occurs to an equal extent in growing point and non-replicating regions of the DNA. Excision repair induced by methyl nitrosourea (MNNU) and methyl nitronitrosoguanidine (MNNG) occurs to a greater extent in growing point regions of the DNA. The overall amount of methyl nitronitrosoguanidine-induced alkylation is the same for replicating and non-replicating regions of the DNA treated in vitro. We conclude that there is some special interaction between methyl-nitronitrosoguanidine and the growing point region in vivo. We suppose that strand displacement and branch migration return DNA lesions at the growing point to a double stranded configuration at which repair is possible.

Topics: Cell Division; DNA Repair; DNA Replication; DNA, Neoplasm; Humans; Lymphoma; Methylnitronitrosoguanidine; Tumor Cells, Cultured

The methylating agents methyl methanesulphonate (MMS) and N-methyl N'-nitro-N-nitrosoguanidine (MNNG), administered by single i.p. injection in mice failed to yield thymic lymphoma at doses around 60% of the LD50 values, in contrast to MNUA which gives a high yield of tumours by this route. Comparison of the tissue distribution and mode of DNA methylation by these agents showed a positive correlation with ability to methylate the 0-6 atom of guanine in DNA of the target tissues thymus and bone marrow and tumorigeneis. MMS gave a low yield of this product due to its relatively low Sn1 reactivity but was able to methylate DNA extensively at other sites in the target tissues and other organs examined. MNNG despite its ability to methylate 0-6 of guanine in DNA in vitro to the same relative extent as the potent carcinogen MNUA, methylated DNA of thymus and bone marrow to a very small extent in vivo but was able to methylate DNA in certain other tissues nearer the site of i.p. injection. These findings contrast with the general relatively extensive methylation of 0-6 of guanine in DNA of the target tissues and other organs by N-methyl-N-nitrosourea (MNUA).

Topics: Animals; Carcinogens; DNA; Female; Guanine; Lymphoma; Mesylates; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Nitrosoguanidines; Thymus Gland; Thymus Neoplasms

An improved method of assay of urinary polyamines (putrescine, spermidine, and spermine) was applied to the study of cancer patients and an experimental gastric tumor of rats. Although total polyamines (putrescine, spermidine, and spermine) in urine of patients with blood and solid cancers were significantly high, putrescine concentrations also increased significantly and were shown to be of diagnostic aid even in solid cancers. A significant increase in putrescine was also noted in the urine of rats with experimental stomach tumors induced by N-methyl-N-nitro-N'-nitrosoguanidine.

Topics: Animals; Female; Humans; Leukemia; Lymphoma; Methylnitronitrosoguanidine; Neoplasms; Neoplasms, Experimental; Polyamines; Pregnancy; Putrescine; Rats; Spermidine; Spermine; Stomach Neoplasms