MNNG Treatment Research Articles

Improvement of in vitro two-stage transformation assay and determination of the promotional effect of cadmium

The classical in vitro two-stage transformation method was modified for high transformation frequency, and the promotional effect of cadmium was evaluated. In this study, we reconfirmed the usefulness of the replating method and the optimal duration time between the initiator and promoter treatments for the optimal transformation of the Balb/3T3 cells. The results also showed that subsequent exposure to CdCl 2 for 2 weeks after initial exposure to N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) markedly enhanced the transformation frequency. At the concentration of 360 ng/ml, the transformation frequency was increased by 35-fold that of the cultures treated only with MNNG, and was higher than that of the positive control group treated with 100 ng/ml 12- O-tetradecanoyl phorbol-13-acetate (TPA) after MNNG treatment. This transformation frequency was higher than that reported previously. Therefore, this in vitro two-stage transformation method can be used efficiently for the screening of genotoxic and non-genotoxic carcinogens and the study of multistage carcinogenesis. These results also indicate that cadmium has a strong potency as a promoter, and the promotional effect of cadmium is higher than that of TPA.

A Simple and Rapid Method for the Detection of Poly(ADP-ribose) by Flow Cytometry

Measurement of poly(ADP-ribose) levels was performed by a new method using a monoclonal antibody against poly(ADP-ribose) and flow cytometry from small amount of cultured cells without the need for isolation of poly(ADP-ribose) polymer. The increase of poly(ADP-ribose)-associated fluorescence intensity was observed in individual human leukemic HL-60 cells when treated with the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and was blocked by the treatment with 3-aminobenzamide before MNNG treatment. It is easy and rapid to detect the time-dependent change of poly(ADP-ribose) levels in HL-60 cells after MNNG treatment. We easily found that the increase of the poly(ADP-ribose) level in nicotinic acid-treated lymphocytes after MNNG treatment was observed, but not in nicotinamide-treated lymphocytes. We investigated the change of poly(ADP-ribose) levels especially in the early phase of apoptosis. Our method is simple and rapid. It is suggested that the investigation of poly(ADP-ribosyl)ation in various fields is possible by using this new method.

Expression of c-jun and c-fos in Apoptotic Cells After DNA Damage

Apoptosis was induced in HeLa cells by exposure to 50 µM N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for various time intervals (up to 120 min). Apoptotic death was confirmed by the microscopic observation of cell blebbing, cell granulation, and cell aggregation. Cells also showed loss of phospholipid symmetry as judged by immunofluorescent microscopy with fluorescently labeled phosphatidyl serine-specific annexin V. In addition, staining of cells with ethidium bromide showed the presence of genomic DNA apoptotic bodies. The protein expression levels of c-jun and c-fos increased in DNA-damaged HeLa cells after MNNG treatment in a time-dependent fashion. Although the levels of c-fos increased rapidly during the first 30 min and remained high for 2 hr, the increase in c-jun expression was more gradual and slower (60–120 min) after MNNG treatment. These results are consistent with the conclusion that c-fos is important in the initial stages (commitment phase) of apoptosis and c-jun is involved in the late stages (execution phase) of apoptosis induced with alkylating agents

17Beta-estradiol modulates gastroduodenal preneoplastic alterations in rats exposed to the carcinogen N-methyl-N'-nitro-nitrosoguanidine.

Gastric cancers are a significant cause of morbidity worldwide. Epidemiological studies and animal models show that males have higher incidences of gastric cancers compared with females, suggesting that sex hormones may modulate gastric cancer risk. An animal model of the initiation phase of gastric cancer was used to determine the effects of systemic estrogen administration on morphological progression of preneoplastic lesions and to define cell populations at which estrogens may act. Preneoplastic progression in antral and duodenal mucosa was examined in male rats that received the chemical carcinogen, N-methyl-N'-nitro-nitrosoguanidine (MNNG), during treatment with implants containing 17beta-estradiol or oil vehicle. Histopathological changes in antral and duodenal gland morphology, numbers of proliferating cells and apoptotic bodies, and antral gastrin cell numbers and protein storage levels were determined 4 weeks later. With MNNG treatment, duodenal villous heights were significantly decreased, and epithelial cells displayed histological features of hyperplasia and dysplasia. Antral glands showed epithelial hyperplasia and dysplasia, increased mucosal height, and decreased mucin levels. Antral gastrin storage protein levels were decreased by MNNG. Systemic treatment with 17beta-estradiol significantly reversed MNNG-induced alterations in duodenal gland heights while increasing mucin and gastrin levels in antral glands. Cell proliferation and apoptosis rates were not significantly different between groups. The present results indicate that systemic 17beta-estradiol treatment influences antral and duodenal gland differentiation during the initiation phase of chemical gastroduodenal carcinogenesis in male rats. These results explain, in part, a potential pathway through which protective effects of estrogens on chemical carcinogenesis are mediated in the upper gastrointestinal tract.

Protein synthesis and transcriptional inhibitors control N-methyl-N'-nitro-N-nitrosoguanidine-induced levels of APC mRNA in a p53-dependent manner.

In the present study, we show that treatment of wild-type (p53+/+) mouse embryonic fibroblast (MEF) cells with a DNA-alkylating agent, N-methyl-N'-nitro-N-nitro-soguanidine (MNNG), resulted in increased levels of adenomatous polyposis coli (APC) mRNA compared to p53 gene-knocked out (p53-/-) MEF cells, indicating that p53 is required for APC expression after alkylation damage. By using HCT-116 colon cancer cells (containing wild-type p53 gene) or p53-/- MEF cells transfected with a pCMV-p53 overexpression plasmid [p53-/-(CMV-p53)], we show that p53 is a labile factor for APC gene expression, and that pretreating HCT-116 cells with a protein synthesis inhibitor, cycloheximide (CHX), inhibited MNNG-induced APC mRNA levels by inhibiting p53 protein synthesis. The effect of CHX on p53 protein synthesis was reversible, as the withdrawal of CHX permitted p53 protein synthesis to resume with a concomitant increase in APC mRNA levels after MNNG treatment. To examine whether p53 regulates APC gene expression at the transcriptional level, we treated HCT-116 or p53-/-(CMV-p53) MEF cells with 5,6-dichloro-1-beta-D-ribofuranosylbenzamidazole (DRB; a transcriptional inhibitor), before the MNNG treatment. Although treatment of cells with DRB resulted in increased p53 protein levels, that the APC mRNA levels were decreased suggests that p53 may enhance APC gene expression upstream of the transcriptional machinery where DRB interacts. That the withdrawal of DRB, and subsequent MNNG treatment, increased the level of APC mRNA indicated that the binding of DRB to the transcriptional machinery was reversible.

Bacterial O6-methylguanine-DNA methyltransferase reduces N-methyl-N'-nitro-N-nitrosoguanidine induction of plasminogen activator in Mer- human glioblastoma A1235 cell line.

The alkylation repair deficient (Mer- phenotype) cells produce high levels of proteolytic enzyme plasminogen activator (PA) after treatment with alkylation agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Both, in Escherichia coli and in mammalian cells O6-methylguanine (O6-MeG) is repaired by analogues O6-methylguanine-DNA methyltransferase (MGMT). In E. coli MGMT is product of ada gene. To investigate the effect of bacterial MGMT expression on the induction of PA activity in human cells, we have transfected ada-alkB operon into Mer- human A1235 cells that are known to produce high levels of PA after MNNG treatment. We have shown here that A4 and A8 transformants that harbour ada gene become resistant to killing by MNNG. In addition, MNNG produced induction of extracellular PA activity was much less pronounced in A4 and A8 transformants (induction ratio 3.42 and 3.74, respectively) than in control A1235 and Aneo-1 cells (induction ratio 11.04 and 9.11, respectively). However, changes of intracellular PA activity were not significant. It appears, therefore, that induction of extracellular PA activity is inversely related to the cell capacity to repair the DNA lesions induced by alkylation agents.

Sister chromatid exchange analysis of human cells carrying retroviral DNA

Chromosomal abnormalities have been seen in human cells following infection with human T lymphotropic virus type I (HTLV-I). Effects on sister chromatid exchange (SCE) in cells carrying HTLV-I related DNA were examined by treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Peripheral blood specimens were obtained from 16 donors. Cells were processed for SCE analysis and sera were tested for HTLV-I antibodies by Western blot analysis. DNA from fresh lymphoid cells was analyzed by PCR-SSCP for HTLV-I genes. Of 16 donors, eight were HTLV-I seropositive and eight seronegative. Five HTLV-I infected lymphocyte cultures were also analyzed for SCE. Cells were stained immunocytochemically with anti-p19 for the viral expression. By MNNG treatment, SCE was significantly enhanced in cells derived from two seropositive donors with HTLV-I genes. In cells of six seropositive and eight seronegative donors with or without HTLV-I related DNA, SCE frequency was influenced insignificantly. Cells from all persons were negative for the p19 expression. SCE was greatly enhanced by MNNG treatment in cells from three productively infected cultures when compared with SCE in two nonproductively infected cultures.

Induction of kinetochore positive and negative micronuclei in V79 cells by N-methyl- N′-nitro- N-nitrosoguanidine: the protective effect of the pyridoindole antioxidant stobadine

The induction of micronuclei by N-methyl- N′-nitro- N-nitrosoguanidine (MNNG) and their reduction by the cardioprotective synthetic antioxidant, stobadine were studied in hamster V79 cells cultured in vitro. The micronuclei derived from acentric fragments or from whole chromosomes were evaluated with the help of an immunofluorescent staining using antikinetochore antibodies from the serum of scleroderma (CREST syndrome) patients. Our results showed that MNNG (0.5 μg/ml) induced mainly kinetochore-negative micronuclei. At 6, 24 and 48 h after MNNG treatment, we measured a 2.7-, 4.3- and 7.0-fold increase, respectively, of kinetochore-negative micronuclei over the controls. The increase of kinetochore-positive micronuclei was rather low and represented at 6, 24 and 48 h, respectively 0.9-, 1.8- and 2.6-fold increases over the controls. Stobadine decreased the level of kinetochore-negative micronuclei at 6, 24 and 48 h to approximately one-half; the frequency of kinetochore-positive micronuclei was reduced only at 6 h. We suppose that the antioxidant stobadine reduces the induction of micronuclei by MNNG by scavenging of MNNG-induced highly reactive OH radicals which cause chromosomal damage.

Activation of Adenomatous Polyposis Coli (APC) Gene Expression by the DNA-alkylating AgentN-MethylN′-nitro-N-nitrosoguanidine Requires p53

Development of colon cancer is a multistep process frequently involving mutations in both the APC and p53 tumor suppressor genes. In this study we treated the HCT-116 colon cancer cell line with alkylating agents including N-methyl-N'-nitro-N-nitrosoguanidine (MNNG),which is known to cause colon cancer in animals, and examined the expression of both APC and p53 genes. Exposure of cells with MNNG caused an 8-12-fold increase in the level of APC mRNA and protein. APC induction was shown to result from increased nuclear transcription of the APC gene and correlated with a concomitant increase in the p53 protein level after MNNG treatment. A necessary role for p53 in APC gene regulation is supported by the failure of MNNG to induce APC expression in cell lines either expressing very low levels of p53 (HeLa cells) or no p53 (K562 erythroleukemia cells). The overexpression of wild-type p53 gene into HCT-116 cells mimicked the effect of MNNG-induced expression of APC mRNA. A direct causal role for p53 in APC gene regulation was further evaluated by transfecting the wild-type p53 gene into K562 cells and observing a 5-fold increase in the APC gene expression. These results support a model featuring a direct link between p53 and APC in response to alkylation-induced DNA damage and suggest a novel role for p53 in a stress-response pathway involving APC.

Low nicotinamide mononucleotide adenylyltransferase activity in a tiazofurin-resistant cell line: effects on NAD metabolism and DNA repair.

Poly(ADP-ribose) polymerase (PADPRP), which uses NAD to synthesize ADP-ribose polymers, is activated by DNA strand breaks and mediates cellular responses to DNA damage. The consequences of low cellular NAD levels in a cell line deficient in nicotinamide mononucleotide adenylyltransferase (NMNAT), an enzyme essential for NAD biosynthesis, were investigated by assessing NAD metabolism and DNA repair after treatment with alkylating agents. A tiazofurin-resistant L1210 cell line (TZR) was isolated. NAD levels were approximately 5933 and 3375 pmol mg(-1) protein for parental (wild type, WT) and TZR cells respectively, and NMNAT levels were reduced by > 95%. TZR cells were more sensitive to temozolomide (TM) and 1-methyl-3-nitro-1-nitroso-guanidine (MNNG), particularly at concentrations that caused > 50% NAD depletion. TM and MNNG treatment decreased NAD levels in both cell lines, but took longer to return to control levels in TZR cells. For example, MNNG (5 microM), depleted NAD levels at 6 h to approximately 4512 (WT) and 1442 (TZR) pmol mg(-1) protein; however, NAD levels had returned to control levels by 8 h in WT cells, but were not restored by 16 h in TZR cells. Both cell lines were equisensitive to the growth-inhibitory effects of NU1025 per se (IC50 370 microM). Co-exposure of the cell lines to TM (100 microM) with increasing concentrations of NU1025 led to a synergistic enhancement of cytotoxicity, with IC50 values for NU1025 decreasing to 17 +/- 4 microM (TZR) and 37 +/- 6 microM (WT). A similar enhanced sensitivity to NU1025 (approximately 2.7-fold) was obtained when TZR cells were co-exposed to MNNG + NU1025. TM-induced DNA strand breaks were increased by co-incubation with NU1025, and again the TZR cell line showed increased sensitivity to NU1025. There were no significant changes in NMNAT activity in response to MNNG treatment over 24 h, either in the presence or in the absence of NU1025. These data demonstrate that modest decreases in cellular NAD levels can sensitize cells to alkylating agents and PADPRP inhibitors.

Enhancement by aminoguanidine of gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in Wistar rats

The effects of prolonged administration of aminoguanidine on gastric carcinogenesis induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG), the ornithine decarboxylase activity of the gastric wall, and the labeling index of the gastric mucosa were investigated in inbred Wistar rats. The rats received 12.5 or 25.0 mg/kg body weight of aminoguanidine s.c. every other day after oral MNNG treatment for 25 weeks. Prolonged administration of aminoguanidine at 25.0 mg/kg body weight, but not at 12.5 mg/kg body weight, significantly increased the incidence of gastric cancers in experimental week 52. However, it did not influence the histologic types of gastric cancers. Aminoguanidine at 25.0 mg/kg body weight also significantly increased the ornithine decarboxylase activity of the antral portion of the gastric wall and the labeling index of the antral epithelial cells. These findings indicate that aminoguanidine enhances gastric carcinogenesis and suggest that its effect may be related to increased proliferation of antral epithelial cells.

Improvement of lysine production by analog-sensitive and auxotroph mutants of the acetylene-utilizing bacterium Gordona bronchialis (Rhodococcus bronchialis).

An acetylene utilizing Gordona (Rhodococcus) bronchialis strain, screened for the production of fine chemicals, was found to be capable of producing small amounts of lysine. Attempts to produce amino-acid analog-resistant and/or sensitive mutants and auxotrophs of this strain with increased lysine production were made following UV-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The bacterium exhibited surprisingly high resistance levels to the aforementioned mutagens which is attributed to highly effective inborn-repair systems. Natural resistance to high levels of S-(2-aminoethyl)-L-cysteine (AEC) (2%) was observed, in contrast with D, L-aspartic acid hydroxamate (AAH), L-lysine hydroxamate (LHX) and beta-fluoropyruvate (FP). A variety of amino-acid analog-resistant (AAHr, LHXr) or analog-sensitive (FPs) mutants were produced following UV-irradiation or MNNG treatment. Similarly, a large number of auxotrophs (68) of different types were also obtained. From these, one FPs mono-auxotroph and two poly-auxotrophs (with at least one requirement for the aspartic acid family) showed an increased lysine production (approximately 1.8 g/L) comparable (4 g/L) to that found in other bacteria capable of utilizing long-chain hydrocarbons (1).

Involvement of human T lymphotropic virus type I in human neoplasia

Of 211 human tissues analyzed by PCR-SSCP and direct sequencing for HTLV-I genes, 165 (78.2%) gave positive signals. Signals detected in family members of adult T cell leukemia (ATL) patients showed mutations distinct from those in ATL. HTLV-I gene sequences showed variability in same persons examined at different times. Lymphocytes with prototypic HTLV-I genes showed higher sister chromatid exchange following MNNG treatment than those without. Nasopharyngeal carcinomas carrying both HTLV-I and Epstein-Barr virus genes showed markedly abnormal gene product expression. HTLV-I genes in human DNA may cause deregulation of host cell genes upon exposure to carcinogens including viruses.

Detection of MNNG-induced DNA lesions in mammalian cells; validation of comet assay against DNA unwinding technique, alkaline elution of DNA and chromosomal aberrations

Human cells (VH10 or Hep G2) and hamster cells V79 were exposed to different concentrations of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and the level of DNA lesions was evaluated by the DNA unwinding technique, alkaline elution of DNA and the comet assay. All three methods were able to detect the effects of MNNG but with a clear difference in sensitivity. At low concentrations of MNNG the most sensitive method appeared to be the comet assay. After the short-term treatment the comet assay was able to detect the lesions induced by MNNG at approx. 0.1 μg/ml, alkaline elution of DNA at 1 μg/ml and DNA unwinding at 1–2 μg/ml. MNNG treated VH10 cells, human lymphocytes and V79 cells were also tested cytogenetically, confirming that MNNG induced chromosomal aberrations at concentrations >1 μg/ml in VH10 cells (short-term treatment); >0.2 μg/ml in V79 cells (long-term treatment) and >8 μg/ml in human lymphocytes (long-term treatment). In some experiments we tried to increase the level of MNNG-induced DNA breaks with help of DNA repair inhibitors cytosine arabinoside (Ara C) and hydroxyurea (HU) which were applied either after or during MNNG treatment. Our results showed that the level of MNNG-induced lesions was increased by simultaneous treatment of cells with MNNG and Ara C and HU. 2×10−5 M Ara C and 2×10−3 M HU were as effective as 10-times higher concentrations of inhibitors. Ara C and HU increased the level of MNNG-induced DNA breaks mainly in combination with lower concentrations of MNNG (<2 μg/ml). Rejoining of DNA breaks was observed in human cells VH10 and Hep G2 as well as in Chinese hamster cells V79 damaged by both lower and higher MNNG-concentrations. All methods showed that MNNG-induced DNA breaks had been gradually rejoined.

Competency in mismatch repair prohibits clonal expansion of cancer cells treated with N-methyl-N'-nitro-N-nitrosoguanidine.

The phenomenon of alkylation tolerance has been observed in cells that are deficient in some component of the DNA mismatch repair (MMR) system. An alkylation-induced cell cycle arrest had been reported previously in one MMR-proficient cell line, whereas a MMR-defective clone derived from this line escapes from this arrest. We examined human cancer cell lines to determine if the cell cycle arrest were dependent upon the MMR system. Growth characteristics and cell cycle analysis after MNNG treatment were ascertained in seven MMR-deficient and proficient cell lines, with and without confirmed mutations in hMLH1 or hMSH2 by an in vitro transcription/translation assay. MMR-proficient cells underwent growth arrest in the G2 phase of the cell cycle after the first S phase, whereas MMR-deficient cells escaped an initial G2 delay and resumed a normal growth pattern. In the HCT116 line corrected for defective MMR by chromosome 3 transfer, the G2 phase arrest lasted more than five days. In another MMR-proficient colon cancer cell line, SW480, cell death occurred five days after MNNG treatment. A competent MMR system appears to be necessary for G2 arrest or cell death after alkylation damage, and this cell cycle checkpoint may allow the cell to repair damaged DNA, or prevent the replication of mutated DNA by prohibiting clonal expansion.

Phosphorylation of T antigen and p53 in carcinogen-treated SV40-transformed Chinese hamster cells.

SV40-transformed Chinese hamster C0631 cells pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) display SV40 DNA amplification. This study shows that following MNNG treatment elevated T antigen synthesis and a 4.5-fold reduction in the extent of its phosphorylation occurred in both pulse-labeled and steady-state-labeled cells. The decrease in phosphorylation was found to be inversely related to carcinogen concentration, i.e. an augmented carcinogen concentration brought about a gradual reduction in T antigen phosphorylation and elevated SV40 DNA amplification. Although the majority of phosphorylation sites on T antigen derived from carcinogen-treated cells were underexpressed, as demonstrated by two-dimensional phosphopeptide mapping, peptide 12 bearing phosphoThrl24, which is known to be essential for DNA replication, was overexpressed. Carcinogen-treated cells showed no changes in p53 synthesis, but it was phosphorylated to a lesser degree. Two-dimensional mapping revealed that the predicted N-terminal major phosphopeptide of p53 extracted from C0631 cells exhibited a lower chromatographic mobility than p53 phosphopeptides from SV40-infected monkey BSC-1 cells. In treated C0631 cells the Rf value of this phosphopeptide was higher than that of control p53. This finding could be ascribed to the failure to phosphorylate the corresponding amino acid residue in this peptide. Moreover, treatment did not affect the halflife of either T antigen or p53 proteins, but caused a dramatic rise in the expression of small t antigen, presumably due to amplification of SV40 DNA.

Invasions of Stomach Tumors in Hypocatalasemic Mice (C3H/Cbs/Gen) Treated with N-Methyl-N-Nitrosourea but Not N-Methyl-N'-Nitro-N-Nitrosoguanidine.

Hypocatalasemic mice were treated with N-methyl-N-nitrosourea (MNU) in their drinking water or gastric intubation for 16 weeks or were given N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in their drinking water for 6 months. With MNU treatment in the drinking water, well differentiated tumors were observed in the glandular stomachs of one male (4%) and 5 female (15%) animals. Sarcomas of the dermis also appeared in 10 males (40%). Adrenal lesions developed in I I females (35%) along with kidney, ovary, and uterus tumors. In the Group given MNU intubation, squamous cell carcinomas of the forestomach appeared from 122 days after the first treatment, reaching incidences of 81% in males, 94% in females, with invasion of the diaphragm in 3 male cases and the diaphragm, liver, pancreas, or adrenal in I I female cases. Glandular stomach tumors occurred in 12 males (52%), with 3 being signet ring cell carcinomas, and in 12 females (35%). In a sub-Group undergoing forestomach removal after MNU intubation esophagectasis appeared from 80 days after the operation at incidences of 63% in males and 74% in females. The glandular stomach tumors including 3 signet ring cell carcinomas were found in 17 of 46 (40%) males, 2 of 18 (11%) females demonstrated lesion, one being a signet ring cell carcinoma. In the Group receiving MNNG treatment, anemia appeared from the 5.5 month time point and no gastric tumors were observed. The present results do not support the conclusion that MNU induction of glandular stomach lesions in hypocatalasemic mice may provide a useful model of signet ring cell carcinomas

Glutamine synthetase/glutamate synthase ammonium-assimilating pathway in Schizosaccharomyces pombe

Kinetic parameters of glutamine synthetase (GS) and glutamate synthase (glutamine-oxoglutarate aminotransferase) (GOGAT) activities, including initial velocity, pH, and temperature optima, as well as Km values, were estimated in Schizosaccharomyces pombe crude cell-free extracts. Five glutamine auxotrophic mutants of S. pombe were isolated following MNNG treatment. These were designated gln1-1,2,3,4,5, and their growth could be repaired only by glutamine. Mutants gln1-1,2,3,4,5 were found to lack GS activity, but retained wild-type levels of NADP-glutamate dehydrogenase (GDH), NAD-GDH, and GOGAT. One further glutamine auxotrophic mutant, gln1-6, was isolated and found to lack both GS and GOGAT but retained wild-type levels of NADP-GDH and NAD-GDH activities. Fortuitously, this isolate was found to harbor an unlinked second mutation (designated gog1-1), which resulted in complete loss of GOGAT activity but retained wild-type GS activity. The growth phenotype of mutant gog1-1 (in the absence of the gln1-6 mutation) was found to be indistinguishable from the wild type on various nitrogen sources, including ammonium as a sole nitrogen source. Double-mutant strains containing gog1-1 and gdh1-1 or gdh2-1 (mutations that result specifically in the abolition of NADP-GDH activity) result in a complete lack of growth on ammonium as sole nitrogen source in contrast to gdh or gog mutants alone.

Identification in rat stomach mucosae of a cell population characterized by a deficiency for the repair of O6-methyldeoxyguanosine from DNA.

Immunohistochemical studies have been used to show the time course for the cell-specific methylation of DNA in the upper gastrointestinal tract of rats treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The doses used were 1, 5, 25 and 50 mg MNNG/kg (i.g.) and tissue samples were analysed at intervals of from 1 to 192 h after treatment. Relatively little reaction with nuclear DNA was observed in the forestomach and still less in the oesophagus. Reaction with DNA was most extensive in the corpus, pylorus and the duodenum, reaching a peak of staining intensity between 2 and 4 h and then declining progressively there-after. Staining for the presence of O6-methyldeoxyguanosine (O6-MedG) in DNA was highly selective and tended to occur in the nuclei of the basal cell of the oesophagus and forestomach and in the cells of the lumenal border of the glands and villi of the corpus, pylorus and duodenum. There were also areas, 5-15, glands apart where staining for O6-MedG in the corpus and pylorus extended as far down as the basal mucosa. From 12 h after MNNG treatment, in the corpus and pylorus, a band of strongly methylated cells became apparent about 3-6 cells deep from the lumen and remained identifiable up to 168 h after treatment with the higher doses. These cells, which apparently have a very low O6-MedG repair capacity, are stationary (i.e. not part of the escalator) and are located in the mesenchymal tissue elements as demonstrated by staining of serial sections with cytokeratin or vementin. The significance of this population of cells is unknown.

Increased DNA-repair capacity and the modulation of 2 proteins in and metallothionein overexpressing Chinese hamster cell line

Elevated intracellular levels of metallothionein have been associated with resistance to the cytotoxic effects of some alkylating agents. In order to study the mechanisms responsible for this resistance, we used a pair of CHO cell lines consisting of normal K1–2 cells and their derivative K1–2MT, which overexpresses the human metallothionein II-A gene (Lohrer et al., 1989). K1–2MT cells were found to be resistant to cadmium chloride and the alkylating agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), but resistance did not extend to the alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea, nor to adriamycin, an inhibitor of DNA synthesis. The DNA damage caused by MNNG, was only marginally less in resistant cells compared with the parental cell line, thus excluding drug scavenging as a possible mechanism for resistance. Also, glutathione S-transferases (GSTs) were present at equal levels in both cell lines (acidic and basic type GST) or slightly reduced in drug resistant K1–2MT cells (neutral type GST), thereby ruling out metabolic inactivation of the alkylating agents. However, the drug resistant phenotype was accompanied by a more efficient block of DNA synthesis after MNNG treatment and by a 3-h delay in the G2 phase of the cell cycle. Using two-dimensional gel electrophoresis of total protein extracts, we identified a 24-kDa protein (MIP1), which is only present in the resistant K1–2MT cells, and a 23.5-kDa protein (MIP2) which is 2–3 times over-synthesized in K1–2MT cells. MNNG treatment reduced the level of both proteins MIP1 and MIP2. These results suggest that the proteins MIP1 and possibly MIP2 may be responsible for the alkylating agent resistant phenotype and are probably modulated by the human metallothionein II-A protein.