methylnitronitrosoguanidine and Neoplasms

The relationship between food and cancer is extremely complex. It is generally accepted that diet is a contributory factor in the aetiology of a large proportion of cancers, but with very few exceptions, we are unable to identify specific causal agents. Many food components have genotoxic potential and more are produced endogenously during digestion. Conversely, there is increasing evidence that consumption of some foods may decrease the risk of cancer, and a number of plant constituents have been shown to have the potential to inhibit various stages of the carcinogenic process. Yet we have little understanding of the interactions between the different food-related genotoxic and protective factors. A further complication is the variation in individual susceptibility and vulnerability. As a result we are still not able to determine the optimal diet for minimising cancer risk. In recognition of these issues, the UK Ministry of Agriculture, Fisheries and Food (MAFF) is funding a number of projects aimed at providing greater mechanistic understanding of the links between food and cancer, in order to offer detailed advice to the public. This report summarises the proceedings of a workshop entitled 'Factors influencing the carcinogenicity of food chemicals', held in London on 1 June 1998, providing overviews of some of the key issues, and demonstrating how the MAFF-funded research is contributing to advances in these areas. It includes discussion of genetic polymorphisms and how they may contribute to individual susceptibility and help to identify causal links between food components and colorectal cancer. Biomarkers of DNA damage in human studies and of inhibition of carcinogen activation and endogenous formation of genotoxic reactive nitrogen species are examined. Also considered are the potential uses of physiologically based pharmacokinetic modelling techniques for providing more accurate estimates of risk and reducing the uncertainty in extrapolation between species and doses. Research now in progress will help to establish the critical risk and protective factors involved in diet-related colorectal cancers, in order to provide a sound scientific basis for formulation of dietary advice to the public.

Topics: Biomarkers; Diet; DNA Adducts; Flavonoids; Genetic Predisposition to Disease; Genotype; Humans; Methylnitronitrosoguanidine; Neoplasms; Phenotype; Polymorphism, Genetic; Risk Assessment

The marginal level of clinical responses to the Chloroethylnitrosoureas (CENU, i.e. BCNU, CCNU, MeCCNU) suggests that there may exist an innate mechanism of resistance in tumors to these chemotherapeutic agents. A decade of research from many laboratories around the world has led to the identification of the mechanisms for tumor cell resistance to the CENU. The ability to prevent the formation of DNA interstrand crosslinks, thought to be the critical lethal lesion induced by these agents, is accomplished in a majority of human tumors by the unique DNA repair protein O-6 methylguanine DNA methyltransferase (MGMT). This review addresses the identification of this mechanism of resistance to therapy, and chemotherapeutic strategies to inhibit this DNA repair system, in an attempt to sensitize resistant tumors to the CENU.

Topics: Animals; DNA Repair; Drug Resistance; Ethylnitrosourea; Guanine; Humans; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methyltransferases; Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Rats; Streptozocin

Treatment of a variety of highly tumorigenic mouse lines in vitro with chemical mutagens, such as ethyl methane sulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), can result in extraordinarily high frequencies (sometimes in excess of 90%) of strongly immunogenic clones unable to grow progressively in normal syngeneic hosts. These clones will, however, grow in immunosuppressed hosts and gradually regain tumorigenic ability in normal mice if maintained in long-term (several months-1 year) culture, i.e., they are often phenotypically unstable. These features--phenotypic drift and high frequency--make it unlikely that point mutations are the underlying mechanism involved in the generation of the variants. Results presented here demonstrate that these observations can be reproduced on the same tumor lines using 5-azacytidine--an analogue of cytidine which can be incorporated into DNA causing subsequent extensive hypomethylation of cytosine residues in the absence of any significant mutagenic effects. Furthermore, 5-azacytidine treatment of a nonmetastatic mouse mammary tumor led to the emergence of a small number of heritable but unstable tumor clones capable of spontaneous metastatic spread. Because it is known that DNA hypomethylation can lead to transcriptional activation of normally silent genes, that altered methylation patterns can be somatically replicated with a high but not perfect fidelity, and that mutagens can cause DNA hypomethylation, we propose that DNA hypomethylation followed by de novo methylation represents a plausible mechanism to account not only for the induction of the nontumorigenic variants but for a number of aspects of tumor progression and tumor heterogeneity, as well. In particular, we refer to heritable phenotypic alterations in tumor cell populations which occur at very high frequency but which are not necessarily stable over very long periods of time.

Topics: Animals; Azacitidine; DNA; Ethyl Methanesulfonate; Humans; Mammary Neoplasms, Experimental; Methylation; Methylnitronitrosoguanidine; Models, Genetic; Mutation; Neoplasm Metastasis; Neoplasms; Neoplastic Stem Cells; Phenotype; Stem Cells

Topics: Animals; Antigens, Neoplasm; Cells, Cultured; Dacarbazine; Histocompatibility Antigens; Humans; Immunization; Leukemia L1210; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Mutagens; Neoplasm Transplantation; Neoplasms; Neoplasms, Experimental; Teratoma; Transplantation Immunology

Chromosomal abnormalities are a frequent concomitant of neoplasia, and although it is tempting to relate these mutations and alterations in chromatin (DNA) function to cancer, their relationship to the initiation or progression of carcinogenesis is unknown. Mammalian cells in culture, after interacting with chemical carcinogens, often exhibit chromosome damage consisting of breaks and exchanges of chromatid material. The pattern of damage of banded metaphases indicates that negative bands are especially vulnerable to the action of chemical carcinogens, probably because of differential chromatin condensation. Damage to individual chromosomes may be random or nonrandom, depending on the species. Cell death can be correlated with chromatid alterations that occur shortly after treatment with chemical carcinogens. There is also a correlation between mutagenic and carcinogenic activity of some chemical carcinogens and the frequency of sister chromatid exchanges. The question of whether specific chromosome changes are absolutely required for neoplastic transformation cannot be answered because of conflicting data and diverse results from studies even with known carcinogens. Cell transformation may occur without any visible chromosome changes. A universal specific numerical or visible structural chromosomal alteration is not necessarily associated with chemical or viral transformation. Chromosome changes are independent of the etiologic agents: different carcinogens may produce transformation associated with the same abnormal chromosomes, but not all transformed lines invariably exhibit the same abnormality, even with the same chemical. In some species, chromosome having nucleolar organizer regions may be more frequently involved in numerical or structural deviations. Progressively growing tumors also may occur as a result of the proliferation of transformed cells without detectable chromosome changes, indicating that tumorigenicity need not be related to an imbalance of chromosome number or structure. Our studies indicate that chromosome changes are not essential for establishment of neoplasms but that karyotypic instability may result in response to selective growth pressures.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Azure Stains; Carcinogens; Cell Line; Cell Nucleolus; Cell Transformation, Neoplastic; Chromatids; Chromosomes; Humans; Karyotyping; Methylnitronitrosoguanidine; Neoplasms; Transformation, Genetic; Trisomy

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinogens; Caspases; Drugs, Chinese Herbal; Gastric Mucosa; Gene Expression Regulation, Neoplastic; Ki-67 Antigen; Male; Methylnitronitrosoguanidine; Neoplasms; Rats; Rats, Wistar; Severity of Illness Index; Signal Transduction; Stomach; Transcription Factor RelA; Tretinoin; Tumor Suppressor Protein p53

Genotoxic stress inflicted by anti-cancer drugs causes DNA breaks and genome instability. DNA double strand breaks induced by irradiation or pharmacological inhibition of Topoisomerase II activate ATM (ataxia-telangiectasia-mutated) kinase signalling pathway that in turn triggers cell cycle arrest and DNA repair. ATM-dependent gamma-phosphorylation of histone H2Ax and other histone modifications, including ubiquitnylation, promote exchange of histones and recruitment of DNA damage response (DDR) and repair proteins. Signal transduction pathways, besides DDR itself, also control expression of genes whose products cause cell cycle arrest and/or apoptosis thus ultimately affecting the sensitivity of cells to genotoxic stress. In this study, using a number of experimental approaches we provide evidence that lysine-specific methyltransferase (KMT) Set7/9 affects DDR and DNA repair, at least in part, by regulating the expression of an E3 ubiquitin ligase, Mdm2. Furthermore, we show that Set7/9 physically interacts with Mdm2. Several cancer cell lines with inverse expression of Set7/9 and Mdm2 displayed diminished survival in response to genotoxic stress. These findings are signified by our bioinformatics studies suggesting that the unleashed expression of Mdm2 in cancer patients with diminished expression of Set7/9 is associated with poor survival outcome.

Topics: Antibiotics, Antineoplastic; Cell Line, Tumor; Cell Survival; Computational Biology; Databases, Genetic; DNA Damage; DNA Repair; Dose-Response Relationship, Drug; Doxorubicin; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone-Lysine N-Methyltransferase; Histones; Humans; Methylnitronitrosoguanidine; Neoplasms; Protein Binding; Proto-Oncogene Proteins c-mdm2; RNA Interference; Signal Transduction; Survival Analysis; Time Factors; Transfection

The major dilemma of cancer chemotherapy has always been a double-edged sword, producing resistance in tumor cells and life-threatening destruction of nontumorigenic tissue. Glioblastoma is the most common form of primary brain tumor, with median survival at 14 months after surgery, radiation and temozolomide (monofunctional alkylator) therapy. Treatment failure is most often due to temozolomide-resistant tumor growth. The underlying basis for development of tumor cell resistance to temozolomide instead of death is not understood. Our current results demonstrate that both cervical carcinoma (HeLa MR) and glioblastoma (U251) tumor cells exposed to an equivalent chemotherapeutic concentration of a monofunctional alkylator undergo multiple cell cycles, maintenance of metabolic activity, and a prolonged time to death that involves accumulation of Apoptosis Inducing Factor (AIF) within the nucleus. A minority of the tumor cell population undergoes senescence, with minimal caspase cleavage. Surviving tumor cells are comprised of a very small subpopulation of individual cells that eventually resume proliferation, out of which resistant cells emerge. In contrast, normal human cells (MCF12A) exposed to a monofunctional alkylator undergo an immediate decrease in metabolic activity and subsequent senescence. A minority of the normal cell population undergoes cell death by the caspase cleavage pathway. All cytotoxic events occur within the first cell cycle in nontumorigenic cells. In summation, we have demonstrated that two different highly malignant tumor cell lines slowly undergo very altered cellular and temporal responses to chemotherapeutic monofunctional alkylation, as compared to rapid responses of normal cells. In the clinic, this produces resistance and growth of tumor cells, cytotoxicity of normal cells, and death of the patient.

Topics: Antineoplastic Agents, Alkylating; Apoptosis Inducing Factor; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Lineage; Dacarbazine; Electrophoretic Mobility Shift Assay; Fluorescent Antibody Technique, Indirect; Humans; Methylnitronitrosoguanidine; Neoplasm Proteins; Neoplasms; Temozolomide

Kinetochore proteins associate with centromeric DNA and spindle microtubules and play essential roles in chromosome segregation during mitosis. In this study, we uncovered a zebrafish mutant, stagnant and curly (stac), that carries the Tol2 transposon element inserted at the kinetochore protein H (cenph) locus. Mutant embryos exhibit discernible cell death as early as 20 hours postfertilization, extensive apoptosis, and upward curly tail during the pharyngula period and deform around 5 days postfertilization. The stac mutant phenotype can be rescued by cenph mRNA overexpression and mimicked by cenph knockdown with antisense morpholinos, suggesting the responsibility of cenph deficiency for stac mutants. We demonstrate that the intrinsic apoptosis pathway is hyperactivated in stac mutants and that p53 knockdown partially blocks excess apoptosis in stac mutants. Mitotic cells in stac mutants show chromosome missegregation and are usually arrested in G(2)/M phase. Furthermore, compared with wild type siblings, heterozygous stac fish develop invasive tumors at a dramatically reduced rate, suggesting a reduced cancer risk. Taken together, our findings uncover an essential role of cenph in mitosis and embryonic development and its association with tumor development.

Topics: Animals; Apoptosis; Cell Cycle Proteins; Cell Division; Chromosome Aberrations; DNA Transposable Elements; Embryo Loss; Embryo, Nonmammalian; Embryonic Development; Female; G2 Phase; Genetic Loci; Genetic Predisposition to Disease; Heterozygote; Male; Methylnitronitrosoguanidine; Mitosis; Mutagenesis; Mutation; Neoplasms; Phenotype; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Tumorigenesis is thought to be the consequence of gene mutation and disordered gene expression. However, the detailed molecular mechanism underlying the development and progress of colon cancer have not been elucidate completely. This study aimed to find out the genes associated with cancer biological pathways involved in transformation and tumorigenesis.. Normal intestinal cell line 6 (IEC-6) cells were transformed to cancer cells by treatment with cancerogenic agent of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and Phorbol 12-myristate 13 acetate (PMA). Then we investigated the altered gene expression of transformed IEC-6 cells by the microarray containing 113 genes associated with cancer pathway. Also the altered miRNAs of transformed IEC-6 cells were analyzed by array hybridization (miRCURY Array v9.2, Exiqon). The levels of acetylated histone H3 in transformed IEC-6 cells was evaluated by western blot.. Cell proliferation was significantly increased as IEC-6 cells were transformed and tumor xenografts could be detected in animals as transformed IEC-6 cells were inoculated subcutaneously in nude mice. Result of microarray showed nine genes were increased and two decreased, as well as 13 miRNA were increased and 97 decreased. Verification by real-time PCR implies that the data obtained from microarray analysis were reliable. Western blot showed the levels of acetylated histone H3 were increased dramatically after MNNG/PMA treatment.. Our results showed many important biological pathways and miRNAs were involved in transformation and tumorigenesis of IEC-6 cells, which suggested the transformation of normal cells was involved with large mount of genetic and epigenetic variation.

Topics: Acetylation; Animals; Cell Line; Gene Expression Regulation, Neoplastic; Histones; Male; Methylnitronitrosoguanidine; Mice; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neoplasms; Oligonucleotide Array Sequence Analysis

Chemical carcinogenicity is of primary interest, because it drives much of the current regulatory actions regarding new and existing chemicals, and its conventional experimental test takes around three years to design, conduct, and interpret as well as the costs of hundreds of millions of dollars, millions of skilled personnel hours, and several animal lives. Both academia and private companies are actively trying to develop alternative methods, such as QSAR models. This paper reports a QSAR study for predicting carcinogenic potency of nitrocompounds bioassayed in female rats. Several different theoretical molecular descriptors, calculated only on the basis of knowledge of the molecular structure and an efficient variable selection procedure, such as Genetic Algorithm, led to models with satisfactory predictive ability. But the best-final QSAR model is based on the GEometry, Topology, and Atom-Weights AssemblY (GETAWAY) descriptors capturing a reasonable interpretation. In fact, structural features such as molecular shape-linear, branched, cyclic, and polycyclic--and bond length are some of the key factors flagging the carcinogenicity of this set of nitrocompounds. This QSAR model, after removal of one identified nitrocompound outlier, is able to explain around 86% of the variance in the experimental activity and manifest good predictive ability as indicated by the higher q(2)s of cross- and external-validations, which demonstrate the practical value of the final QSAR model for screening and priority testing. This model can be applied to nitrochemicals different from the studied nitrocompounds (even those not yet synthesized) as it is based on theoretical molecular descriptors that might be easily and rapidly calculated.

Topics: Animals; Drug Evaluation, Preclinical; Female; Neoplasms; Nitro Compounds; Quantitative Structure-Activity Relationship; Rats

Global genomic changes, including DNA aneuploidy, may be necessary for carcinogenesis; however, such genomic changes in precancerous cells have not been studied extensively. To identify early global genotypic changes associated with precancerous lesions, a non-transformed human liver epithelial cell line, THLE-3, was treated with benzo[a]pyrene or N-methyl-N-nitro-N-nitrosoguanidine, then by 12-O-tetradecanoyl-phorbol-13-acetate, resulting in morphological transformation of cells. We examined genotypic changes of the transformed cells by laser scanning cytometry, fluorescence in situ hybridization, and comparative genomic hybridization. Transformed fusiform cells displayed tetraploidy, chromosomal instability, DNA copy number aberrations. Cells with these changes were still in the precancerous stage. However, it is suggested that these global genomic changes including tetraploidization provide cells with genetic alterations leading to cancer.

Topics: Benzo(a)pyrene; Carcinogens; Cell Line; Cell Transformation, Neoplastic; Chromosomes, Human, Pair 11; Chromosomes, Human, Pair 17; DNA; DNA Damage; G1 Phase; Gene Expression Regulation, Neoplastic; Genome, Human; Genotype; Humans; In Situ Hybridization, Fluorescence; Laser Scanning Cytometry; Liver; Methylnitronitrosoguanidine; Neoplasms; Nucleic Acid Hybridization; Phenotype; Ploidies; Resting Phase, Cell Cycle; Tetradecanoylphorbol Acetate; Time Factors

The 39-kDa DNA polymerase beta (pol beta) is an essential enzyme in short-patch base excision repair pathway. A wild-type and a truncated forms of pol beta proteins are expressed in primary colorectal and breast adenocarcinomas and in a primary culture of renal cell carcinoma. To test whether pol beta has a contributory role in tumorigenicity of human tumor cell lines, we have undertaken a study to determine expression of pol beta in colon, breast, and prostate tumor cell lines. Unlike primary colon tumor cells, three types of pol beta mRNA have been identified in HCT116, LoVo, and DLD1, colon tumor cell lines. A 111-bp-deleted pol beta transcript was expressed in MCF7, a breast tumor cell line, but not in primary breast tumor cells. An expression of a smaller pol beta transcript has been revealed in DU145, a prostate tumor cell line, whereas, a single base (T) deletion in mRNA at codon 191 was found in prostate cancer tissue. Interestingly, a wild-type pol beta transcript was also expressed in all tumor cell lines similar to primary tumor cells. Furthermore, the cell extract of LoVo exhibited highest gap-filling synthesis function of pol beta when the extract of DU145 showed lowest activity. MNNG, a DNA alkylating agent, enhanced the gap-filling synthesis activity in extracts of LoVo cell line. Furthermore, the cellular viability of LoVo and HCT116 cells is sensitive to MNNG when DU145 cells are resistant. These results demonstrate heterogeneity in pol beta mRNA expression, which may be a risk factor related to tumorigenic activities of tumor cell lines.

Topics: Breast Neoplasms; Cell Survival; Colonic Neoplasms; DNA Polymerase beta; DNA Repair; DNA, Neoplasm; Female; Gene Expression; Humans; Male; Methylnitronitrosoguanidine; Neoplasms; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Stem Cell Assay

The activities and the expression of 3-methyladenine glycosylase (3-meAde gly) and O6-alkylguanine-DNA-alkyltransferase (O6 ATase) were investigated in ten human cancer cell lines. Both 3-meAde gly and O6 ATase activities were variable among different cell lines. mRNA levels of the O6 ATase gene, appeared to be related to the content of O6 ATase in different cell lines, whereas no apparent correlation was found between mRNA of 3-meAde gly and the enzyme activity. No correlation was found between the activity of the two enzymes and the sensitivity to alkylating agents of different structures such as CC-1065, tallimustine, dimethylsulphate (DMSO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cis-diamminedichloroplatinum (cDDP) and melphalan (L-PAM). The most striking finding of this study is that a correlation exists between the activity of O6 ATase and 3-meAde gly in the various cell lines investigated (P<0.01), suggesting a common mechanism of regulation of two DNA repair enzymes.

Topics: Alkylating Agents; Antineoplastic Agents, Alkylating; Carcinogens; Cell Division; Cisplatin; Distamycins; DNA Glycosylases; Humans; Melphalan; Methylnitronitrosoguanidine; Methyltransferases; N-Glycosyl Hydrolases; Neoplasms; Nitrogen Mustard Compounds; O(6)-Methylguanine-DNA Methyltransferase; RNA, Messenger; Sensitivity and Specificity; Sulfuric Acid Esters; Tumor Cells, Cultured

Topics: Adaptor Proteins, Signal Transducing; Base Sequence; Carcinogens; Carrier Proteins; Chromosomes, Human, Pair 3; DNA Repair; DNA Replication; Humans; Methylnitronitrosoguanidine; Microsatellite Repeats; Molecular Sequence Data; Mutagens; MutL Protein Homolog 1; Neoplasm Proteins; Neoplasms; Nuclear Proteins; Phenotype; Tumor Cells, Cultured

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.

Topics: Alkylating Agents; Carcinoma; Colonic Neoplasms; DNA Damage; DNA Repair; DNA-Binding Proteins; Female; G2 Phase; Humans; Methylnitronitrosoguanidine; Models, Genetic; MutS Homolog 2 Protein; Neoplasms; Ovarian Neoplasms; Proto-Oncogene Proteins; RNA, Messenger; Sequence Deletion; Stem Cells; Tumor Cells, Cultured

Humans show heterogeneous susceptibility to cancer development, suggesting the involvement of various genetic backgrounds in control of the production of endogenous carcinogens, the metabolism of carcinogens, the repair of DNA damage, cell proliferation and defence mechanisms including immune reactions. Gastric cancer is the major cancer in Japan. However, little is known about the genes linked with its development. In 1967, we found that N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced gastric cancers in Wistar rats. Subsequently the Buffalo strain of rats was reported to be resistant to MNNG stomach carcinogenesis, while ACI rats were very sensitive. In a carcinogenesis study using F1 and F2 rats, we suggested that this trait of MNNG stomach carcinogenesis-resistance was regulated by a single autosomal dominant allele. The O6-methylguanine adduct levels in gastric mucosa induced by MNNG were the same in Buffalo and ACI rats, but cell proliferation induced by MNNG was much higher in ACI than Buffalo animals. Chromosome mapping of the gene responsible for susceptibility to MNNG-induced carcinogenesis is now in progress and its identification will hopefully give us clues to the involvement of genetic traits in susceptibility to gastric cancer in humans. In addition, the genetic background of susceptibility to breast cancer is also being studied. In Japan, about 5% of all cases of breast cancer are familial. We have studied BRCA1, the breast cancer susceptibility gene, as a determinant of susceptibility to breast cancer by linkage analyses in 11 families, but our results indicate that BRCA1 may not be important for development of familial breast cancer in Japanese.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; BRCA1 Protein; Breast Neoplasms; Female; Gastric Mucosa; Genetic Predisposition to Disease; Humans; Japan; Lod Score; Methylnitronitrosoguanidine; Middle Aged; Neoplasm Proteins; Neoplasms; Ovarian Neoplasms; Polymorphism, Genetic; Rats; Rats, Inbred ACI; Rats, Inbred BUF; Rats, Wistar; Species Specificity; Stomach Neoplasms; Transcription Factors

Methylating agents are widely distributed environmental mutagens. To identify mechanisms that protect cells against the toxic effects of methylating compounds Chinese hamster ovary (CHO) cells were transfected with human DNA and selected for resistance to methylating agents. From methylation-resistant transfectants, human DNA was cloned that conferred methylation resistance to CHO cells. A human cDNA was isolated (designated c81) encoding a protein of 410 amino acids which is partially identical to human lamp-2 encoding a lysosomal membrane glycoprotein. Transfection of c81, but not of the neo gene or a c81 antisense construct, into CHO cells gave rise to significant increases of survivors after methylating treatments. c81 mRNA is expressed both in human, CHO parental, and transfected cells, albeit in different amounts. The methylation-resistant transfectants lacked alkyltransferase activity (Mex- phenotype), did not repair O6-methylguanine in vivo, and neither showed reduced initial DNA methylation level nor increase of removal of 3-methyladenine and 7-methylguanine from DNA. They are therefore representatives of the "tolerance" phenotype. The transfectants also showed a higher serum requirement, flat growth, and a reduced tumor forming ability. The data suggest a link between altered regulation of cell proliferation and acquired resistance to methylating DNA damaging agents.

Topics: Amino Acid Sequence; Animals; Blood; CHO Cells; Cloning, Molecular; Cricetinae; DNA; DNA Repair; Drug Resistance; HeLa Cells; Humans; Methylation; Methylnitronitrosoguanidine; Methylnitrosourea; Molecular Sequence Data; Mutagens; Neoplasms; Nucleic Acid Hybridization; Phenotype; Sequence Homology, Amino Acid; Transfection

DNA probes prepared from human O6-methylguanine-DNA methyltransferase complementary DNA were hybridized to mRNA isolated from human liver and fifteen human tumor cell lines proficient (Mer+) or deficient (Mer-) in transferase activity. Liver and Mer+ cells contained levels of transferase-specific mRNA that correlated with their transferase activity levels, whereas Mer- cells contained undetectable amounts of transferase mRNA. The mRNA levels were not induced in human cells by treatments that induce other DNA damage-inducible genes. These results demonstrate that in human cells the transferase gene is constitutively expressed, that its expression is related to activity levels, and that in Mer- tumor cells the expression of the transferase gene is probably blocked at the level of mRNA production.

Topics: Cell Line; Gene Expression; Humans; Methyl Methanesulfonate; Methylnitronitrosoguanidine; Methyltransferases; Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Phenotype; RNA, Messenger; X-Rays

Transformation experiments have been carried out on human diploid fibroblasts derived from normal individuals and those from 2 groups with dominantly inherited cancer predisposition, familial polyposis coli (FPC), and multiple endocrine neoplasia, type 2 (MEN-2). Treatment with a single or multiple doses of the carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), resulted in limited anchorage-independent (AI) growth in both normal and FPC cultures; no permanent cell lines were produced but FPC cells showed increased proliferation with low doses of the carcinogen. Carcinogen treatment followed by application of the tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), for 38 weeks was insufficient to cause full transformation in cultures derived from normal people or MEN-2 patients although AI growth was induced in all 3 cell types. Three FPC cultures exhibited an extended life span over the solvent controls. Two of these are still actively dividing and have a clonal pseudodiploid karyotype.

Topics: Adenomatous Polyposis Coli; Cell Division; Cell Transformation, Neoplastic; Fibroblasts; Humans; Kinetics; Methylnitronitrosoguanidine; Multiple Endocrine Neoplasia; Neoplasms; Skin; Tetradecanoylphorbol Acetate

In two recent reports we have shown that pretreatment of MER+ cells [cells proficient at: reactivating N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated adenovirus; removing O-6 methylguanine from their DNA; and preventing DNA interstrand crosslinks produced by the chloroethylnitrosoureas (CNUs)] with MNNG apparently inhibits the repair process that these cells utilize to prevent CNU-induced DNA interstrand crosslinking. The MNNG pretreatment, accompanied by a subsequent CNU treatment, resulted in a synergistic increase in cell kill of 2-3 logs. In the present study we have examined whether or not conditions which inhibit the ability of a cell to prevent CNU-induced DNA interstrand crosslinking can also prevent DNA interstrand crosslinking induced by four clinically used alkylating anti-tumor agents. The agents used in the present study include cis-diamminedichloroplatinum(II) (cis-Pt), L-phenylalanine mustard (L-PAM), nitrogen mustard (HN-2) and 4-S-(propionic acid)-sulfidocyclophosphamide (C-2), a derivative of cyclophosphamide. Alkaline elution analysis was used to measure DNA interstrand crosslinking, and colony formation assays to estimate cell survival. Unlike the CNUs, all four agents produced DNA interstrand crosslinks in a Mer+ cell line in the absence of MNNG pretreatment. MNNG pretreatment did not alter the levels of DNA interstrand crosslinks formed. Similar results were seen with a Mer- cell line. In cytotoxicity studies, in contrast to the CNUs, MNNG pretreatment did not appreciably increase the cell kill produced by the four agents. Since all four agents studied are thought to react primarily at the N-7 position in guanine, these data suggest that: the DNA repair system which prevents CNU-induced crosslinking is specific for methyl, ethyl, and chloroethyl monoadducts; this DNA repair system is specific for adducts only at the O-6 position of guanine and does not recognize and remove adducts at other positions in DNA; or a combination of the two explanations.

Topics: Alkylating Agents; Antineoplastic Agents; Cell Line; Cell Survival; Cross-Linking Reagents; DNA; DNA Repair; Ethylnitrosourea; Humans; Methylnitronitrosoguanidine; Neoplasms; Nitrosourea Compounds

Among 15 human tumor cell strains from Japanese patients, one strain derived from a patient with thyroid cancer showed inability to support the growth of adenovirus 5 treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When plated on this Mer- strain, adenovirus 5 showed 3-4 times higher sensitivity to MNNG-induced killing than when plated on any of the other 14 Mer+ tumor cell strains. Biochemical analysis showed that the Mer- strain was defective in demethylation repair of O6-methylguanine produced by MNNG treatment. The sensitivities of 12 of the 15 human tumor strains, including the Mer- strain, to MNNG were compared by measuring their colony-forming abilities. All the strains tested showed the Rem- phenotype (having higher sensitivity to MNNG-produced cell killing than normal fibroblasts). The differential killing effects of MNNG on Mer- and Mer+ tumor cells under in vivo conditions were tested using the Mer+ HeLa S3 strain and its Mer- variant. Mer+ cells and Mer- cells were implanted subcutaneously into the left and right flanks, respectively, of 10 nude mice and the next day, MNNG solution (0.25 ml at 1 mg/ml) was injected into the implantation sites of eight mice. Mer- tumor cells in six of eight treated mice showed no growth and those in the other two mice did grow, but regressed after approximately 3 weeks. In contrast, Mer+ tumor cells continued to grow in all the eight mice treated, indicating that Mer- tumor cells may be selectively inactivated by suitable therapeutic regimens with appropriate methylating drugs.

Topics: Adenoviruses, Human; Animals; Cell Line; Colony-Forming Units Assay; DNA Repair; DNA, Neoplasm; Humans; Japan; Methylnitronitrosoguanidine; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Phenotype; Viral Plaque Assay

Juvenile medaka were exposed to N-methyl-N'-nitro-N-nitrosoguanidine in water under static renewal conditions for 28 days. Two groups of 134 fish each were pulsed 3 times weekly at nominal concentrations of 1.0 and 0.5 mg/liter with N-methyl-N'-nitro-N-nitrosoguanidine dissolved in dimethylformamide. A third group of 134 fish was exposed to the solvent control, 0.01% dimethylformamide in water. Following the 28-day exposure, and during the recovery period, fish were sampled at intervals of approximately 0, 3, 6, and 9 months and examined grossly. Selected tissues were evaluated microscopically. Many tumor types developed in both N-methyl-N'-nitro-N-nitrosoguanidine exposure groups, but only the gill lesions will be discussed. Approximately 50% of the fish in both treatment groups died from gill damage in the second to third month of the recovery period. More than 90% of the surviving treated fish displayed gill lesions, which progressed from mild epithelial hyperplasia of gill filaments at 0-months recovery to epitheliomatous hyperplasia at 3 months and advanced to a more focal nodular appearance of gill filaments at 6 months. Eight to 9 months after the treatment period, at least four fish displayed branchial blastomas. The control fish had no gill lesions. Chemically induced gill tumors have not been previously observed in fish. Even gill tumors of unknown origin are very rare.

Topics: Animals; Disease Models, Animal; Fish Diseases; Fishes; Gills; Hyperplasia; Methylnitronitrosoguanidine; Neoplasms; Time Factors

Two DNA repair assays were used to group human cells. (a) The first assay, survival of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated adenovirus infecting cellular monolayers, was previously used to define the Mer phenotype of the strain. Strains that supported the growth of MNNG-treated viruses as well as did human fibroblasts were "Mer+"; those that gave rise to clearly less virus survival were "Mer-." (b) The second assay, data from which are presented in this paper, was that of post-MNNG colony-forming ability, and defined the Rem phenotype of the strain. Strains having post-MNNG colony-forming ability like that of human fibroblasts were "Rem+"; more sensitive strains were "Rem-". In all, 22 human cell strains were analyzed for their post-MNNG colony-forming ability. The most resistant strains (eight Mer+ Rem+ strains) had an average inactivation slope of 0.32 "lethal hit"/microM and were those fully able to repair O6-methylguanine (O6mGua) produced in their DNA by a 5 microM dose of MNNG. The most sensitive strains (9 Mer- Rem- strains) had an average inactivation slope of 7.0 "lethal hits"/microM, and were strains that failed to repair O6mGua. Five strains of intermediate sensitivity (Mer+ Rem-) had an average inactivation slope of 0.93 "lethal hit"/microM and were able to repair some labeled O6mGua produced by a 5 microM dose of labeled MNNG, but they repaired significantly less labeled O6mGua if pretreated with unlabeled MNNG. Representative strains from each group were treated with MNNG and assayed for ability: (a) to perform DNA repair synthesis (and DNA repair replication); (b) to support the growth of MNNG-treated adenoviruses; and (c) to restore control levels of tertiary structure to their DNA as assayed by nucleoid sedimentation. The results support the hypothesis that a lesion (both produced by agents that produce O6mGua and repaired by cell strains that repair O6mGua, but not by those that do not) is a lesion lethal to Mer- Rem- strains. This lesion may also initiate induction of excess DNA repair synthesis, the relaxed conformation of nucleoids, the reduced ability to repair MNNG-treated adenovirus, and sister chromatid exchanges as well.

Topics: Adenoviridae; Cell Line; Cell Survival; Cell Transformation, Viral; DNA Repair; DNA Replication; Humans; Methylnitronitrosoguanidine; Neoplasms; Phenotype

The repair of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-produced O6-methylguanine (O6-MeG) in DNA and its correlation with MNNG-produced cell-killing and sister chromatid exchange (SCE) induction were compared in mouse and reference human tumor cell strains. As a result, mouse cell strains were divided into three groups: (i) cells proficient in O6-MeG-repair and insensitive to MNNG similar to human Mer+ Rem+ strains; (ii) cells deficient in O6-MeG-removal and sensitive to MNNG similar to human Mer-Rem- strains; (iii) cells deficient in O6-MeG-removal but insensitive to MNNG similar to some SV40-transformed human strains. Attempts at correlating lack of capacity for O6-MeG-removal, MNNG-sensitivity and high SCE induction showed that O6-MeG in DNA may be a lesion common to cell-killing and SCE induction only in mouse cells of groups i and ii. Levels of O6-MeG-DNA methyltransferase activity in mouse cells were measured and the enzyme had the same molecular weight as that in human cells.

Topics: Animals; Cell Line; Cell Survival; Cells, Cultured; DNA Repair; Embryo, Mammalian; Female; Guanine; Humans; Kinetics; Methylation; Methylnitronitrosoguanidine; Mice; Mice, Inbred Strains; Neoplasms; Neoplasms, Experimental; Species Specificity

Topics: Adenocarcinoma; Animals; Antioxidants; Ascorbic Acid; Carcinoma, Squamous Cell; Male; Methylnitronitrosoguanidine; Neoplasms; Rats; Rats, Inbred F344; Sarcoma; Stomach Neoplasms

The ability of extracts of human tumor cells to demethylate O6-methylguanine (O6-MeG) in DNA was assayed using the synthetic DNA polymer poly(dC,dG,m6dG). Cell strains proficient in repair of O6-MeG in vivo (Mer+ phenotype) contained a methyltransferase activity while repair deficient cells (Mer- phenotype) had little or no activity. Mixing extracts of different Mer- strains did not result in the appearance of the activity. Extracts of Mer- cells did not inhibit the activity in extracts of Mer+ cells. Both Mer+ and Mer- strains contained methylnitrosourea-damage-specific endonuclease activity. The data suggest that the Mer- strains are deficient in methyltransferase and that this is the fundamental reason for their hypersensitivity to the cytotoxic effects of DNA alkylation. The activity was partially purified from a Mer+ colon carcinoma cell strain. Its kinetics parallel the repair of O6-MeG in DNA in vivo and suggest that the activity is inactivated during repair of DNA.

Topics: DNA Repair; DNA, Neoplasm; Female; Guanine; Humans; Kinetics; Methylnitronitrosoguanidine; Methyltransferases; Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Phenotype

A microcrystal (ca 5 micrograms) of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 4-nitroquinoline-1-oxide (4NQO) was directly administered to the regeneration blastema on day 7 after amputation of a forelimb in the newt in order to analyze the effect of such potent carcinogenic substances on regeneration cells. Although neither MNNG nor 4NQO arrested regeneration completely, they caused great retardation of the regeneration cone formation followed by various abnormalities in the bony structures. Abnormal regenerants could be classified into the following four categories; (1) complete absence of both ulna and radius; (2) subregeneration or superregeneration of carpals and digits; (3) multiple disorganization of skeletal elements; (4) arrest of regeneration at the stage of regeneration cone. The polarity of regenerants developed after application of MNNG or 4NQO was very often shifted, during which the regeneration cone was always formed from the site where a microcrystal of the carcinogens was administered. The secondary regeneration initiated by reamputation of the regenerating limb, which had received the carcinogens at the early blastema stage, proceeded in the same way as observed in the case of a simple amputation. This suggested local and temporal effects of the carcinogens applied. Nevertheless, tumor formation has not induced in the newt limb so far. We can learn from these data that both MNNG and 4NQO only alter behavior of the newt regeneration cells without excreting their carcinogenic effects on them, and that the newt cells are highly resistant and stable against the above-mentioned carcinogens.

Topics: 4-Nitroquinoline-1-oxide; Abnormalities, Drug-Induced; Animals; Bone and Bones; Forelimb; Methylnitronitrosoguanidine; Neoplasms; Nitroquinolines; Regeneration; Triturus

Cancers are clones of abnormal cells, arising presumably as the result of mutational or epignetic alterations of gene expression. The kinetics of appearance of spontaneous cancers in populations of multiplying cells (i.e. the relation between age and cancer incidence) will therefore depend, among other things, on how these populations are organized and, in general, on the kinetics of the response of cells to prolonged mutagenesis. The organization of cell renewal in epithelia (i.e. the arrangement of cell lineages) is still rather obscure; in particular, it is not known to what extent the properties and organization of the stem cells tend to protect them from accumulating mutations. We have tried to mimic the arrangement of epithelia by attaching multiplying bacteria to filters. Study of mutagenesis in long-term cultures of such anchored bacterial has led to the discovery of some additional pathways for DNA repair which also appear to operate in mammalian cells.

Topics: Adaptation, Biological; Alkylating Agents; Animals; Bacteria; Bacteriological Techniques; Biotransformation; Carcinogens; Cells, Cultured; Cricetinae; Cricetulus; Dimethylnitrosamine; DNA Repair; Escherichia coli; Methylnitronitrosoguanidine; Mutagens; Mutation; Neoplasms; Research Design

We have identified a group of 8 (among 39) human tumour cell strains deficient in the ability to support the growth of adenovirus 5 preparations treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but able to support the growth of non-treated adenovirus normally. This deficient behaviour defines the Mer- phenotype. Strains having the Mer- phenotype were found to arise from tumours originating in four different organs. Relative to Mer+ strains, Mer- tumour strains showed greater sensitivity to MNNG-produced killing, greater MNNG-stimulated "DNA repair synthesis and a more rapid MNNG-produced decrease in semi-conservative DNA synthesis. Here we report that (1) Mer- strains are deficient in removing O6-methylguanine (O6-MeG) from their DNA after [Me-14C]MMNG treatment (Table 1); (2) Mer- tumour strains originate from tumours arising in patients having Mer+ normal fibroblasts (Fig. 1a, b); (3) SV40 transformation of (Mer+) human fibroblasts often converts them to Mer- strains (Fig. 1c, d); (4) MNNG produces more sister chromatid exchanges (SCEs) in Mer- than in Mer+ cell strains (Fig. 2).

Topics: Adenoviruses, Human; Cells, Cultured; DNA Repair; Guanine; Humans; Methylation; Methylnitronitrosoguanidine; Neoplasms; Simian virus 40; Sister Chromatid Exchange

Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.

Topics: Animals; Humans; Immune Sera; Methylnitronitrosoguanidine; Neoplasms; Rats; Sarcoma, Experimental; Thymus Gland

The theory of cancer induction of ultimate carcinogens by parametric excitation is presented. Attention is drawn to the requirement of an energy transfer in perturbing DNA. The concept of energy transfer by resonance is rejected in favor of energy transfer by parametric excitation of which resonance is a special case. The result, reported here, is a remarkable agreement between the Raman vibrational bands of these ultimate carcinogens, various hypochromic bands of DNA and the predictions of parametric excitation theory. This result suggests that the peculiar relation of an ultimate carcinogen and DNA results in parametrically induced energy transfer in the vibrational mode. The problem, then, of cancer induction becomes one of analytical mechanics.

Topics: Carcinogens; DNA; Energy Transfer; Humans; Methylnitronitrosoguanidine; Models, Biological; Neoplasms; Spectrum Analysis, Raman

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