methylnitronitrosoguanidine and Adenomatous-Polyposis-Coli

Experimental chemical carcinogenesis in the digestive tract is reviewed, mainly on the basis of information obtained in the laboratories of the National Cancer Center Research Institute. It is generally accepted that cancer is the outcome of DNA damage, resulting in mutation, loss, amplification and recombination of genes. Gastric cancer is no exception. It was shown very early that cancer of the glandular stomach can be produced in rats by administration of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a widely used mutagen. However, this depends on the genotype. Whereas the ACI rat is susceptible to MNNG, the Buffalo rat is resistant and this is a dominantly inherited trait. Genes responsible for the sensitivity to gastric cancer induction are at present under investigation by linkage analysis of rat genome markers. With regard to cancer in humans, our finding that cooked proteinaceous foods can give rise to a series of heterocyclic amines (HCAs) is of major significance. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the most abundant, causes colon cancers in male rats, whereas in females it induces breast cancers. The colon cancers induced by PhIP feature a deletion of G as represented by 5-GGGA-3-->5-GGA-3 in the Apc gene, resulting in a truncated Apc molecule. Microsatellite mutations have also been found in PhIP-induced colon tumors, as in human hereditary non-polyposis colorectal cancer cases. Similarly to the case of gastric cancer production by MNNG, there is a genetic component and F344 rats are more susceptible to PhIP colon carcinogenesis than the ACI/N strain and the gene responsible is being sought. Since carcinogenesis proceeds with accumulation of genetic alteration, often involving genomic instability, exposure to any kind of carcinogenic substances, either xeno- or autobiotics, needs to be reduced as far as possible, taking account of inconvenience at the individual and socio-economical levels.

Topics: Adenomatous Polyposis Coli; Animals; Carrier Proteins; Colonic Neoplasms; DNA, Neoplasm; Gene Deletion; Genes, APC; Humans; Male; Methylnitronitrosoguanidine; Molecular Chaperones; Nerve Tissue Proteins; Rats; Rats, Inbred ACI; Rats, Inbred BUF; Rats, Inbred F344; Rats, Wistar; Receptors, Aryl Hydrocarbon; Stomach Neoplasms

Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10(-6). Beginning with about 10(8) human cells we first enrich for the desired nuclear sequence 10,000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin-streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC-->AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 x 10(-6) and 9 x 10(-6). The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC-->TA transversions at a mutant fraction equivalent to approximately 10(-6) in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of approximately 10(8) cells.

Topics: Adenomatous Polyposis Coli; Adenomatous Polyposis Coli Protein; Base Sequence; Cell Line; Cytoskeletal Proteins; DNA Mutational Analysis; DNA, Complementary; Electrophoresis, Capillary; Genome, Human; Humans; Methylnitronitrosoguanidine; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Denaturation; Point Mutation; Polymerase Chain Reaction; Reproducibility of Results; Sensitivity and Specificity

In a search for hypersensitivity to chromosome breaking agents, lymphocytes and fibroblasts from patients with the precancerous condition adenomatous polyposis coli (APC) were treated with N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) and bleomycin. The APC cells showed a significantly increased level of chromatid-type damage following MNNG treatment (5 micrograms/ml for lymphocytes, 1 or 2 micrograms/ml for fibroblasts). No such differential effect was noted for bleomycin, but a threefold increase in "pulverized" cells was seen in APC lymphocyte cultures treated in G0, that is before cells have entered the cell cycle. Increased spontaneous and induced chromosome instability appears to be an important effect of the APC mutant gene as molecular evidence suggests that chromosomal mechanisms are likely to play a major role in tumorigenesis both in this condition and in sporadic colorectal cancer.

Topics: Adenomatous Polyposis Coli; Bleomycin; Chromosome Aberrations; Chromosomes, Human; Fibroblasts; Humans; Lymphocytes; Methylnitronitrosoguanidine; Reference Values; Skin

Carcinogenesis is considered to be a multi-step process comprising 'initiation', 'promotion' and 'conversion' events. Skin fibroblasts from patients with hereditary retinoblastoma (RB) and familial polyposis coli (FPC) were chosen for study since their predisposition to the tumour may be due to an inherited 'initiation' event which is present in every cell. Experiments involving skin fibroblasts from FPC patients showed certain of these cells to grow in semi-solid medium following treatment with the complete carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) alone. When tested prior to the commencement of the experiments the FPC patient cell populations had shown no strong predisposition to malignant transformation as assessed by increased saturation densities, reduced serum requirements, altered migration patterns in collagen gels, anchorage-independent growth and tumourigenicity in nude mice. Following carcinogen or promoter treatment, apart from exhibiting low-level frequencies of anchorage-independent growth, the cells appeared no more transformed than they were before. Parallel cytogenetic studies showed TPA to increase both tetraploidy and the chromosome-aberration frequency during the course of these transformation studies. However, the FPC cell clones induced by TPA to grow in semi-solid medium were, at best, considered to be only partially transformed when their properties were compared with those of tumour-derived cell lines.

Topics: Adenomatous Polyposis Coli; Cell Adhesion; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Chromosome Aberrations; Eye Neoplasms; Female; Fibroblasts; Humans; Male; Methylnitronitrosoguanidine; Ploidies; Retinoblastoma; Skin

Skin fibroblasts derived from three patients with familial polyposis coli (FPC) were treated in vitro with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) alone or in combination with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). None of the cultures treated four times with MNNG alone (1 microgram/ml) or in combination with TPA (eight applications, 0.1 microgram/ml each) showed either morphological transformation or anchorage-independent growth for 18 months after treatments. FPC cells treated with MNNG alone showed cell growth inhibition, breakage and loss of chromosomes, as well as the deletion of 5.7 kilobase (kb) EcoRI fragment in the c-K-ras locus as detected by Southern blot analysis with v-K-ras specific probe (clone KBE-2). The control cells contained two EcoRI fragments of 5.7 and 4.2 kb. On the other hand, cells treated with MNNG first and then followed by treatment with TPA not only showed an increase in the rate of cell growth but also exhibited two novel EcoRI fragments of 9.4 and 12 kb. Treatment with TPA alone appeared to stimulate cell division long after application and to induce chromosomal aberrations but had no effect on c-K-ras sequences. The most likely explanation for the appearance of chromosome pulverization and hyperploidy in FPC cells treated with TPA is the induction of premature chromosome condensation of the S phase cells due to fusion with the mitotic chromosomes.

Topics: Adenomatous Polyposis Coli; Adolescent; Adult; Cell Division; Cells, Cultured; Child; Chromosome Aberrations; Deoxyribonuclease EcoRI; Fibroblasts; Genes, ras; Humans; Methylnitronitrosoguanidine; Ploidies; Skin; Tetradecanoylphorbol Acetate

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