methylnitronitrosoguanidine and Colorectal-Neoplasms

Colorectal cancer (CRC) is the third most frequent malignancy worldwide. Coffee is the second most consumed drink in the globe and suggested to decrease the CRC risk. Here, we explored whether coffee, decaffeinated coffee, or caffeine impact on the development of colorectal carcinogenesis induced by the direct carcinogen N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in rats. To this end, sixty-four young male Wistar rats were divided into eight groups of eight animals each. We analyzed the frequency of dysplastic crypts and expression of metallothionein as a biomarker of the cancer risk, as well the expression of phosphorylated H2A histone family/member X (γH2AX) for DNA damage and cyclooxygenase-2 (COX-2) for inflammatory response. We also studied the oxidative stress profile in hepatic and colonic frozen samples (malondialdehyde [MDA], glutathione [GSH], and α-tocopherol). We found that coffee but neither decaffeinated coffee nor caffeine decreased the development of dysplastic crypts in MNNG-exposed rats. All treatments reduced DNA damage intensity in colonocytes. Only decaffeinated coffee increased the numbers of metallothionein positive crypts in comparison with coffee-treated rats. Coffee and caffeine inhibited COX-2 expression in the colon. Both decaffeinated coffee and caffeine decreased hepatic α-tocopherol levels. We suggest that coffee may have other compounds that elicit greater chemoprotective effects than caffeine reducing the CRC risk.

Topics: alpha-Tocopherol; Animals; Anticarcinogenic Agents; Caffeine; Carcinogens; Coffee; Colon; Colorectal Neoplasms; Cyclooxygenase 2; DNA Damage; Histones; Male; Metallothionein; Methylnitronitrosoguanidine; Oxidative Stress; Rats, Wistar

The relationships between environmental factors and the genetic abnormalities that drive carcinogenesis are supported by experimental and epidemiologic evidence but their molecular basis has not been fully elucidated. At the genomic level, most human cancers display either chromosomal (CIN) or microsatellite (MIN) instability. The molecular mechanisms through which normal cells acquire these forms of instability are largely unknown. The arylamine 4-aminobiphenyl (4-ABP) is a tobacco smoke constituent, an environmental contaminant, and a well-established carcinogen in humans. Among others, bladder, lung, colon, and breast cancers have been associated with 4-ABP. We have investigated the effects of 4-ABP and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on genetically stable colorectal (HCT116) and bladder (RT112) cancer cells. Cells were treated with carcinogens to generate resistant clones that were then subjected to genetic analysis to assess whether they displayed either CIN or MIN. We found that 50% to 60% of cells treated with 4-ABP developed CIN but none developed MIN as confirmed by their ability to gain and lose chromosomes. In contrast, all MNNG-treated clones (12/12) developed MIN but none developed CIN as shown by the microsatellite assay. The mismatch repair protein expression analysis suggests that the acquired mechanism of MIN resistance in the HCT116 MNNG-treated cells is associated with the reduction or the complete loss of MLH1 expression. By providing a mechanistic link between exposure to a tobacco constituent and the development of CIN, our results contribute to a better understanding of the origins of genetic instability, one of the remaining unsolved problems in cancer research.

Topics: Adaptor Proteins, Signal Transducing; Aminobiphenyl Compounds; Carcinogens; Chromosomal Instability; Colorectal Neoplasms; DNA Mismatch Repair; Humans; Immunoblotting; In Situ Hybridization, Fluorescence; Methylnitronitrosoguanidine; Microsatellite Instability; MutL Protein Homolog 1; Nicotiana; Nuclear Proteins; Smoke; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Sporadic microsatellite mutations are frequently observed in lung, bladder, and head and neck tumors with intact DNA mismatch repair. AAAG tetranucleotide repeats appear to be especially prone to the accumulation of these mutations. We hypothesized that occurrences of microsatellite mutations in these cancers may be linked to DNA damage caused by exposure to carcinogens in tobacco smoke. To test this hypothesis, we developed a model system based on reactivation of green fluorescent protein (GFP) in which a plasmid vector carries a microsatellite repeat that places the GFP sequence out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the GFP reading frame and become detectable by flow cytometry as GFP-positive cells. Pools of stably transfected RKO cells were treated at four dose levels each of gamma-irradiation, benzo(a)pyrene diol epoxide, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), t-butyl hydrogen peroxide, and UV irradiation and assayed for GFP-positive cells 48 h later. We studied the microsatellite repeats AAAG, ATAG, CAGT, and CA, as well as a control sequence lacking any repetitive elements. A log-linear regression approach was used to discriminate between the effects of repeat unit and dose for each agent. A statistically significant increase in GFP-positive cells was found with increasing dose with all agents, although repeat unit-specific response patterns were only observed with MNNG, t-butyl hydrogen peroxide, and UV irradiation. With MNNG, significant differences in response were observed between dinucleotide and tetranucleotide repeat units. The effects of UV irradiation were consistent with the predicted number of pyrimidine dimers/repeat unit, with higher GFP activation in repeats that had large numbers of adjacent pyrimidines. We found no evidence to indicate that the AAAG repeat responded to any of the DNA-damaging agents with higher levels of GFP activation than other repeat units. These results provide evidence that DNA damage can induce slippage mutations and increase mutation rates in repeated sequences and that there are sequence-specific responses to different types of DNA damage. Our results are compatible with the hypothesis that sporadic microsatellite mutations in human cancer may reflect DNA damage caused by carcinogen exposure.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Carcinogens; Colorectal Neoplasms; DNA Damage; Flow Cytometry; Green Fluorescent Proteins; Humans; Luminescent Proteins; Methylnitronitrosoguanidine; Microsatellite Repeats; Mutagens; tert-Butylhydroperoxide; Transfection; Tumor Cells, Cultured; Ultraviolet Rays

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatments for a long period induced morphological and molecular alterations in the benign human colorectal polyps which were maintained in the severe combined immunodeficient C.B17/N-scid/scid mice. Thirty four xenografts of colorectal polyps from five solitary polyp and three familial polyposis patients were examined for K-ras and p53 mutations. Six K-ras mutations were induced in 16 grafts treated with MNNG more than five times, while no K-ras mutations were detected in 14 untreated grafts (P<0.05). Additional and new K-ras mutations were also induced in two polyps in which K-ras mutation had pre-existed. p53 mutations were not observed in both MNNG-treated and untreated groups. The mutations in K-ras gene were induced at codon 12 (GGT-->GAT) except one at codon 13 (GGC-->GGT). The results indicate that K-ras mutation plays an important role in human colorectal carcinogenesis as is the case in experimental animals.

Topics: Animals; Codon; Colonic Polyps; Colorectal Neoplasms; Disease Models, Animal; Genes, p53; Genes, ras; Humans; Methylnitronitrosoguanidine; Mice; Mice, SCID; Mutagenesis; Mutagens; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Time Factors; Transplantation, Heterologous

An association between low selenium intake and the incidence or prevalence of cancers is well known. Selenium in the form of selenomethionine supplemented in drinking water has been found to be highly effective in reducing tumour incidence and preneoplastic foci during the development of hepatocarcinogenesis in rats in our previous studies. Here, an attempt has been made to investigate whether the dose and form of selenium found to be effective during hepatocarcinogenesis is equally effective in N-methylnitronitrosoguanidine-induced colorectal carcinogenesis in terms of antioxidant defence enzyme systems, DNA chain breaks and incidences of aberrant crypt foci. Treatment with selenomethionine either on initiation or on selection/promotion, or during the entire experiment showed that selenomethionine was most effective in regulating the cellular antioxidant defence systems, DNA chain break control and reducing aberrant crypt foci in the colorectal tissues of rats. Our results also confirm that selenium is particularly effective in limiting the action of the carcinogen during the initiation phase of this colorectal carcinogenesis, just as we found with hepatocarcinogenesis in our previous studies.

Topics: Animals; Antioxidants; Carcinogens; Cell Transformation, Neoplastic; Chemoprevention; Colorectal Neoplasms; DNA Damage; Male; Methylnitronitrosoguanidine; Rats; Rats, Sprague-Dawley; Selenomethionine

The human colorectal tumor cell line LoVo has a homozygous deletion in the hMSH2 gene from exon 3 to exon 8, is deficient in mismatch repair (MMR) activity, and exhibits microsatellite instability. To determine whether the introduction of a wild type hMSH2 into LoVo restores MMR activity and stabilizes microsatellite loci, we transferred a chromosome 2 fragment containing hMSH2 into a homologous recombination-proficient chicken DT40/human hybrid (DT40 2C) and a human chromosome 2 in a mouse A9 hybrid to LoVo. Transfers of these chromosomes into LoVo resulted in LoVo both with and without a wild-type hMSH2. Complete correlation was found between the presence of the wild-type hMSH2 and hMSH2 expression, an increased stability in microsatellite loci, and competency in MMR. The hMSH2-positive LoVo hybrids also showed an increased sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine. This enhanced toxicity is associated with G(2) cell-cycle arrest followed by premature mitosis and cell death. These results suggest that hMSH2 may be responsible for complementing mutator and drug-resistant phenotypes in chromosome 2-transferred LoVo cells. To test whether the hMSH2 in DT40 2C cells can be modified by homologous recombination, we transfected DT40 2C with a targeting vector containing an hMSH2 exon 4 disrupted by the zeocin-resistant gene. The results showed that the hMSH2 locus in DT40 2C was efficiently targeted by an exogeneously transfected homologous sequence, suggesting that transfer of a modified hMSH2 from DT40 2C to LoVo via chromosome transfer could be used to determine the function of hMSH2.

Topics: Animals; Base Sequence; Cell Death; Chromosomes, Human, Pair 2; Colorectal Neoplasms; DNA Primers; DNA-Binding Proteins; G2 Phase; Gene Targeting; Genetic Complementation Test; Humans; Methylnitronitrosoguanidine; Mice; Microsatellite Repeats; MutS Homolog 2 Protein; Proto-Oncogene Proteins

Mutations of the mismatch repair genes hMSH2 and hMLH1 have been found in a high proportion of individuals with hereditary nonpolyposis colon cancer (HNPCC), establishing the link between mismatch repair and cancer. Tumor cell lines that are deficient in mismatch repair develop a mutator phenotype that appears to drive the accumulation of mutations required for tumor development. However, mutations of other mismatch repair genes such as hPMS2 can lead to a mutator phenotype, although inherited mutations of these genes are rare in HNPCC families. Here, we show that overexpression of hMSH2 or hMLH1 but not of hMSH3, hMSH6, or hPMS2 induces apoptosis in either repair-proficient or -deficient cells. Furthermore, primary mouse embryo fibroblasts derived from Msh2-deficient mice lose their ability to undergo apoptosis after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These results suggest that the mismatch repair proteins hMSH2 and hMLH1 may be components of a pathway that influences apoptosis. We consider the possibility that loss of apoptosis as a result of hMSH2 or hMLH1 deficiency may be an additional factor in cancer predisposition in HNPCC.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Animals; Apoptosis; Base Pair Mismatch; Carrier Proteins; Cell Line; Cells, Cultured; Colorectal Neoplasms; DNA Repair; DNA-Binding Proteins; Endometrial Neoplasms; Female; Humans; In Situ Nick-End Labeling; Methylnitronitrosoguanidine; Mice; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nuclear Proteins; Proto-Oncogene Proteins; Recombinant Proteins; Transfection; Tumor Cells, Cultured; Uterine Neoplasms

Topics: 1,2-Dimethylhydrazine; Adenocarcinoma; Adenoma; Animals; Carcinogens; Colorectal Neoplasms; Dietary Fats; Dimethylhydrazines; Fruit; Intestinal Neoplasms; Male; Methylnitronitrosoguanidine; Rats; Rats, Wistar; Vegetables

In eukaryotic cells, DNA polymerase beta (polbeta) carries out base-excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. A specific deletion in one allele in the coding sequence of the polbeta gene occurs in colorectal and breast carcinomas. The 87-bp deleted region encodes amino acid residues 208-236 in the catalytic domain of the enzyme. Here, we report evidence for expression of the wild-type (WT) and the truncated polbeta proteins in colorectal tumors. To elucidate the potential functional consequences of polbeta truncation, stable HeLa cell lines were established from cloned WT and variant polbetaDelta208-236. Cells expressing the variant protein exhibited substantially decreased BER activity. To test our hypothesis that truncated polbeta may disrupt the function of the WT enzyme, we stably transfected mouse embryonic fibroblast 16.3 cells with polbetaDelta208-236 cDNA. Reverse transcription-PCR and Western blot analyses showed that the new cell line, 16.3DeltaP, expresses the WT and the truncated polbeta mRNA and proteins. BER and binding activities were undetectable in these cells. Furthermore, in vivo the 16.3DeltaP cells were more sensitive to N-methyl-N'-nitro-N-nitrosoguanidine than the 16.3 cells. On adding increasing amounts of 16.3DeltaP protein extracts, the BER and DNA binding activities of extracts of the parent 16.3 cell line progressively declined. These results strongly suggest that truncated polbeta acts as a dominant negative mutant. The defective polbeta may facilitate accumulation of mutations, leading to the expression of a mutator phenotype in tumor cells.

Topics: Blotting, Western; Cell Line; Cell Survival; Chromatography, Gel; Colorectal Neoplasms; DNA Polymerase I; DNA-Binding Proteins; Embryo, Mammalian; Fibroblasts; HeLa Cells; Humans; Methylnitronitrosoguanidine; Mutation; Polymerase Chain Reaction

The modulation of a vegetables-fruit mixture on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced colorectal carcinogenesis was studied in rats maintained on a low- or a high-fat diet. For this purpose, 120 rats received a semisynthetic diet without (Groups A and C) or with a vegetables-fruit mixture (19.5% wt/wt, Groups B and D) for 35 weeks. Diets of Group A and B contained 20 (low) energy percent (20e%) fat, whereas diets of Groups C and D contained 40e% (high) fat. Between Weeks 4 and 9 the animals were given weekly intrarectal instillations of 6 mg MNNG/kg body wt. The colorectal adenocarcinoma incidences showed a significant decrease in animals fed high-fat diets with a vegetables-fruit mixture compared with animals fed a high-fat diet alone. Furthermore, without a vegetables-fruit mixture, diets high in fat caused a significant increase in adenocarcinoma incidence compared with diets low in fat. Although not significant, the adenoma incidences tended to be lower in animals fed a vegetables-fruit mixture than in animals maintained on a diet without this mixture. The results demonstrate that a vegetables-fruit mixture has a significant inhibitory potency on the development of colorectal tumors induced by MNNG in rats fed diets high in fat.

Topics: Administration, Rectal; Animals; Carcinogens; Colorectal Neoplasms; Dietary Fats; Fruit; Male; Methylnitronitrosoguanidine; Rats; Rats, Wistar; Vegetables

Loss of DNA mismatch repair is a common finding in many types of sporadic human cancers as well as in tumors arising in patients with hereditary nonpolyposis colon cancer. The effect of the loss of DNA mismatch repair activity on sensitivity to a panel of commonly used chemotherapeutic agents was tested using one pair of cell lines proficient or deficient in mismatch repair due to loss of hMSH2 function and another due to loss of hMLH1 function. 6-Thioguanine and N-methyl-N'-nitro-N-nitrosoguanidine, to which these cells are known to be resistant, were included in the panel as controls. The results were concordant in both pairs of cells. Loss of either hMSH2 or hMLH1 function was associated with low level resistance to cisplatin, carboplatin, and etoposide, but there was no resistance to melphalan, perfosfamide, 5-fluorouracil, doxorubicin, or paclitaxel. The results are consistent with the concept that the DNA mismatch repair proteins function as a detector for adducts produced by 6-thioguanine, N-methyl-N'-nitro-N-nitrosoguanidine, cisplatin, and carboplatin but not for melphalan and perfosfamide. They also suggest that these proteins play a role in detecting the DNA damage produced by the binding of etoposide to topoisomerase II and propagating signals that contribute to activation of apoptosis.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Antineoplastic Agents; Carboplatin; Carrier Proteins; Cisplatin; Colorectal Neoplasms; Cyclophosphamide; DNA Adducts; DNA Damage; DNA Repair; DNA-Binding Proteins; DNA, Neoplasm; Doxorubicin; Drug Resistance, Neoplasm; Endometrial Neoplasms; Etoposide; Female; Fluorouracil; Humans; Melphalan; Methylnitronitrosoguanidine; Mutagenesis; MutL Protein Homolog 1; MutS Homolog 2 Protein; Neoplasm Proteins; Nuclear Proteins; Paclitaxel; Proto-Oncogene Proteins; Thioguanine; Tumor Cells, Cultured