methylnitronitrosoguanidine and Mouth-Neoplasms

Oral cancer is a common malignancy, ranking first among all cancers in Western and Asian countries. Use of tobacco is regarded as a major risk factor, along with age and gender. Oral cancer is preceded by some benign lesions or conditions, which are termed pre-cancerous. Only one-third of people at the pre-cancerous stage of disease succumb to cancer. No biomarker is available to identify people with pre-cancerous lesions or conditions at high risk of developing cancer. The focus of this study was to explore such biomarkers. The study included 129 untreated people with cancer, 138 untreated people at the pre-cancerous stage and 176 control participants. For statistical analysis of this data, analysis of variance and t-test were used. Three biomarkers (i.e. micronucleus test [MNT], comet assay and challenge comet assay were used. MNT and comet assay were carried out on buccal epithelial cells. In addition, challenge comet assay was carried out on peripheral blood leucocytes by using mutagen MNNG sensitivity of DNA after DNA repair. A significant stepwise increase in the DNA damage (basal/MNNG-treated/post-repair) was observed in buccal epithelial cells and peripheral blood leucocytes from control to pre-cancer patients and from pre-cancer to cancer patients. Micronucleus frequency also increased in the same way. Considerable inter-individual and inter-cellular variability in DNA damage was observed, which increased from control to pre-cancer patients and from pre-cancer to cancer patients. The outliers among patients with pre-cancerous states were identified on the basis of more than mean +2 SD limits for comet tail length, as well as mean percentage of micronuclei. Hence, those participants whose cells showed high basal DNA damage, extreme sensitivity to MNNG and reduced repair were identified as high-risk individuals.

Topics: Adolescent; Adult; Aged; Chromosome Aberrations; Comet Assay; Disease Progression; DNA Damage; DNA Repair; DNA, Neoplasm; Female; Humans; Leukocytes; Male; Methylnitronitrosoguanidine; Micronucleus Tests; Middle Aged; Mouth Mucosa; Mouth Neoplasms; Precancerous Conditions; Risk Assessment

The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) modulates the effectiveness of alkylating agents. However, the relationship between MGMT and the sensitivities to other agents has not been explored. In the present study, the association between MGMT expression and the cellular sensitivity to the platinum agent, CDDP was examined in four human oral cancer cell lines. CDDP depleted MGMT protein and mRNA levels in all four cell lines. Two cell lines with low MGMT expression were sensitive to an alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine and CDDP, whereas two other cell lines with high MGMT expression were resistant to both agents. Furthermore, the addition of the MGMT inhibitor, O6-benzylguanine (O6-BG), invariably enhanced CDDP sensitivity. CDDP depleted MGMT expression, and CDDP sensitivity was enhanced by O6-BG. These results provide valuable information about the relationship between MGMT expression and CDDP sensitivity in oral cancer chemotherapy.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Line, Tumor; Cisplatin; DNA Repair; Docetaxel; Drug Resistance, Neoplasm; Guanine; Humans; Methylnitronitrosoguanidine; Mouth Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Taxoids

Many studies have indicated that cancer cells expressing mutant (mt) p53 are resistant to genotoxic stress such as chemotherapy and radiation therapy. Inasmuch as most human oral cancer cells either express mt p53 or are infected with human papillomavirus (HPV), we determined the sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a genotoxic chemical carcinogen, the activity of a DNA repair enzyme O6-methylguanine methyl transferase (MGMT) and the status of p53 in 11 human oral cancer cell lines, 2 HPV-immortalized human oral keratinocytes and 3 normal human oral keratinocyte (NHOK) cultures. Ten cancer cell lines demonstrated significantly higher level of MGMT activities compared to normal or immortalized non-tumorigenic cells, while one cancer cell line showed negligible MGMT activity. All immortalized cells and NHOK showed very low MGMT activity. Interestingly, all cancer cells with high MGMT activity expressed either mt p53 or harbored HPV DNA. However, infection of one cancer cell line (that does not demonstrate the MGMT activity) with retrovirus expressing an mt p53 protein did not alter the sensitivity of cells to MNNG and the enzyme activity. These data indicate that most human oral cancer cells contain moderately high DNA repair enzyme activity, and in doing so may be resistant to genotoxic stress, but the association of p53 with MGMT activities should further be investigated.

Topics: Blotting, Western; Cell Line; DNA Repair; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Humans; Keratinocytes; Methylnitronitrosoguanidine; Mouth Neoplasms; O(6)-Methylguanine-DNA Methyltransferase; Papillomaviridae; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Tumor Cells, Cultured; Tumor Suppressor Protein p53

We immortalized oral keratinocytes by transfecting them with recombinant human papillomavirus (HPV) type 18 DNA and established three cell lines. These lines were morphologically different from their normal counterpart, contained integrated entire HPV-18 DNA, and expressed the viral E6/E7 genes. The cells contained less p53 protein and more c-myc mRNA than normal cells. However, they proliferated only in keratinocyte growth medium (KGM) containing low calcium and were not tumorigenic in nude mice. To test the hypothesis that tumors result from the combined effect of a "high-risk" HPV and chemical carcinogens in the human oral cavity, we exposed the immortalized cells to the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Three chemically transformed cell colonies were isolated. These cells (a) proliferated well in both KGM and Dulbecco's modified minimum essential medium containing physiological levels of calcium; (b) were capable of proliferating in nude mice; (c) contained intact, integrated HPV-18 sequences; (d) transcribed substantially more HPV-18 E6/E7, transforming growth factor-alpha, and c-myc than the immortalized counterpart; and (e) contained, like the immortalized counterpart, less wild-type p53 protein and DCC message. These data indicate that human oral keratinocytes can be transformed by sequential exposure of normal keratinocytes to a "high-risk" HPV and chemical carcinogens.

Topics: Animals; Base Sequence; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; ErbB Receptors; Gene Expression; Genes, myc; Genes, p53; Humans; Keratinocytes; Methylnitronitrosoguanidine; Mice; Mice, Nude; Molecular Sequence Data; Mouth Neoplasms; Papillomaviridae; RNA, Messenger; RNA, Viral; Transcription, Genetic; Transfection; Transforming Growth Factor alpha

We previously immortalized human oral keratinocytes by transfection with recombinant human papillomavirus type 16 (HPV-16) DNA and established two cell lines. These transfected cells were morphologically different from the normal counterpart, contained intact HPV-16 DNA in an integrated form, and expressed numerous viral genes. These cells contained lower levels of wild-type p53 protein and higher levels of c-myc mRNAs compared to normal cells. However, they proliferated only in keratinocyte growth medium containing a low level of calcium and were not tumorigenic in nude mice. A HPV-16-immortalized cell line was exposed to either 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone or N-methyl-N'-nitro-N-nitrosoguanidine. Four chemically transformed cell colonies were isolated. These cells proliferated well in Dulbecco's minimum essential medium containing a physiological level of calcium. They contained, similar to the immortalized counterpart, integrated HPV-16 sequences and lower levels of both wild-type p53 protein and DCC messages compared to normal cells. Among the chemically transformed cells, two colonies obtained from 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone exposure demonstrated an enhanced proliferation capacity in nude mice and transcribed a substantially higher amount of HPV-16 E6/E7, epidermal growth factor receptors, and c-myc genes compared with the immortalized counterpart. These experiments indicate that malignant transformation of oral keratinocytes can be caused by a sequential combined effect of "high risk" HPV and tobacco-related carcinogens.

Topics: Base Sequence; Carcinogens; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Colonic Neoplasms; DNA Primers; ErbB Receptors; Exons; Genes, myc; Genes, p53; Genes, ras; Humans; Keratinocytes; Male; Methylnitronitrosoguanidine; Molecular Sequence Data; Mouth Neoplasms; Mutagenesis; Nicotiana; Nitrosamines; Oligonucleotides, Antisense; Papillomaviridae; Plants, Toxic; Polymerase Chain Reaction; Transforming Growth Factor alpha

Of 158 channel catfish (Ictalurus punctatus) exposed to N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) CAS:70-25-7] in water for 28 days, 2 developed disseminated lymphosarcoma. One fish was necropsied at 12 months and another at 18 months following exposure. Both fish had a massive neoplastic infiltration of the bilateral pairs of head and trunk kidneys from which the neoplastic cells appeared to originate. The neoplastic infiltration was also observed in the following: thymus, gills, oral mucosa, liver, skin, skeletal muscle of head-neck region, and to a lesser extent spleen and bone marrow. This is probably the first report of lymphosarcoma in channel catfish. Although the occurrence of lymphosarcoma in these 2 catfish appeared to be related to exposure to MNNG, the exact role MNNG played in the tumor formation was not determined.

Topics: Animals; Carcinoma; Carcinoma, Squamous Cell; Fish Diseases; Fishes; Kidney Neoplasms; Lipoma; Lymphoma, Non-Hodgkin; Methylnitronitrosoguanidine; Mouth Mucosa; Mouth Neoplasms; Papilloma; Water Pollutants, Chemical

Topics: Animals; Dog Diseases; Dogs; Eye Neoplasms; Eyelid Neoplasms; Male; Methylnitronitrosoguanidine; Mouth Neoplasms; Papilloma; Papillomaviridae