methylnitronitrosoguanidine and Head-and-Neck-Neoplasms

Major risk factors for cancer of the oral cavity, pharynx and larynx are smoking and excess alcohol consumption. Since long-term survival rates of head and neck cancer patients have not substantially been improved, new preventive strategies including the use of cancer chemopreventive agents have to be developed. With the aim of developing biomarkers which can verify the efficacy of chemopreventive interventions, a standardised alkaline microgel electrophoresis (MGE) assay was applied as a sensitive and rapid tool to detect DNA damage on a single cell level. Macroscopically normal laryngeal mucosa biopsies obtained by surgery from head and neck cancer patients (n=29) and from hospital controls (n=22) were analysed by MGE in a pilot study. As compared to controls, cells from head and neck cancer patients showed a significantly elevated DNA damage without any further genotoxic treatment (P<0.01). We conclude that this increased background DNA damage in laryngeal epithelia could result from genetic alterations caused by smoking and alcohol leading, in accord with the field cancerisation hypothesis, to a gradual decrease of genomic stability and malignancy. MGE should now be explored as a rapid screening method in larger clinical studies: (i) to identify high-risk subjects carrying cells with decreased genomic stability and (ii) to verify the efficacy of chemopreventive regimens to prevent or slow down the development of head and neck cancer in high-risk persons.

Topics: Adult; Alcohol Drinking; Dimethylnitrosamine; DNA Damage; Electrophoresis; Female; Head and Neck Neoplasms; Humans; Laryngeal Mucosa; Laryngoscopy; Male; Methylnitronitrosoguanidine; Mutagens; Pilot Projects; Risk Factors

The GADD153 gene is strongly transcriptionally activated by many types of cellular injury and the magnitude of the change in GADD153 expression is proportional to the extent of damage. We developed a novel reporter system in which a chimeric gene containing the GADD153 promoter linked to the coding region of an enhanced green fluorescent protein (EGFP) gene was stably integrated into the genome of a clone of UMSCC10b head and neck carcinoma cells. Activation of the exogenous GADD153 promoter was quantified using flow cytometric measurement of EGFP expression following drug exposure. The exogenous GADD153 promoter in this clone was activated by N-methl-N'-nitro-N-nitrosoguanidine (MNNG) in a concentration-dependent manner with kinetics that closely paralleled perturbation of cell cycle phase distribution. EGFP expression was strongly activated by a variety of genotoxic agents including DNA cross-linking and methylating agents, oxygen free radicals, DNA intercalator, UV and gamma-radiation and hypoxia. When grown as a xenograft in nude mice, the stably transfected clone also demonstrated dose-dependent EGFP expression when measured 4 days after cisplatin treatment. The reporter system accurately categorized the relative potency of adducts produced by 6 related platinum-containing drugs. In conclusion, this reporter system can facilitate in vitro and in vivo screening for agents capable of producing cytotoxicity via a wide variety of different mechanisms, and can be utilized to investigate the relative potency of structurally related DNA adducts.

Topics: Animals; Antineoplastic Agents; CCAAT-Enhancer-Binding Proteins; Cell Cycle; Cisplatin; Cross-Linking Reagents; DNA; DNA Adducts; DNA Methylation; Dose-Response Relationship, Drug; Flow Cytometry; Gamma Rays; Genes, Reporter; Genetic Vectors; Genome; Green Fluorescent Proteins; Head and Neck Neoplasms; Humans; Hypoxia; Kinetics; Luminescent Proteins; Methylnitronitrosoguanidine; Mice; Mice, Nude; Plasmids; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transcription Factor CHOP; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tumor Cells, Cultured; Ultraviolet Rays