title: Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes             in head and neck squamous cell carcinoma: pharmacological genome demethylation             reduces proliferation of head and neck squamous carcinoma cells.
abstract: Promoter hypermethylation of tumor suppressor genes (TSGs) is a common feature             of primary cancer cells. However, to date the somatic epigenetic events that occur             in head and neck squamous cell carcinoma (HNSCC) tumorigenesis have not been well-defined.             In the present study, we analyzed the promoter methylation status of the genes             mutL homolog 1 (MLH1), Ras-association domain family member 1 (RASSF1A) and O-6-methylguanine-DNA             methyltransferase (MGMT) in 23 HNSCC samples, three control tissues and one HNSCC             cell line (UM-SCC 33) using methylation-specific PCR (MSP). The expression of             the three proteins was quantified by semi-quantitative immunohistochemical analysis.             The cell line was treated with the demethylating agent 5-azacytidine (5-Aza) and             the methylation status after 5-Aza treatment was analyzed by MSP and DNA sequencing.             Proliferation was determined by Alamar blue staining. We found that the MGMT promoter             in 57% of the analyzed primary tumor samples and in the cell line was hypermethylated.             The MLH promoter was found to be methylated in one out of 23 (4%) tumor samples             while in the examined cell line the MLH promoter was unmethylated. The RASSF1A             promoter showed methylation in 13% of the tumor samples and in the cell line.             MGMT expression in the group of tumor samples with a hypermethylated promoter             was statistically significantly lower compared to the group of tumors with no             measured hypermethylation of the MGMT promoter. After treatment of the cell line             with the demethylating agent 5-Aza no demethylation of the methylated MGMT and             RASSF1A genes were determined by MSP. DNA sequencing verified the MSP results,             however, increased numbers of unmethylated CpG islands in the promoter region             of MGMT and RASSF1A were observed. Proliferation was significantly (p<0.05)             reduced after treatment with 5-Aza. In summary, we have shown promoter hypermethylation             of the tumor suppressor genes MGMT and RASSF1A in HNSCC, suggesting that this             epigenetic inactivation of TSGs may play a role in the development of HNSCC. 5-Aza             application resulted in partial demethylation of the MGMT and RASSF1A TSGs and             reduced proliferation of the tumor cells suggesting further evaluation of 5-Aza             for HNSCC treatment.
authors.affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, University Medical Center of the Johannes Gutenberg University Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. koutsimpelas@hno.klinik.uni-mainz.de
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Koutsimpelas
authors.forename: Warut
authors.initials: W
authors.lastname: Pongsapich
authors.forename: Ulf
authors.initials: U
authors.lastname: Heinrich
authors.forename: Sylvia
authors.initials: S
authors.lastname: Mann
authors.forename: Wolf J
authors.initials: WJ
authors.lastname: Mann
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brieger
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism; D000368:Aged; D000369:Aged, 80 and over; D001374:Azacitidine / Q000494:pharmacology*; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects*; D015254:DNA Modification Methylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D003640:Dealkylation; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D016147:Genes, Tumor Suppressor; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D017422:Sequence Analysis, DNA; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; C501486:MLH1 protein, human; D009687:Nuclear Proteins; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D000070957:MutL Protein Homolog 1; D045643:DNA Repair Enzymes; D001374:Azacitidine