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

• Published in: Oncology reports Vol. 27; no. 4; pp. 1135 - 1141
• Main Authors: KOUTSIMPELAS, DIMITRIOS, PONGSAPICH, WARUT, HEINRICH, ULF, MANN, SYLVIA, MANN, WOLF J, BRIEGER, JÜRGEN
• Format: Journal Article
• Language: English
• Published: Athens D.A. Spandidos 01-04-2012; Spandidos
• Subjects: 5-azacytidine; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Aged; Aged, 80 and over; Azacitidine - pharmacology; Base Sequence; Biological and medical sciences; Carcinoma, Squamous Cell - genetics; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; Case-Control Studies; Cell Line, Tumor; Cell Proliferation - drug effects; CpG Islands; Dealkylation; DNA Methylation - drug effects; DNA Modification Methylases - antagonists & inhibitors; DNA Modification Methylases - genetics; DNA Modification Methylases - metabolism; DNA Repair Enzymes - antagonists & inhibitors; DNA Repair Enzymes - genetics; DNA Repair Enzymes - metabolism; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors - pharmacology; Female; Genes, Tumor Suppressor; Head and Neck Neoplasms - genetics; Head and Neck Neoplasms - metabolism; Head and Neck Neoplasms - pathology; head and neck squamous cell carcinoma; Humans; Immunohistochemistry; Male; Medical sciences; Middle Aged; Molecular Sequence Data; mutL homolog 1; MutL Protein Homolog 1; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; O-6-methylguanine-DNA methyltransferase; Otorhinolaryngology (head neck, general aspects and miscellaneous); Otorhinolaryngology. Stomatology; Polymerase Chain Reaction; Promoter Regions, Genetic - drug effects; Ras association domain family member 1; Sequence Analysis, DNA; tumor suppressor gene; Tumor Suppressor Proteins - antagonists & inhibitors; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism; Tumors; Antineoplastic agent; Cell proliferation; Transcription promoter; mutL homolog 1; Demethylation; Azanucleoside; Cancerology; Azacitidine; ENT disease; Head and neck squamous cell carcinoma; Protooncogene; Tumor suppressor gene; Enzyme; Ras association domain family member 1; Transferases; 5-azacytidine; Malignant tumor; O-6-methylguanine-DNA methyltransferase; Methylated-DNA-protein-cysteine S-methyltransferase; Antimetabolic; Methyltransferases; C-Onc gene; Triazine derivatives; Head and neck cancer; Ras gene; Genome; Methylation; Cancer
• ISSN: 1021-335X; 1791-2431
• DOI: 10.3892/or.2012.1624

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.