Epigenetic regulation of O6-methylguanine-DNA methyltransferase gene expression by histone acetylation and methyl-CpG binding proteins.

Transcriptional silencing of the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT) in a proportion of transformed cell lines is associated with methylated CpG hotspots in the MGMT 5' flank. The goal of the study was to evaluate the mechanism by which CpG methylation of theMGMT promoter region influenced silencing of the gene. Analysis of histone acetylation status in two regions of the promoter using chromatin immunoprecipitation assay showed that a higher level of histone acetylation was associated with expression in three MGMT-expressing cell lines (HeLa CCL2, HT29, and Raji) compared with three MGMT-silenced cell lines (HeLa S3, BE, and TK6). To determine how the modulation of CpG methylation and histone acetylation influenced MGMT expression, we exposed the cells to 5-aza-2'deoxycytidine (5-Aza-dC), inhibitor of DNA methylation, which strongly up-regulated MGMT expression in three MGMT-silenced cell lines whereas trichostatin A, inhibitor of histone deacetylase, weakly induced MGMT. However, combined treatment with 5-Aza-dC and trichostatin A significantly up-regulated MGMT RNA expression to a greater extent than in cells treated with either agent alone suggesting that histone deacetylation plays a role in MGMT silencing but that CpG methylation has a dominant effect. Consistent with enhanced MGMT expression, 5-Aza-dC increased the association of acetylated histone H3 and H4 bound to the MGMT promoter. Chromatin immunoprecipitation analysis of methyl-CpG binding domain containing proteins detected a greater amount of MeCP2, MBD1, and CAF-1 bound to the MGMT promoter in MGMT-silenced cells. Our findings implicate specific MBD proteins in methylation-mediated transcriptional silencing of MGMT.

O6-methylguanine-DNA methyltransferase gene: epigenetic silencing and prognostic value in head and neck squamous cell carcinoma.

BACKGROUND: Alkylating N-nitroso compounds can interact directly with DNA, forming O(6)-alkylguanine, a DNA adduct proved to be mutagenic and carcinogenic if not sufficiently repaired. A specific DNA repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT), can remove the alkyl group from the O(6)-position of the guanine, thereby preventing its mutagenic and carcinogenic effects. Inactivation of the MGMT gene in association with promoter hypermethylation results in persistence of O(6)-alkylguanine in DNA, leading to G:C to A:T transition mutation and these G:C to A:T transition mutations can inactivate p53 tumor suppressor gene or activate ras proto-oncogene. METHODS: We analyzed MGMT promoter hypermethylation and protein expression patterns in 94 cases of primary head and neck squamous cell carcinoma (HNSCC) by methylation-specific PCR (MSP) and immunohistochemical staining. The results were then correlated with clinical follow-up data. RESULTS: MGMT promoter hypermethylation was present in 17 of 94 patients (18.1%) and apparent loss of protein expression was seen in 19 of 93 HNSCC patients (20.4%). The presence of MGMT promoter hypermethylation was significantly correlated with loss of MGMT protein expression in HNSCC. Both MGMT promoter hypermethylation and loss of protein expression were significantly correlated to increased tumor recurrences and decreased patient survival, independent of other risk factors, such as tumor site, tumor size, nodal status, age, and chemoradiation therapy. CONCLUSIONS: MGMT promoter hypermethylation and apparent loss of protein expression are reliable and independent prognostic factors in HNSCC. The above study may also provide guideline or basis for applying alkylating antitumor agents to patients with HNSCC that display MGMT promoter hypermethylation and/or loss of MGMT protein expression.

Successful treatment of murine beta-thalassemia using in vivo selection of genetically modified, drug-resistant hematopoietic stem cells.

Successful gene therapy of beta-thalassemia will require replacement of the abnormal erythroid compartment with erythropoiesis derived from genetically corrected, autologous hematopoietic stem cells (HSCs). However, currently attainable gene transfer efficiencies into human HSCs are unlikely to yield sufficient numbers of corrected cells for a clinical benefit. Here, using a murine model of beta-thalassemia, we demonstrate for the first time that selective enrichment in vivo of transplanted, drug-resistant HSCs can be used therapeutically and may therefore be a useful approach to overcome limiting gene transfer. We used an oncoretroviral vector to transfer a methylguanine methyltransferase (MGMT) drug-resistance gene into normal bone marrow cells. These cells were transplanted into beta-thalassemic mice given nonmyeloablative pretransplantation conditioning with temozolomide (TMZ) and O6-benzylguanine (BG). A majority of mice receiving 2 additional courses of TMZ/BG demonstrated in vivo selection of the drug-resistant cells and amelioration of anemia, compared with untreated control animals. These results were extended using a novel gamma-globin/MGMT dual gene lentiviral vector. Following drug treatment, normal mice that received transduced cells had an average 67-fold increase in gamma-globin expressing red cells. These studies demonstrate that MGMT-based in vivo selection may be useful to increase genetically corrected cells to therapeutic levels in patients with beta-thalassemia.