Methylthioadenosine phosphorylase gene is silenced by promoter hypermethylation in human lymphoma cell line DHL-9: another mechanism of enzyme deficiency

Abstract

Methylthioadenosine phosphorylase (MTAP) involved in the metabolism of purine and polyamine has been known to be deficient in a variety of tumors. Although this enzyme deficiency was reportedly caused by partial or total deletion of the MTAP gene, human MTAP-deficient lymphoma cell line DHL-9 has the intact MTAP gene. In order to determine the mechanism of MTAP deficiency in DHL-9, we carried out methylation-specific PCR analysis of sodium bisulfite-treated genomic DNA followed by DNA sequence analysis. Following incubation with various concentrations of 5-Aza-2'-deoxycytidine, DHL-9 cells were subjected to RT-PCR and an immunoblot analysis for MTAP expression. MTAP promoter in DHL-9 cells was methylated at cytosine of all CpG dinucleotides analyzed. Moreover, 5-Aza-2'-deoxycytidine treatment induced DHL-9 cells to express MTAP mRNA and protein. Taken together, MTAP deficiency in DHL-9 was caused by transcriptional silencing due to promoter methylation. Promoter methylation of the MTAP gene was also found in DNA samples from adult T-cell leukemia patients. These results indicated that promoter hypermethylation is another mechanism of MTAP deficiency in human malignancy. Thus, immunological diagnostics will be needed for an accurate evaluation of MTAP expression at the protein level.