title: 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.
authors.affiliation: The Second Department of Surgery, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Ishii
authors.forename: Keiko
authors.initials: K
authors.lastname: Nakazawa
authors.forename: Hideo
authors.initials: H
authors.lastname: Wada
authors.forename: Junji
authors.initials: J
authors.lastname: Nishioka
authors.forename: Kaname
authors.initials: K
authors.lastname: Nakatani
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Yamada
authors.forename: Shimeru
authors.initials: S
authors.lastname: Kamihira
authors.forename: Masato
authors.initials: M
authors.lastname: Kusunoki
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Nobori
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D019175:DNA Methylation; D000077209:Decitabine; D020868:Gene Silencing; D006801:Humans; D008223:Lymphoma / Q000235:genetics* / Q000473:pathology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011683:Purine-Nucleoside Phosphorylase / Q000172:deficiency* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D000077209:Decitabine; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D001374:Azacitidine