title: Azacitidine induces demethylation of p16INK4a and inhibits growth in adult T-cell leukemia/lymphoma.
abstract: Adult T-cell leukemia/lymphoma (ATL) is one of the peripheral T-cell malignant neoplasms strongly associated with human T-cell leukemia virus type-I (HTLV-I). Although the viral transactivator protein Tax has been proposed to play a critical role in leukemogeneis, additional cellular events are required for the development of ATL. One of the genetic events of the disease is inactivation of tumor suppressor genes. The CDKN2A locus on chromosome 9p encodes 2 cell cycle regulatory proteins, p14ARF and p16INK4a, which share exon 2 using different reading frames. The p14ARF and p16INK4a genes have been implicated as tumor suppressor genes by their frequent mutation, deletion or promoter hypermethylation in a variety of human tumors. In this report, we describe the expression status of p14ARF and p16INK4a in 9 ATL cell lines (MT1, MT2, OKM3T, F6T, K3T, Oh13T, S1T, Su9T01 and HUT102). By reverse transcription polymerase chain reaction (RT-PCR), expression of p14ARF was not detected in one cell line (OKM3T), while expression of p16INK4a was not detected in 6 cell lines (OKM3T, MT1, MT2, Oh13T, S1T and Su9T01). In the OKM3T cell line, the shared exon 2 of the p14ARF/p16INK4a gene was deleted; however, the p16INK4a gene, was epigenetically inactivated in 5 other cells lines. In primary tumor cells obtained from ATL patients, p14ARF expression was absent in 6 of the 11 samples. We confirmed the methylation of the p16INK4a gene in MT1 and MT2 cells using the methylation-specific PCR (MSP) method. Treatment with 2.0 µM of Azacitidine (AZA), a demethylating agent, for 72 h restored p16INK4a transcript expression and induced growth inhibition in MT2 cells. Our results demonstrate that p16INK4a is epigenetically silenced in ATL. AZA offers a potential new therapeutic approach to improve the poor outcomes associated with ATL.
authors.affiliation: Department of Hematology and Rheumatology, Nihon University, School of Medicine, Itabashi-ku, Tokyo, Japan.
authors.forename: Kumi
authors.initials: K
authors.lastname: Uenogawa
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hatta
authors.forename: Naomichi
authors.initials: N
authors.lastname: Arima
authors.forename: Satoshi
authors.initials: S
authors.lastname: Hayakawa
authors.forename: Umihiko
authors.initials: U
authors.lastname: Sawada
authors.forename: Shin
authors.initials: S
authors.lastname: Aizawa
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Jin
authors.initials: J
authors.lastname: Takeuchi
mesh_terms: D001374:Azacitidine / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000378:metabolism*; D008745:Methylation / Q000187:drug effects*; D016133:Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D001374:Azacitidine