Methylation Silencing of the Apaf-1 Gene in Acute Leukemia

• Published in: Molecular cancer research Vol. 3; no. 6; pp. 325 - 334
• Main Authors: Furukawa, Yutaka, Sutheesophon, Krittaya, Wada, Taeko, Nishimura, Miki, Saito, Yasushi, Ishii, Hideshi, Furukawa, Yusuke
• Format: Journal Article
• Language: English
• Published: United States American Association for Cancer Research 01.06.2005
• Subjects: 5' Untranslated Regions; Antimetabolites, Antineoplastic - pharmacology; Apoptotic Protease-Activating Factor 1; Azacitidine - analogs & derivatives; Azacitidine - pharmacology; Cell Line, Tumor; CpG Islands - genetics; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases - metabolism; DNA Methylation; DNA methylation/epigenetics; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Jurkat Cells; K562 Cells; Leukemia, Myeloid, Acute - genetics; Leukemia, Myeloid, Acute - pathology; Leukemia, Myeloid, Acute - therapy; Leukemias and lymphomas; Promoter Regions, Genetic; Proteins - genetics; Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Silencing and reactivation of gene expression; Transcriptional control of apoptosis; U937 Cells
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-04-0105

Apaf-1 is important for tumor suppression and drug resistance because it plays a central role in DNA damage–induced apoptosis. Inactivation of the Apaf-1 gene is implicated in disease progression and chemoresistance of some malignancies. In this study, we attempted to clarify the role of Apaf-1 in leukemogenesis. Apaf-1 mRNA levels were below the detection limit or very low in 5 of 20 human leukemia cell lines (25%) and 5 of 12 primary acute myeloblastic leukemia cells (42%). There were no gross structural abnormalities in the Apaf-1 gene in these samples. Expression of factors regulating Apaf-1 transcription, such as E2F-1, p53, and Sp-1, did not differ between Apaf-1-positive and Apaf-1-negative cells. Methylation of CpG in the region between +87 and +128 of the Apaf-1 gene was almost exclusively observed in Apaf-1-defective cell lines. Treatment of these cells with 5-aza-2′-deoxycytidine, a specific inhibitor of DNA methylation, restored the expression of Apaf-1. Furthermore, we showed that the region between +87 and +128 could act as a repressor element by recruiting corepressors such as methylated DNA-binding domain 2 and histone deacetylase 1 upon methylation. Overexpression of Dnmt1, a mammalian maintenance DNA methyltransferase, was associated with Apaf-1 gene methylation. DNAs from Dnmt1-overexpressing cells were more resistant to digestion with methylation-sensitive enzyme Hpa II than those from cells with low Dnmt1 expression, suggesting that Dnmt1 mediates aberrant methylation of multiple genes. In conclusion, methylation silencing is a mechanism of the inactivation of Apaf-1 in acute leukemia, and Dnmt1 overexpression may underlie hypermethylation of the Apaf-1 gene.