Methylation Profiling of Apoptotic Genes in Leukemia Cell Lines and Patients.

^equal contribution Apoptosis is a morphologically and biochemically distinct form of eukaryotic cell death that occurs under a variety of physiological and pathological conditions. Apoptosis is executed via 2 main pathways that lead to the activation of caspases: the death receptor (DR) pathway and the mitochondrial pathway. Defects in apoptotic mechanisms play an important role in enabling neoplastic cells to survive by ignoring death inducing signals leading to protection from the lack of survival factors. DNA methylation is a reversible mechanism of gene inactivation that might be responsible for impaired apoptosis in leukemia cells. Methylation profiling of apoptotic genes can identify possible gene targets for epigenetic therapy using demethylating agents like 5-aza-deoxycytidine and azacytidine. It can also be used for classification and prognosis. We performed a comprehensive DNA methylation analysis of apoptosis pathway genes with CpG islands in promoter/exon 1 regions in 5 leukemic cell lines (TF1, K562, Raji, HL60 and Jurkat), 20 patients with acute myelogenous leukemia (AML), 20 patients with acute lymphoblastic leukemia (ALL), and 6 control samples (normal peripheral blood cells). We used bisulfite treatment of DNA, followed by PCR and pyrosequencing to quantitatively measure levels of cytosine methylation in promoter-associated CpG islands close to transcription start sites. Nonparametric tests were used for statistical analysis. Our analysis covered a total of 25 genes: 11 in the intrinsic pathway (BAX, BAK, HRK, CYC1, DIABLO, ATM, APAF1, MAP3K5, CHEK2,PAK1, and BNIP 3), 4 in the extrinsic pathway (TRADD,PYCARD, CARD 10 and BID), 5 genes of the Caspase family (Caspase 2, 3, 6, 7, 9) in addition to 4 of the programmed cell death genes (PDCD 2, 4, 6, and 11). By using a 15% cutoff level to call a gene methylated, methylation was observed in 7 genes in at least one cell line: BNIP3 (Raji), PYCARD (K562), HRK (Tf1, Raji and Jurkat), CARD10 (Raji and HL-60), BID (Raji), PAK1 (Raji) and Casp 6 (Raji). We then performed methylation analysis of 4 genes (BNIP3, PYCARD, PAK1 and CARD 10) in AML and ALL patient samples. Methylation frequencies over a 15% cutoff are summarized in Table 1. We conclude that DNA methylation-associated epigenetic silencing is likely to play a role in modulating apoptosis in a subset of AML and ALL patients, with PYCARD being the most frequent target.