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. 2015 Jan;9(1):399-404.
doi: 10.3892/ol.2014.2677. Epub 2014 Nov 6.

Methylation analysis of the DAPK1 gene in imatinib-resistant chronic myeloid leukemia patients

Selcen Celik  1 , Dilara Akcora  2 , Tulin Ozkan  1 , Nuray Varol  3 , Sena Aydos  3 , Asuman Sunguroglu  3
Affiliations

Methylation analysis of the DAPK1 gene in imatinib-resistant chronic myeloid leukemia patients

Selcen Celik et al. Oncol Lett. 2015 Jan.

Abstract

Death-associated protein kinase-1 (DAPK1) is a pro-apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation-specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region-Abelson murine leukemia (BCR-ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non-resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR-ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non-resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.

Keywords: DAPK1; DNA methylation; chronic myeloid leukemia; imatinib resistance; methylation-specific polymerase chain reaction.

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Figures

Figure 1
Figure 1
Analysis of DAPK1 methylation. (Aa) DAPK primer sequences used for methylation analysis performed using methylation-specific PCR following bisulfite conversion. (Ab) Representative PCR products for U and M alleles of DAPK1 in resistant (samples 4 and 5) and non-resistant (samples 1 and 2) patients, in a control sample from healthy individuals (sample 3), as well as in control U, M and W DNA samples. (B) The total number of samples and the number of samples exhibiting DAPK1 methylation. (C) The proportion (%) of samples with DAPK1 methylation is significantly greater in CML patients compared with the healthy controls. (D) The proportion (%) of patients with DAPK1 methylation varied among different mutations. The majority of patients with M351T demonstrated DAPK1 methylation; however, none of patients with T315I or E255K were methylated, and no difference was detected between the patients with triple (T315I-M351T-E255K) and double (T315I-M351T) mutations (P>0.05). Furthermore, a relatively low number of non-resistant patients demonstrated DAPK1 methylation (P<0.05). *P<0.05. DAPK1, death-associated protein kinase-1; PCR, polymerase chain reaction; U, unmethylated; M, methylated; W, wild type; CML, chronic myeloid leukemia.
Figure 2
Figure 2
Analysis of breakpoint cluster region-Abelson murine leukemia mutations. Imatinib resistance in chronic myeloid leukemia patients was detected using allele-specific oligonucleotide PCR. Representative PCR products are indicated for the (A) T315I, (B) M351T and (C) E255K mutations. Mutant samples have a normal and mutant allele, but normal samples only have a normal allele. PCR, polymerase chain reaction; Positive cntrl, samples from patients known to be resistant to imatinib treatment; Negative ctrl, samples from healthy individuals; Cntrl, control.
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