Abstract
Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.
Highlights
The reciprocal translocation t(9:22) in chronic myeloid leukemia (CML) generates a BCR-ABL fusion gene that results in the expression of a leukemia-specific oncoprotein, BCR-ABL
suppressor of cytokine signaling-3 (SOCS-3) provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients
We investigated the DNA methylation profiles of suppressors of cytokine signaling (SOCS)-1 and SOCS-3 genes, their expression analysis and effects on JAK/ STAT pathway in both parental and resistant cells to reveal the relationship between DNA methylation and the mechanism of resistance
Summary
The reciprocal translocation t(9:22) in chronic myeloid leukemia (CML) generates a BCR-ABL fusion gene that results in the expression of a leukemia-specific oncoprotein, BCR-ABL. A tyrosine kinase inhibitor is used as the treatment of CML targets BCR-ABL protein and induces hematological remission in these patients (Melo et al, 2007). Expression profiling and in vitro studies predict the involvement of additional genes in imatinib resistance and disease progression (Radich et al, 2006) Most of those genes have not been thoroughly investigated or described in clinical specimens from CML patients. Imatinib mesylate is a tyrosine kinase inhibitor that targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients
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