Silencing of Suppressor of Cytokine Signaling-3 due to Methylation Results in Phosphorylation of STAT3 in Imatinib Resistant BCR-ABL Positive Chronic Myeloid Leukemia Cells

Al-Jamal, Hamid AN; Jusoh, Siti Asmaa Mat; Yong, Ang Cheng; Asan, Jamaruddin Mat; Hassan, Rosline; Johan, Muhammad Farid

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자료유형: 학술저널

저자정보: Al-Jamal, Hamid AN (Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia) Jusoh, Siti Asmaa Mat (Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia) Yong, Ang Cheng (Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia) Asan, Jamaruddin Mat (Department of Immunology, School of Medical Sciences, Universiti Sains Malaysia) Hassan, Rosline (Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia) Johan, Muhammad Farid (Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia)

저널정보: 아시아태평양암예방학회 Asian Pacific journal of cancer prevention : APJCP Asian Pacific journal of cancer prevention : APJCP 제15권 제11호

발행연도: 2014.1

수록면: 4,555 - 4,561 (7page)

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Background: 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. Materials and Methods: 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 $IC_{50}$ 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. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM 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. Conclusions: 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.

#Methylation #SOCS-3 #STAT3 #imatinib #BCR-ABL #K562

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