title: 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.
abstract: 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.
  
  
  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 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.
  
  
  RESULTS
  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.
  
  
  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.
authors: Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, Kelantan, Malaysia E-mail : faridjohan@usm.my.
authors: Hamid A N
authors: HA
authors: Al-Jamal
authors: Siti Asmaa Mat
authors: SA
authors: Jusoh
authors: Ang Cheng
authors: AC
authors: Yong
authors: Jamaruddin Mat
authors: JM
authors: Asan
authors: Rosline
authors: R
authors: Hassan
authors: Muhammad Farid
authors: MF
authors: Johan
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics; D001549:Benzamides / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D020868:Gene Silencing; D016313:Genes, abl / Q000235:genetics*; D006801:Humans; D000068877:Imatinib Mesylate; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics*; D010879:Piperazines / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology*; D050796:STAT3 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; C106644:SOCS3 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D000068877:Imatinib Mesylate