Imatinib inhibits the expression of SCO2 and FRATAXIN genes that encode mitochondrial proteins in human Bcr–Abl + leukemia cells Lefkothea C. Papadopoulou, Angeliki V. Kyriazou, Ioannis D. Bonovolias, Asterios S. Tsiftsoglou ⁎ Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki (A.U.TH.), GR-54124 Thessaloniki, Macedonia, Greece abstract article info Article history: Submitted 3 January 2014 Revised 6 March 2014 Available online 13 April 2014 (Communicated by H. E. Broxmeyer, Ph.D., 06 March 2014) Keywords: Bcr–Abl Imatinib COX SCO2 FXN Imatinib mesylate (IM/Gleevec®), a selective inhibitor of chimeric Bcr–Abl tyrosine kinase, was developed as a first line drug to treat CML and ALL Ph + patients. Earlier studies have shown that hemin counteracts the IM-induced cell killing in human K-562 CML cells. In this study, we investigated whether IM disrupts the heme-dependent Cytochrome c Oxidase (COX) Biosynthesis and Assembly Pathway (HDCBAP) in Bcr–Abl + and Bcr–Abl - cells by affecting the expression of key-genes. Cells were exposed to IM and evaluated at time intervals for cell growth, cell death, expression of various genes by RT-PCR analysis as well as Sco2 mature protein levels by western blot analysis and COX enzymatic activity. IM at 1 μM induced extensive cell growth inhibition and cell death as well as marked suppression of the expression of SCO2 and FRATAXIN (FXN) genes in human K-562 and KU-812 Bcr–Abl + CML cells. IM also reduced the protein level of mature Sco2 mitochondrial protein as well as COX activity in these cell lines. However, treatment of human MOLT-4 Bcr–Abl - cells with 1 μM and even with higher concentrations (4 × 10 -5 M) of IM neither reduced the expression of SCO2 and FXN genes nor suppressed the protein level of mature Sco2 protein and COX activity. Our findings indicate that SCO2 and FXN genes, involved in HDCBAP, are repressed by IM in human Bcr–Abl + CML cells and may represent novel target sites in leukemia therapy. © 2014 Elsevier Inc. All rights reserved. Introduction Imatinib mesylate (IM/Gleevec®) is a selective inhibitor of chimeric Bcr–Abl tyrosine kinase, developed to treat human Bcr–Abl + [Philadelphia positive (Ph + )] chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) [1–3]. “Targeted therapy” with IM, despite achieving great progress, promotes acquired resistance [4–7] and may cause cardiovascular toxicity [8,9]. Earlier studies have shown that IM markedly induced killing of human Bcr–Abl + CML cells by repressing the expression of BCL-2a and BCL-2b as well as other genes [10]. Hemin (Fe 3+ –protoporphyrin-IX), on the other hand, was found to encounter the IM-induced effect, prevent cell death and maintain cell survival in human Bcr–Abl + cells [10]. This effect of hemin may occur either via HMOX (Heme Oxygenase) induction [11,12] or via activation of BCL-2 or other related genes [10,13]. Moreover, IM was shown to cause Cytochrome c Oxidase (COX) deficiency in human K-562 CML cells by suppressing the expression of mitochondrial SCO2 gene that encodes the Sco2 COX assembly protein [14]. Furthermore, recombinant fusion Sco2 protein, transduced via the protein transduction domain (PTD) technology in those cells, led to partial recovery of COX activity [14]. These observations prompted us to extend our previous work and further explore the possible effects of IM on genes encoding proteins involved in HEME-dependent COX Biosynthesis and Assembly Pathway (HDCBAP). COX is the terminal mitochondrial respiratory chain enzyme, complex IV of the mitochondrial oxidative phosphorylation system (OXPHOS). COX is composed of 13 subunits, encoded by both mitochondrial (COX1, COX2, COX3) and nuclear (COX4, COX5A, COX5B, COX6A, COX6B, COX6C, COX7A, COX7B, COX7C, COX8) genes [15] and is embedded in the inner mitochondrial membrane, where it catalyzes the electron transfer from reduced cytochrome c to dioxygen. COX bio- genesis is a multiple step process that occurs via discrete intermediates (S1, S2, S3)(Fig. 1) [15,16]. Basically, the COX biosynthesis depends on: (a) proteins involved in iron metabolism and heme biosynthesis, such as Frataxin [(Fxn), and involved in iron–sulfur cluster biogenesis] and Ferrochelatase [(Fech), an iron–sulfur cluster enzyme catalyzing iron in- corporation into the protoporphyrin-IX ring system] [17–19]; (b) COX structural subunits (Cox1, Cox2, Cox3, Cox4, Cox5a, Cox5b, Cox6a, Cox6b, Cox6c, Cox7a, Cox7b, Cox7c, Cox8); and (c) COX assembly pro- teins, including Sco1, Sco2, Cox11, Cox17, Cox10, Cox15 and Surf1 [15, Blood Cells, Molecules and Diseases 53 (2014) 84–90 Abbreviations: ALL, acute lymphocytic leukemia; COX, cytochrome oxidase; CML, chronic myelogenous leukemia; CS, citrate synthase; FECH, ferrochelatase; FXN, FRATAXIN; HDCBAP, heme-dependent cytochrome c oxidase biosynthesis and assembly pathway; HMOX, heme oxygenase; IM, imatinib; OXPHOS, oxidative phosphorylation. ⁎ Corresponding author. Fax: +30 2310 997618. E-mail address: tsif@pharm.auth.gr (A.S. Tsiftsoglou). http://dx.doi.org/10.1016/j.bcmd.2014.03.001 1079-9796/© 2014 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd