Arsenic-trioxide-induced apoptosis of chronic lymphocytic leukemia cells O. BAIREY, A. VANICHKIN, O. SHPILBERG INTRODUCTION Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature B cells that co-express CD5 and CD23 antigens. CLL is considered mainly an accumulative disorder: the malignant cells increase in peripheral blood, bone marrow, and peripheral lym- phoid organs because defective apoptosis causes their extended survival (Caligaris-Cappio & Hamblin, 1999). The cells are arrested in the G0/G1-phase of the cell cycle and do not display a high proliferative capacity. Of the numerous anti-apoptotic proteins expressed at high levels by long-lived circulating CLL cells, the Bcl-2 family plays a key role (Reed, 1998). This resistance to apoptosis is responsible for the inability of conventional therapeutic approaches to cure patients with CLL. Researchers are therefore seeking new options based on our improved under- standing of the molecular biology of CLL (Reed & Pellecchia, 2005). Institute of Hematology, Rabin Medical Center, Beilinson Hospital, Petah Tiqwa, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel Correspondence : Osnat Bairey, Institute of Hematol- ogy, Rabin Medical Center, Beilin- son Hospital, Petah Tiqwa 49100, Israel. Tel.: +972 3 937 8008/09; Fax: +972 3 9378046; E-mail: obairey@post.tau.ac.il doi:10.1111/j.1751-553X.2008.01134.x Received 19 August 2008; accepted for publication 18 November 2008 Keywords Chronic lymphocytic leukemia, ar- senic trioxide, apoptosis, Bcl-2, cas- pase-3 SUMMARY Chronic lymphocytic leukemia (CLL) cells are characterized by defective apoptosis which leads to their extended survival. Arsenic trioxide (As 2 O 3 ) was reported to induce cell death in many malignant cells, but the specific pathway of As 2 O 3 -induced apoptosis/necrosis remains controversial. Our aim was to determine if As 2 O 3 kills CLL cells through apoptosis and whether this is accompanied by reduction in Bcl-2 levels. Cells from nine patients with CLL were incubated with increasing concentrations of As 2 O 3 (0.5–2 lm) for 2, 7, or 14 days. Cells viability was measured using Alamar Blue assay and apoptosis using human Annexin V-FITC and propidium iodine (PI) kit (BMS306FI; Bender MedSystems, Vienna, Austria). Intracellular Bcl-2, Bax, and caspase-3 levels were measured by flow cytometry. As 2 O 3 significantly reduced CLL cell viability (P < 0.01) and induced apoptotic cell death in a time- and dose-dependent manner. After 7 days, CLL cells showed a significant decrease in mean fluorescence intensity (MFI) of Bcl-2 on flow cytometry study. Bax and caspase-3 levels showed significant decrease in MFI only after prolonged incubations (7 and 14 days) and mostly at higher concentrations of As 2 O 3 . The mechanism underlying the reduction in viability of CLL cells incubated with As 2 O 3 is mediated by induction of apoptosis maybe through the down-regulation of Bcl-2. Further studies are needed to elucidate the potential therapeutic role of As 2 O 3 in CLL. ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY Ó 2009 The Authors Journal compilation Ó 2009 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2010, 32, e77–e85 e77 International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology