Leukemic transformation seems to be caused by deregulated proliferation and inhibition of apoptosis of circulating chronic lymphocytic leukemia (CLL) cells. Apoptosis, a programmed cell death, is a physio- logical process occurring via intrinsic and extrinsic pathways. Expression of appropriate genes responsi- ble for the physiological activation of apoptosis leads to changes in expression levels of specific proteins. Among many CLL prognostic factors, the IGVH mutational status is considered as one of the strongest clinical course predictors. Patients with IGVH mutated gene (IGVH+) have better prognosis than those with IGVH unmutated one (IGVH-) [1-2]. In recent years, both preclinical and clinical studies have confirmed efficacy of bendamustine (BENDA), especially in combination with rituximab (RIT) - anti- CD20 monoclonal antibody, in the treatment of hema- tological diseases, particularly indolent non-Hodgkin lymphomas. BENDA combines the properties of cyto- toxic alkylating agents and anti-metabolites leading to a mitotic catastrophe, and cell death by necrosis, and on the other hand to the induction of apoptosis [3-6]. The influence of BENDA and RIT on the expres- sion of apoptosis-regulating markers depending on the IGVH mutational status has not been as yet reported. Therefore, the aim of our study was to determine if BENDA alone or BENDA+RIT influence the expres- sion of P53, PUMA, BAX, BCL-2 and APAF-1 pro- teins, involved in the intrinsic apoptotic pathway, and of FADD involved in the extrinsic one, depending on the IGVH mutational status of CLL cells. INTRODUCTION AND AIM The influence of bendamustine used alone or in combination with rituximab on apoptosis and expression of some apoptosis-regulating proteins depending on the IGVH mutational status of CLL cells Anna Korycka-Wolowiec 1 , Ewelina Ziolkowska 1 , Barbara Cebula-Obrzut 2 , Jerzy Z. Blonski 1 , Ewa Lech-Maranda 3 , Piotr Smolewski 2 , Tadeusz Robak 1 1 Department of Hematology, Medical University, Lodz, Poland; 2 Department of Experimental Hematology, Medical University, Lodz, Poland; 3 Department of Hematology, Institute of Hematology and Transfusion Medicine, Warsaw, Poland PCR products subjected to sequencing on an automated se- quencer and analyzed using the international information sys- tem (IMGT) database and tools. The sequences with a germline identity ≥ 98% - unmutated and < 98% - mutated. Cell culture conditions CLL cells incubated for 48 hrs in RPMI 1640 medium contain- ing 10% (v/v) heat inactivated fetal calf serum (HI-FCS) + 10% (v/v) autologous serum (AS), at 37°C, in the atmosphere of 5% CO 2 and full humidity, with or without (control) tested drugs. Assessmet of the influence of drugs on apoptosis The percentage of apoptotic cells measured after 48 hrs by flow cytometry using Annexin V (Ann-V) and propidium iodide (PI). Drug-induced apoptosis (DIA) = (% of apoptotic cells af- ter culture with drug) - (% of apoptotic cells in control culture). Drug-induced necrosis (DIN) = (% of necrotic cells after cul- ture with drug) - (% of necrotic cells in control culture). Disruption of m low Changes in  m low evaluated using MitoTracker Red CMX Ros kit (30 min., 37°C) and analysed by flow cytometry (FACSCalibour, Becton Dickinson, San Diego, USA). Drug- induced changes in mitochondrial transmembrane potential (DI m ) = (% of cells with  m changes after culture with drug) - (% of cells with  m changes in control culture). Detection of the active forms of caspase-3, caspase-9 and caspase-8 The caspases activity assessed by flow cytometry, according to the manufacturer’s protocols, using appropriate Caspase-9, -3, -8 Kits . Detection of apoptosis-regulatory proteins Cells collected after 48 hrs, fixed, permeabilized and incubated with specific antibodies: P53, BAX, PUMA, BCL-2, APAF-1, FADD. The fluorescence measured by flow cytometer. Drug- induced expression of active forms of caspases (DIE) = (% of cells with expression of caspases after culture with drug) - (% of cells with expression of caspases in control culture). Statistical analysis Statistical differences evaluated by Wilcoxon signed-rank test, with p ≤ 0.05 considered as statistically significant. We demonstrated that PUMA seems to play an important role in BENDA-induced intrinsic apoptosis pathway. Moreover, BENDA used either alone or in combination with RIT induced apoptosis as well as expression of the apoptosis-involved proteins in both IGVH (+) and IGVH (-) patients. There were no statistically significant differences in the effect of those drugs on the expression of the apoptosis-involved proteins studied, between the IGVH (+) and IGVH (-) groups. The role of the IGVH mutational status in the BENDA-induced expression of apoptosis-regulating factors deserves further studies. CONCLUSIONS Patients 26 newly diagnosed CLL patients (11 F, 15 M) aged 65 yrs (range 24-84). The IGVH gene mutated (IGVH+) in 13, and unmutated (IGVH-) in 13 patients. Approved by the Local Ethics Committee. Therapeutics RIT (Roche, Switzerland) - commercially available. Final concen- tration 10µg/ml. BENDA - kindly supplied by Mundipharma Research LTD. Fi- nal concentration 40μg/ml. Isolation of PBMNCs. Peripheral blood mononuclear cells (PBMNCs) isolated by centri- fuging in a density gradient. PBMNCs concentration-1.0x10 6 cells/ml. Mean B-cell (CD19+) purity >95%. IgVH mutational status analysis Polymerase chain reaction (PCR) amplification performed on DNA extracted from PBMNCs of each patient. MATERIAL AND METHODS Fig. 2. Percentage of early apoptotic (A), late apoptotic (B), total apoptotic (C) and necrotic (D) cells under influence of BENDA used alone and in combination with RIT depending on IGVH mutational status. Fig.4. Mitochondrial potential changes (m low ) under influence of BENDA used alone and in combination with RIT depending on IGVH mutational status. Fig. 3. Caspase-9 (A), caspase-3 (B) and caspase-8 (C) activity under influence of BENDA used alone and in combination with RIT depending on IGVH mutational status. Fig. 5. Percentage of cells expressing P53 (A), PUMA (B), BAX (C), BCL-2 (D), APAF-1 (E) and FADD (F) proteins under influence of BENDA used alone and in combination with RIT depending on IGVH mutational status. 1. Parker TL, Shrout MP. Chronic Lymphocytic Leukemia: Prognostic Factors and Imapact on Treatment. Discovery Medicine, 2011; 11: 115-123 2. Yu J, Zhang L. PUMA, a potent killer with or without p53. Oncogene, 2008; 27(1): 71-83 3. Robak T. Novel monoclonal antibodies for the treatment of chronic lymphocytic leukemia. Current Cancer Therapy Reviews, 2008; 8: 156-171 4. Leoni LM, Baliey B, Reifert J, et al. Bendamustine (Treanda) dis- plays a distinct pattern of cytotoxicity and unique mechanism fea- tures compared with other alkylating agent. Clin Cancer Res 2008;14: 309-317 5. Cheson BD, Rummel MJ. Bendamustine: Rebirth of an old drug. J Clin Oncol, 2009; 27: 1492-1501 6. Korycka-Wolowiec A, Robak T. Pharmacokinetic Evaluation and Therapeutic Activity of Bendamustine in B-Cell Lymphoid Malig- nancies. Expert Opinion on Drug Metabolism & Toxicology, 2012; 8 (11): 1455-1468 REFERENCES CONTACT INFORMATION: E-mail: anna.korycka-wolowiec@umed.lodz.pl ewelina.ziolkowska@onet.com.pl The work was supported by Mundipharma Research LtD. RESULTS Fig. 1. Mechanism of action of bendamustine.