Alternative pathways of programmed cell death are activated in cells with defective caspase-dependent apoptosis

Available online at www.sciencedirect.com

Leukemia Research 32 (2008) 599–609

Alternative pathways of programmed cell death are activated in cells with defective caspase-dependent apoptosis a,∗ ˇ Eva Ondrouˇskov´a a , Karel Souˇcek b , Viktor Horv´ath b , Jan Smarda a b

Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotl´arˇsk´a 2, 611 37 Brno, Czech Republic Laboratory of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Kr´alovopolsk´a 135, 612 65 Brno, Czech Republic Received 21 February 2007; received in revised form 11 May 2007; accepted 19 May 2007 Available online 6 July 2007

Abstract Loss of programmed cell death pathways is one of the features of malignancy that complicate the response of cancer cells to a therapy. Activation of alternative cell death pathways offers a promising approach to enhance efficiency of cancer chemotherapy. We analysed programmed cell death pathways of v-myb-transformed BM2 monoblasts induced by arsenic trioxide, cycloheximide and camptothecin with U937 promonocytes as a reference cell line. We show that induced death of BM2 cells is not executed by caspases but rather by alternative cell death pathways. Camptothecin induces the lysosome-dependent cell death, arsenic trioxide induces autophagy, and most of cycloheximidetreated BM2 cells die by necrosis. The fact that alternative cell death pathways can be switched in cells with defects in activation and/or function of caspases suggests that understanding and targeting of these pathways could improve therapy of cancer cells suffering from defective apoptosis. © 2007 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Autophagy; Programmed cell death

1. Introduction Programmed cell death (PCD) is a process of genetically regulated elimination of specific cells during development, disease or following exposure to cytotoxic agents. A cell can activate various pathways of self-destruction, including the most rapid and effective one, the apoptosis. The cells undergoing apoptosis shrink, decay and exhibit multiple specific features, such as chromatin condensation, cytoplasmic membrane blebbing, cytoskeleton protein cleavage and nuclear DNA fragmentation [1]. A family of cysteine-aspartyl proteases, caspases, is responsible for execution of the apoptotic cell death [2]. Under specific conditions, particularly in the absence of functional caspase-dependent pathway, activation of several alternative cell death models has been proposed, ∗

Corresponding author. E-mail addresses: [email protected] (E. Ondrouˇskov´a), [email protected] (K. Souˇcek), [email protected] (V. Horv´ath), ˇ [email protected] (J. Smarda). 0145-2126/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2007.05.012

including autophagy, paraptosis, mitotic catastrophe and the descriptive model of apoptosis-like and necrosis-like PCDs [3]. The caspase-independent execution of cell death is usually mediated by cathepsins, calpains or AIF proteases [4–6]. Autophagy is the most frequent non-caspase PCD pathway. Cells undergoing autophagy internalize cytoplasmic components into autophagic vacuoles that are targeted to lysosomes for digestion. Autophagy can be induced by starvation [7], pathogens, toxins [8] and by other death stimuli in cells with defective caspases [9,10]. Rarely, caspases can also participate in regulation of autophagic cell death, as described for Drosophila [11]. Lysosomes mediate the execution phase of the apoptotic as well as other cell deaths. Lysosomal proteases actively participate in cell death induced by oxidative stress [12] or chemotherapeutic drugs [13]. Increased volume of acidic compartment (VAC) precedes the apoptosislike changes in cell morphology and DNA-fragmentation despite the lack of detectable caspase activation in MCF-7 cells [14]. While partial selective permeabilization of lysosomes triggers apoptotic-like PCD, massive

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breakdown of lysosomes results in unregulated necrosis [15]. In this study, we analysed cell death pathways induced by arsenic trioxide, cycloheximide and camptothecin in the line of v-myb-transformed chicken monoblasts BM2 and human promonocytes U937. While U937 cells died by the caspasemediated apoptosis, the programmed cell death of BM2 cells was not executed by caspases. In response to arsenic trioxide, BM2 cells activated autophagic cell death pathway, whereas camptothecin induced cell death pathway typified by increase of VAC. Induction of the alternative cell death pathways can be an important tool for therapy of cancer cells evading apoptosis.

2. Material and methods 2.1. Cell culture, cultivation and transfection Human monocytic U937 cells (European Collection of Cell Cultures (ECACC; Salisbury, Wiltshire, UK)) were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA, USA) and gentamycin (50 ␮g/ml, Serva; Heidelberg, Germany) in a humidified atmosphere of 5% CO2 at 37 ◦ C. v-mybtransformed chicken BM2 monoblasts (a gift from J. Lipsick) [16] were cultured as described elsewhere [17]. To obtain BM2 cells expressing the GFP-LC3 protein chimera, BM2 cells (2 × 106 ) were transfected with 2 ␮g of the GFP-LC3 vector [18] using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. Next day, the cells were washed with phosphate-buffered saline (PBS), and Geneticin (G418, Invitrogen) was added to the final concentration of 500 ␮g/ml. Pool of G418-resistant cells was cloned by limiting dilution. 2.2. Induction of cell death Camptothecin (CAM, Fluka Chemie; Buchs, Switzerland), arsenic trioxide (As2 O3 ) and cycloheximide (CHX, Sigma–Aldrich, Prague, Czech Republic) were used for induction of PCD of BM2 and U937 cells in concentrations ranging from 0.095 to 100 ␮M (serial dilution factor 2) during 24 h. A ratio of living/dead cells upon treatment with As2 O3 and CHX was determined using calcein and propidium iodide (PI) double staining. The cells were incubated with PI (5 ␮g/ml) (Sigma–Aldrich), and calcein AM (200 nM) (Invitrogen) for 15 min at 37 ◦ C. At least 30,000 cells were analysed using FACSCalibur flow cytometry. The cells negative for calcein fluorescence, and emitting fluorescence of PI were considered as dead. CellQuest 3.0 software (Beckton Dickinson; San Jose, CA, USA) was used for data analysis. Viability of cells treated with CAM was determined using MTT test of cytotoxicity. The cells were seeded in 96-well tissue culture plates in triplicate and treated with camptothecin for 24 h. Then, 10 ␮l of the tetrazolium salt MTT (2.5 mg/ml;

Sigma–Aldrich) was added to each well and incubated for 4 h. Next, the formazan product was dissolved in 50 ␮l of 10% Triton X-100 in 0.1M HCl, and quantified using a microplate spectrophotometer reader (DigiScan Reader, ASYS Hitech; Eugenford, Austria) at 570 nm. All experiments were performed at least three times. LD50 for As2 O3 and CHX, and IC50 for CAM were determined from concentration-response curves using Sigma Plot 8.0 software. 2.3. Analysis of mitochondrial membrane potential Changes of the mitochondrial membrane potential (Ψ m) were measured using tetramethylrhodamine ethyl ester perchlorate (TMRE; Invitrogen). Cells were cultured in the presence or absence of the PCD inducers for 4 and 16 h. Pelleted cells were washed twice with Hank’s Balanced Salt Solution lacking calcium and magnesium ions (HBSS), resuspended in TMRE (100 nM) in HBSS (approximately 1 × 106 cells/ml), and kept light-protected at room temperature for 20 min. Then, the cells were washed with HBSS, resuspended in a total volume of 500 ␮l, and at least 30,000 cells were analysed using FACSCalibur flow cytometry. Data were evaluated as percentage of cells with negative TMRE fluorescence from population with scatter characteristics of viable cells. CellQuest 3.0 software (Beckton Dickinson) was used for data analysis. 2.4. Caspase assay The cells (3–5 × 106 ) were lysed in 100 ␮l of lysis buffer (250 mM HEPES; 25 mM CHAPS; 25 mM DTT) in icebath for 20 min and centrifuged (15,000 × g for 15 min) at 4 ◦ C. The proteins present in supernatants were quantified using Bradford protocol [19], and samples were diluted to get an equal protein concentration. The 50 ␮g protein samples were incubated in the assay buffer (40 mM HEPES; 20% glycerol; 4 mM DTT) containing 50 ␮M of caspase3 (Ac-DMQD-AMC) and/or caspase-9 (Ac-LEHD-AMC) substrates (Alexis, Lausen, Switzerland) at 37 ◦ C for 16 h in parallels. The level of fluorescence was determined using microplate reader (FluostarGalaxy; 355 nm excitation, 460 nm emission; BMG Labtechnologies GmbH; Offenburg, Germany). 2.5. Immunoblotting The cells were cultured in the presence or absence of PCD inducers for 16 h. Harvested cells (3 × 106 ) were pelleted by centrifugation, washed with PBS, and resuspended in reducing loading buffer. The cell extracts were boiled, and centrifuged prior to the loading of the equal amount of proteins to 8 or 10% sodium dodecylsufate (SDS) polyacrylamide gels, resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and electroblotted on a PVDF membrane (Millipore; Billerica, MA, USA). The blots were probed using a rabbit polyclonal antiserum directed against

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PARP (sc-7150, Santa Cruz Biotechnology; Santa Cruz, CA, USA) or goat polyclonal antiserum directed against lamin B1 (sc-6217, Santa Cruz Biotechnology), and developed using the horseradish peroxidase-conjugated anti-rabbit or antigoat antibodies using Enhanced Chemiluminescence (ECL Plus) Western Blotting Detection Reagents (GE Healthcare Life Science; Buckinghamshire, UK). For control of protein loading, the PVDF membranes were stained with 0.1% amidoblack 10B (Fluka Chemie).

Software (Scion Corporation; Frederick, MA, USA) in the original 400× magnification. For detection of lysosomes, BM2 cells treated with PCD inducers were incubated with 1 ␮M of LysoSensor DND-189 (Invitrogen) in standard growth media for 30 min at 37 ◦ C. Then, the cells were washed with fresh media, mounted under coverslip and immediately observed by fluorescence microscopy.

2.6. Inhibition of caspases and determination of viability

The BM2GFP-LC3 cells of clone F3 were cultured in the presence or absence of PCD inducers for 24 h. Then, the cells were washed with PBS and resuspended in PBS/Mowiol 488 in a ratio 1:1, dropped under coverslips and immediately analysed using an inverted Nikon fluorescence miscroscope (Diaphot). For analysis of cell ultrastructure, the cells were fixed with 2% glutaraldehyde and post-fixed with 1% osmium tetroxide. The cell pellets were embedded in LR White Resin and cut to 56 nm sections using an ultramicrotome. The sections were stained with 2.5% uranylacetate and lead citrate, and analysed by transmission electron microscopy (model Morgagni 268, Fei; Hillsboro, Oregon, USA).

The cells were cultured in the presence of pan-caspase inhibitor Z-VAD-FMK (10 ␮M, 550,377, BD Pharmingen; San Diego, CA, USA) for 1 h before adding the PCD inducers for next 16 h. Then, the cells were harvested and dead cells marked by permeabilized membranes were stained with propidium iodide. At least 10,000 cells were analysed using FACSCalibur flow cytometer. 2.7. Analysis of viability and nuclear morphology by fluorescence microscopy The cells were cultured in the presence or absence of PCD inducers for 16 h and stained with Hoechst 33342 (1 ␮g/ml, Sigma–Aldrich) for the last 15 min and PI (2 ␮g/ml, Sigma–Aldrich) for the last 5 min of cultivation. Then, the cell suspension was dropped onto the slide and analysed by fluorescence microscopy. The cells having intact blue nuclei and PI-negative cytoplasms were considered as viable, the cells having blue condensed and/or fragmented nuclei and PI-negative cytoplasms were considered as cells undergoing PCD. The cells having red cytoplasms and blue condensed and/or fragmented nuclei were considered as secondary necrotic. The red-stained cells lacking condensed nuclei were considered as necrotic. For analysis of cell viability in the presence of antioxidant, the cells were pre-treated with 10 mM N-acetylcysteine (NAC) (Fluka Chemie) for 1 h, and cultured in the presence of As2 O3 , CHX and CAM for 24 h. Living cells were determined by eosin dye exclusion, and enumerated using light microscopy.

2.9. Detection of autophagic vacuoles

3. Results 3.1. Camptothecin, cycloheximide and arsenic trioxide induce cell death of BM2 and U937 cells To induce cell death, BM2 and U937 cells were treated with camptothecin (CAM), cycloheximide (CHX) and arsenic trioxide (As2 O3 ). In order to determine the effective concentrations of these drugs, the cells were cultured in increasing doses of CAM, CHX and As2 O3 for 24 h as described in Section 2. Dose-response curves and concentrations of the drugs causing 50% reduction of viability (LD50) or metabolic activity (IC50) are shown in Fig. 1 These concentrations of drugs effectively killed the cells within 3 days.

2.8. Detection of lysosomal vacuoles

3.2. Arsenic trioxide, cycloheximide but not camptothecin decrease the mitochondrial membrane potential in BM2 cells

The cells were cultured in the presence or absence of PCD inducers for 24 h. One-hundred and twenty minutes prior to the harvest, the cells were incubated with 50 ␮M monodansylcadaverine (MDC) (Fluka Chemie) at 37 ◦ C. For visualization of MDC-labeled vacuoles, the cells were fixed with 2% paraformaldehyde for at least 30 min at 4 ◦ C. Fixed cells were washed three times with PBS and mounted in Mowiol 4-88 (Calbiochem, San Diego, CA, USA) under coverslips. The samples were analysed using an inverted Olympus IX-70 microscope, fluorescence images were captured using a CCD camera (model 4912, Cohu, Inc., San Diego, CA, USA), VG-5 frame grabber and Scion Image

A decrease of the mitochondrial membrane potential (Ψ m) can be either associated with early events of apoptosis or it can result from general collapse of cellular homeostasis in late stages of cell death. In order to evaluate Ψ m, BM2 and U937 cells were treated with CAM, CHX and As2 O3 for 4 and 16 h, and Ψ m was analysed in individual cells using TMRE by flow cytometry. Frequency of U937 as well as BM2 cells with collapsed mitochondrial membrane potential significantly increased upon treatment with CHX and As2 O3 in both intervals. In contrast, the effect of CAM was the cell type-specific. While significant fraction of CAM-treated U937 cells decreased Ψ m (35.7% in 4 h,

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Fig. 1. Determination of effective drug concentrations. BM2 and U937 cells were treated with arsenic trioxide (As2 O3 ), cycloheximide (CHX) and camptothecin (CAM) in concentration range from 0.095 to 100 ␮M (serial dilution factor: 2) for 24 h. Ratios between dead and living cells were determined using calcein and propidium iodide double staining (As2 O3 , CHX) and/or MTT test of cytotoxicity (CAM) as described in Section 2. Data indicate mean ± standard deviations from at least three independent experiments. LD50 for As2 O3 and CHX and IC50 for CAM were determined for both cell lines from concentration-response curves using Sigma Plot 8.0 software.

30.5% in 16 h), BM2 cells did not (1.9 and 0.3%) (Fig. 2). These results document that the death pathways induced by As2 O3 and CHX in BM2 and by As2 O3 , CHX and CAM in U937 cells include the mitochondrial membrane depolarization. In contrast, CAM-treated BM2 cells use alternative death pathway. 3.3. Programmed cell death of BM2 cells does not dependent on caspase-9 and caspase-3 activities Activation of mitochondria-dependent pathway of apoptosis is generally associated with activation of caspase-9 and subsequent activation of the execution caspase-3. Therefore, we wished to determine the activities of caspase-9 and caspase-3 in BM2 and U937 cells undergoing PCD. The cells were treated with CAM, CHX, and As2 O3 for 16 h

and the caspase activity was measured using specific fluorogenic substrates by fluorescence photometry. Activities of both caspases increased in control U937 cells upon treatment with CAM, CHX as well as As2 O3 (Fig. 3A). However, only weak increase of caspase-3 and -9 activities was detected in identically treated BM2 cells suggesting that caspase activity is less important for execution of PCD in BM2 cells than in U937 cells. In order to address the role of caspases in the death processes induced by CAM, CHX and As2 O3 in BM2 and U937 cells in more detail, we compared viability of these cells cultured in the presence and absence of the pan-caspase inhibitor, the Z-VAD-FMK. The cells were pre-treated with Z-VAD-FMK for 1 h prior cultivation in the presence of the PCD-inducers for next 16 h. The cell viability was analysed by propidium iodide staining using flow cytometry. The

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Fig. 2. Changes of mitochondrial membrane potential. BM2 and U937 cells were treated with CAM, CHX and As2 O3 for 4 and 16 h or left untreated (control). Changes of the mitochondrial membrane potential (Ψ m) were evaluated using the TMRE probe by flow cytometry. Histograms represent typical results. Numbers indicate the mean percentage ± standard deviations of the cells with collapsed Ψ m from three independent experiments.

pan-caspase inhibitor increased number of viable U937 cells treated with CHX and CAM. In contrast, the effect of Z-VAD-FMK in As2 O3 -, CAM- and CHX-treated BM2 cells was not significant (Fig. 3B). These results further support the hypothesis that the death pathways induced by As2 O3 , CAM and CHX in BM2 cells are less dependent on activity of caspases than in U937 cells. Poly(ADP) ribose polymerase (PARP) and lamin B1 belong to a number of substrates that are targeted and cleaved by execution caspase-3 and -6. In order to confirm that PCD of BM2 cells is less caspase-dependent than in U937 cells, we addressed cleavage of the PARP and lamin B1 proteins in BM2 and U937 cells treated with CAM, CHX and As2 O3 for 16 h by SDS-PAGE followed by immunoblotting. PARP

as well as lamin B1 were clearly truncated in U937 cells treated with the death inducers, thus forming fragments of expected molecular weights: the 89 kDa-fragment derived from PARP and the 45 kDa-fragment derived from lamin B1 (Fig. 3C). PARP and lamin B1 were also truncated in BM2 cells upon treatment with CAM, CHX and As2 O3 , but the molecular weights of resulting fragments were different: PARP was truncated to about 55 kDa-, and lamin B1 to about 65 kDa-fragments. These results indicate that protein degradation during PCD of BM2 cells is preferentially executed by proteases that do not possess caspase-specific cleavage pattern. In contrast to U937 cells, caspase-3 and -9 are not activated in BM2 cells treated with inducers of PCD suggesting that they are not significantly involved in the execution phase of the cell death.

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Fig. 3. Activity of caspases in BM2 cells undergoing PCD is low. (A) BM2 and U937 cells were treated with As2 O3 , CHX and CAM for 16 h or left untreated. Activity of caspases was determined using caspase-3 (Ac-DMQD-AMC) and caspase-9 (Ac-LEHD-AMC) fluorogenic substrates using fluorometry. The bars represent relative caspase activities as percentages of untreated controls indicating the means of three independent experiments. Error bars indicate standard deviations. (B) BM2 and U937 cells were pre-treated with pan-caspase inhibitor Z-VAD-FMK (10 ␮M) for 1 h. Then, As2 O3 , CHX and CAM were added for next 24 h. Number of dead cells was determined by propidium iodide staining, at least 10,000 cells were analysed using flow-cytometry. Error bars indicate standard deviations. Asterisk (*) denotes significant (P < 0.05) changes in number of living cells in the presence of Z-VAD-FMK. (C) The cells were treated with As2 O3 , CHX and CAM for 16 h or left untreated. Proteins purified from harvested cells were resolved by SDS-PAGE, electroblotted and probed with PARP- and lamin B1-specific antibodies. Arrows indicate the positions of full length and truncated proteins, numbers represent their molecular weights (kDa).

3.4. CHX induces rather necrosis than PCD of BM2 cells CHX and As2 O3 caused mitochondrial membrane depolarization in BM2 cells. This feature marks the cells undergoing PCD. On the other hand, the other hallmark of apoptosis, upregulation of the caspase activity did not occur in these cells. To distinguish necrotic cells from cells undergoing PCD, we stained BM2 and U937 cells treated with CAM, CHX and As2 O3 for 16 h with two DNA-binding dyes: the membrane-permeable Hoechst 33342, and the membranenon-permeable propidium iodide. Fractions of viable cells, the cells undergoing PCD, and the cells dying from necrosis were enumerated by fluorescence microscopy. PCD was induced preferentially in U937 cells. Typical nuclear morphology and staining pattern of apoptosis exhibited 37% of As2 O3 -treated, 62% of CAM-treated and 63% of CHX-

treated U937 cells, while frequency of cells with necrotic morphology did not exceed 15% (Fig. 4). In BM2 cells, frequency of PCD reached 35% upon treatment with As2 O3 , and 12% upon treatment with CAM, and CHX. Necrotic morphology was detected in 41% of CHX-treated, 28% of As2 O3 -treated and 20% of CAM-treated BM2 cells. These results indicate that programmed cell death is less frequent in BM2 than in U937 cells and that necrosis became the predominant type of cell death of CHX-treated BM2 cells. 3.5. Camptothecin induces VAC increase of BM2 cells CAM-induced PCD of BM2 cells lacks some typical features of apoptosis, such as decrease of the mitochondrial membrane potential and activation of caspases. Alternative cell death pathways are often based on degradation of cell components in acidic compartments, such as lysosomes. In

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and lysosomes [20,21]. We detected the MDC-stained vacuoles only in CAM-treated BM2 cells (Fig. 5A) but not in any other sample. Next, we used the lysosome-specific probe, Lysotracker DND-189 instead of MDC. Again, only the CAM-treated BM2 cells exhibited strong Lysotracker staining pattern (Fig. 5B). These results document that CAMtreated BM2 cells increase content of lysosomes that are involved in degradation processes. 3.6. Arsenic trioxide induces autophagic cell death of BM2 cells

Fig. 4. Necrosis is enhanced in drug-treated BM2 cells. The cells were treated with the drugs for 16 h and then stained with Hoechst 33,342 and propidium iodide. Nuclear morphology and viability of at least 800 cells were analysed by fluorescence microscopy. The results of four independent experiments are presented. Error bars represent standard deviations.

order to assess the presence of these cell compartments, we observed CAM-, CHX- and As2 O3 -treated U937 and BM2 cells stained with monodansylcadaverine (MDC) probe by fluorescence microscopy. MDC has been shown to nonspecifically stain the acidic vacuoles, the autolysosomes

BM2 cells treated with As2 O3 partially die by necrosis and partially by programmed cell death that does not include caspase activation and increase of the acidic cell compartments. To further characterize the way of PCD of As2 O3 -treated BM2 cells, we followed the presence of the autophagic vacuoles. We used two approaches: location of the LC3-II fragment by fluorescent microscopy and analysis of cell ultrastructure by transmission electron microscopy. The cells undergoing autophagy cleave the LC3 protein to the LC3-II fragment localized in the membranes of autophagosomes [18]. To visualize the LC3 protein, we prepared the derivatives of BM2 cells expressing the GFP-LC3 protein chimera. Redistribution of GFP-LC3 from cytoplasma to vacuolar-like dots was clearly detectable in As2 O3 -treated BM2 GFP-LC3 cells by fluorescence microscopy. In contrast, the GFP-LC3 protein did not form dots and rather remained

Fig. 5. Acidic vacuoles are formed in CAM-treated BM2 cells. BM2 and U937 cells were treated with As2 O3 , CHX and CAM or left untreated (control) for 24 h. For staining of acidic vacuoles, the cells were incubated with monodansylcadaverine (MDC) for 1 h (A) or with LysoSensor probe (B). The cells were examined by fluorescence microscopy using the same (400×) magnification.

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diffused in cytoplasm of CAM- and CHX-treated BM2 cells (Fig. 6A). To confirm the presence of vacuoles in the cytoplasm of As2 O3 -treated BM2 cells, we performed analysis of cell ultrastructure by transmission electron microscopy. Number and size of vacuoles was markedly increased in cytoplasm of As2 O3 - but not CHX- and CAM-treated BM2 cells (Fig. 6B). Frequency of As2 O3 -treated BM2 cells containing more than ten vacuoles reached 35%, while it did not exceed 10% in untreated, CAM- and CHX-treated BM2 cells. The presence of GFP-LC3 dots and autophagic vacuoles documents that arsenic trioxide induces autophagy in BM2 cells. 3.7. Reactive oxygen species are involved in As2 O3 -induced cell death CAM and As2 O3 were described as agents increasing the level of reactive oxygen species (ROS) in mammalian cells [22,23]. In order to assess the role of ROS in induction of PCD of U937 and BM2 cells, we exposed them to As2 O3 , CHX or CAM in the presence of antioxidant, the N-acetylcysteine. NAC significantly (P  0.05) improved viability of both BM2 and U937 cells treated with As2 O3 causing increase of viable cell fraction from 62 to 92% in BM2 and from 63 to 83% in U937 cells during 24 h. In contrast, viability of the cells treated with CAM and CHX was not affected by the

presence of NAC (Fig. 7). These results suggest that cytotoxicity of As2 O3 results from increased ROS formation in BM2 and U937 cells. Therefore, the ROS may participate in induction of apoptosis of U937 and autophagy of BM2 cells treated with As2 O3 .

4. Discussion Programmed cell death is essential for normal development, homeostasis and also for effective treatment of cancer. Malfunction of PCD programs can induce resistance of cancer cells to therapy. In this work, we studied the death pathways activated by three drugs commonly used for induction of PCD in various cancer cell types: camptothecin (CAM), cycloheximide (CHX) and arsenic trioxide (As2 O3 ). CAM generates free radicals [23] and inhibits DNA topoisomerase I, thus inducing DNA-damage [24,25]. CHX can induce apoptosis by blocking synthesis of proteins [26]. As2 O3 induces PCD in leukemic cells by increasing the level of hydrogen peroxide or by direct inhibition of GTPtubulin interaction [22,27]. Effects of these PCD inducers were tested in cells of promonocytic cell lines U937 and BM2. Human U937 cells were obtained from a patient with histiocytic lymphoma [28]. Chicken BM2 cells are monoblasts that constitutively express the v-myb oncogene of avian

Fig. 6. Autophagic vacuoles are formed in As2 O3 -treated BM2 cells. (A) BM2GFP-LC3 cells were cultured in the presence of As2 O3 , CHX and CAM or left untreated (control) for 24 h. Cells were examined by fluorescence microscopy using the same (400×) magnification. Arrows indicate the cells containing dot-like structures of GFP-LC3 in the membranes of autophagosomes. (B) BM2 cells were treated with As2 O3 , CHX and CAM or left untreated (control) for 24 h and observed using electron microscopy (magnifications 2200× and 4400× are indicated).

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Fig. 6. (Continued).

myeloblastosis virus (AMV) [16]. U937 cells were described to undergo apoptosis marked by membrane blebbing, formation of apoptotic bodies and activation of caspases upon treatment with PCD inducers [22,26]. Details of the death pathways activated in BM2 cells have not been described yet. One of the early events in cells undergoing PCD can be a decrease of the mitochondrial membrane potential (Ψ m) [29]. As expected, Ψ m decreased in U937 cells upon treatment with CAM, CHX and As2 O3 . Similar results were also obtained in BM2 cells treated with As2 O3 and CHX. However, no significant change of Ψ m was detected in CAM-treated BM2 cells. Mitochondria-dependent apoptosis is usually associated with activation of caspase-9 and

subsequent activation of execution caspases-3 and -6 [30]. Activities of caspase-9 and -3 clearly increased in U937 cells exposed to cytotoxic drugs causing extensive fragmentation of specific fluorogenic substrates; however, only weak activation was observed in similarly treated BM2 cells. Since the chicken caspases exhibit the same sequence-specificity as the human ones, this difference cannot result from the inter-species variability [31]. Furthermore, the pan-caspase inhibitor Z-VAD-FMK did not enhance viability of the drugtreated BM2 cells, thus supporting the caspase-independent model of cell death in BM2 cells. One of the proteases that can be involved in the execution of PCD in the absence of active caspases is Ca2+ -dependent calpain protease [32]. However, we did not detect any calpain activity in BM2 cells

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Fig. 7. Antioxidant NAC increases viability of As2 O3 -treated but not CAMand CHX-treated BM2 and U937 cells. The cells were pre-treated with NAC (10 mM) for 1 h and then cultured in the presence of As2 O3 , CHX and CAM for 24 h. Viability of at least 300 cells per sample was determined by eosin dye exclusion. The results of three independent experiments are presented. Error bars represent standard deviations Asterisk (*) denotes significant (P  0.05) changes in number of living cells in the presence of NAC.

undergoing PCD (not shown). PARP and lamin B1 are typical substrate proteins that are targeted by caspases during the execution phase of PCD [33–35]. Activation of caspases in U937 cells caused truncation of both PARP and lamin B1 to fragments of expected molecular weights. The same proteins were also degraded in As2 O3 -, CHX- and CAMtreated BM2 cells but the cleavage pattern was different. Truncation of PARP and lamin B1 resulted in formation of ∼55 and ∼65 kDa-fragments, respectively. Because both PARP and lamin B1 exhibit the same cleavage pattern in human and chicken cells [36,37], the differential fragmentation of PARP and lamin B1 in BM2 and U937 cells cannot result from species-specific effects but rather from activity of other proteases. Truncation of the PARP protein to ∼55 kDa-fragment can also mark necrosis [38]. Indeed, large fraction of CHX-treated BM2 cells was found necrotic as documented by analysis of nuclear morphology and viability. Furthermore, the predominant necrotic morphology was confirmed in CHX-treated BM2 cells using electron microscopy. The proteosynthesis is necessary for alternative types of cell death (autophagy) [39]. When it is inhibited in the presence of cycloheximide, the cells presumably cannot activate alternative cell death pathways and die from necrosis. It has been postulated that the dominant cell death phenotype is determined by the relative speed of the available cell death programs; although characteristics of several death pathways can be displayed, only the fastest and most effective one, i.e. apoptosis is usually evident [15]. Nevertheless, there are more types of cell death that are activated under specific conditions or when the caspase-dependent apoptotic pathway is defective. CAM-induced PCD of BM2 cells did not include change of the mitochondrial membrane potential

and caspases activity, but formation of cytoplasmic acidic organelles that we detected using two probes; the MDC and LysoSensor [20,21]. Therefore, we can suppose that lysosomes participate in caspase-independent cell death of BM2 cells induced by CAM. As2 O3 can induce both the caspase-dependent and caspase-independent programmed cell death pathways [40]. The presence of autophagic vacuoles marking PCD by autophagy was detected in BM2 cells treated with As2 O3 using both electron microscopy and analysis of intracellular location of the GFP-LC3 protein. These two methods are fundamental for identification of autophagic vacuoles [18,21]. The role of reactive oxygen species in induction of PCD of mammalian cells upon treatment with As2 O3 was described [22]. We confirmed a significant role of ROS in the cell death pathway induced by As2 O3 using a scavenger of oxygen radicals, the N-acetylcysteine. NAC significantly decreased mortality of As2 O3 -treated cells, thus suggesting a substantial role of ROS in induction of apoptosis of U937 cells and interestingly also in induction of autophagy in BM2 cells. Capability of As2 O3 to efficiently kill even chemoresistant tumor cells with disturbed apoptotic signaling and caspase activity, a frequent finding in malignancy, highlights its significance for the cancer treatment. This is especially the case in hematologic malignances because this agent has been associated with high rates of hematologic and molecular remissions [41]. Successful treatment of cancer cells with chemotherapeutic drugs is largely dependent on their ability to trigger cell death. Cell lines defective in apoptosis but capable of alternative cell death pathways, such as the line of vmyb-transformed chicken monoblasts BM2 can represent valuable tools in research of alternative cell death pathways. Understanding of the molecular mechanisms controlling alternative types of cell death can then provide new targets and approaches to the therapy of cancer cells that are often resistant to the standard inducers of apoptosis.

Acknowledgements We thank Alois Kozub´ık and Jiˇrina Hofmanov´a for kind provision of FACSCalibur, Tamotsu Yoshimori and Noboru Mizushima for kind provision of the GFP-LC3-expressing ˇ vector, Jan Smarda, Sen., Roman Janish and Dobromila Klemov´a for help with electron microscopy, Alena Vaculov´a for help with the caspase assay, Jarmila Navr´atilov´a for help ˇ c´ıkov´a for corrections with viability assays and Sabina Sevˇ of English. Financial support: This work was supported by a grant of the Grant Agency of the Czech Republic (no. 301/06/0036), a grant of the Ministry of Education, Youth and Sports of the Czech Republic (MSM0021622415), a grant of the Czech Science Foundation (no. 204/07/0834) and the Research Plan AVOZ50040507 of Academy of Sciences of the Czech Republic.

E. Ondrouˇskov´a et al. / Leukemia Research 32 (2008) 599–609

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