Leukemia Research 28 (2004) 53–61 AAF-cmk sensitizes tumor cells to trail-mediated apoptosis Izabela Młnarczuk a,b,c , Paweł Mróz a , Grażyna Hoser d , Dominika Nowis a , Łukasz P. Biały b , Halina Ziemba f , Tomasz Grzela b , Wojciech Feleszko e , Jacek Malejczyk b , Cezary Wójcik b , Marek Jakóbisiak a , Jakub Goł˛ab a,∗ a b Department of Immunology, Center of Biostructure, The Medical University of Warsaw, Chałubińskiego 5, 02004 Warsaw, Poland Department of Histology and Embryology, Center of Biostructure, The Medical University of Warsaw, Chałubińskiego 5, 02004 Warsaw, Poland c Institut fur Biochemie, Medical Faculty, Charite, Humboldt University, Monbijoustrasse 2, 10117 Berlin, Germany d Department of Clinical Cytology, Postgraduate Center of Medical Instruction, Marymoncka 99, 01813 Warsaw, Poland e Department of Pediatric Pneumonology, Allergic Diseases and Hematology, The Medical University Children’s Hospital, Działubińdowska 1, 01184 Warsaw, Poland f Department of Peridontology, The Medical University of Warsaw, Miodowa 18, 02246 Warsaw, Poland Received 13 December 2002; accepted 30 April 2003 Abstract TRAIL is a member of the tumor necrosis factor (TNF) superfamily. This cytokine is cytotoxic for a high proportion of tumor cells, but could be also toxic for normal cells. There is a need to find other agents able to potentiate the antitumor effects of this cytokine. In our study, we found that Ala-Ala-Phe-chloromethylketone (AAF-cmk) augmented cytotoxic activity of TRAIL or TNF against human leukemic cells. Flow cytometry studies and electron microscopy revealed that apoptosis was primarily responsible for this potentiation. Altogether, our studies indicate that AAF-cmk might effectively sensitize human leukemia cells to apoptosis induced by TRAIL and TNF. © 2003 Elsevier Ltd. All rights reserved. Keywords: AAF-cmk; TRAIL; TNF; Lymphoma; Apoptosis; Tripeptidylpeptidase II 1. Introduction Regulated cytosolic proteolysis is indispensable for many cellular functions, including activation of transcription factors, regulation of the cell cycle, processing of peptides presented by the major histocompatibility complex (MHC) class I molecules and removal of incorrectly folded or damaged proteins . The ubiquitin- and proteasome-dependent system of protein degradation is thought to be the principal machinery responsible for the cytosolic proteolysis. The culture of tumor cells in the presence of proteasome inhibitors (PSI) induces apoptosis in these cells [2,3] and causes a block in G1 phase of the cell cycle . Clinical trials with at least one class of proteasome inhibitors are already being conduced in terminally ill cancer patients [5–7]. However, not all tumor cell lines are equally susceptible to proteasome inhibitors [8,9]. Even those tumor cells, which are susceptible, may become resistant to normally lethal doses of proteasome inAbbreviations: FBS, fetal bovine serum; TPPII, tripeptidylpeptidase II Corresponding author. Tel.: +48-22-622-63-06; fax: +48-22-622-63-06. E-mail address: [email protected] (J. Goł˛ab). ∗ 0145-2126/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0145-2126(03)00122-X hibitors as was shown with EL-4 cells growing in the constant presence of low doses of proteasome inhibitors . It was reported that the function of proteasome could be replaced by tripeptidylpeptidase II (TPPII) in cells adapted to high doses of proteasome inhibitors [11,12], but some more recent findings revealed the residual activity of proteasomes remains essential for protein degradation, antigen presentation and survival. Therefore, the physiological role of this protease still awaits explanation. It was hypothesized that in the cell TPPII further degrades oligopeptides produced by the proteasome into tripeptides. The latter are then proteolysed into free aminoacids by aminopeptidases , that can be used for new protein synthesis or other metabolic purposes. The native form of TPPII has a remarkably high molecular mass (>1000 kDa) and, like the proteasome, can assemble into a higher-order structure composed of multiple 138 kDa subunits that has an internal channel . The enzyme has been isolated from, for example human erythrocytes , rat brain , pig  and Drosophila melanogaster . Thus, like the proteasome, the enzyme appears to have a ubiquitous distribution and its structure is highly conserved . TPPII and the proteasome have different substrate 54 I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 spectrum and inhibition patterns. It was suggested that the activity of TPPII may be required for the survival of Burkitt’s lymphoma cells, and inhibition of TPPII induced apoptosis in these cells . Also TPPII could be responsible for the breakdown of myofibrillar proteins during muscle cachexia . Ala-Ala-Phe-chloromethylketone (AAF-cmk) has been used in various studies as an irreversible and specific inhibitor of TPPII, however more recent data indicate that it also blocks to some extent the chymotrypsin-like activity of the proteasome [11,20]. Tumor necrosis factor (TNF) is one of the main inflammatory cytokines, capable of inducing apoptosis in susceptible tumor cells. However, its administration in patients is limited by profound toxicity. More promising antitumor agent is the recently discovered TNF-related apoptosis inducing ligand (TRAIL), which belongs to the TNF ligand superfamily and induces apoptosis in a broad range of tumor cells with apparently no cytotoxic activity against most non-transformed cells [21–23]. The apoptosis induced by TRAIL and TNF can be blocked by a pan-caspase inhibitor, benzoxycarbonyl–VAD–fluoromethylketone (zVAD-fmk) [24,25]. Recombinant soluble TRAIL has shown promising antitumor effectiveness in numerous human and murine tumor cell lines and in some in vivo models [21,23,26]. However, it was reported that TRAIL could be toxic for normal human hepatocytes , therefore there is an urgent need for agents, which could synergistically potentate antitumor activity of TRAIL reducing its effective doses and thus attenuating its toxicity. Since we have recently shown that a proteasome inhibitor (N-benzyloxycarbonyl-Ile-Glu-(o-t-butyl)-Ala-leucinal) is capable of sensitizing tumor cells to the cytotoxic effects of TRAIL  or TNF , we have now decided to evaluate the antitumor effectiveness of these cytokines in combination with an inhibitor of tripeptidylpeptidase II, a protease that can either replace or complement the function of proteasome. 2. Materials and methods 2.1. Reagents Ala-Ala-Phe-chloromethylketone (Sigma, St. Louis, MO) was dissolved in dimethylsulfoxide, DMSO and stored as a 10 mM stock solution at −20 ◦ C. Recombinant human LZ-TRAIL, thereafter referred to as TRAIL, was a gift from the Immunex Corporation, Seatle, WA . Stock solution was prepared according to the manufacturer instruction and kept at −70 ◦ C. Recombinant human TNF (rhTNF produced in Escherichia coli was kindly provided by Dr. W. Stec (Department of Bioorganic Chemistry, Center of Molecular and Macromolecular Studies, Łódź, Poland). The specific activity of the rhTNF was (4.3 ± 1.1) × 107 U/mg. The broad-spectrum cas- pase inhibitor benzoyxycarbonyl-VAD-fluoromethylketone (Sigma) was dissolved in DMSO and kept as a 20 mM stock solution at −70 ◦ C . 2.2. Cell culture U937 human promonocytic leukemia cells were routinely cultured in a humidified atmosphere of 5% CO2 at 37 ◦ C in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), antibiotics, and l-glutamine (2 mM) (all from Gibco BRL, Paisley, UK). Cells were kept in 25 cm2 tissue flasks (Nunc, Roskilde, Denmark) and passaged every 2–3 days. 2.3. Cytostatic/cytotoxic assay The cytostatic/cytotoxic effects of TRAIL or TNF with/without AAF-cmk on U937 cells in vitro was assayed by oxidation of the tetrasolium salt MTT (Sigma). Leukemia cells (5 × 103 ) in 100 l of medium were dispensed into 96-well microtiter plates (Corning, Bibby Sterlin Ltd., Staffordshire, UK). Then, the serial dilutions of AAF-cmk (50 l; final concentration 5–20 M) and TRAIL (50 l; final concentration 10–100 ng/ml) or human TNF (50 l; final concentration 125–500 ng/ml) were added in quadruplicate to a final volume of 200 l. Appropriate volumes of culture medium, supplemented with DMSO (<0.1%) were added as controls. After an incubation period of 24 h with AAF-cmk and for additional 24 h with TRAIL/TNF and/or AAF-cmk, a standard MTT assay was performed. After solubilization of formazan crystals in DMSO, the plates were read on an ELISA reader (SLT-Lab Instruments Ges. M.b.H., Salzburg, Austria) using a 550 nm filter. Cytostatic/cytotoxic effect was expressed as relative viability of U937 cells (percentage of control U937 cells incubated with medium only) and was calculated as follows: relative viability = (Ae − Ab ) × 100/(Ac − Ab ), where Ab is background absorbance, Ae is experimental absorbance and Ac is the absorbance of untreated controls. Similar treatment regimen was applied to the experiments with normal human monocytes isolated from peripheral blood using a Ficoll gradient. Monocytes were plated in triplicate at 4 × 104 cells per well. Cytostatic/cytotoxic effect was assessed by crystal violet staining. The tetrapeptide caspase inhibitor, zVAD-fmk was added to the cells at a final concentration of 10 M 4 h before AAF-cmk, and then the same conditions as described above for the cytotoxicity measurements were applied and the results were obtained using MTT assay. 2.4. Drug interaction analysis To determine whether AAF-cmk and TRAIL exert synergistic cytostatic/cytotoxic effects against U937 cells the isobolanalysis by Berenbaum as described elsewhere was I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 used . Briefly, inhibition of cell proliferation was determined using MTT assay as described above. Equi-effective concentrations (concentrations of either drug alone or in combination, which gave equivalent inhibition of cell proliferation as compared with untreated controls at P < 0.05 Student’s t-test) were analyzed. The interaction index for two-drug combination was computed by applying the following equation: interaction index = (TRAILc /TRAILe ) + (AAF-cmkc /AAF-cmke ), where TRAILe and AAF-cmke are concentrations of both drugs that produce some specified effect when used alone and TRAILc and AAF-cmkc are concentrations of the drugs that produce the same effect when used in combination. According to this analysis, synergy occurs when the interaction index is less than 1.0. 2.5. Apoptosis measurement using flow cytometry Cells were treated for 24 h with AAF-cmk (20 M) and then for additional 24 h with either RPMI alone or TRAIL (30 ng/ml) alone or with AAF-cmk. Phosphatidylserine externalization of apoptotic cells was visualized by staining with annexin V-FITC according to the manufacturer’s instruction (BioSource, Camarillo, CA). Cell death was assessed by the uptake of propidium iodide, PI (Sigma). Briefly, the U937 cells were washed in PBS, resuspended in 300 l of a calcium containing binding buffer supplied by the manufacturer (diluted 1:10 in distilled water) with the addition of 3 l annexin V-FITC (BioSource) and 50 l PI (100 g/ml). After 15 min incubation at room temperature, flow cytometry was performed. Live cells are negative for both annexin V and PI, early apoptotic cells are positive for annexin V only and late apoptotic and necrotic cells are positive for both dyes [33,34]. The probes were analyzed by FACSCalibur flow cytometer (Beckton Dickinson, San Diego, CA) and analysed by CellQuest software. Argon laser excitation wavelength was 488 nm, while emission data were acquired at wavelength 520 nm for fluorescein and 580 nM FL-2 and 650 nM FL-3 for PI. Statistical significance was calculated using the chi-square test. 2.6. Transmission electron microscopy For electron microscopy after twice washing in PBS the cells were immediately fixed in 2.5% glutaraldehyde EM grade (Merck, Darmstadt, Germany) and 2% paraformaldehyde (Sigma) in 0.1 M cacodylate buffer (pH 7.4) for 1.5 h, postfixed in 1% OsO4 in 0.1 M cacodylate buffer (pH 7.4) for 1 h, dehydrated in graded alcohol and embedded in Spurr resin (Sigma). Ultrathin sections were cut with OMU-3 ultratome (C. Reichert AG, Vienna, Austria) and put onto copper grids and contrasted with uranyl acetate and lead citrate (Merck). The sections were observed at 5500–6000× primary magnification with a JEOL JEM 100 S electron microscope (Jeol Ltd., Tokyo, Japan) at 80 kV. The apoptotic cells were detected according to the ultrastructural criteria of alterations in the nuclei and in the cy- 55 toplasm. These alterations consisted of chromatin condensation, formation of nuclear blebs and buds with condensed chromatin that pinched off to produce components of apoptotic bodies, leaks in nuclear envelopes, and vacuolization of the cytoplasm [35–38]. The sequence of ultrastructural findings allowed us to divide this process into two stages: the first—early apoptosis and the second—advanced apoptosis. Early apoptosis was characterized by chromatin condensation in peripheral regions of the nuclei and by the presence of numerous vacuoles in the cytoplasm, while the cytoplasm itself was electron dense but the number of its organelles was reduced. In advanced apoptosis the condensation of chromatin was more pronounced the nuclei were either forming buds pinching off to form components of apoptotic bodies or were already breaking up. In advanced apoptosis the tendency to enlargement and clustering of vacuoles could be observed, while the cytoplasm was disorganized. 2.7. SDS-PAGE and Western blotting Western blotting analysis was performed as described elsewhere . Briefly, leukemic cells (4 × 106 ) were lysed in SDS-sample buffer. Equal amounts of protein were separated by 7.5% SDS-PAGE on MiniProtean II electrophoresis system (Biorad, Hercules, CA). As molecular weight marker, kaleidoscope prestained marker set was used (Biorad). Semi-dry electrophoretic transfer was made and membranes were blocked with 5% non-fat dry milk in TBST with 0.5% fetal calf serum, overnight and probed with the rabbit polyclonal anti poly(ADP-ribose) polymerase (PARP) antibody, (Santa Cruz, CA, USA). After washing in TBST, the membranes were incubated with alkaline phosphatase conjugated donkey anti-rabbit polyclonal serum 1:2500 (Jackson Immunoresearch Laboratories Inc., West Grove, PN). Alkaline phosphatase was detected by a standard method using BCIP/NBT (Sigma). After drying, the membranes were scanned with Plustek Opticpro 9636T flatbed scanner. 2.8. Statistical analysis Data are presented as means ± standard deviation (S.D.). Differences between groups were analyzed for significance by the Student’s t-test. Significance was defined as a two-sided P < 0.05. The statistical analysis for flow cytometry results was performed with use of the two-sided chisquare test. A series of independent experiments were performed and the results presented in the paper are representative. 3. Results 3.1. Potentiated antitumor effects of AAF-cmk used in combination with TRAIL or TNF U937 leukemia cells were found to be susceptible to the cytostatic/cytotoxic activity of both TRAIL (Fig. 1A) 56 I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 Fig. 2. Isobologram analysis depicting the interaction between AAF-cmk and TRAIL in inhibiting the growth of U937 cells. Solid line concentrations of both drugs that cause a 60% inhibition of cell proliferation (IC60 ). Hyphenated line, the hypothetical amounts of both drugs required to cause the same decrease in cell proliferation as if the interactions were additive. The number in the box indicate the interaction index calculated as described in “Section 2”. Fig. 1. U937 cells were exposed for 24 h to various concentrations of AAF-cmk followed by 24 h incubation with AAF-cmk and/or TRAIL (A) or with TNF (B). Cytostatic/cytotoxic effects were tested in a standard MTT assay. Data are presented as a relative viability (percentage of untreated controls). Each bar represents mean ± S.D. All groups marked with (∗) are significantly different from the controls (P < 0.05). and human TNF (Fig. 1B) as well as AAF-cmk (Fig. 1A and B). Preincubation of U937 cells with AAF-cmk for 24 h followed by incubation with both AAF-cmk and each of the two cytokines for additional 24 h resulted in the potentiation of growth inhibitory cytostatic/cytotoxic effects of TRAIL as well as TNF (Fig. 1A and B). Isobologram analysis according to Berenbaum revealed synergistic activity of the combination of TRAIL and AAF-cmk at the synergism index of 0.55 (Fig. 2). Importantly, the combination treatment with AAF-cmk and TRAIL was not toxic towards normal human monocytes (Fig. 3). 3.2. AAF-cmk increases TNF- and TRAIL-induced apoptosis To investigate whether the cytotoxic/cytostatic effect of AAF-cmk with TRAIL was due to increased apoptosis, the U937 cells were labeled with annexin V-FITC and PI before FACScan analysis. TRAIL in experimental concentrations induced 19.4% apoptosis while the inhibitor of TPPII alone induced 24.7% apoptosis in U937 cells. The combination of AAF-cmk/TRAIL increase the percentage of apoptotic cells to 55.8%, however there were also some necrotic cells in the combination group. The combined treatment left 17.9% of viable U937 cells (Table 1). Similar results were obtained for AAF-cmk/TNF treatment (data not shown). The electron microscopy analysis of U937 cells was provided according to the ultrastructural apoptotic criteria as described in Section 2. This analysis revealed increased number of cells with characteristic apoptotic morphology in groups treated with AAF-cmk and both TRAIL or TNF compared to each group treated alone. Moreover, these findings were more pronounced and advanced in cells undergoing the combination treatment (Fig. 4). 3.3. A pan-caspase inhibitor zVAD-fmk prevents the cytotoxic effects of the combination of AAF-cmk with either TRAIL or TNF To determine whether the cytostatic/cytotoxic activity of the combination of AAF-cmk with either TRAIL or TNF was mediated through caspase activation, the U937 cells were incubated with the drugs in the presence or absence of a pan-caspase inhibitor, zVAD-fmk. Caspase inhibition by zVAD-fmk significantly reduced the cytotoxicity of AAF-cmk at 10 M in combination with TRAIL at 30 ng/ml against U937 cells (cell viability averaged 35 ± 13.6 and 77 ± 4.6% in the absence and presence of zVAD-fmk, respectively; n = 4) (Fig. 5A). Preincubation of the cells in I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 57 Fig. 3. Normal human monocytes were exposed for 24 h to various concentrations of AAF-cmk followed by 24 h incubation with AAF-cmk and/or TRAIL. Cytostatic/cytotoxic effect was assessed by crystal violet staining. Data are presented as a relative viability (percentage of untreated controls). Each bar represents mean ± S.D. All groups marked with (∗) are significantly different from the controls (P < 0.05). the presence of the pan-caspase inhibitor zVAD-fmk also diminished the cytotoxicity caused by the combination of AAF-cmk at 10 M with TNF at 500 ng/ml in U937 cells (cell viability averaged 40 ± 7.6 and 70 ± 3.5% in the absence and presence of zVAD-fmk, respectively; n = 4) (Fig. 5B). lanes 3 and 6). Cleavage of PARP from 116 to 85 kDa, was also the strongest in the combined group. Each drug given alone in experimental concentrations showed only weak or no degradation of PARP (Fig. 6, lanes 2 and 5). 4. Discussion 3.4. PARP cleavage analysis Cleavage of the nuclear repair enzyme poly(ADP-ribose) polymerase is considered a very sensitive maker of caspase activation and apoptosis . To examine whether PARP cleavage is enhanced following combination treatment of AAF-cmk with TRAIL or TNF, an immunoblotting analysis was performed. The cells constitutively expressed PARP, but after treatment with combination of AAF-cmk with TRAIL or TNF, proteolytic fragments of PARP were found (Fig. 6, Table 1 Percentage of apoptotic, necrotic and living U937 cells, after 24 h pretreatment with AAF-cmk and 24 h incubation with a control solvent or AAF-cmk and/or TRAIL Living Apoptotic Necrotic Control AAF-cmk (20 M) TRAIL (30 ng/ml) TRAIL + AAF-cmk 95.8 2.8 1 70.1 24.7 4.1 74.2 19.4 5.52 17.9 55.8 26.3 Data was obtained in each group for 10 000 cells separated from other events based on light scatter characteristics. Apoptotic, necrotic and living cells were discriminated as described in “Section 2”. The combinations of AAF-cmk and TRAIL were significantly different from single agent-treated groups (chi square <0.005). In this report, we demonstrate that AAF-cmk, an inhibitor of TPPII, potentiates cell death induced by TNF and TRAIL in human leukemic U937 cells. Increased cell death is primarily caused by an increased apoptosis induction as shown by flow cytometric analysis, caspase inhibitor studies, PARP cleavage and electron microscopy. Necrosis was also shown in the combination group as shown in Table 1, which could mean that after 24 h treatment the apoptoic cells deteriorated to the extent that they took up propidium iodide. The viability of tumor cells treated with AAF-cmk is somewhat lower in cytostatic/cytotoxic assays with MTT (Fig. 1A and B) as compared to cytometric analyses (Table 1). This discrepancy can be explained by the fact that MTT assay shows both antiproliferative and cytotoxic effects of AAF-cmk, while cytometric analysis refers only to the latter. The physiological role of TPPII in mammalian cells is still unresolved. It was suggested that this protease could replace some of the functions of proteasome [11,12]. Although very attractive, this hypothesis was recently challenged [20,40]. However, it could be suspected that the function of TPPII inhibitors could also imitate the activity of proteasome inhibitors. Moreover, inhibition of the proteasome by PSI potentiated the antitumor effects of both TRAIL and TNF 58 I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 Fig. 4. Ultrastucture of U937 cells treated for 24 h with AAF-cmk (20 M) followed by 24 h incubation with AAF-cmk and/or TRAIL (100 ng/ml) and/or with TNF (500 ng/ml) (magnification 5200×). Control group (A): The utlrastructure of U937 cell—the polymorphic nucleus with eu- and heterochromatin and the cytoplasm with well preserved organelles. TRAIL group (B): The apoptotic cell with peripheral nuclear chromatin condensation and reduced amount of organelles in cytoplasm. TNF group (C): The cell in more advanced apoptosis than in Fig. 4B. The chromatin is condensed the cytoplasm is vacuolised with few organelles. AAF-cmk/TRAIL group (D): Two apoptotic cells, left—the early apoptotic cell with peripheral chromatin condensation and cytoplasm vacuolization; right—the advanced apoptotic cell with condensed chromatin and disorganized cytoplasm. AAF-cmk/TNF group (E): The advanced apoptotic cell. The nucleus is budding and breaking up to form components of apoptotic bodies. AAF-cmk group (F): The U937 cell with no evident ultrastructural features of apoptosis. I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 Fig. 5. The influence of caspase inhibition with zVAD-fmk on the AAF-cmk/TRAIL (A) and the AAF-cmk/TNF (B) mediated cytostatic/cytotoxic activity revealed by a standard MTT assay on U937 cells. Data are presented as a relative viability (percentage of untreated controls). Each bar represents mean ± S.D. All groups marked by (∗) are significantly different from the control (P < 0.05). 59 . Despite unknown physiological role of TPPII, there is some data indicating that it could complement or mimic the proteolytic function of proteasome. Thus, it can be speculated that inhibition of TPPII could also induce similar antitumor effects as proteasome inhibitors. AAF-cmk irreversibly blocks TPPII and reversibly the lysosomal TPPI . It also blocks, to a certain degree, the chymotrypsin-like activity of the proteasome . When TPPII is blocked and oligopeptides are not effectively degraded it could lead to accumulation of these peptides and disturb the cell’s environment and interfere with cellular metabolic pathways. In our experiments, the cytostatic/cytotoxic activity of AAF-cmk with TRAIL and AAF-cmk with TNF could be reduced by incubation with zVAD-fmk suggesting that activation of caspases could be responsible for the cell death induced by this combination treatment. The results obtained with flow cytrometric studies found also confirmation in ultrastructural studies where increased number of apoptotic cells has been observed in the combination groups. Altogether, our studies indicate that AAF-cmk is capable of sensitizing human leukemic cells to proapoptotic effects of TRAIL and TNF. The present study provides the first evidence that, in addition to the proteasome, also TPPII might play a role in TRAIL and TNF mediated apoptotic pathways. Whatever mechanism is responsible for this activity, AAF-cmk seems to be a potent sensitiser for antitumor effects of TNF-family cytokines. Fig. 6. SDS-PAGE (7.5%) of the caspase-specific substrate PARP in either control U937 whole lysates (lane 1), or the same cells treated with: 100 ng/ml TRAIL, lane 2; AAF-cmk (20 M), lane 4; TNF 500 ng/ml), lane 5 and combination of TRAIL/AAF-cmk, lane 3; as well as combination of TNF with AAF-cmk, lane 6. Treatment of U937 cells by combination of AAF-cmk and TRAIL/TNF correlated with PARP cleavage. Filled arrows indicate uncleaved forms of PARP and opened arrows indicate the cleaved forms of these protein. Actin was used as the loading control. 60 I. Młnarczuk et al. / Leukemia Research 28 (2004) 53–61 Acknowledgements This work has been supported by KBN Grant 4 P05A 084 16 to CW, by the Medical University Grant 01-1M15NS2-2002 to IM and by the CMKP grant 501-2-1-03-48/02 to GH. References  Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem 1998;67:425.  Wójcik C, Stokłosa T, Giermasz A, Goł˛ab J, Zagożdżon R, Kawiak J, et al. Apoptosis induced in L1210 leukemia cells by an inhibitor of the chymotrypsin-like activity of the proteasome. Apoptosis 1997;2:455.  Wójcik C. Proteasomes in apoptosis: villains or guardians? Cell Mol Life Sci 1999;56:908.  Wójcik C, Schroeter D, Stoehr M, Wilk S, Paweletz N. 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