Interferon-alpha suppresses proliferation of chronic myelogenous leukemia cells K562 by extending cell cycle S-phase without inducing apoptosis

Blood Cells, Molecules, and Diseases 32 (2004) 262 – 269 www.elsevier.com/locate/ybcmd

Interferon-alpha suppresses proliferation of chronic myelogenous leukemia cells K562 by extending cell cycle S-phase without inducing apoptosis Dana Grebenˇova´, a Katerˇina Kuzˇelova´, a Ota Fuchs, a Petr Halada, b Vladimı´r Havlı´cˇek, b Iuri Marinov, a and Zbyneˇk Hrkal a,* a

Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, 128 20, Prague-2, Czech Republic b Institute of Microbiology AS CR, Prague-2, Czech Republic Submitted 17 September 2003; revised 23 October 2003 (Communicated by M. Lichtman, M.D., 30 October 2003)

Abstract We examined the effects of interferon-alpha (IFN-a) treatment on the growth, cell cycle, proliferation, and apoptotic parameters as well as adhesive properties and proteome of chronic myelogenous leukemia (CML)-derived K562 cells. IFN-a treatment (200 to 600 U/ml, 24 to 72 h) suppressed growth and caused accumulation of K562 cells in the S-phase of cell cycle (increase in S-phase cells by up to 52% in comparison with the untreated controls) at the expenses of cells in G1-phase. No transition of cells to G0-phase occurred as followed from Ki-67 protein determination. Although the level of chimeric gene product, BCR-ABL mRNA coding for BCR-ABL protein with antiapoptotic properties, decreased by 30%, apoptosis was not triggered as judged from Annexin-V, APO2.7, and TUNEL assays. Adhesion of K562 cells to fibronectin-coated surfaces increased by up to 52% as determined by calcein assay. The proteomic analysis (2-D electrophoresis in combination with mass spectrometry, MALDI-MS) revealed a single protein, ubiquitine cross-reactive protein (UBCR), whose level markedly increased due to IFN-a treatment. The ubiquitination-like directed degradation processes may thus play a role in the mechanism of IFN-a antiproliferative effects. D 2003 Elsevier Inc. All rights reserved. Keywords: K562; IFN-a; Cell cycle; Apoptosis; Adhesion; Proteomic analysis

Introduction IFN-a belongs to a family of proteins secreted by eukaryotic cells following challenge by viruses and other infectious agents [1]. In addition to the antiviral effects [2], interferon-alpha (IFN-a) exerts pleiotropic cellular effects including inhibition of cell proliferation [3], induction of apoptosis [4,5], and modulation of the immune system [6]. IFN-a is able to induce a state of tumor dormancy and to control the chronic phase of CML. IFN-a causes complete genetic remission in 10% to 20% patients with chronic phase CML [7], in some patients (approximately 7%) even without further therapy [8]. The main problems encountered * Corresponding author. Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, U Nemocnice 1, 128 20 Prague-2, Czech Republic. Fax: +420-224918390. E-mail address: [email protected] (Z. Hrkal). 1079-9796/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2003.10.008

with IFN-a therapy are its toxicity at the optimal therapeutic doses and low efficiency in blast phase of CML. Although the therapeutic efficacy of IFN-a for the treatment of chronic phase CML is well established, the mechanism by which IFN-a exerts the direct antitumor action on CML cells is an open question. It has been reported that IFN-a binding to type I IFN receptor activates JAK/STAT pathway leading to the formation of interferon-stimulated gene factor 3 (ISGF3) complex, which activates the interferon-stimulated responsive element (ISRE) sequences on the related genes [9]. Induction of cell cycle inhibitors is the commonly accepted mechanism of IFN-a antiproliferative effects [10]. Frequently IFN-a treatment leads to G1 or G0 arrest in IFNa responsive cells or induces apoptosis [5,10]. The chronic myelogenous leukemia (CML)-derived K562 cells [11] bear an abnormal Ph chromosome characterized by the abnormal fusion gene BCR-ABL of the b3a2 type [12]. This gene expresses mRNA, which is translated to

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the chimeric 210-kDa fusion protein BCR-ABL whose constitutively elevated Abl kinase activity is responsible for the known resistance of K562 cells to anticancer druginduced apoptosis [13]. In addition to abnormal gene BCRABL, K562 cells also have mutated gene for the p53 protein [14] whose proapoptotic or cell cycle regulatory function is thus annulled. These combined mutations make of K562 cells a suitable model for the ‘in vitro’ studies of CML blast phase treatment. In our recent paper, we deal with the mechanism of cytotoxic effects imposed on K562 cells by the photodynamic treatment showing that an early apoptotic process, which is triggered by this procedure, does not proceed to the terminal phase and is followed by the cell necrosis [15]. In this communication, we report on the growth inhibitory effect imposed on wild-type K562 cells by IFN-a treatment and its mechanism.

Materials and methods Chemicals Human recombinant interferon-aA/D, propidium iodide, RPMI-1640, mouse monoclonal antibodies to cyclin A, hactin, and p21WAF1/Cip1, rabbit antibodies to cyclins D1 and E, were purchased from Sigma (Prague, Czech Republic) and rabbit antibody to retinoblastoma protein (pT821) was purchased from BioSource Europe, S.A., Belgium. The APO 2.7 kit was purchased from Coulter/Immunotech A.S., Prague, Czech Republic; FITC-conjugated mouse anti-human Ki-67 antibody set was purchased from BD Biosciences; Annexin-V-FLUOS staining kit and In situ cell death detection kit, fluorescein, were purchased from Roche Applied Science (Mannheim, Germany); Vybrant (TM) cell adhesion kit was purchased from Molecular Probes Europe, BV (Leiden, The Netherlands); and FIX and PERM cell permeabilization kit was purchased from An Der Grub, Kaumberg, Austria. Cell culture K562 cells were purchased from the European Collection of Animal Cell Cultures (Salisbury, UK) and cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 units/ml penicillin, 50 Ag/ml streptomycin, and 2 mM L-glutamine at 37jC in 5% CO2 humidified atmosphere. Cells were diluted to a density of 2  105 cells per ml three times a week. Cell viability The effect of IFN-a treatment on the cell viability was assessed by flow cytometry of propidium iodide (PI)-stained cells using Coulter Epics XL flow cytometer as previously described for photodynamic therapy experiments [16] and by visual cell counting (trypan blue exclusion test).

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3H-Thymidine proliferation assay Four sample replicates of IFN-a-treated cells as well as of the appropriate controls (without IFN-a) were pulsed with 24 kBq (6-3H)-thymidine (Institute for Research, Development and Application of Radioisotopes, Prague, Czech Republic, specific activity 980 Gbq/mmol) for 4 h and the cells were collected using Scatron cell harvester. The incorporated radioactivity into the newly synthesized DNA was measured with the beta scintillation counter. The mean values of the quadruplicates expressed in counts per minute were calculated, and the suppression of DNA synthesis caused by IFN-a was given as the percentage of control values [17]. Cell cycle analysis The cells (1  106) were collected by centrifugation, suspended in 70% ethanol, and incubated for 30 min at room temperature in 1 ml of the modified Vindelovs propidium iodide buffer (10 mM Tris, pH 8, 1 mM NaCl, 0.1% Triton X-100, 20 Ag/ml PI, and 10 Kunitz units of ribonuclease A). The red fluorescence excited at 488 nm was then measured using Coulter Epics XL flow cytometer. The histograms were analyzed using the G1/G2M Only Fit method. Detection of cell cycle antigen Ki-67 Ki-67 protein expression was analyzed using the direct immunofluorescence method. After the treatment with IFNa for defined periods (0 to 72 h), 1  106 cells were harvested and stained following the protocol for Intracellular Staining With FIX and PERM (BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany) using FITC-conjugated anti-Ki-67 antibody. The fraction of Ki-67 antigen expressing cells was determined by flow cytometry. Exposition of mitochondrial membrane antigen 7A6 Expression of mitochondrial membrane neoantigen 7A6 appearing in apoptotic cells was measured in IFN-a-treated cells as well in appropriate controls by flow cytometry employing antibody APO 2.7 [18]. Assessment of apoptosis by TUNEL assay The TUNEL assay was performed employing the In situ cell death detection kit, fluorescein (Roche Applied Science), following the standard manufacturer’s protocol. The extent of DNA labeling with fluorescein-dUTP was determined by flow cytometry. Annexin-V apoptosis assay The analysis of phosphatidylserine exposition on the outer side of plasma membrane of IFN-a-treated cells as

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well as of controls was performed using Annexin-V-FLUOS staining kit (Roche Applied Science), employing flow cytometry. The kit consists of Annexin-V-fluorescein and propidium iodide for the differentiation of apoptotic and necrotic cells. The recommended manufacturer’s protocol was obeyed. Electrophoresis and Western blotting The cells (5  106) were suspended in a lysis buffer containing 0.15 M NaCl, 1 mM PMSF (phenylmethanesulfonyl fluoride), 1 Al/100 Al of phosphatase inhibitor cocktail 2, and 0.5% Triton X-100 (Sigma) and kept for 20 min on ice. Protein samples were heated to 100jC for 4 min in the presence of 5% 2-mercaptoethanol, chilled, and subjected to one-dimensional electrophoresis (PAGE) in 12% or 15% gel with SDS according to Laemmli [19]. As a rule, 10 Ag total protein was applied to each well. Proteins were transferred to Hybond-ECL membrane at 10 V for 30 min. Nitrocellulose membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated for 1 h with the appropriate antibody in TBS-T. Immunoblots were reacted in parallel with anti-h-actin as the IFN-a unaffected controls. After washes with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies. The antigens were detected using the enhanced chemiluminescence Western blotting detection system ECL + PLUS (Amersham Pharmacia Biotech, UK) according to the manufacturer’s instructions and visualized by autoradiography on X-ray film. Intensity of protein bands was compared by densitometry evaluation using AIDA Version 2.1, 1D-Evaluation system (Raytest GmbH, Germany). Cell adhesion assay Adhesion of IFN-a-treated K562 as well as control cells to fibronectin surface was determined using Vybrantk cell adhesion kit employing the manufacturer’s protocol. The microtitration plate wells (Microfluor 2 Flat Bottom Microtiter Plates, Dynex Technologies, Chantilly, VA, USA) were coated with fibronectin (100 Al fibronectin, 25 Ag/ml in PBS) at 4jC overnight. The cells (5  106/ml) were labeled with calcein-AM (5 AM, 37jC, 30 min). The cell density was adjusted to 1  106 cells/ml and 100 Al aliquots of cell suspension were transferred to the wells and incubated for 2 h at 37jC. Finally, fluorescence of adhering cells was measured (excitation 485 nm, emission 520 nm) using FluoStar Galaxy fluorescence microplate reader (BMG Labtechnologies, GmbH, Offenburg, Germany).

performed on cDNA templates generated by reverse transcription (Superscript II, Gibco, Life Technologies, Rockville, MD, USA) from 2 Ag of DNase I treated total RNA in a 20-Al reaction volume. The BCR-ABL fusion transcript was amplified using bcr-2-ex-sense primer 5V-TTCAGAAGCTTCTCCCTG-3V and abl-2-ex-antisense primer 5V-CTCCACTGGCCACA-AAT-3V. For PCR amplifications, the PTC-200 Peltier Thermal Cycler (MJ Research Inc., Waltham, Massachusetts) was employed. PCR products were examined after electrophoresis in ethidium bromide stained 1.5% agarose gel, and fluorescence signals were evaluated with Fuji FLA-2000 phosphoimager (Raytest GmbH) and quantitated by AIDA version 2.1 software (Raytest GmbH). The results are the average of three independent experiments. Two-dimensional electrophoresis (2-DE) The linear immobilized pH gradient gels (180  3.3  0.5 mm), ReadyStripk IPG strips pH 3 to 10 (BioRad Hercules, CA, USA), were placed in the Immobiline DryStrip Reswelling Tray (Amersham Biosciences, Vienna, Austria) and rehydrated overnight in 315 Al of the cell lysate containing 150 Ag proteins, 8 M urea, 2% CHAPS, 65 mM DTT, 0.2% Bio-Lyte 3-10, and 0.01% bromphenol blue. Isoelectric focusing (IEF) was performed using Multiphor II system (Amersham Biosciences) at 6000 V for a total 70 kV/h at 20jC. After the first dimension run, the strips were equilibrated with a solution containing Tris – HCl pH 8.8 (0.375 mM), urea (6 M), glycerol (20% v/v), DTT (2% w/v), SDS (2% w/v), and 0.01% bromphenol blue. Resulting free SH groups were blocked in the second equilibration step in which DTT was replaced with iodoacetamide (2.5% w/v). The second dimension run was performed using a Protean II xi vertical electrophoresis system (BioRad Hercules), gel size 200  200  1.5 mm, in a Laemmli-SDSdiscontinuous buffer system [19] and polyacrylamide gel gradient (9% to 16% T, 2.6% C). The equilibrated first dimension gel was placed directly onto the second dimension gel and overlaid with a solution containing 0.4% agarose in Tris – glycine – SDS pH 8.3 buffer (25 mM Tris, 192 mM glycine, 0.1% w/v SDS) heated to 70jC. Electrophoresis was performed at a constant current 35 mA/gel for 5 h at 10jC. The gels were fixed in a solution containing ethanol (40% v/v) and acetic acid (10% v/v) before staining with silver nitrate. The analytical gels (protein load 150 Ag) were silver stained as described by Bjellqvist et al. [20] and the preparative gels (protein load 1500 Ag) according to Schevchenko et al. [21]. 2-D PAGE gel imaging

Analysis of BCR-ABL gene transcript Total RNA was isolated from K562 cells using RNazol B (Tel-Test Inc, Friendswood, TX, USA) according to manufacturer’s recommendations. Semiquantitative RT-PCR was

Silver-stained gels were scanned using a scanner UMAX PowerLook III (1200  2400 dpi) (Umax Systems GmbH, Germany). The 2-DE image computer analysis was carried out employing AIDA Proteomix 2D Protein Gel Matching

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Fig. 1. Effect of IFN-a on the growth of K562 cells. The cells were cultured in the absence and presence of 200 to 800 U/ml IFN-a for 72 h and regularly counted.

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acetate, 1 mM CaCl2, 10% acetonitrile (MeCN), and sequencing grade trypsin (50 ng/Al; Promega, Madison, WI, USA). The digestion was carried out overnight and resulting peptides were extracted with 30% MeCN/0.5% trifluoroacetic acid [22]. One microliter aliquot of each peptide mixture was deposited on the target, and 1 Al of a matrix solution (saturated a-cyano-4-hydroxycinnamic acid in 30% MeCN/0.2% trifluoroacetic acid) was added. Positive ion mass spectra were measured on an MALDI reflectron timeof-flight mass spectrometer BIFLEX (Bruker-Franzen, Bremen, Germany) equipped with a nitrogen laser (337 nm) and gridless delayed extraction ion source. Protein spots were identified by searches of peptide mass maps in a nonredundant database NCBI using the search program ProFound (http://prowl.rockefeller.edu/cgi-bin/ProFound) [22].

Results Version 6.00 software (Raytest GmbH). Individual gels of IFN-a-treated cells were compared with the gels of untreated cells and vice versa. The differences in spot volumes greater than 50% (integrated optical density over spot area) were considered significant. The findings are based on the evaluation of at least ten 2-D PAGE experiments. The isoelectric points and molecular weights of individual proteins were approximated using polypeptide 2-D SDS PAGE Standards (BioRad, Richmond, USA). MALDI mass spectrometry and protein identification Silver-stained protein spots were excised from the gel and the gel pieces reconstituted in a cleavage buffer containing 2-sulfanylethan-1-ol, 50 mM 4-ethylmorpholine

We examined the effects of IFN-a treatment on the growth of wild-type K562 cells. In Fig. 1, the time dependence of cell density is shown for several IFN-a doses. It follows that doses of 200 U/ml and higher suppressed progressively the cell growth. The decrease in cell growth was not due to the IFN-a cytotoxicity since fractions of propidium iodide-positive cells (around 5%) as well as number of Trypan blue-positive cells (3% to 4%) found in untreated controls did not increase during 48 h incubation with IFN-a at concentrations 200 to 800 U/ml. To examine the effect of IFN-a on the proliferation of K562 cells, we used 3H-thymidine incorporation into the cell DNA. The extent of 3H-thymidine incorporation was reduced by 40% to 50% following 48 h and by approxi-

Fig. 2. Effect of IFN-a on the proliferation of K562 cells assayed by 3H-thymidine incorporation. The cells were incubated with 200 to 800 U/ml IFN-a for up to 72 h, and the cell aliquots in quadruplicates were incubated with 3H-thymidine for 4 h. The cells were harvested and the 3H-thymidine radioactivity was measured in counts/min. The data are normalized to the untreated controls (100%).

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mately 75% following 72 h incubation with 200 to 800 U/ml IFN-a in comparison with the untreated controls (Fig. 2). As apoptosis is considered a possible mechanism by which IFN-a executes its anticancer effects, we determined several apoptotic parameters of K562 cells during their treatment with IFN-a. The amount of TUNEL-positive cells

was approximately 1% in the control and their number did not increase during 72 h cultivation in the presence of 200 and 400 U/ml IFN-a. Similarly, the subG1 peak was not observed on cell cycle histograms (see Fig. 3). Further, almost no AnnexinV-positive cells have been detected by flow cytometry (fractions of AnnexinV-positive cells were

Fig. 3. DNA content analysis of the controls (panels 1 to 4) and IFN-a-treated K562 cells (panels 5 to 7). The cells were incubated with 200 U/ml IFN-a for up to 72 h, and in time intervals the flow cytometry histograms were obtained of permeabilized cells stained with propidium iodide solution (containing RNAse). The control cells were treated equally with the exception of IFN-a addition. The cell cycle analysis was performed by G1/G2M Only Fit method (see enclosures).

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about 1%), and the fraction of APO2.7-positive cells (cells exhibiting the mitochondrial apoptosis-related antigen 7A6) was only 3% to 5% in both cases after 48 h incubation with 200 to 800 U/ml IFN-a. We conclude that apoptosis is not a mechanism responsible for the suppressed growth of wildtype K562 cells caused by IFN-a treatment. Another possible mechanism by which cell growth can be limited is the transition of cells from G1 to G0 phase. We therefore determined the fraction of cells in the overall active phases of cell cycle (G1, S, G2, and M) by flow cytometry using the antibody against the proliferation-related protein Ki-67 (MKI-67 protein). It followed that the active phases of cell cycle were not affected by IFN-a treatment as the fraction of Ki-67-positive cells represented 91% and 96% both in the control and IFN-a-treated cells (200 U/ml, 48 and 72 h). Thus, no transition of cells from G1 to G0 phase occurred. Subsequently, we examined the effect of IFN-a treatment on the cell cycle distribution of K562 cells. In Fig. 3, a series of flow cytometry histograms are presented showing changes in DNA distribution during K562 treatment with 200 U/ml IFN-a. Analysis of DNA distribution by G1/G0-G2/M Only Fit method showed pronounced changes that occurred during IFN-a treatment. The fractions of cells in G1 as well as G2/M phase decreased while the fraction in S phase increased (see enclosures). Since we assumed that suppressed proliferation of K562 cells may be due to the perturbation of certain cell cycle regulatory proteins, we determined by Western blotting the effects of IFN-a treatment on the expression of cyclins D1, E, and A, the cell cycle inhibitor, protein p21, and on the phosphorylation state of Rb protein (Fig. 4). We found no increased expression of cyclins D1 and E and the cell cycle inhibitor p21. On the other hand, an increase by 30% in the level of cyclin A as well as in the phosphorylation state of Rb protein has been observed.

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Fig. 5. Effect of IFN-a on the adhesive properties of K562 cells. The cells were treated with IFN-a (200 and 400 U/ml) for 72 h. In time intervals, the relative numbers of cells adhering to microtitration plate wells previously coated with fibronectin were determined by fluorescence measurement of calcein-AM-stained cells (Vybrant cell adhesion assay). The data were normalized to the adherent fractions of untreated K562 cells (100%).

The clinically relevant question, whether IFN-a can affect expression of BCR-ABL gene in blast phase CML cells, has been addressed by determining BCR-ABL mRNA level following bcr-abl gene amplification by PCR. The amounts of BCR-ABL mRNA decreased by 30.4% F 4.4% following K562 cells IFN-a treatment (200 U/ml, 48 h). We further investigated the effect of IFN-a treatment on the adhesive properties of K562 cells. The fraction of cells adhering to fibronectin surface increased by up to 53% due to IFN-a treatment in dependence of IFN-a concentration and incubation time (Fig. 5). Finally we attempted by 2-D electrophoresis to uncover proteins that were differentially expressed in K562 cells due to the IFN-a treatment. Fig. 6 shows the section of 2-D electrophoretic polypeptide map of

Fig. 4. Effect of IFN-a on the expression of cell cycle proteins and inhibitors determined by Western blotting. The cells were incubated with IFN-a for up to 72 h, they were lysed, subjected to SDS electrophoresis, and blotted to nitrocellulose membrane. The blots were incubated with specific primary antibodies, developed with peroxidase-conjugated secondary antibody, and detected by ECL.

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Fig. 6. Proteomic analysis of IFN-a-treated K562 cells. Two-dimensional electrophoresis (a section) of the lysate of K562 cells untreated (panel A) and treated with IFN-a (panel B; 200 U/ml, 48 h) stained with silver. The protein spot newly appearing on the electrophoreogram of IFN-a-treated cells is depicted by an arrow.

K562 cells that reveals a newly appearing protein spot after treatment with 200 U/ml IFN-a for 48 h. This protein was identified by mass spectrometry (MALDI-MS) as the ubiquitine cross-reactive protein (UCRP).

Discussion We employed the K562 cell line as the model of CML blast phase, as this cell line not only bears the Philadelphia chromosome and expresses BCR-ABL gene, but it also possesses an additional mutation in gene p53 resulting in the expression of nonfunctional protein p53. This protein normally functions as the anti-oncogenic compound inducing G1/S cell cycle arrest following DNA damage. IFN-a treatment suppressed growth of K562 cells; however, neither apoptosis was triggered nor necrosis occurred. In the recent paper dealing with IFN-a pro-apoptotic effects on K562 cells [23], the authors have shown pronounced apoptosis in these cells due to IFN-a treatment and even in the untreated controls. In contrast to these results, we did not find any increase of apoptosis above the (low) level

found in untreated controls. The explanation we offer is that cell lines behavior is much dependent on the particular strand employed; our cells obtained from European Collection of Animal Cell Cultures, UK, are probably closer to the original type established by Lozzio and Lozzio [11]. Possible transition of cells from G1 to G0 phase has been excluded by the measurements of proliferation-related protein Ki-67, which is present in all active phases of the cell cycle. The fraction of cells expressing this protein approached completion and was not affected by IFN-a treatment, suggesting that no transition of cells to G0 phase occurred. The suppressive effect must therefore rely in a perturbation of the cell cycle. The decrease in the G1 phase as well as G2/M phase cells due to IFN-a treatment was compensated by an increase of S-phase cells. Since the cell density as well as overall DNA replication rate decreased, the possible explanation is that the cells were either arrested in S-phase or the DNA replication was slowed down. We tried to uncover the molecular mechanism, which is behind the antiproliferative effects of IFN-a. However, neither cyclins D1 and E, which drive cells through G1/S phase restriction point, nor the cell cycle inhibitor protein p21 were up-regulated. Thus, the IFN-a antiproliferative mechanism does not involve the G1 phase of the cell cycle. To the contrary, an increase of S-phase cells supports the idea on slowing down the DNA replication leading to accumulation of cells in S-phase. In accordance with this theory, we observed an increased level of cyclin A and increased phosphorylation of retinoblastoma protein due to IFN-a treatment. The decrease of G2/M phase cells (Fig. 3) resulting from the delay of cells in S phase leads to the lower number of replicating cells and therefore to the suppressed proliferation. Cell adhesion to extracellular matrix is mediated by the transmembrane proteins, the integrins. Following interaction with extracellular ligands h1-integrin associates with several cytoskeletal proteins including a-actinin, talin, vinculin, and tensin to form multimolecular ‘focal adhesions’ complex. Abnormal circulation and unregulated proliferation of CML progenitors is related, at least in part, to BCR-ABL proteininduced abnormalities in h1 integrin-mediated adhesion and signaling [24]. CML progenitors were shown to adhere significantly less to a4h1 and a5h1 binding regions of fibronectin, indicating that h1 integrin receptor function is abnormal in CML. The BCR-ABL protein exhibits enhanced binding to F-actin, which may alter the integrin – cytoskeletal interactions [25]. The decreased level of BCRABL kinase in K562 cells due to IFN-a treatment leads presumably to partial restoration of h1 integrin –cytoskeletal interactions, which contribute to their increased adhesion to fibronectin surfaces. Employing two-dimensional electrophoresis, we revealed the single distinct protein spot in K562 cell lysate, which was markedly enhanced on IFN-a treatment. Using mass spectrometry (MALDI-MS), this protein was identified as the ubiquitine cross-reacting protein (UCRP/ISG15). Induc-

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tion of this 17 kDa ubiquitine-like protein and its subsequent conjugation to the cellular targets is the earliest response of cells to the type I interferons [26]. Proteins conjugated to UCRP are either modulated or targeted for processing through the proteasome [27]. It was reported that conjugates of UCRP distribute in a cytoskeletal pattern in both uninduced and interferon-treated A549 lung carcinoma cells, while significant increase in the sequestration of UCRP conjugates on intermediate filaments accompanied interferon induction [28]. It can be speculated that increased expression of UCRP in K562 cells following IFN-a treatment may be related to IFN-a antiproliferative activity mediated by a specific degradation of intermediate filaments protein compounds.

Acknowledgments The authors wish to thank Mrs. J. Sedlmaierova´ for the expert technical assistance. The work was supported by grants of the Grant Agency of the Czech Republic No 303/ 01/1445 and Internal Grant Agency of the Ministry of Health, Czech Republic No NL-7681-3.

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