Potential Pathogenetic Implications of Cyclooxygenase-2 Overexpression in B Chronic Lymphoid Leukemia Cells
2005; Elsevier BV; Volume: 167; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)61244-8
ISSN1525-2191
AutoresPaola Secchiero, Elisa Barbarotto, Arianna Gonelli, Mario Tiribelli, Carlotta Zerbinati, Claudio Celeghini, Claudio Agostinelli, Stefano Pileri, Giorgio Zauli,
Tópico(s)NF-κB Signaling Pathways
ResumoEvidence suggests that cyclooxygenase-2 (COX-2) increases tumorigenic potential by promoting resistance to apoptosis. Because B chronic lymphoid leukemia (B-CLL) cells exhibit a defective apoptotic response, we analyzed CD19+ B lymphocytes purified from the peripheral blood of B-CLL patients. Microarray analysis showed a variable (up to 38-fold) increase in the steady-state mRNA levels of COX-2 in B-CLL lymphocytes compared with normal CD19+ B lymphocytes. The up-regulation of COX-2 in B-CLL cells was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. Moreover, immunohistochemical analysis of B-CLL bone marrow infiltrates confirmed clear expression of COX-2 in leukemic cells. Ex vivo treatment with the COX-2 inhibitor NS-398 significantly decreased the survival of leukemic cells by increasing the rate of spontaneous apoptosis in 13 of 16 B-CLL samples examined, but it did not affect the survival of normal lymphocytes. Pretreatment with NS-398 significantly potentiated the cytotoxicity induced by chlorambucil in 8 of 16 B-CLL samples examined. Moreover, although recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Apo2L showed little cytotoxic effect in most B-CLL samples examined, pretreatment with NS-398 sensitized 8 of 16 B-CLL samples to TRAIL-induced apoptosis. Taken together, our data indicate that COX-2 overexpression likely represents an additional mechanism of resistance to apoptosis in B-CLL and that pharmacological suppression of COX-2 might enhance chemotherapy-mediated apoptosis. Evidence suggests that cyclooxygenase-2 (COX-2) increases tumorigenic potential by promoting resistance to apoptosis. Because B chronic lymphoid leukemia (B-CLL) cells exhibit a defective apoptotic response, we analyzed CD19+ B lymphocytes purified from the peripheral blood of B-CLL patients. Microarray analysis showed a variable (up to 38-fold) increase in the steady-state mRNA levels of COX-2 in B-CLL lymphocytes compared with normal CD19+ B lymphocytes. The up-regulation of COX-2 in B-CLL cells was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. Moreover, immunohistochemical analysis of B-CLL bone marrow infiltrates confirmed clear expression of COX-2 in leukemic cells. Ex vivo treatment with the COX-2 inhibitor NS-398 significantly decreased the survival of leukemic cells by increasing the rate of spontaneous apoptosis in 13 of 16 B-CLL samples examined, but it did not affect the survival of normal lymphocytes. Pretreatment with NS-398 significantly potentiated the cytotoxicity induced by chlorambucil in 8 of 16 B-CLL samples examined. Moreover, although recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL)/Apo2L showed little cytotoxic effect in most B-CLL samples examined, pretreatment with NS-398 sensitized 8 of 16 B-CLL samples to TRAIL-induced apoptosis. Taken together, our data indicate that COX-2 overexpression likely represents an additional mechanism of resistance to apoptosis in B-CLL and that pharmacological suppression of COX-2 might enhance chemotherapy-mediated apoptosis. Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease with one-third of patients never requiring treatment, whereas in other patients, the disease progresses at a variable rate.1Dighiero G Binet JL When and how to treat chronic lymphocytic leukemia.N Engl J Med. 2000; 343: 1799-1801Crossref PubMed Scopus (111) Google Scholar, 2Pangalis GA Vassilakopoulos TP Dimopoulou MN Siakantaris MP Kontopidou FN Angelopoulou MK B-cronic lymphocytic leukemia: practical aspects.Hematol Oncol. 2002; 20: 103-146Crossref PubMed Scopus (45) Google Scholar CLL represents a quintessential example of human malignancies that are caused primarily by defects in apoptosis.3Masdehors P Merle-Beral H Maloum K Omura S Magdelenat H Delic J Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes.Blood. 2000; 96: 269-274Crossref PubMed Google Scholar, 4Reed JC Kitada S Kim Y Byrd J Modulating apoptosis pathways in low-grade B-cell malignancies using biological response modifiers.Semin Oncol. 2002; 29: 10-24Crossref PubMed Scopus (46) Google Scholar Defects in apoptotic pathways contribute to chemoresistance, rendering tumor cells less sensitive to the cytotoxic actions of currently available anticancer drugs. Cyclooxygenase (COX)-1 and -2, also known as prostaglandin H synthases (PTGSs), catalyze one of the rate-limiting steps in the prostanoids biosynthesis.5Ueno N Murakami M Tanioka T Fujimori K Tanabe T Urade Y Kudo I Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.J Biol Chem. 2001; 276: 34918-34927Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, 6FitzGerald GA Patrono C The coxibs, selective inhibitors of cyclooxygenase-2.N Engl J Med. 2001; 345: 433-442Crossref PubMed Scopus (1429) Google Scholar COX isoenzymes, which are encoded by two different genes, possess the same oxygenase and peroxidase activities and catalyze the formation of prostaglandin H2 from arachidonic acid. Despite the remarkable structural and functional homologies, COX-1 and COX-2 have been shown to preferentially couple with different isoforms of prostagladin synthases and subserve distinct functions even within the same cell.5Ueno N Murakami M Tanioka T Fujimori K Tanabe T Urade Y Kudo I Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.J Biol Chem. 2001; 276: 34918-34927Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar In particular, it has been clearly established that the COX-2 (or PTGS2) gene behaves like an immediate-early gene, being rapidly induced in response to mitogenic or inflammatory stimuli.5Ueno N Murakami M Tanioka T Fujimori K Tanabe T Urade Y Kudo I Coupling between cyclooxygenase, terminal prostanoid synthase, and phospholipase A2.J Biol Chem. 2001; 276: 34918-34927Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar The premise that COX-2 is involved in growth and progression of several types of solid cancers is strongly supported by both epidemiological and animal studies.7Kutchera W Jones DA Matsunami N Groden J McIntyre TM Zimmerman GA White RL Prescott SM Prostaglandin H synthase 2 is expressed abnormally in human colon cancer: evidence for a transcriptional effect.Proc Natl Acad Sci USA. 1996; 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345: 433-442Crossref PubMed Scopus (1429) Google Scholar, 12Hara A Yoshimi N Niwa M Ino N Mori H Apoptosis induced by NS-398, a selective cyclooxygenase-2 inhibitor, in human colorectal cancer cell lines.Jpn J Cancer Res. 1997; 88: 600-604Crossref PubMed Scopus (111) Google Scholar, 13Erickson BA Longo WE Panesar N Mazuski JE Kaminski DL The effect of selective cyclooxygenase inhibitors on intestinal epithelial cell mitogenesis.J Surg Res. 1999; 81: 101-107Abstract Full Text PDF PubMed Scopus (77) Google Scholar, 14Sawaoka H Kawano S Tsuji S Tsujii M Gunawan ES Takei Y Nagano K Hori M Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice.Am J Physiol. 1998; 274: G1061-G1067PubMed Google Scholar, 15Liu XH Yao S Kirschenbaum A Levine AC NS398, a selective cyclooxygenase-2 inhibitor, induces apoptosis and down-regulates bcl-2 expression in LNCaP cells.Cancer Res. 1998; 58: 4245-4249PubMed Google Scholar Although the role of COX-2 in lymphoid carcinogenesis is poorly defined, high levels of prostaglandins have been found in patients with lymphoma, and elevated levels of COX-2 protein have been detected in lymphoma cell lines.16Sebahoun G Maraninchi D Carcassonne Y Increased prostaglandin E production in malignant lymphomas.Acta Haematol. 1985; 74: 132-136Crossref PubMed Scopus (8) Google Scholar, 17Wun T McKnight H Tuscano JM Increased cyclooxygenase-2 (COX-2): a potential role in the pathogenesis of lymphoma.Leuk Res. 2004; 28: 179-190Crossref PubMed Scopus (96) Google Scholar Microarray technology has been previously used to profile gene expression in B-CLL and to subcharacterize patients with heterogeneous clinical outcome.18Klein U Tu Y Stolovitzky GA Mattioli M Cattoretti G Husson H Freedman A Inghirami G Cro L Baldini L Neri A Califano A Dalla-Favera R Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.J Exp Med. 2001; 194: 1625-1638Crossref PubMed Scopus (783) Google Scholar, 19Rosenwald A Alizadeh AA Widhopf G Simon R Davis RE Yu X Yang L Pickeral OK Rassenti LZ Powell J Botstein D Byrd JC Grever MR Cheson BD Chiorazzi N Wilson WH Kipps TJ Brown PO Staudt LM Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.J Exp Med. 2001; 194: 1639-1647Crossref PubMed Scopus (932) Google Scholar, 20Vallat L Magdelenat H Merle-Beral H Masdehors P Potocki de Montalk G Davi F Kruhoffer M Sabatier L Orntoft TF Delic J The resistance of B-CLL cells to DNA damage-induced apoptosis defined by DNA microarrays.Blood. 2003; 101: 4598-4606Crossref PubMed Scopus (85) Google Scholar, 21Wright G Tan B Rosenwald A Hurt EH Wiestner A Staudt LM A gene expression-based method to diagnose clinically distinct subgroups of diffuse large B cell lymphoma.Proc Natl Acad Sci USA. 2003; 100: 9991-9996Crossref PubMed Scopus (834) Google Scholar, 22Gricks CS Zahrieh D Zauls AJ Gorgun G Drandi D Mauerer K Neuberg D Gribben JG Differential regulation of gene expression following CD40 activation of leukemic compared to healthy B cells.Blood. 2004; 104: 4002-4009Crossref PubMed Scopus (37) Google Scholar, 23Stratowa C Loffler G Lichter P Stilgenbauer S Haberl P Schweifer N Dohoner H Wilgenbus KK cDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.Int J Cancer. 2001; 91: 474-480Crossref PubMed Scopus (105) Google Scholar Because it has been clearly shown that B-CLL cells are defective in their apoptotic response, in searching for new potential therapeutic targets, we have analyzed by cDNA microarray the expression profile of a set of stress- and toxicity-associated genes, including COX-2/PTGS2, in B-CLL samples in comparison with normal B lymphocytes. Because COX-2 was identified as one of the up-regulated genes, we have further characterized the expression of COX-2 protein in B-CLL and its role on cell viability. Moreover, to investigate the therapeutic potential of a strategy based on COX-2 inhibition, viability of B-CLL cells was analyzed in response to NS-398, a selective pharmacological inhibitor of COX-2 activity, used alone or in association with chemotherapeutic agents or recombinant tumor necrosis factor-related apoptosis inducing ligand (TRAIL). Peripheral blood (PB) samples were collected from 10 healthy human blood donors and 16 B-CLL patients after informed consent, in agreement with institutional guidelines. The diagnosis of B-CLL was made by PB morphology and immunophenotyping. Patients were staged according to the Rai system (Table 1). Two independent blood collections were obtained from each B-CLL patient within a clinical follow-up time of 6 to 18 months. During this period, patients were clinically stable, with a lymphocyte doubling time of >12 months, free from infectious complications, and with no evidence of disease progression. Moreover, none of the patients received cytoreductive chemotherapy both before entering this study and during the follow-up period.Table 1Clinical Characteristics of the B-CLL PatientsPatientSex/AgeMonths since diagnosisWBC*×103 cells/ml.Stage†Rai classification.Doubling time‡Months.1F/7417129.71122M/652025.60203M/738342.84204F/605947.60295F/841628.80306M/712718.61337M/7510742.81368M/53812.10459M/613646.914610M/613446.314611M/736435.804712M/634025.914913F/655031.506014M/636424.609615M/6918147.4012516M/5815827.72130* ×103 cells/ml.† Rai classification.‡ Months. Open table in a new tab PB mononuclear cells (PBMCs) were separated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane Laboratories, Hornby, Ontario, Canada). T lymphocytes and monocytes were depleted from normal PBMCs and, in some cases, from B-CLL with immunomagnetic microbeads (MACS microbeads; Miltenyi Biotech, Auburn, CA), resulting in purity of >90% CD19+ B cells, as assessed by flow cytometry. After purification, CD19+ B lymphocytes were resuspended at a cell density of 1 × 106 cells/ml in RPMI supplemented with 10% fetal calf serum (Gibco-BRL, Grand Island, NY), 2 mmol/L l-glutamine, and 40 mg/ml gentamicin sulfate in the absence of exogenous cytokines. Surface expression of CD19 and CD5 was evaluated by direct staining with fluorescein isothiocyanate (FITC)-CD19 (Becton Dickinson Biosciences, San Jose, CA) and PE-CD5 (Immunotech, Marseille, France) monoclonal antibodies (mAbs). Surface expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was evaluated by staining with primary mAbs (all from Alexis Biochemical, Lausen, Switzerland) followed by PE-conjugated anti-mouse secondary Ab (Immunotech). Nonspecific fluorescence was assessed using isotype-matched Abs. Flow cytometry analyses were performed by FACScan (Becton Dickinson). Total RNA was extracted from purified normal or B-CLL CD19+ cells by using the Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) according to the supplier's instructions. Three micrograms of total RNA were transcribed into cDNA using GEArray AmpoLabeling-LPR kit (Superarray Bioscience Corporation, Frederick, MD). An in vitro linear polymerase reaction was then performed to generate biotinylated cRNA. Labeled cDNA was hybridized with a customized cDNA microarray containing a panel of genes associated to stress and toxicity response (GEArray HS-603; SuperArray Bioscience Corporation). Hybridization was revealed by alkaline phosphatase-conjugated streptavidin, using a chemiluminescent detection kit (Superarray Bioscience Corporation). Signal intensity was measured for each microarray, the minimal intensity was used for background subtraction, and the values were normalized to the median signal value for each array. Expression levels were compared between the leukemic samples and normal CD19+ B lymphocytes, and then data were filtered for the genes whose expression level increased by at least twofold, that is, filtering the ratio for values ≥2.0. To validate the accuracy of the genes selected on the microarray, the same RNA samples used for microarray hybridization were analyzed by semiquantitative RT-PCR. In particular, COX-2 mRNA amplification was performed using the following primers: forward, 5′-TCCTGGCGCTCAGCCATACAG-3′; reverse, 5′-GTAGCCATAGCTAGC-ATTGTA-3′. Western blot was performed on approximately 5 to 10 × 106 cells/sample. To obtain cell lysates, cell suspensions were mixed with a lysing buffer containing 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS and protease inhibitors (Protease inhibitor cocktail P8340; Sigma Chemical, St. Louis, MO). Protein determination was performed by Bradford assay (Bio-Rad, Richmond, CA). Equal amounts of proteins for each sample were migrated in 10% polyacrylamide gel electrophoresis and blotted onto nitrocellulose filters. Blotted filters were blocked for 60 minutes in a 3% suspension of dried skimmed milk in phosphate-buffered saline and incubated overnight at 4°C with Abs anti-COX-2 (Cayman Chemicals, Ann Arbor, MI), caspase-3, poly (ADP-ribose) polymerase (PARP), and tubulin (Santa Cruz Biotechnology, Santa Cruz, CA). Filters were washed and further incubated for 1 hour at room temperature with 1:1000 dilution of peroxidase-conjugated anti-mouse IgG (Sigma). Specific reactions were revealed with the ECL Western blotting detection reagent (Amersham Corp., Arlington Heights, IL). Needle biopsies taken from bone marrow of three B-CLL patients at Rai stage 2 were used for immunohistochemical analysis. Sections were processed as previously described.24Pileri SA Sabattini E Agostinelli C Bodega L Rossi M Zinzani PL Marafioti T Histopathology of B-cell chronic lymphocytic leukemia.Hematol Oncol Clin N Am. 2004; 18: 807-826Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Briefly, antigen retrieval was performed by microwaving (three cycles lasting 5 minutes each at 900W) in 1 mmol of ethylenediamine tetraacetic acid (pH 8.0), and a three-layer alkaline phosphatase-anti alkaline phosphatase (APAAP) technique was applied for antigen detection. For COX-2 immunohistochemistry, the anti-COX-2 mAb (Cayman Chemicals; 1:20 dilution) was used. The specificity of the staining was ensured by using isotype-matched irrelevant antibody, as a substitute for primary antibody, and the immuno-alkaline phosphatase was adopted to avoid any disturbance from the endogenous peroxidase activity. Slides were analyzed blinded by two independent people. NS-398, a relative specific pharmacological inhibitor of COX-2 isoenzyme (Biomol, Plymouth Meeting, PA) was dissolved in ethanol, and stock solution was stored at −20°C for no more than 3 months. NS-398 was used at the final concentrations of 12.5 to 200 μmol/L. DuP697, an unrelated pharmacological inhibitor of COX-2 (Sigma Chemical) was dissolved in ethanol and used at the final concentration of 100 μmol/L. Chlorambucil and Fludarabine (F-ara-A) (Sigma) were added at the final concentration of 10 μmol/L. Recombinant histidine 6-tagged TRAIL(114–281), produced in bacteria and purified by chromatography, as described previously,25Secchiero P Gonelli A Celeghini C Mirandola P Guidotti L Visani G Capitani S Zauli G Activation of the nitric oxide synthase pathway represents a key component of the tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematological malignancies.Blood. 2001; 98: 2220-2228Crossref PubMed Scopus (70) Google Scholar was added at the final concentration of 1 μg/ml. PGE2 levels were measured in the supernatants of cell cultures using the prostaglandin E2 EIA kit-monoclonal (Cayman Chemicals), following manufacturer's instructions. Cell viability was assessed by trypan blue dye exclusion, and the degree of apoptosis was assessed by annexin V-FITC/propidium iodide double staining (Trevigen Inc., Gaithersburg, MD) followed by flow cytometric analysis and in parallel by analyzing cell lysates for PARP cleavage in Western blot. Box plots were used to show the median, minimum, maximum values and 25th to 75th percentiles of the RNA levels for each group of data. Correlation coefficients were calculated by Spearman's method. Results were evaluated by using analysis of variance with subsequent comparisons by Student's t-test and with the Mann-Whitney rank-sum test. Statistical significance was defined as P < 0.05. All B-CLL patients who entered this study (Table 1) were previously untreated, and we did not pre-select any subgroup of B-CLL patients to represent the heterogeneity of this disease course. RNA was extracted from eight freshly purified B-CLL PBMCs containing an excess of B leukemic cells (>85% of CD19+/CD5+ B cells) and from three normal B lymphocyte samples (>90% CD19+) and analyzed by cDNA microarray for a set of stress and toxicity associated genes (GEArray HS-603; SuperArray Bioscience Corporation), which included PTGS2/COX-2. The concept of peripheral blood B cells being appropriate controls for B-CLL cells in microarray analysis has been recently validated by different authors.18Klein U Tu Y Stolovitzky GA Mattioli M Cattoretti G Husson H Freedman A Inghirami G Cro L Baldini L Neri A Califano A Dalla-Favera R Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells.J Exp Med. 2001; 194: 1625-1638Crossref PubMed Scopus (783) Google Scholar, 22Gricks CS Zahrieh D Zauls AJ Gorgun G Drandi D Mauerer K Neuberg D Gribben JG Differential regulation of gene expression following CD40 activation of leukemic compared to healthy B cells.Blood. 2004; 104: 4002-4009Crossref PubMed Scopus (37) Google Scholar Genes up-regulated in all B-CLL included IL-1β, IL-8, BCL3, and NOS2A in agreement with previous studies.23Stratowa C Loffler G Lichter P Stilgenbauer S Haberl P Schweifer N Dohoner H Wilgenbus KK cDNA microarray gene expression analysis of B-cell chronic lymphocytic leukemia proposes potential new prognostic markers involved in lymphocyte trafficking.Int J Cancer. 2001; 91: 474-480Crossref PubMed Scopus (105) Google Scholar, 26Francia di Celle P Mariani S Riera L Stacchini A Reato G Foa R Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.Blood. 1996; 87: 4382-4389Crossref PubMed Google Scholar, 27Hulkkonen J Vilpo J Vilpo L Koski T Hurme M Interleukin-1beta, interleukin-1 receptor antagonist and interleukin-6 plasma levels and cytokine gene polymorphisms in chronic lymphocytic leukemia: correlation with prognostic parameters.Haematologica. 2000; 85: 600-606PubMed Google Scholar, 28Levesque MC Misukonis MA O'Loughlin CW Chen Y Beasley BE Wilson DL Adams DJ Silber R Weinberg JB IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells.Leukemia. 2003; 17: 442-450Crossref PubMed Scopus (38) Google Scholar For the purpose of this study, however, it is particularly remarkable that PTGS2/COX-2 was also consistently up-regulated, at variable levels (mean fold of increase compared CD19+ B lymphocytes, 12.4; range, 2 to 33.3) in all of the B-CLL samples examined. Validation of the COX-2 microarray results was performed by RT-PCR on the same samples analyzed by microarray approach (Figure 1A). Furthermore, quantitative analysis of the steady-state mRNA levels of COX-2 was extended to a total of 16 B-CLL samples and 10 normal B cell samples (Figure 1B). Overall, a significant (P < 0.01) increase of COX-2 mRNA levels was demonstrated in the B-CLL samples examined over normal B lymphocytes (Figure 1B). Because COX-2 is irreversibly inactivated following catalysis, it is assumed that COX activity is determined by the amount of the enzyme protein and is regulated exclusively at the levels of transcription and translation. Consistent with this hypothesis, the results of RT-PCR were further confirmed by analyzing COX-2 protein at Western blot. In fact, as shown in Figure 1C, whereas COX-2 protein was virtually undetectable in normal B cells, it was detected, at variable levels, in the majority of B-CLL samples examined. The relative expression levels of COX-2 protein, determined by densitometry analysis, significantly correlated (r = 0.81, P < 0.01) with the steady-state mRNA levels. In additional experiments, COX-2 expression was examined in biopsies obtained from B-CLL patients with leukemic bone marrow infiltrates. As shown in a representative sample (Figure 2A), the leukemic bone marrow infiltrate displayed a nodular pattern mainly consisting of small lymphocytes and pro-lymphocytes. These cells exhibited the characteristic B-CLL immunophenotype, ie, positivity for CD79a, CD23, and CD5. When the bone marrow biopsies were immunostained for COX-2 antigen, COX-2 expression was observed in normal erythroblasts and megakaryocytes (Figure 2B, left panel), as expected on the basis of previous studies of our and other groups of investigators.29Rocca B Secchiero P Ciabattoni G Ranelletti FO Catani L Guidotti L Melloni E Maggiano N Zauli G Patrono C Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets.Proc Natl Acad Sci USA. 2002; 99: 7634-7639Crossref PubMed Scopus (310) Google Scholar, 30Rocca B Secchiero P Celeghini C Ranelletti FO Ciabattoni G Maggiano N Habib A Ricerca BM Barbarotto E Patrono C Zauli G Modulation of the expression and activity of cyclooxygenases in normal and accelerated erythropoiesis.Exp Hematol. 2004; 32: 925-934Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 31Tanaka N Sato T Fujita H Morita I Constitutive expression and involvement of cyclooxygenase-2 in human megakaryocytopoiesis.Arterioscler Thromb Vasc Biol. 2004; 24: 607-612Crossref PubMed Scopus (30) Google Scholar More importantly, clear expression of COX-2 protein was detected also in the leukemic infiltrate (Figure 2B, right panel). In the next group of experiments, B-CLL cells were cultured ex vivo for 72 hours in the absence or presence of the pharmacological inhibitor of COX-2, NS-398. This time frame was chosen taking into account that in the absence of exogenous cytokines, which are known to prolong the in vitro lifespan of B-CLL cells,26Francia di Celle P Mariani S Riera L Stacchini A Reato G Foa R Interleukin-8 induces the accumulation of B-cell chronic lymphocytic leukemia cells by prolonging survival in an autocrine fashion.Blood. 1996; 87: 4382-4389Crossref PubMed Google Scholar, 27Hulkkonen J Vilpo J Vilpo L Koski T Hurme M Interleukin-1beta, interleukin-1 receptor antagonist and interleukin-6 plasma levels and cytokine gene polymorphisms in chronic lymphocytic leukemia: correlation with prognostic parameters.Haematologica. 2000; 85: 600-606PubMed Google Scholar, 28Levesque MC Misukonis MA O'Loughlin CW Chen Y Beasley BE Wilson DL Adams DJ Silber R Weinberg JB IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells.Leukemia. 2003; 17: 442-450Crossref PubMed Scopus (38) Google Scholar leukemic cells did not proliferate ex vivo and the number of viable cells remained relatively constant, never dropping below 80% of the total cell number seeded at time 0 (Figure 3A). The effect of NS-398 on cell viability was monitored every 24 hours, as exemplified in Figure 3A for one B-CLL sample. In some B-CLL cultures (n = 3), a structurally unrelated COX-2 inhibitor (DuP697; 100 μmol/L) was also used, to rule out possible bystander effects of NS-398 on B-CLL cell viability. As shown in Figure 3A, also DuP697 induced a significant decrease in the number of viable cells, comparable with that observed with NS-398. The susceptibility of each B-CLL sample to the ex vivo NS-398 treatment is reported for the end point (72 hours) in Figure 3B. In 13 of 16 B-CLL patients, NS-398 induced a significant (P < 0.01, cut-off >20%) decline in viability with respect to untreated control cultures (32 ± 16% of cytotoxicity, means ± SD of 13 cases), whereas no significant variations were observed in terms of viable cell number in only 3 of 16 B-CLL samples, as well as in normal purified CD19+ B lymphocytes (n = 4) and in total PBMCs (n = 8) (Figure 3B). There were no apparent correlations between sensitivity to NS-398 and clinical characteristics of the B-CLL patients. Moreover, analysis of the samples examined by cDNA microarray did not reveal any correlation between sensitivity to NS-398 and the steady-state mRNA levels of Bcl-2 (r = 0.48, P > 0.05). Cell viability was next measured in either B-CLL or primary normal PBMCs incubated with various concentrations of NS-398 for 72 hours (Figure 4A). NS-398 induced a dose-dependent cytotoxic effect when added to B-CLL leukemic cells, whereas normal PBMCs were refractory to NS-398 cytotoxicity even at the highest drug concentrations (200 μmol/L) (Figure 4A). It should be noted that a 24-hour treatment with the concentration of NS-398 used in this and in the following set of experiments (100 μmol/L) was able to completely block the production and the release in culture of prostaglandins (PGE2) in five different B-CLL samples examined (Figure 4B). Concurrently, we sought to elucidate whether the cytotoxicity of NS-398 toward B-CLL cells was caused by apoptosis. For this purpose, B-CLL cell lysates were examined for the levels of PARP, a downstream target of activated caspase-3 that is typically cleaved in the setting of caspase-mediated apoptosis. In NS-398-treated B-CLL cells, Western blot analysis revealed the increase of the 85-kd cleaved product of PARP (Figure 5A). Moreover, exposure to NS-398 resulted in a significant (P < 0.05) increase in the percentage of Annexin V-positive cells compared with untreated cultures (Figure 5B). Taken together, these findings suggest that COX-2 overexpression plays a role in counteracting the apoptotic pathway in B-CLL cells and demonstrate that B-CLL cells are more susceptible to NS-398-induced cell death compared with normal purified CD19+ B lymphocytes and PBMCs. We next invest
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