Phosphodiesterase Type 4 Inhibitor Suppresses Expression of Anti

Leukemia (2001) 15, 1564–1571 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Phosphodiesterase type 4 inhibitor suppresses expression of anti-apoptotic members of the Bcl-2 family in B-CLL cells and induces caspase-dependent apoptosis B Siegmund1, J Welsch1, F Loher1, G Meinhardt2, B Emmerich2, S Endres1 and A Eigler1 1Division of Clinical Pharmacology and 2Division of Haematology and Oncology, Medizinische Klinik Innenstadt, Klinikum of the Ludwig- Maximilians-University Munich, Munich, Germany B cell chronic lymphocytic leukemia (B-CLL) is an incurable synergizes with chlorambucil in vitro and a phase II clinical clonal disease which shows initial responsiveness to a number trial suggested that this synergism may be clinically rel- of chemotherapeutic drugs. However, in most patients the dis- 13,14 ease becomes resistant to treatment. Rolipram, a specific evant. However, therapy with broad-spectrum PDE inhibi- inhibitor of phosphodiesterase (PDE) type 4, the PDE predomi- tors such as theophylline or pentoxifylline in patients with B- nantly expressed in B-CLL cells, has been shown to induce CLL is limited due to side-effects partially mediated by their cAMP-dependent apoptosis in these cells. In the present study, activity as adenosine receptor antagonists.15 The expression of we demonstrate that the extent of rolipram-induced apoptosis specific phosphodiesterases in B-CLL cells favors a more tar- is similar to fludarabine-induced apoptosis in vitro. The combi- geted strategy by using specific PDE inhibitors. Kim and nation of rolipram and fludarabine results in an enhancement 16 in the number of apoptotic cells compared to apoptosis Lerner described recently that CLL cells contain transcripts induced by either agent alone. Second, rolipram suppresses for PDE 1B1, PDE 4A and PDE 4B. They further demonstrated the expression of anti-apoptotic members of the Bcl-2 family the cAMP-dependent induction of apoptosis by the PDE 4 and induces the pro-apoptotic protein Bax, thereby shifting the inhibitor rolipram in B-CLL cells.16 New PDE 4 inhibitors have balance between pro- and anti-apoptotic members of the Bcl- a favorable ratio of desired action and side-effects, and are 2 family towards a pro-apoptotic direction. Finally rolipram- under clinical investigation in phase III trials for chronic induced apoptosis is caspase-dependent. PDE 4 inhibitors are currently under investigation for chronic obstructive pulmon- obstructive pulmonary disease and asthma, with encouraging 17 ary disease and asthma in phase III clinical trials showing preliminary results. promising results with tolerable side-effects. In conclusion, by To evaluate the function of PDE in the pathogenesis of B- inducing apoptosis, by enhancing apoptosis induced by fluda- CLL and the therapeutic potency of PDE 4 inhibitors such as rabine, by suppressing Bcl-2, Bcl-X and by inducing Bax rolipram further in vitro investigations are needed. A clinically expression, PDE 4 inhibitors may add a new therapeutic option relevant question is, whether rolipram synergizes with cur- for patients with B-CLL. Leukemia (2001) 15, 1564–1571. Keywords: rolipram; Bcl-X; Bax; apoptosis; B-CLL; PDE 4 rently used chemotherapy in induction of apoptosis. Combi- nation with PDE 4 inhibitors could allow a dose reduction of toxic cytostatic drugs. The resistance to chemotherapy in B- CLL can partly be mediated by the high expression of anti- Introduction apoptotic proteins such as Bcl-2 in B-CLL. If rolipram shifts the balance of pro- and anti-apoptotic members of the Bcl-2 B cell chronic lymphocytic leukemia (B-CLL) is the most com- family towards apoptosis cytotoxic agents could become mon adult leukemia in the western hemisphere and accounts more effective. for 25% of all leukemias. The indolent natural course of both In the present study we investigated the apoptosis-inducing early stage and smoldering CLL has left many physicians with potency of rolipram alone and in combination with either the perception that because of this ‘favorable course’ the dis- fludarabine or mitoxantrone as determined by annexin- ease can be ignored until the advanced stage at which time V/propidium iodide flow cytometric analysis and by DNA palliative therapy is indicated.1,2 A diverse interpretation of fragmentation. We studied the expression of Bcl-2 and Bcl-X the clinical data on CLL regards this disease as incurable, with expression as anti-apoptotic members and Bax expression advanced stage patients having a median survival of 18 as a pro-apoptotic member of the Bcl-2 family either by months to 3 years.3–5 The majority of circulating cells are non- flow cytometry or by Western blot analysis. We further investi- dividing, and it has been shown that the clonal excess of B gated the role of caspase activation in rolipram-induced cells results from decreased cell death rather than prolifer- apoptosis. ation.6,7 In lymphoid cells, cytolysis induced by phosphodiesterase (PDE) inhibition results from an increase in protein kinase A- mediated phosphorylation of unknown lymphoid target pro- Materials and methods teins which eventually induces apoptosis.8,9 Cyclic AMP is Ј Ј Ј catabolized within cells to 5 -AMP by 3 :5 cAMP-PDE. The Patients family of PDE includes 10 classes of enzymes which are differ- entially expressed in diverse cell types.10,11 The unspecific Nineteen patients (13 men and six women) with B-CLL who phosphodiesterase inhibitor theophylline has been reported to had not received treatment for the previous 6 months with a induce apoptosis in B-CLL cells.12 In addition, theophylline median age of 69 years (range, 53 to 79 years) were studied. B-CLL had been diagnosed according to standard clinical and laboratory criteria. Blood sampling was approved by the local Correspondence: S Endres, Division of Clinical Pharmacology, Mediz- ethics committee. The median peripheral blood leukocyte inische Klinik Innenstadt, Klinikum of the Ludwig-Maximillians-Uni- × 9 × 9 versity of Munich, Ziemssenstraβe 1, 80336 Mu¨nchen, Germany; Fax: count was 76 10 leukocytes/l (range, 12 to 159 10 089–5160–4406 leukocytes/l). The median ␤2-microglobulin concentration Received 20 November 2001; accepted 21 May 2001 was 4 mg/l (range, 2 to 14 mg/l) and the median thymidine Rolipram-induced apoptosis in CLL B Siegmund et al 1565 kinase concentration was 18 U/l (range 5 to 80 U/l) both inhibitor zVAD-fmk from R&D Systems (Wiesbaden, determined as negative predictors for disease-free Germany). survival.18–20 Leukemic cells were positive in all cases for CD5 and CD19 by flow cytometry. According to Binet’s classi- fication,2 at the time of inclusion, seven patients were in stage A, three patients were in stage B, and nine patients were at Analysis of apoptosis by annexin binding stage C (Table 1). Exposure of phosphatidylserine at the outer plasma cell mem- brane of apoptotic cells was quantified by surface annexin V staining as described.23 Briefly, one million cells were incu- Preparation of B-CLL cells bated for 48 h with the substances to be studied. Cells were then washed in phosphate-buffered saline (PBS), were resus- Mononuclear cells were isolated from peripheral blood by pended in 200 ␮l binding buffer (10 mmol/l Hepes, pH 7.4, gradient centrifugation over Ficoll–Hypaque (Biochrom, 2.5 mmol/l CaCl2, 140 mmol/l NaCl) and were incubated with Berlin, Germany), as described previously.21,22 As a modifi- 0.5 ␮g/ml of annexin V-fluorescein isothiocyanate (FITC; cation of the protocol, the isolation was performed in tubes Bender MedSystem, Vienna, Austria) for 15 min in the dark. containing a horizontal porous filter disc over the Ficoll layer Cells were washed again, were resuspended in binding buffer (Leucosep tubes; Greiner, Frickenhausen, Germany) in order and propidium iodide (PI, 5 ␮g/ml) was added. Samples were to facilitate layering of blood. RPMI 1640 culture medium was analyzed by a FACSCalibur (Becton Dickinson, Heidelberg, supplemented with 2 mML-glutamine, 10 mM Hepes buffer, Germany). For the determination of CD19 and CD5 100 U/ml penicillin and 100 ␮g/ml streptomycin (all from expression cells were labeled with either a FITC- or PE-labeled Sigma, Munich, Germany). The cells were suspended and antibody (Becton Dickinson). Data were analyzed using were cultured immediately after isolation at a final concen- FlowJo software (Version 2.7.8). tration of 2.5 × 106 cell/ml in RPMI 1640 culture medium further supplemented with 2% heat-inactivated sterile human serum. B-CLL cells were incubated at 37°C in a humidified Bcl-X analysis atmosphere containing 5% carbon dioxide. One million cells were fixed using a commercially available Fix and Perm kit (Caltag, Burlingame, CA, USA), washed in PBS and centrifuged at 300 g for 5 min. The pelleted cells Preparation of compounds were resuspended in permeabilization solution and incubated with 10 ␮l of anti-Bcl-XL (clone 7B2.5; epitope Bcl-XS; South- Rolipram (racemate of 4-[3Ј-cyclopentyloxy-4Ј-methoxy- ern Biotechnology Associates, Birmingham, AL, USA) or iso- phenyl]-2-pyrrolidone, from Schering, Berlin, Germany), sup- type-negative control (clone B 10; Southern Biotechnology plied in powder form, was dissolved in RPMI 1640 medium Associates). The cells were washed, centrifuged at 300 g for by vigorous vortexing. Fludarabine was obtained from Scher- 5 min and resuspended in 0.5 ml of 1% paraformaldehyde. ing, chlorambucil and mitoxantrone were obtained from Led- All samples were studied using a FACS Calibur (Becton erle Laboratories (Gosport, UK). Propidium

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