Phosphodiesterase IV Inhibition by Piclamilast Potentiates the Cytodifferentiating Action of Retinoids in Myeloid Leukemia Cells
2004; Elsevier BV; Volume: 279; Issue: 40 Linguagem: Inglês
10.1074/jbc.m406530200
ISSN1083-351X
AutoresEdoardo Parrella, Maurizio Gianni’, Virginia Cecconi, Elisa Agnese Nigro, Maria Monica Barzago, Alessandro Rambaldi, Cécile Rochette‐Egly, Mineko Terao, Enrico Garattini,
Tópico(s)Acute Myeloid Leukemia Research
ResumoInhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor α (RARα), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARα through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARα. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + piclamilast. Initial in vivo studies conducted in severe combined immunodeficiency mice transplanted with NB4 leukemia cells indicate that the enhancing effect of piclamilast on ATRA-induced myeloid maturation translates into a therapeutic benefit. Inhibition of phosphodiesterase IV by N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (piclamilast) enhances the myeloid differentiation induced by all-trans-retinoic acid (ATRA), retinoic acid receptor α (RARα), or retinoic acid receptor X agonists in NB4 and other retinoid-sensitive myeloid leukemia cell types. ATRA-resistant NB4.R2 cells are also partially responsive to the action of piclamilast and retinoic acid receptor X agonists. Treatment of NB4 cells with piclamilast or ATRA results in activation of the cAMP signaling pathway and nuclear translocation of cAMP-dependent protein kinase. This causes a transitory increase in cAMP-responsive element-binding protein phosphorylation, which is followed by down-modulation of the system. ATRA + piclamilast have no additive effects on the modulation of the cAMP pathway, and the combination has complex effects on cAMP-regulated genes. Piclamilast potentiates the ligand-dependent transactivation and degradation of RARα through a cAMP-dependent protein kinase-dependent phosphorylation. Enhanced transactivation is also observed in the case of PML-RARα. In NB4 cells, increased transactivation is likely to be at the basis of enhanced myeloid maturation and enhanced expression of many retinoid-dependent genes. Piclamilast and/or ATRA exert major effects on the expression of cEBP and STAT1, two types of transcription factors involved in myeloid maturation. Induction and activation of STAT1 correlates directly with enhanced cytodifferentiation. Finally, ERK and the cAMP target protein, Epac, do not participate in the maturation program activated by ATRA + piclamilast. Initial in vivo studies conducted in severe combined immunodeficiency mice transplanted with NB4 leukemia cells indicate that the enhancing effect of piclamilast on ATRA-induced myeloid maturation translates into a therapeutic benefit. All-trans-retinoic acid (ATRA) 1The abbreviations used are: ATRA, all-trans-retinoic acid; APL, acute promyelocytic leukemia; ERK, extracellular signal-regulated kinase; EMSA, electromobility shift assay; 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate; O-Me cAMP, 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate; Rp-8Br-cAMP, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rpisomer; AML, acute myeloid leukemia; PDEIV, phosphodiesterase IV; NBT-R, nitro blue tetrazolium reductase; PKA, cAMP-dependent protein kinase; MCP-1, monocyte chemotactic peptide 1; PKA-rIα and -rIβ, regulatory subunits I α and β of PKA; PKA-rIIα, regulatory subunit II α of PKA; PKA-cIα, catalytic subunit I α of PKA; TNF, tumor necrosis factor α; CREB, cAMP-responsive element-binding protein; RAR, retinoic acid receptor; RXR, retinoic acid receptor X; CAT, chloramphenicol acetyltransferase; RARE, retinoic acid-responsive element; PRAM, protein regulated by retinoic acid and PML-RAR; ctsD, cathepsin D; pLym, putative lymphocyte G0-G1 switch gene; BTG, B cell translocation gene 2 protein; THBS, thrombospondin; EGR1, early growth factor-regulated 1; cEBPα, -β, and -γ, CAAT element-binding proteins α, β, and γ, respectively; STAT1, signal transducer and activator of transcription 1; SCID, severe combined immunodeficiency; Epac, exchange protein directly activated by cAMP; MAP, mitogen-activated protein; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; RT, reverse transcriptase; MST, median survival time.1The abbreviations used are: ATRA, all-trans-retinoic acid; APL, acute promyelocytic leukemia; ERK, extracellular signal-regulated kinase; EMSA, electromobility shift assay; 8-CPT-cAMP, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate; O-Me cAMP, 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate; Rp-8Br-cAMP, 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Rpisomer; AML, acute myeloid leukemia; PDEIV, phosphodiesterase IV; NBT-R, nitro blue tetrazolium reductase; PKA, cAMP-dependent protein kinase; MCP-1, monocyte chemotactic peptide 1; PKA-rIα and -rIβ, regulatory subunits I α and β of PKA; PKA-rIIα, regulatory subunit II α of PKA; PKA-cIα, catalytic subunit I α of PKA; TNF, tumor necrosis factor α; CREB, cAMP-responsive element-binding protein; RAR, retinoic acid receptor; RXR, retinoic acid receptor X; CAT, chloramphenicol acetyltransferase; RARE, retinoic acid-responsive element; PRAM, protein regulated by retinoic acid and PML-RAR; ctsD, cathepsin D; pLym, putative lymphocyte G0-G1 switch gene; BTG, B cell translocation gene 2 protein; THBS, thrombospondin; EGR1, early growth factor-regulated 1; cEBPα, -β, and -γ, CAAT element-binding proteins α, β, and γ, respectively; STAT1, signal transducer and activator of transcription 1; SCID, severe combined immunodeficiency; Epac, exchange protein directly activated by cAMP; MAP, mitogen-activated protein; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; RT, reverse transcriptase; MST, median survival time. and retinoids exert their antileukemic activity through activation of three processes (i.e. cytodifferentiation, growth inhibition, and apoptosis). ATRA induces complete clinical remission in the majority of acute promyelocytic leukemia (APL) patients (1Chomienne C. Fenaux P. Degos L. FASEB J. 1996; 10: 1025-1030Crossref PubMed Scopus (121) Google Scholar, 2Warrell Jr., R.P. Semin. Hematol. 1994; 31: 1-13PubMed Google Scholar) and is part of the standard protocol used for the management of this type of leukemia (3Fenaux P. Chastang C. Chevret S. Sanz M. Dombret H. Archimbaud E. Fey M. Rayon C. Huguet F. Sotto J.J. Gardin C. Makhoul P.C. Travade P. Solary E. Fegueux N. Bordessoule D. Miguel J.S. Link H. Desablens B. Stamatoullas A. Deconinck E. Maloisel F. Castaigne S. Preudhomme C. Degos L. Blood. 1999; 94: 1192-1200Crossref PubMed Google Scholar). Paradoxically, APL is characterized by the prototypical t15:17 translocation, which involves the nuclear retinoic acid receptor RARα and leads to the expression of the aberrant fusion protein PML-RARα (1Chomienne C. Fenaux P. Degos L. FASEB J. 1996; 10: 1025-1030Crossref PubMed Scopus (121) Google Scholar). The success of ATRA in APL represents proof of principle that cytodifferentiation therapy (4Garattini E. Terao M. Curr. Opin. Pharmacol. 2001; 1: 358-363Crossref PubMed Scopus (12) Google Scholar) can be applied clinically. However, a more general use of ATRA at the clinical level is limited by toxicity and natural or induced resistance (5Biesalski H.K. Toxicology. 1989; 57: 117-161Crossref PubMed Scopus (124) Google Scholar, 6Gallagher R.E. Leukemia. 2002; 16: 1940-1958Crossref PubMed Scopus (118) Google Scholar, 7Cote S. Rosenauer A. Bianchini A. Seiter K. Vandewiele J. Nervi C. Miller Jr., W.H. Blood. 2002; 100: 2586-2596Crossref PubMed Scopus (64) Google Scholar).With this in mind, it would be desirable to devise strategies aimed to increase the efficiency of the intracellular signals regulating the cytodifferentiating, growth-inhibitory, and apoptotic action of retinoids. The identification of agents capable of augmenting the therapeutic index of ATRA may lead to the development of useful pharmacological combinations in the treatment of APL and other acute myeloid leukemias (AMLs). In addition, dissection of the molecular mechanisms underlying the interactions between these agents and retinoids in AML cells is likely to cast light on the process of myeloid maturation.Analogs of cAMP (8Gianni M. Terao M. Norio P. Barbui T. Rambaldi A. Garattini E. Blood. 1995; 85: 3619-3635Crossref PubMed Google Scholar, 9Garattini E. Gianni M. Leuk. Lymphoma. 1996; 23: 493-503Crossref PubMed Scopus (21) Google Scholar, 10Ruchaud S. Duprez E. Gendron M.C. Houge G. Genieser H.G. Jastorff B. Doskeland S.O. Lanotte M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8428-8432Crossref PubMed Scopus (109) Google Scholar, 11Quenech'Du N. Ruchaud S. Khelef N. Guiso N. Lanotte M. Leukemia. 1998; 12: 1829-1833Crossref PubMed Scopus (36) Google Scholar, 12Koyama T. Hirosawa S. Kawamata N. Tohda S. Aoki N. Blood. 1994; 84: 3001-3009Crossref PubMed Google Scholar), interferons (13Gianni M. Zanotta S. Terao M. Rambaldi A. Garattini E. Int. J. Cancer. 1996; 68: 75-83Crossref PubMed Scopus (19) Google Scholar, 14Garattini E. Mologni L. Ponzanelli I. Terao M. Leuk. Lymphoma. 1998; 30: 467-475Crossref PubMed Scopus (15) Google Scholar, 15Matikainen S. Ronni T. Hurme M. Pine R. Julkunen I. Blood. 1996; 88: 114-123Crossref PubMed Google Scholar), granulocyte colony-stimulating factor (16Gianni M. Terao M. Zanotta S. Barbui T. Rambaldi A. Garattini E. Blood. 1994; 83: 1909-1921Crossref PubMed Google Scholar), and a novel class of experimental compounds, bis-indols (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar), enhance the cytodifferentiating activity of ATRA in AML cell lines. Enhanced cytodifferentiation by combinations of bis-indols and ATRA translates into a therapeutically significant effect, as assessed in animal models of APL (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). The cross-talk between the cAMP signaling pathway and ATRA is particularly attractive in terms of the development of pharmacological combinations to be used in the cytodifferentiating treatment of AML. In fact, the cAMP pathway is modulated by various pharmacological agents (18Guillemin M.C. Raffoux E. Vitoux D. Kogan S. Soilihi H. Lallemand-Breitenbach V. Zhu J. Janin A. Daniel M.T. Gourmel B. Degos L. Dombret H. Lanotte M. de The H. J. Exp. Med. 2002; 196: 1373-1380Crossref PubMed Scopus (92) Google Scholar, 19Moon E.Y. Lerner A. Blood. 2003; 101: 4122-4130Crossref PubMed Scopus (70) Google Scholar, 20Tucholski J. Johnson G.V. J. Biol. Chem. 2003; 278: 26838-26843Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), some of which are already in the clinics for the treatment of asthma (21Barnette M.S. Prog. Drug Res. 1999; 53: 193-229Crossref PubMed Google Scholar, 22Bundschuh D.S. Eltze M. Barsig J. Wollin L. Hatzelmann A. Beume R. J. Pharmacol. Exp. Ther. 2001; 297: 280-290PubMed Google Scholar). In this report, we demonstrate that inhibition of phosphodiesterase IV (PDEIV), the enzyme that hydrolyzes cAMP to the corresponding nucleoside monophosphate, by piclamilast, potentiates the retinoid-dependent granulocytic maturation of the APL-derived NB4 and other AML cell lines. The molecular mechanisms underlying this potentiating effect are investigated in the NB4 model. Furthermore, we present initial evidence that the combination of ATRA and piclamilast is more effective than the single components in prolonging the life span of SCID mice transplanted with APL cells.EXPERIMENTAL PROCEDURESChemicals—ATRA, rolipram, H89, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (8-CPT-cAMP), dibutyryl-cAMP, H89, and 8-bromoadenosine-3′,5′-cyclicmonophosphorothioate-Rp-isomer (Rp-8-BrcAMP) were from Sigma. AM580, CD2809, and CD2915 were obtained from CIRD-Galderma (Sophia Antipolis, France). N-(3,5-dichloropyrid-4-yl)-3-cyclopentyloxy-4-methoxybenzamide (piclamilast) was synthesized by Altana Pharma GmbH (Konstanz, Germany). 8-(4-chlorophenylthio)-2′-O-methyladenosine 3′,5′-cyclic monophosphate (O-Me cAMP) was from BIOLOG Life Science Institute (Bremen, Germany).Cell Cultures, Intracellular cAMP, cAMP-dependent Protein Kinase (PKA) Activity, Electromobility Shift Assay (EMSA), Nitro Blue Tetrazolium Reductase (NBT-R) Activity, and Transfections—NB4 (23Lanotte M. Martin-Thouvenin V. Najman S. Balerini P. Valensi F. Berger R. Blood. 1991; 77: 1080-1086Crossref PubMed Google Scholar), NB4.R2 (24Roussel M.J. Lanotte M. Oncogene. 2001; 20: 7287-7291Crossref PubMed Scopus (57) Google Scholar), U937 (25Larrick J.W. Fischer D.G. Anderson S.J. Koren H.S. J. Immunol. 1980; 125: 6-12PubMed Google Scholar), and HL-60 (ATCC, Manassas, VA) cells and blasts from one AML patient (FAB classification M5) were cultured as described (16Gianni M. Terao M. Zanotta S. Barbui T. Rambaldi A. Garattini E. Blood. 1994; 83: 1909-1921Crossref PubMed Google Scholar). Cell number and viability were determined following staining with erythrosin (Sigma) (26Garattini E. Parrella E. Diomede L. Gianni' M. Kalac Y. Merlini L. Simoni D. Zanier R. Ferrara F.F. Chiarucci I. Carminati P. Terao M. Pisano C. Blood. 2004; 103: 194-207Crossref PubMed Scopus (62) Google Scholar).Intracellular cAMP and PKA activity were determined with the cAMP Biotrak EIA (Amersham Biosciences) and PKA PepTag Assay (Promega, Madison, WI) kits.EMSAs were conducted as described (26Garattini E. Parrella E. Diomede L. Gianni' M. Kalac Y. Merlini L. Simoni D. Zanier R. Ferrara F.F. Chiarucci I. Carminati P. Terao M. Pisano C. Blood. 2004; 103: 194-207Crossref PubMed Scopus (62) Google Scholar) with an oligodeoxynucleotide representing the DNA sequence for the binding of cAMP-responsive element-binding protein (5′-GAGAGATTGCCTGACGTCAGAGAGGTAG-3′).Nitro blue tetrazolium reductase (NBT-R) activity was measured in cell extracts, and the number of NBT-R+ cells was determined as detailed (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). To determine the morphology of NB4 cells quantitatively, cytospins were stained with May-Grunwald-Giemsa. Approximately 100 cells/microscopic field were scored for the presence of morphologically differentiated cells (27Charrad R.S. Gadhoum Z. Qi J. Glachant A. Allouche M. Jasmin C. Chomienne C. Smadja-Joffe F. Blood. 2002; 99: 290-299Crossref PubMed Scopus (103) Google Scholar).COS-7 cells (ATCC) were grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and transfected with RARα, PMLRARα, or the mutant RARαS369A cDNAs (28Rochette-Egly C. Oulad-Abdelghani M. Staub A. Pfister V. Scheuer I. Chambon P. Gaub M.P. Mol. Endocrinol. 1995; 9: 860-871Crossref PubMed Google Scholar) in the presence of the CREB-dependent Som-CAT plasmid (containing 1.4 kb of 5′-flanking region of the somatostatin gene promoter) (29Leonard J. Serup P. Gonzalez G. Edlund T. Montminy M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6247-6251Crossref PubMed Scopus (102) Google Scholar) or the RARE-containing β2RARE-CAT, DR5-tk-CAT, or TRE-tk-CAT reporter constructs and the normalization plasmid pCH110 (containing the bacterial β-galactosidase cDNA) (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). CAT and β-galactosidase activities were measured in cell extracts as detailed (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar).RNA Purification, Nuclear Extracts, RT-PCR, Northern Blot, ELISA, Western Blot, and Fluorescence-activated Cell Sorting (FACS) Analyses—For the amplification of the cDNAs coding for human PDEIV, total RNA (1 μg) was subjected to reverse transcription. cDNAs were amplified by PCR with the following couples of amplimers: PDEIVA, 5′-TGAGCTGACGCTGGAGGAGG-3′ (nucleotides 1735-1754 of the PDEIVA cDNA, NCBI accession number BC038234) and 5′-CCACCAGGACGTGGGAGTGC-3′ (complementary to nucleotides 2222-2241); PDEIVB, 5′-GACCTGAACAAATGGGGTCT-3′ (nucleotides 985-1004 of the PDEIVB3 cDNA, U85048) and 5′-TTCAGGTCTGCCAGCAGGCT-3′ (complementary to nucleotides 1525-1544); PDEIVC, 5′-AGGCAGAGAACTGGCATATC-3′ (nucleotides 1520-1539 of the PDEIVC cDNA, NM_000923) and 5′-GATTGTCCTCCAGCGTGTCC-3′ (complementary to nucleotides 1984-2003); PDEIVD, 5′-ATGGTGAGTCAGACACGGAA-3′ (nucleotides 1753-1772 of the PDEIVD cDNA, BC036319) and 5′-TTGTCAGCTCTACCAAGCTG-3′ (complementary to nucleotides 2189-2208).Northern blot analysis was performed as detailed (8Gianni M. Terao M. Norio P. Barbui T. Rambaldi A. Garattini E. Blood. 1995; 85: 3619-3635Crossref PubMed Google Scholar) with specific human cDNA probes obtained following RT-PCR amplification of the relevant transcripts. The couples of amplimers used for the amplification of the cDNAs were as follows: vinculin (5′-ATGCCAGTGTTTCATACGCG-3′, nucleotides 51-70; 5′-CTAAGATCTGTCTGATGGCC-3′ complementary to nucleotides 940-960 of the vinculin cDNA, M33308); thrombospondin (THBS) (5′-CAGCTTTCCGCATGCAGGAT-3′, nucleotides 281-300; 5′-AGGAGCCCTCACATCGGTTG-3′ complementary to nucleotides 1341-1360 of the THBS cDNA, NM_003246); early growth factor-regulated 1 (EGR1) (5′-AGCAGCAGCAGCACCTTCAA-3′, nucleotides 511-530; 5′-CCGCAAGTGGATCTTGGTAT-3′ complementary to nucleotides 1511-1530 of the EGR1 cDNA, NM_001964); B cell translocation gene 2 protein (BTG) (5′-TTTTGGGACCCAAAGAGTATCCAC-3′, nucleotides 1356-1379; 5′-GGATTGTACTGAGACAGACAGCAA-3′ complementary to nucleotides 1877-1900 of the BTG cDNA, NM_006763); CD38 (5′-ACTGCCAAAGTGTATGGGATGCTT-3′, nucleotides 311-334; 5′-CAGATGTGCAAGATGAATCCTCAG-3′ complementary to nucleotides 941-964 of the CD38 cDNA, NM_001775); sialoadhesin (5′-CACATGGCTCTGTTCATCTGCAC-3′, nucleotides 2431-2453; 5′-CATCCCGATACCAACGATATGAG-3′ complementary to nucleotides 2778-2800 of the sialoadhesin cDNA, NM_023068); protein regulated by retinoic acid and PML-RAR (PRAM) (5′-GCAGAGATGAAGCGCCCTCAGTT-3′, nucleotides 1778-1800; 5′-CAGAAGTCGACATCATCGTACAC-3′ complementary to nucleotides 2158-2180 of the PRAM cDNA, NM_032152); cathepsin D (ctsD) (5′-GCACAAGTTCACGTCCATCC-3′, nucleotides 211-230; 5′-ACACCTTCTCACAGGGGATC-3′ complementary to nucleotides 1111-1130 of the ctsD cDNA, NM_001909); pLym (5′-GAGATGATGGCCCAGAAGCGCA-3′, nucleotides 199-221; 5′-ACCGTGTCCTGCTGCTTGCCTTT-3′ complementary to nucleotides 409-431 of the pLym cDNA, NM_015714); CAAT element-binding protein α (cEBPα) (5′-CCAAGAAGTCGGTGGACAAGAACA-3′, nucleotides 820-843; 5′-TAGAGACCCTCCACCTTCATGTAG-3′ complementary to nucleotides 1637-1660 of the cEBPα cDNA, NM_004364); CAAT element-binding protein β (cEBPβ) (5′-AAACCAACCGCACATGCAGATGGG-3′, nucleotides 1417-1440; 5′-GATTCCCAAAATATACAGACGCCT-3′ complementary to nucleotides 1751-1774 of the cEBPβ cDNA, NM_005194); CAAT element binding protein epsilon (cEBPϵ) (5′-GCTAGGGGACATGTGTGAGCATGA-3′, nucleotides 261-284; 5′-CTCTGCCATGTACTCCAGCACCTT-3′ complementary to nucleotides 887-900 of the cEBPϵ cDNA, NM_001805). The granulocyte colony-stimulating factor receptor probe is a full-length human cDNA (16Gianni M. Terao M. Zanotta S. Barbui T. Rambaldi A. Garattini E. Blood. 1994; 83: 1909-1921Crossref PubMed Google Scholar).Protein extracts of NB4 cells were prepared as described (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). Nuclear and cytosolic fractions were isolated with nuclear extract kit (Active Motif Europe, Rixensart, Belgium). The nuclear extracts are essentially free of cytosolic contamination. Similarly, cytosolic extracts (cytosol) are not significantly contaminated with cell nuclei. In fact, the protein band corresponding to the nuclear protein marker histone H3 is present only in the nuclear fraction, whereas the cytosolic marker caspase-3 is evident only in the cytosolic fraction (see Supplemental Fig. 1S).EMSAs using a CREB-specific oligonucleotide were conducted on nuclear extracts as reported (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). Western blot analyses were performed on total or nuclear extracts (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). The antibodies directed against ERK-1/ERK-2, CREB, STAT1, and the corresponding phosphorylated forms were from Cell Signaling Technology (Beverly, MA). The antibodies recognizing cEBPα (sc-9315), cEBPβ (sc-150), cEBPϵ (sc-158), PKA-rIα (sc-18798), PKA-rIIα (sc-908), PKA-cα (sc-903), histone H3 (sc-10809), cathepsin D (sc-6486), and β-actin (sc-8432) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The anti-RARα polyclonal antibodies were used as described (30Gianni' M. Kalac Y. Ponzanelli I. Rambaldi A. Terao M. Garattini E. Blood. 2001; 97: 3234-3243Crossref PubMed Scopus (62) Google Scholar). CD11b, CD11c, CD38, and CD33 fluorescein-conjugated antibodies (Becton Dickinson Europe, Erembodegem, Belgium) were used for FACS, as described (16Gianni M. Terao M. Zanotta S. Barbui T. Rambaldi A. Garattini E. Blood. 1994; 83: 1909-1921Crossref PubMed Google Scholar). ELISAs were used to determine secreted monocyte chemotactic peptide 1 (MCP-1) (31Peri G. Milanese C. Matteucci C. Ruco L. Zhou D. Sozzani S. Coletta I. Mantovani A. J. Immunol. Methods. 1994; 174: 249-257Crossref PubMed Scopus (64) Google Scholar) and TNF (32Sironi M. Milanese C. Vecchi A. Polenzani L. Guglielmotti A. Coletta I. Landolfi C. Soldo L. Mantovani A. Pinza M. Int. J. Clin. Lab. Res. 1997; 27: 118-122Crossref PubMed Scopus (43) Google Scholar) (Endogen, Woburn, MA).In Vivo Experiments—For the in vivo experiments, cells were suspended in 199 Hanks' medium, and 0.1 ml (1 × 106 cells/mouse) were intraperitoneally inoculated in SCID mice (17Pisano C. Kollar P. Gianni M. Kalac Y. Giordano V. Ferrara F.F. Tancredi R. Devoto A. Rinaldi A. Rambaldi A. Penco S. Marzi M. Moretti G. Vesci L. Tinti O. Carminati P. Terao M. Garattini E. Blood. 2002; 100: 3719-3730Crossref PubMed Scopus (27) Google Scholar). Piclamilast was dissolved in 0.5% carboxyl methyl cellulose, 0.01% Tween 80 solution and injected at a dose of 10 mg/kg. ATRA (Sigma) was dissolved in the same solution and injected at a dose of 15 mg/kg. Drugs were administered intraperitoneally in a volume of 100 μl/animal 1 day after the inoculation, and the treatment continued for 13 days (5 daily injections/week). Data on the survival of animals were analyzed considering the following parameters: median survival time (MST) and percentage increase in life span (MST-treated/MST control × 100) - 100). Statistical treatment of the results was conducted according to the Cox regression model (33Cox D.R. J. R. Stat. Soc. 1972; B34: 187-220Google Scholar).RESULTSInhibition of PDEIV Enhances and Accelerates the Differentiation of the ATRA-sensitive NB4 Cell Line—As illustrated in Fig. 1A, upon RT-PCR analysis, the APL-derived NB4 blast synthesizes detectable amounts of the transcripts coding for members of the PDEIV A, B, and D families. PDEIVC substitutes PDEIVB in the monoblastic cell line U937, which is used as a positive control (34MacKenzie S.J. Houslay M.D. Biochem. J. 2000; 347: 571-578Crossref PubMed Scopus (127) Google Scholar) in these experiments. Treatment of NB4 cells with ATRA for 4 days does not affect the levels of the various PDEIV transcripts.Challenge with combinations of ATRA and various PDEIV inhibitors, such as rolipram and piclamilast, enhances the ATRA-dependent maturation of NB4 cells (data not shown). Since piclamilast belongs to a chemical series of promising PDEIV inhibitors undergoing phase II/III clinical trials (22Bundschuh D.S. Eltze M. Barsig J. Wollin L. Hatzelmann A. Beume R. J. Pharmacol. Exp. Ther. 2001; 297: 280-290PubMed Google Scholar), all subsequent experiments were conducted with this molecule.The effect of piclamilast on the morphological differentiation of NB4 cells is illustrated in Fig. 1B. Relative to what was observed with ATRA alone, NB4 cultures treated for 3 days with ATRA (0.1 μm) + piclamilast (30 μm) showed an increased proportion of cells with lobated nuclei, cytoplasmic granules, and/or elevated cytoplasm/nucleus volume ratios, three parameters that are associated with granulocytic maturation (27Charrad R.S. Gadhoum Z. Qi J. Glachant A. Allouche M. Jasmin C. Chomienne C. Smadja-Joffe F. Blood. 2002; 99: 290-299Crossref PubMed Scopus (103) Google Scholar). No detectable myeloid maturation is evident upon treatment with the PDEIV inhibitor alone. Enhanced morphological maturation by ATRA + piclamilast is associated with a parallel stimulation of the synthesis of ctsD, a retinoid-regulated and lysosomal differentiation marker (35Levy J. Kolski G.B. Douglas S.D. Infect. Immun. 1989; 57: 1632-1634Crossref PubMed Google Scholar). The enhancing effect of piclamilast is not limited to ctsD and extends to the other surface differentiation marker, CD11b, as demonstrated by the FACS analysis shown in Fig. 1C. Maximal induction of CD11b is observed at the highest concentration of ATRA considered (0.01 μm), both in terms of the number of positive cells and mean associated fluorescence. At 0.001 μm ATRA, the addition of piclamilast results in a doubling of the number of CD11b-positive cells. At 0.01 μm ATRA, the PDEIV inhibitor maintains a significant stimulating effect on CD11b mean associated fluorescence. Similar effects are not evident in the case of CD11c and CD38, two other ATRA-regulated surface antigens. As expected (16Gianni M. Terao M. Zanotta S. Barbui T. Rambaldi A. Garattini E. Blood. 1994; 83: 1909-1921Crossref PubMed Google Scholar), undifferentiated and ATRA-, piclamilast-, or ATRA + piclamilast-treated NB4 cells are positive for CD33.Induction of the cell membrane enzymatic complex, NBT-R, is another popular myeloid marker used to assess granulocytic maturation of APL cells. Piclamilast (30 μm) enhances the ATRA-dependent induction of NBT-R in NB4 cells. Enhanced NBT-R is evident both when the enzymatic activity is measured in cell extracts and when the number of NBT-R+ cells is counted (data not shown). The enhancing effect of piclamilast is similar to that observed with the cAMP analogue, 8-CPT-cAMP (data not shown). Fig. 2A demonstrates that treatment with piclamilast (30 μm) does not result in a significant increase in the number of NBT-R+ NB4 at any of the time points considered. However, when the PDEIV inhibitor is added to ATRA, the retinoid-dependent increase of NB4 positive cells is enhanced and accelerated. Enhanced NBT-R activity is not only time-dependent but also dose-dependent relative to ATRA (Fig. 2B) and piclamilast (Fig. 2C). When ATRA is present in the medium at a concentration of 0.1 μm, a significant increase in the levels of retinoid-induced NBT-R is already observed with 1 μm piclamilast, and the effect plateaus at 30 μm. In the absence of ATRA, even high concentrations of piclamilast (100 μm) are ineffective in inducing NBT-R. Piclamilast has a growth-inhibitory action at all of the concentrations tested, and the action is augmented by the presence of ATRA. When the concentration of piclamilast is kept constant at 30 μm, enhancement of the ATRA-dependent induction of NBT-R is evident at all of the concentrations of the retinoid. A similar dose-response curve is observed in the case of the antiproliferative effect of ATRA + piclamilast.Fig. 2Time- and concentration-dependent effects of piclamilast and ATRA on NBT-R activity.A, NB4 cells (150,000/ml) were treated with vehicle (Me2SO), ATRA, piclamilast, or the combin
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