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• W2014197880 abstract "The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells. The mitogen-activated protein (MAP) kinase family is activated in response to a wide variety of external stress signals such as UV irradiation, heat shock, and many chemotherapeutic drugs and leads to the induction of apoptosis. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in chronic myelogenous leukemia (CML) cells, which are resistant to many chemotherapeutic agents. In this study we have delineated part of the mechanism by which a representative compound known as PBOX-6 induces apoptosis. We have investigated whether PBOX-6 induces activation of MAP kinase signaling pathways in CML cells. Treatment of K562 cells with PBOX-6 resulted in the transient activation of two JNK isoforms, JNK1 and JNK2. In contrast, PBOX-6 did not activate the extracellular signal-regulated kinase (ERK) or p38. Apoptosis was found to occur independently of the small GTPases Ras, Rac, and Cdc42 but involved phosphorylation of the JNK substrates, c-Jun and ATF-2. Pretreatment of K562 cells with the JNK inhibitor, dicoumarol, abolished PBOX-6-induced phosphorylation of c-Jun and ATF-2 and inhibited the induced apoptosis, suggesting that JNK activation is an essential component of the apoptotic pathway induced by PBOX-6. Consistent with this finding, transfection of K562 cells with the JNK scaffold protein, JIP-1, inhibited JNK activity and apoptosis induced by PBOX-6. JIP-1 specifically scaffolds JNK, MKK7, and members of the mixed-lineage kinase (MLK) family, implicating these kinases upstream of JNK in the apoptotic pathway induced by PBOX-6 in K562 cells. Mitogen-activated protein (MAP) 1The abbreviations used are: MAPmitogen-activated proteinPBOXpyrrolo-1,5-benzoxazepineCMLchronic myelogenous leukemiaJNKc-Jun N-terminal kinaseJIPJNK-interacting proteinMLKmixed-lineage kinaseGCKgerminal center kinaseERKextracellular signal-regulated kinaseMAPKKMAP kinase kinaseMAPKKKMAPKK kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCRIBCdc42/Rac interactive binding domainPMAphorbol 12-myristate 13-acetate 1The abbreviations used are: MAPmitogen-activated proteinPBOXpyrrolo-1,5-benzoxazepineCMLchronic myelogenous leukemiaJNKc-Jun N-terminal kinaseJIPJNK-interacting proteinMLKmixed-lineage kinaseGCKgerminal center kinaseERKextracellular signal-regulated kinaseMAPKKMAP kinase kinaseMAPKKKMAPKK kinaseTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineCRIBCdc42/Rac interactive binding domainPMAphorbol 12-myristate 13-acetate kinases are a group of protein serine/threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction cascades that play an important regulatory role in cell growth, differentiation, and apoptosis (1Chan-Hui P.Y. Weaver R. Biochem. J. 1998; 336: 599-609Crossref PubMed Scopus (69) Google Scholar). In mammalian systems, the biochemical properties of three MAP kinases have been characterized in detail, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) also referred to as stress-activated protein kinase (SAPK), and the p38 MAP kinase (2Ichijo H. Oncogene. 1999; 18: 6087-6093Crossref PubMed Scopus (473) Google Scholar). The ERK subgroup of MAP kinases is activated primarily by mitogenic stimuli such as growth factors (3Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar). In contrast, the JNK and p38 pathways are activated primarily by a diverse array of cellular stresses including UV irradiation, hydrogen peroxide, DNA damage, heat, and osmotic shock (2Ichijo H. Oncogene. 1999; 18: 6087-6093Crossref PubMed Scopus (473) Google Scholar). mitogen-activated protein pyrrolo-1,5-benzoxazepine chronic myelogenous leukemia c-Jun N-terminal kinase JNK-interacting protein mixed-lineage kinase germinal center kinase extracellular signal-regulated kinase MAP kinase kinase MAPKK kinase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Cdc42/Rac interactive binding domain phorbol 12-myristate 13-acetate mitogen-activated protein pyrrolo-1,5-benzoxazepine chronic myelogenous leukemia c-Jun N-terminal kinase JNK-interacting protein mixed-lineage kinase germinal center kinase extracellular signal-regulated kinase MAP kinase kinase MAPKK kinase N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Cdc42/Rac interactive binding domain phorbol 12-myristate 13-acetate All MAP kinases are activated in response to their phosphorylation within a T XY motif, by dual specificity MAP kinase kinases (MAPKKs). The MAPKKs form a highly conserved group, which are activated through phosphorylation of conserved serine and threonine residues by a somewhat more diverse group of MAP kinase kinase kinases (MAPKKKs). In the JNK subgroup, three genes (jnk1, jnk2, and jnk3) have been described. Of these, JNK1 and JNK2 protein kinases are expressed ubiquitously, while JNK3 is expressed primarily in the brain (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar). JNK is activated by two distinct MAPKKs, MKK4 (5Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar) or MKK7 (6Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). A number of MAPKKKs have been identified as upstream activators of the JNK pathway, including MEKK (MEKK-1, -2, -3, and -4), the mixed-lineage protein kinases MLK-2, MLK-3, DLK, and LZK (7Leung I.W. Lassam N. J. Biol. Chem. 2001; 276: 1961-1967Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and Tpl-2, a member of the Raf family (8Tibbles L.A. Woodget J.R. Cell Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (554) Google Scholar). A number of factors have been implicated in the coupling between activating stimuli and the MAPKKKs. For example, receptor-mediated signaling by growth factors involves autophosphorylation on tyrosine residues (3Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar). Alternatively, small GTP-binding protein such as Ras (9Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar) and members of the Rho family of GTPases, Rac and Cdc42, (10Murasawa S. Matsubara H. Mori Y. Masaki H. Tsutsumi Y. Shibasaki Y. Kitabayashi I. Tanaka Y. Fujiyama S. Koyama Y. Fujiyama A. Iba S. Iwasaka T. J. Biol. Chem. 2000; 275: 26856-26863Abstract Full Text Full Text PDF PubMed Google Scholar, 11Puls A. Eliopoulos A.G. Nobes C.D. Bridges T. Young L.S. Hall A. J. Cell Sci. 1999; 112: 2983-2992Crossref PubMed Google Scholar, 12Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar) have been found to couple external stimuli to MAP kinase activation. In addition, a subgroup of the Ste20-like serine/threonine protein kinases, known as germinal center kinases (GCKs), have also been found to activate JNK through the activation of members of the MLK family (7Leung I.W. Lassam N. J. Biol. Chem. 2001; 276: 1961-1967Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 13Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G., Xu, Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 14Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett J.R. Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (204) Google Scholar). The targeting of certain signals to specific MAP kinase modules may be regulated by the presence of scaffold proteins, such as JNK-interacting protein (JIP), which facilitates signal transduction (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar). JIP has been shown to interact with the MAPKK MKK7 but not MKK4 and to interact with MAPKKKs from the mixed-lineage kinase family, but not the MAPKKK MEKK1 or MEKK4. Therefore, the function of JIP as a scaffold protein is selective for the MLK-MKK7-JNK MAP kinase module (15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (588) Google Scholar). Activation of the JNK signaling pathway leads to phosphorylation of a number of targets including the transcription factors, activating transcription factor 2 (ATF-2) (16Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar) and c-Jun (17Seimiya H. Mashima T. Toho M. Tsuruo T. J. Biol. Chem. 1997; 272: 4631-4636Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) resulting in an increase in their transcriptional activity. Overexpression of JIP proteins causes cytoplasmic retention of JNK, thereby inhibiting gene expression mediated by JNK signaling pathways (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar). Human chronic myelogenous leukemia (CML) is a malignancy of pluripotent hematopoietic cells. 95% of CML cases display a distinctive cytogenetic abnormality known as the Philadelphia (Ph) chromosome, which results from a reciprocal translocation between the long arms of chromosome 9 and 22 [t(9;22) (q34;q11)] (18Clarkson B.D. Strife A. Wisniewski D. Lambek C. Carpino N. Leukemia. 1997; 11: 1404-1428Crossref PubMed Scopus (69) Google Scholar). This translocation results in the production of a p210 Bcr-Abl fusion protein with increased tyrosine kinase activity (19Faderl S. Talpaz M. Estrov Z. Kantarjian H.M. Ann. Intern. Med. 1999; 131: 207-219Crossref PubMed Scopus (380) Google Scholar). Clinically CML follows a triphasic course; an initial chronic phase followed by an accelerated phase, which subsequently leads to blast crisis. Blast crisis is accompanied by the appearance of poorly differentiated myeloid or lymphoid blast cells in the bone marrow and is unresponsive to conventional doses of chemotherapy (20Fernandes R.S. Gorman A.M. McGahon A. Lawlor M. McCann S. Cotter T.G. Leukemia. 1996; 10: S17-S21PubMed Google Scholar). K562 cells, which are derived from the pleural effusion of a patient in terminal blast crisis and express the p210 Bcr-Abl fusion protein (21Lozzio C.B. Lozzio B.B. Blood. 1975; 45: 321-334Crossref PubMed Google Scholar), are particularly resistant to the induction of apoptosis by various agents including camptothecin, ara-C, etoposide, paclitaxel, staurosporine, and anti-Fas antibodies (22Kang C.D. Yoo S.D. Hwang B.W. Kim K.W. Kim D.W. Kim C.M. Kim S.H. Chung B.S. Leuk. Res. 2000; 24: 527-534Crossref PubMed Scopus (92) Google Scholar). Down-regulation of Bcr-Abl using antisense treatment restores the sensitivity of CML cells to apoptosis-inducing chemotherapeutic agents (23McGahon A. Bissonnette R. Schmitt M. Cotter K.M. Green D.R. Cotter T.G. Blood. 1994; 83: 1179-1187Crossref PubMed Google Scholar). We have recently found that some members of a novel series of pyrrolo-1,5-benzoxazepines potently induce apoptosis in a number of CML cell lines, such as K562, KYO.1, and LAMA 84, by bypassing the apoptotic suppressor, Bcr-Abl (24Mc Gee M.M. Campiani G. Ramunno A. Fattorusso C. Nacci V. Lawler M. Williams D.C. Zisterer D.M. J. Pharmacol. Exp. Ther. 2001; 296: 31-40PubMed Google Scholar). An insight into the mechanism of action of many novel drugs has helped in establishing their therapeutic potential in the treatment of various diseases. In this study we sought to investigate the mechanism by which a representative compound from this series, known as PBOX-6, induces apoptosis in CML cells. We provide evidence that activation of the stress-activated protein kinase, JNK, is an essential part of the mechanism by which this compound induces apoptosis. Apoptosis is accompanied by phosphorylation of the JNK substrates, c-Jun and ATF-2. We show that PBOX-6-induced apoptosis occurs independently of small G-proteins, Ras, Rac, and Cdc42 and is likely to involve phosphorylation and activation of MKK-7 and members of the MLK and GCK families, which occurs upstream of JNK activation. K562 human chronic myelogenous leukemia cells were gratefully received from Dr. Mark Lawler (St. James's Hospital, Dublin). The pyrrolobenzoxazepine 7-[(dimethylcarbamoyl)oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1Chan-Hui P.Y. Weaver R. Biochem. J. 1998; 336: 599-609Crossref PubMed Scopus (69) Google Scholar, 5Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar)-benzoxazepine (PBOX-6) was synthesized as described previously (25Zisterer D.M. Hance N. Campiani G. Garofalo A. Nacci V. Williams D.C. Biochem. Pharmacol. 1998; 55: 397-403Crossref PubMed Scopus (25) Google Scholar). The RapiDiff kit was obtained from Diagnostic Developments (Burscough, Lancashire, UK). FuGENE 6 transfection reagents was from Roche Molecular Biochemicals (Mannheim, Germany). Anti-JNK and anti-ERK and anti-p38-phosphospecific polyclonal antibodies, anti-ATF-2-phosphospecific, and anti-c-Jun-phosphospecific antibodies were purchased from New England Biolabs (Hertfordshire, UK). Dicoumarol was obtained from Sigma (Poole, Dorset, UK). The ATF-2- and c-Jun-luciferase reporting systems, (PathDetectTM) were purchased from Stratagene (La Jolla, CA). The constitutively active mutants (RacV12 and RasV12), and dominant negative (DN) mutants (RacN17 and RasN17), along with the JIP-1 plasmid and the β-galactosidase expression vector were kindly provided by Professor Luke O'Neill (Biochemistry Department, Trinity College, Dublin). Wild type and dominant negative versions of cdc42 were generous gifts from Professor Alan Hall (University College, London). Unless stipulated, all other reagents were from Sigma. K562 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, gentamycin (0.1 mg/ml), and l-glutamate (2 mm) and incubated in a humidified atmosphere of 95% O2 and 5% CO2 at 37 °C. Cells were seeded at a density of 3 × 105 cells/ml, and following treatment with PBOX-6 (10 μm), an aliquot (100 μl) was cytocentrifuged onto glass slides. They were then stained with the RapiDiff kit (eosin/methylene blue) under conditions described by the manufacturer. The degree of apoptosis and necrosis was determined by counting ∼300 cells under a light microscope. At least 3 fields of view per slide, with an average of ∼100 cells per field, were counted and the percent apoptosis and necrosis was determined as previously described (26Zisterer D.M. Campiani G. Nacci V. Williams D.C. J. Pharmacol. Exp. Ther. 2000; 293: 48-59PubMed Google Scholar). Transient transfection of K562 cells was performed with FuGENE 6 transfection reagent, using 400 ng of DNA in 100 μl of serum-free RPMI 1640 medium combined with 1.5 μl of FuGENE 6 in 20 μl of serum-free RPMI 1640. The DNA/FuGENE mixture was incubated for 15 min at room temperature and was then added dropwise to 4 × 105 cells in 400 μl of RPMI 1640 containing 20% fetal calf serum in a 24-well dish. Following overnight incubation, cells were stimulated with either vehicle (1% (v/v) ethanol) or PBOX-6 (10 μm). Cells were harvested for luciferase and β-galactosidase assays. A luciferase assay were performed by centrifugation of the cells at 500 × g for 5 min, and the pellets were lysed using passive lysis buffer (Promega) followed by gentle agitation at room temperature for 15 min. Luciferase activity was assayed by the addition of luciferase assay mix (40 μl) (20 mm Tricine, 1.07 mm(MgCO3)4Mg(OH)2·5H20, 2.67 mm MgSO4, 0.1 mm EDTA, 33.3 mm dithiothreitol, 270 mm coenzyme A, 470 mm luciferin, 530 mm ATP) to each sample, and luminescence was read using a luminometer. β-galactosidase activity was assayed by the addition of o- nitrophenylgalactosidase to an aliquot of cell lysate (20 μl) in a 96-well plate followed by the addition of β-galactosidase buffer (23 mmNaH2PO4, 77 mmNa2HPO4, 0.1 mm MnCl2, 2 mm MgSO4, 40 mmβ-mercaptoethanol, pH 7.3) and the plate was incubated at 37 °C overnight and absorbance was read at 405 nm. All transfections were performed at least in triplicate, and all luciferase values were normalized according to β-galactosidase readings. Cells were collected by centrifugation at 500 × g, washed with ice-cold phosphate-buffered saline and lysed on ice for 20 min in a buffer containing 150 mm NaCl, 50 mm Tris/Cl, pH 8.0, 0.1% (v/v) SDS, 1.0% (v/v) Triton X-100, sodium orthovanadate (1 mm), phenylmethylsulfonyl fluoride (1 mm) supplemented with leupeptin (1 μg/ml) and aprotinin (10 μg/ml), followed by centrifugation at 20,000 × g for 10 min, and the supernatants were collected. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane by electroblotting. Membranes were probed with phosphospecific antibodies to JNK, ERK, p38, ATF-2, or c-Jun according to the manufacturers' instructions. Proteins were visualized using enhanced chemiluminesence reageants (ECL from AmershamBiosciences). MAP kinases represent one of the most important signaling cascades in response to extracellular stimuli and it is the dual phosphorylation of MAP kinases by upstream kinases that results in their activation (1Chan-Hui P.Y. Weaver R. Biochem. J. 1998; 336: 599-609Crossref PubMed Scopus (69) Google Scholar). The ability of PBOX-6 to activate intracellular MAP kinase pathways in K562 cells was assessed following treatment with either vehicle or PBOX-6 for various lengths of time. Whole cell extracts were prepared and Western blotting was performed using antibodies against the active/phosphorylated form of the MAP kinases, ERK, JNK, and p38. Fig. 1 A demonstrates that PBOX-6 induces the transient activation of two JNK isoforms, JNK1 and JNK2, in K562 cells. JNK activation becomes visible following a 15-min treatment with PBOX-6, peaks between 30–45 min, and slowly declines to basal levels over 8 h. In contrast, whereas UV irradiation of Jurkat cells for 2 min activates p38, PBOX-6 treatment of K562 cells for up to 8 h fails to activate p38 (Fig. 1 B). Similarly, PMA treatment of K562 cells for 30 min activates the ERK MAP kinase whereas PBOX-6 treatment of cells for up to 8 h has no effect (Fig. 1 C). These results suggest that activation of the JNK MAP kinase and not the p38 or ERK MAP kinase may be an important intermediate in the pathway by which PBOX-6 induces apoptosis in K562 cells. Furthermore, it has been previously shown that only some members of this series of pyrrolo-1,5-benzoxazepines induce apoptosis in CML cells (24Mc Gee M.M. Campiani G. Ramunno A. Fattorusso C. Nacci V. Lawler M. Williams D.C. Zisterer D.M. J. Pharmacol. Exp. Ther. 2001; 296: 31-40PubMed Google Scholar). In this study we have found that the pro-apoptotic members result in activation of JNK whereas the non-apoptotic members failed to activate JNK in K562 cells (data not shown). It is widely reported that the JNK MAP kinase phosphorylates the transcription factors c-Jun and ATF-2 resulting in increased transcriptional activity (3Minden A. Karin M. Biochim. Biophys. Acta. 1997; 1333: F85-104PubMed Google Scholar, 16Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar, 27Fuchs S.Y. Tappin I. Ronai Z. J. Biol. Chem. 2000; 275: 12560-12564Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Results from Western blotting using phosphospecific antibodies demonstrate that PBOX-6 induces a dose- and time-dependent phosphorylation of c-Jun (Fig. 2, A and B) and ATF-2 (Fig. 2, C and D) in K562 cells. Taken together these results demonstrate that PBOX-6-induced activation of JNK and its downstream substrates, c-Jun and ATF-2, in K562 cells may be important in the mechanism in which this compound induces apoptosis. It has recently been shown that the quinone reductase inhibitor, dicoumarol, specifically inhibits activation of the JNK MAP kinase in response to a variety of stress stimuli (28Cross J.V. Deak J.C. Rich E.A. Qian Y. Lewis M. Parrott L.A. Mochida K. Gustafson D. Vande Pol S. Templeton D.J. J. Biol. Chem. 1999; 274: 31150-31154Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 29Krause D. Lyons A. Fennelly C. O'Connor R. J. Biol. Chem. 2001; 276: 19244-19252Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To determine whether activation of JNK is directly associated with the pro-apoptotic activity of PBOX-6 in K562 cells, we sought to block JNK activity using dicoumarol and to determine the effect on the extent of apoptosis induced by PBOX-6. Cells were treated with PBOX-6 (10 μm) for 16 h in the absence and presence of dicoumarol, and cell lysates were prepared. Western blot analysis reveals that dicoumarol inhibits c-Jun and ATF-2 phosphorylation in response to PBOX-6 (Fig. 3 A). To determine the effect of dicoumarol on PBOX-6-induced apoptosis, K562 cells were pretreated with dicoumarol for 1 h prior to treatment with PBOX-6 for a further 16 h, followed by RapiDiff staining of the cells, and the extent of apoptosis was determined by morphological examination. Results shown in Fig. 3 B illustrate that although dicoumarol itself does not induce apoptosis in K562 cells, pretreatment with dicoumarol completely abolishes PBOX-6-induced apoptosis in these cells. These results confirm earlier findings that dicoumarol inhibits JNK activity and suggests that c-Jun and ATF-2 phosphorylation in K562 cells occurs as a direct consequence of JNK activation. In addition, the ability of dicoumarol to completely abrogate apoptosis suggests that JNK activation is essential in the pathway by which PBOX-6 induces apoptosis in K562 cells. To confirm the importance of JNK activation during PBOX-6-induced apoptosis and to further delineate the signal transduction cascade induced, transient transfection assays using a luciferase reporting system were carried out. Overexpression of the JNK scaffold protein, JIP-1, has been previously shown to inhibit the downstream signaling of JNK (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar). K562 cells were transiently transfected with a c-Jun- or ATF-2-dependent luciferase reporting system. Activation of c-Jun and ATF-2, expressed as -fold stimulation over unstimulated cells, shows an approximate 4-fold increase in c-Jun activity and a 7-fold increase in ATF-2 activity in response to PBOX-6 treatment, whereas ethanol (1%) had no effect (Fig. 4 A). These results confirm the earlier findings from Western blotting outlining that PBOX-6 causes phosphorylation of the JNK substrates, c-Jun and ATF-2. To confirm the essential requirement of JNK activation during PBOX-6-induced apoptosis, K562 cells, transfected with the ATF-2 luciferase reporting system, were co-transfected with JIP-1, and the effect on PBOX-6 activity was determined. Results shown in Fig. 4 B demonstrate that while PBOX-6 stimulates a 5-fold increase in ATF-2-dependent reporter gene activity, co-transfection of the JIP-1 protein into K562 cells completely inhibits PBOX-6-induced stimulation of ATF-2 activity. In addition, K562 cells were transfected with empty vector (PCDNA3) or JIP-1 followed by treatment with PBOX-6 for a further 16 h and the extent of apoptosis was determined by morphological examination. The results shown in Fig. 4 C demonstrate that co-transfection of JIP-1 into K562 cells significantly reduces the extent of apoptosis induced by PBOX-6, which is consistent with a 50% transfection efficiency. These findings confirm earlier results, which suggest that JNK activation is essential during PBOX-6-induced apoptosis in K562 cells. A number of upstream components are implicated in activation of the JNK signaling cascade, including the small GTP-binding proteins Ras, Rac, and Cdc42 (9Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 10Murasawa S. Matsubara H. Mori Y. Masaki H. Tsutsumi Y. Shibasaki Y. Kitabayashi I. Tanaka Y. Fujiyama S. Koyama Y. Fujiyama A. Iba S. Iwasaka T. J. Biol. Chem. 2000; 275: 26856-26863Abstract Full Text Full Text PDF PubMed Google Scholar, 12Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 30Bagrodia S. Derijard B. Davis R.J. Cerione R.A. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar). To determine whether these small G-proteins are activated upstream of JNK during PBOX-6-induced apoptosis in K562 cells, cells were co-transfected with constitutively active (V12) and dominant negative (N17) mutants of these proteins, and the effect on PBOX-6-induced phosphorylation of ATF-2-dependent luciferase reporter gene was determined. Results shown in Fig. 5indicate that co-transfection with constitutively active versions of Ras, Rac, and Cdc42 cause an increase in ATF-2 phosphorylation, confirming that these proteins are capable of activating the JNK signaling pathway. Upon treatment of cells with PBOX-6 an additive increase in ATF-2 activity is observed as PBOX-6 also drives the JNK signaling pathway. On the other hand, cells transfected with RasN17, RacN17, or Cdc42N17 alone have no effect on ATF-2 activation, whereas following treatment with PBOX-6, DN mutants fail to inhibit PBOX-6-induced activation of ATF-2, suggesting that activation of these small G-proteins does not lie upstream of JNK in the signal transduction pathway by which PBOX-6 induces apoptosis in K562 cells. The results presented herein provide an insight into the mechanism whereby a novel apoptotic agent, known as PBOX-6, induces apoptosis in the extremely drug-resistant K562 cell line. PBOX-6 was found to activate the JNK signaling pathway, but not the ERK or p38 MAP kinases pathways, in CML cells. Although the three MAP kinase pathways share structural similarities, the outcome of activation is quite different. ERK stimulation by mitogenic and trophic agents results in cell division and differentiation whereas activation of p38 and JNK results in growth arrest and cell death (31Zanke B.W. Boudreau K. Rubie E. Winnett E. Tibbles L.A. Zon L. Kyriakis J. Liu F.F. Woodgett J.R. Curr. Biol. 1996; 6: 606-613Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar). The same signaling pathway can often have different functions depending on the cell context. However, because JNK is activated by a variety of cellular stresses, it has been proposed to serve as the major apoptosis switch. For example, activation of the JNK signaling pathway has been shown to result in apoptosis in response to cis-platinum and heat shock (31Zanke B.W. Boudreau K. Rubie E. Winnett E. Tibbles L.A. Zon L. Kyriakis J. Liu F.F. Woodgett J.R. Curr. Biol. 1996; 6: 606-613Abstract Full Text Full Text PDF PubMed Scopus (438) Google Scholar), DNA damaging agents such as anisomycin and UV irradiation (28Cross J.V. Deak J.C. Rich E.A. Qian Y. Lewis M. Parrott L.A. Mochida K. Gustafson D. Vande Pol S. Templeton D.J. J. Biol. Chem. 1999; 274: 31150-31154Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 32Shaulian E. Karin M. J. Biol. Chem. 1999; 274: 29595-29598Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), chemotherapeutic agents such as etoposide and camptothecin (17Seimiya H. Mashima T. Toho M. Tsuruo T. J. Biol. Chem. 1997; 272: 4631-4636Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), and vinblastine and adriamycin (33Osborn M.T. Chambers T.C. J. Biol. Chem. 1996; 271: 30950-30955Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Pathologically, failure of an apoptosis program often leads to an imbalance in cell number, which in turn leads to tumorigenesis. Therefore, control of apoptosis has emerged as an important strategy for clinical cancer therapy (17Seimiya H. Mashima T. Toho M. Tsuruo T. J. Biol. Chem. 1997; 272: 4631-4636Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Although a number of chemotherapeutic agents have been used in the treatment of leukemia, many forms such as chronic myeloid leukemia are resistant to the induction of apoptosis (22Kang C.D. Yoo S.D. Hwang B.W. Kim K.W. Kim D.W. Kim C.M. Kim S.H. Chung B.S. Leuk. Res. 2000; 24: 527-534Crossref PubMed Scopus (92) Google Scholar). Recently we have shown that some members of a novel series of pyrrolo-1,5-benzoxazepine compounds potently induce apoptosis in CML cells (24Mc Gee M.M. Campiani G. Ramunno A. Fattorusso C. Nacci V. Lawler M. Williams D.C. Zisterer D.M. J. Pharmacol. Exp. Ther. 2001; 296: 31-40PubMed Google Scholar), and they do so by bypassing the apoptotic suppressor Bcr-Abl, indicating the potential of these novel compounds in the treatment of CML and related disorders. In the present study we set out to delineate the apoptotic signaling pathway induced in CML cells by a representative compound from this novel series, called PBOX-6. Our findings that PBOX-6 induces that transient activation of two JNK isoforms, JNK1 and JNK2, in K562 cells supports a role for this stress pathway in the induction of apoptosis by PBOX-6. The quinone reductase inhibitor, dicoumarol, has been shown to inhibit JNK activation in response to the TNFα receptor-interacting protein, TRAF2, and in response to anisomycin, sorbitol, UV irradiation, and ceramide, while having no effect on the mitogen-activated pathways (28Cross J.V. Deak J.C. Rich E.A. Qian Y. Lewis M. Parrott L.A. Mochida K. Gustafson D. Vande Pol S. Templeton D.J. J. Biol. Chem. 1999; 274: 31150-31154Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In this study, inhibition of JNK activity in K562 cells by dicoumarol, together with its inhibitory effect on the extent of apoptosis induced by PBOX-6 confirms the importance of JNK activation in the apoptotic pathway induced by PBOX-6. In addition, we have previously reported that only some members of this novel series of pyrrolo-1,5-benzoxazepines induce apoptosis in CML cells (24Mc Gee M.M. Campiani G. Ramunno A. Fattorusso C. Nacci V. Lawler M. Williams D.C. Zisterer D.M. J. Pharmacol. Exp. Ther. 2001; 296: 31-40PubMed Google Scholar). We have found that all the members that induce apoptosis in CML cells result in activation of JNK, whereas the non-apoptotic members failed to activate JNK (data not shown), lending further support to the suggestion that JNK activation, which occurs within 15 min, is an important part of the mechanism by which these novel compounds induce apoptosis in CML cells. The phosphorylation of several transcription factors, such as c-Jun and ATF-2, in response to JNK activation, has been well documented. In agreement with this, PBOX-6 induces phosphorylation and thus activation of c-Jun and ATF-2, in K562 cells, in a time- and dose-dependent manner, which further outlines the importance of the JNK signaling pathway during PBOX-6-induced apoptosis in K562 cells. JNK activation occurs because of dual phosphorylation by the upstream kinases, MKK4 or MKK7. These are phosphorylated and activated by further upstream kinases known as MAPKKK. A number of different MAPKKK have been shown to activate JNK, and these include members of the MEKK and MLK families. For example it has been shown that overexpression of MLK-3 was sufficient to activate JNK in COS-7 cells (12Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). Although it is clear that the MLKs play an important role in transmitting a variety of different signals to JNK activation, the mechanisms that directly regulate MLK activity are not well understood. It is however believed that in some cases, members of the MLK family are regulated by the small GTP-binding proteins, Rac and Cdc42, and by a subgroup of the Ste20-related kinase family known as germinal center kinases (GCKs) (34Diener K. Wang X.S. Chen C. Meyer C.F. Keesler G. Zukowski M. Tan T.H. Yao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9687-9692Crossref PubMed Scopus (117) Google Scholar). For example, MLK has been shown to bind Cdc42 and Rac in vivo, through a Cdc42/Rac interactive binding domain (CRIB), and a dominant negative mutant of MLK3 abolishes activation of JNK by Cdc42 and Rac (12Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). In addition, a GCK family member known as HGK has been found to induce JNK activation in human embryonic kidney cells, which was inhibited by dominant negative MKK4 and MKK7 mutants (6Yao Z. Zhou G. Wang X.S. Brown A. Diener K. Gan H. Tan T.H. J. Biol. Chem. 1999; 274: 2118-2125Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In the present study we found that the small GTP-binding proteins, Ras, Rac, and Cdc42 are not involved in the upstream signaling pathway leading to JNK activation upon PBOX-6 treatment of K562 cells. In addition, the synergy observed between constitutively active G-proteins and PBOX-6 would suggest that the GTPases and PBOX-6 activate JNK via independent signaling pathways. These results are in agreement with many reports, which demonstrate that JNK activation may occur independently of the GTP-binding proteins (9Minden A. Lin A. McMahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1011) Google Scholar, 35Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P., Xu, N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1567) Google Scholar, 36Davis L. Stephens L.R. Hawkins P.T. Saklatvala J. Biochem. J. 1999; 338: 387-392Crossref PubMed Scopus (29) Google Scholar). For example, dominant negative Rac and Cdc42 had no effect on anisomycin-induced JNK activation in COS-7 cells (35Coso O.A. Chiariello M. Yu J.C. Teramoto H. Crespo P., Xu, N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1567) Google Scholar), or on IL-1 and PDGF-induced JNK activation in PAE cells (36Davis L. Stephens L.R. Hawkins P.T. Saklatvala J. Biochem. J. 1999; 338: 387-392Crossref PubMed Scopus (29) Google Scholar). Therefore, it seems that GTPases may only modulate JNK activity in certain cell types and under certain conditions. In support of this concept, the MLK family members DLK and LZK have been shown to lack a CRIB domain suggesting that MLKs can be regulated in a GTPase independent pathway (37Merritt S.E. Mata M. Deepak N. Zhu C., Hu, X. Holzman L.B. J. Biol. Chem. 1999; 274: 10195-10202Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Germinal center kinases which are located upstream of JNK, lack a CRIB domain and are therefore not under the control of the small GTPases. It has indeed been reported that dominant negative forms of MLK-3 block JNK activation mediated through the Ste20 homologues GCK and HPK (7Leung I.W. Lassam N. J. Biol. Chem. 2001; 276: 1961-1967Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). JIP-1 has been identified as a scaffold protein that interacts in a specific fashion with specific kinases and leads to activation of the JNK signaling pathway. Components of the complex mediated by JIP-1 have been used to determine the specificity of the stress-activated kinase signaling pathway. In addition, overexpression of JIP-1 inhibits the downstream JNK signaling pathway, due to cytoplasmic retention of the JNK signaling module (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar). Data obtained in the present study using JIP-1 have been used to delineate the signaling pathway induced by PBOX-6. We have found that co-transfection of JIP-1 into K562 cells completely abolishes PBOX-6-induced JNK activity and inhibits apoptosis. These data correlate with results obtained earlier using dicoumarol, which indicate that JNK activity is essential during PBOX-6-induced apoptosis in K562 cells. JIP-1 selectively binds components of the JNK signaling module. It has been shown that JIP-1 binds to members of the MLK family, but not to MEKK1 or MEKK4 (15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (588) Google Scholar). In addition, JIP-1 interacts with HPK1, a member of the GCK family, MKK7 from the MAPKK tier of kinases but it does not interact with MKK4 or the small GTP-binding proteins, Rac and Cdc42 (4Yasuda J. Whitmarsh A.J. Cavanagh J. Sharma M. Davis R.J. Mol. Cell. Biol. 1999; 19: 7245-7254Crossref PubMed Scopus (408) Google Scholar, 15Whitmarsh A.J. Cavanagh J. Tournier C. Yasuda J. Davis R.J. Science. 1998; 281: 1671-1674Crossref PubMed Scopus (588) Google Scholar). Consistent with this, it has been reported that although MLK-3 and MLK-2 can activate JNK via MKK4 and MKK7, these kinases show preferential association with MKK7. In addition, DLK activates JNK through MKK7 only (7Leung I.W. Lassam N. J. Biol. Chem. 2001; 276: 1961-1967Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Therefore JIP-1 selectively scaffolds the MLK-MKK7-JNK kinase module. The ability of JIP-1 to block PBOX-6-induced apoptosis in K562 cells supports the finding that apoptosis occurs in a GTPase-independent fashion and strongly supports the hypothesis that members of the GCK and MLK families, together with MKK7 are involved in the upstream apoptotic pathway induced by PBOX-6 in K562 cells. Events occurring upstream of GCKs and leading to JNK activation have not been fully determined; however, some reports suggest that activation of GCK family members occurs following recruitment, via adaptor proteins, to receptor tyrosine kinases such as the epidermal growth factor receptor (38Anafi M. Kiefer F. Gish G.D. Mbamalu G. Iscove N.N. Pawson T. J. Biol. Chem. 1997; 272: 27804-27811Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Further work to investigate this hypothesis is currently underway. In summary, we have found that the novel apoptotic agent, PBOX-6, induces the transient activation of the JNK signaling pathway in K562 cells, an event that is crucial for its apoptotic activity. In an attempt to further delineate the signaling pathway activated by PBOX-6, we conclude that apoptosis occurs independently of the GTP-binding proteins Ras, Rac, and Cdc42 and is likely to involve activation of members of the GCK and MLK families, together with MKK7, thus leading to JNK activation." @default.
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• W2014197880 title "Activation of the c-Jun N-terminal Kinase (JNK) Signaling Pathway Is Essential during PBOX-6-induced Apoptosis in Chronic Myelogenous Leukemia (CML) Cells" @default.
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