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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Biochemistry, Trinity College, Dublin, Ireland (M.M.M.G., L.M.G., S.L., D.C.W., D.M.Z.); Dipartimento Farmaco Chimico Tecnologico, Universita'delgi Studi di Siena, Siena, Italy (G.C., V.N.); and Department of Hematology, Institute of Molecular Medicine, Trinity Centre, St. James's Hospital, Dublin, Ireland (M.L.)
Received February 25, 2004; accepted May 12, 2004.
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; Deininger et al., 2000). For example, it has been reported that Bcr-Abl acts through expression of antiapoptotic proteins Bcl-2 and Bcl-XL (Sanchez-Garcia and Grutz, 1995; Oetzel et al., 2000). Members of the Bcl-2 family together with mitochondria and cysteine proteases are essential components of many intracellular signaling pathways, although the mechanisms by which they do so remain uncertain.
Bcl-2, the prototypic member of the Bcl-2 family of proteins, was cloned as a result of the t(14; 18) chromosomal translocation in human follicular B cell lymphoma, which results in its overexpression (Baell and Huang, 2002). At least 16 Bcl-2 homologs are found in humans, as defined by sequence similarity to some or all four Bcl-2 homology domains in Bcl-2, and the family comprises both pro- and antiapoptotic regulators (Tsujimoto and Shimizu, 2000). Antiapoptotic Bcl-2 proteins can protect cells from death induced by many stimuli, suggesting that they control a crucial step in the final common pathway for cell death (Martin et al., 1995; Keogh et al., 2000); however, the molecular mechanism involved remains incompletely defined. A striking feature of Bcl-2 proteins is their ability to form homodimers and heterodimers and heterodimerization between opposing family members is thought to inhibit the biological activity of their partners (Sattler et al., 1997). In addition to heterodimerization, alternative independent mechanisms of cell death regulation also exist including inhibition of caspase activity (Chinnaiyan et al., 1996) and the release of mitochondrial proteins such as cytochrome c and apoptosis-inducing factor (Susin et al., 1999). The proapoptotic Bcl-2 family member Bax normally resides in the cytoplasm and translocates to the mitochondria where it undergoes oligomerization and/or a conformational change in response to apoptotic stimuli (Nomura et al., 1999; Nouraini et al., 2000). Bcl-2 counteracts the proapoptotic effect of Bax in some systems by preventing its translocation to the mitochondrial membrane (Mikhailov et al., 2001). In addition, emerging data implies that Bcl-2 proteins are targets for phosphorylation, suggesting another mechanism of control although the functional significance of phosphorylation remains controversial (Tsujimoto, 1998). Phosphorylation of Bcl-2 occurs within an unstructured loop and involves several sites, the principal sites being serine 70, serine 87, and threonine 69 (Yamamoto et al., 1999; Srivastava et al., 1999). In some instances, phosphorylation of Bcl-2 results in its inactivation, rendering the cells more sensitive to the induction of apoptosis, whereas other reports suggest that Bcl-2 phosphorylation can enhance its antiapoptotic effect (Ruvolo et al., 1998).
Much interest has focused on the specific signaling pathway leading to Bcl-2 phosphorylation during drug-induced apoptosis. A family of mitogen-activated protein kinase signaling proteins transduce signals from the cell membrane to the nucleus during apoptosis. One family member, known as c-Jun NH2-terminal kinase (JNK), becomes activated after its phosphorylation by a variety of upstream kinases, including mitogen-activated protein kinase kinase kinase and members of the mixed lineage protein kinase (MLK) family (Xu et al., 2001). Activated JNK phosphorylates downstream substrates such as activating transcription factor-2, c-Jun, and members of the Bcl-2 family, thereby regulating their expression (Fan et al., 2000; Barr and Bogoyevitch, 2001).
We have previously described how some members of a novel series of pyrrolo-1,5-benzoxazepine compounds potently induce apoptosis in a variety of cancerous cells, including chemotherapy-resistant CML cells, and the human breast carcinoma, MCF-7 cells, indicating their potential in the treatment of both diffuse and solid tumors (Zisterer et al., 2000; Mc Gee et al., 2002b). We have shown that PBOX-6 induces apoptosis in K562 cells in a mechanism that bypasses the apoptotic suppressor Bcr-Abl (Mc Gee et al., 2001). We have shown that JNK activation is essential during PBOX-6-induced apoptosis, whereas the activation of caspases is dispensable (Mc Gee et al., 2002a). In this study, we show that PBOX-6 targets antiapoptotic Bcl-2 proteins, resulting in their phosphorylation and inactivation leading to the induction of apoptosis in resistant tumor cells, whereas it has no cytotoxic effect on normal peripheral blood mononuclear cells (PBMCs). Furthermore, we confirm the involvement of JNK in the signaling pathway leading to Bcl-2 phosphorylation, and we have identified further potential targets for therapeutic intervention.
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). The JNK inhibitor CEP-11004 was generously provided by Cephalon, Inc. (West Chester, PA). Dicoumarol, etoposide, actinomycin D, and MTT were all purchased from Sigma Chemical. Anti-Bcl-2 and anti-JNK antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-Bcl-XL antibody was purchased from BD Biosciences PharMingen (San Diego, CA), and anti-Bax antibody was purchased from BD Transduction Laboratories (Lexington, KY). The phospho-specific JNK antibody and GST-c-Jun substrate were purchased from New England Biolabs (Hertfordshire, UK). Lymphoprep was purchased from Unitech (Dublin, Ireland). The RapiDiff kit was obtained from Diagnostic Development (Burscough, Lancashire, UK), and the Lactate Dehydrogenase kit was obtained from Promega (Madison, WI). The enhanced chemiluminescence reagent and the [
-32P]ATP were obtained from Amersham Biosciences Inc. (Aylesbury, UK).
Cell Lines. K562 cells were described previously (Mc Gee et al., 2001). CEM-Neo and CEM-Bcl-2 cells were generously supplied by Professor Seamus Martin (Department of Genetics, Trinity College Dublin, Ireland). Jurkat-Neo, Jurkat-WT Bcl-2, and Jurkat AA/A Bcl-2 cells were a kind gift from Professor Stanley Korsmeyer (Harvard Medical School and Dana-Farber Cancer Institute, Boston, MA).
Cell Culture and Apoptosis Assay. Cells were grown in RPMI 1640 medium supplemented with fetal calf serum (10%), gentamicin (0.1 mg/ml), and L-glutamine (2 mM). Cells were maintained at 37°C in a humidified incubator with 95% air and 5% CO2. The apoptosis assay was performed as described previously (Mc Gee et al., 2002a). Briefly, cells were seeded at 3 x 105 cells/ml and after the relevant treatment, an aliquot of cells (150 µl) was cytocentrifuged onto a glass slide and stained using the RapiDiff kit as described by the manufacturer. The degree of apoptosis and necrosis was determined by counting 
300 cells under a light microscope. At least three fields of view per slide, with an average of 100 cells per field, were counted, and the percentage of apoptosis and necrosis was calculated.
Normal PBMCs. Peripheral blood was obtained with informed consent from normal volunteers, in syringes containing preservativefree heparin, diluted 1:2 with sterile phosphate-buffered saline, and layered over a cushion of 15 ml of Lymphoprep in sterile 50-ml centrifuge tubes. After centrifugation for 30 min at 400g at room temperature, the interface layer, consisting of mononuclear cells, was extracted with a sterile Pasteur pipette and diluted in fresh RPMI 1640 medium containing 10% fetal calf serum. Trypan blue dye exclusion was used to check viability. Only samples containing greater than 90% viable cells were included in the study.
MTT Assay. The cytotoxic activity of PBOX-6 on normal PBMCs was measured using the MTT dye reduction assay. The assay is based on the principle that only viable cells are able to reduce MTT to a colored formazan product that can be determined spectrophotometrically. Optical density is linearly related to the number of viable cells. Unfractionated PBMCs (100 µl) consisting of a mixed population of mononuclear cell types, including lymphocytes, monocytes, and natural killer cells, were seeded in 96-well plates at a density of 2 x 106 cells/ml. Cells were treated with either medium alone, vehicle [1% (v/v) ethanol] or PBOX-6 (10 µM) for 48 h. After treatment, MTT solution (5 mg/ml in phosphate-buffered saline) was added (20 µl/well), and plates were incubated for a further 3 h at 37°C. The purple formazan crystals were dissolved in 200 µl of DMSO. After 20 min, the plates were read on an automated microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at 595 nm, reference filter 690 nm. Assays were performed in triplicate on three independent blood donors. Complete medium (100 µl), MTT stock (20 µl), and DMSO (200 µl) were used as a blank solution. Statistical analysis was performed using a two-tailed Student's t test.
JNK Kinase Assay. JNK activity was measured by immunocomplex assay with anti-JNK1 antibody and GST-c-Jun as a substrate. Cells (6 x 106) were lysed in 400 µl of IP buffer [50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (w/v) Tween 20, 1 mM dithiothreitol, 1 mM sodium fluoride] supplemented with 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, and 0.1 mM sodium orthovanadate. Protein concentration of the cell lysates was normalized to contain 300 µg of protein and incubated for 3 h at 4°C in the presence of anti-JNK antibody. Immune complexes were recovered with the aid of protein A-Sepharose beads followed by two washes with IP buffer and one wash with kinase reaction buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 2.5 mM EGTA, 0.1 mM dithiothreitol, and 0.1 mM sodium orthovanadate). A kinase reaction was performed by resuspending the beads with 30 µl of kinase reaction buffer containing 2.5 µg of GST-c-Jun, 20 µM ATP, and 2 µCi of [-32P]ATP, for 30 min at room temperature. The reaction was terminated by boiling the samples in Laemmli sample buffer for 3 min. Proteins were resolved by SDS-PAGE and the incorporation of 32P was visualized by autoradiography.
Subcellular Fractionation of Cells. Cells (2 x 106) were collected by centrifugation at 500g for 5 min and resuspended in isotonic lysis buffer (100 µl) (200 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 10 mM HEPES, pH 6.9). Cells were ruptured by drawing repeatedly through a 21-gauge needle followed by incubation on ice for 10 min. Unbroken cells, nuclei, and heavy membranes were separated by centrifugation at 1000g for 5 min. The supernatant was centrifuged at 12,000g for 20 min at 4°C to produce cytosolic and mitochondrial enriched fractions. The supernatant was collected and represents the cytosolic fraction. The mitochondria-enriched pellet was washed once with isotonic lysis buffer (200 µl) followed by centrifugation at 12,000g for 5 min and resuspended in hypotonic lysis buffer (20 µl) (10 mM Tris-Cl, pH7.5, 5 mM MgCl2, 1 mM EGTA, and 1 mM dithiothreitol) supplemented with protease inhibitors (5 µg/ml pepstatin A, 2 µg/ml leupeptin, and 2 µg/ml aprotinin) and the protein concentration was determined using a Bradford assay.
Western Blotting. Cells (2 x 106) were collected by centrifugation at 500g for 5 min, and whole cell lysates were prepared by resuspending the pellet in SDS lysis buffer (100 µl) [62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (w/v) glycerol, 50 mM dithiothreitol, and 0.1% (w/v) bromphenol blue]. Extracts were sonicated for 15 s and heated to 95°C for 5 min. An equal amount of sample (20 µl) was resolved on an SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membrane. The membrane was blocked overnight at 4°C in Tris-buffered saline containing 5% (w/v) dried milk followed by incubation with primary antibody for 1 h at room temperature. After incubation, the membrane was washed with Tris-buffered saline containing 0.1% (w/v) Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h and enhanced chemiluminescence was used to visualize the proteins.
Lactate Dehydrogenase (LDH) Assay. The assay was performed under the conditions described by the manufacturer.
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). We have verified that CEM-Bcl-2 cells overexpress Bcl-2 by Western blot analysis (Fig. 1A). In agreement with Martin et al. (1995), we have found that CEM-Neo cells are susceptible to apoptosis induced by etoposide, actinomycin D, and UV irradiation, whereas CEM-Bcl-2 cells are not (Fig. 1, B and C). In contrast, we have found that PBOX-6 induces apoptosis in both CEM-Neo and CEM-Bcl-2 cells (Fig. 1, B and C, respectively). Apoptosis was induced with similar potency in CEM-Neo and CEM-Bcl-2 cells and occurred in a dose- and time-dependent manner after PBOX-6 treatment (Fig. 2, A and B). The morphological features of apoptosis, which include cell shrinkage, chromatin condensation, nuclear fragmentation, and the appearance of apoptotic bodies were absent from vehicle-treated cells but were visible in CEM-Neo and CEM-Bcl-2 cells after treatment with PBOX-6 (Fig. 2C). These results suggest that PBOX-6 can overcome Bcl-2-mediated resistance to apoptosis and highlights its potential in the treatment of tumor cells that overexpress Bcl-2.
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Lack of Cytotoxicity toward Normal PBMCs after PBOX-6 Treatment. To evaluate the effect of PBOX-6 on normal mononuclear cells, PBMCs were isolated from normal healthy volunteers and treated with either vehicle [1% (v/v) ethanol] or PBOX-6 (10 µM) up to 48 h. Cell viability was assessed using an MTT assay as well as by cytocentrifugation of the cells onto slides and examination of cell morphology. The MTT assay measures the reduction of a tetrazolium salt by viable cells and absorbance is directly proportional to cell viability. Therefore, reduced cell viability due to apoptosis or necrosis is reflected by reduced absorbance.
An MTT assay was performed on PBMCs obtained from three volunteers. Statistical analysis of data obtained from all samples was carried out using the Instat computer program and it was found that PBOX-6 (10 µM) did not display any significant cytotoxicity toward normal PBMCs up to 48 h after treatment. Results from a representative sample are shown in Fig. 3A. RapiDiff staining of cells has shown that normal PBMCs remained healthy after treatment with PBOX-6 for 24 h (data not shown). In agreement with this finding from the MTT assay, examination of PBMC morphology after treatment with ethanol or PBOX-6 for 48 h does not display any characteristic features of apoptosis or necrosis (Fig. 3, B and C, respectively). Collectively these results illustrate the noncytotoxic effect of PBOX-6 toward normal mononuclear cells and suggest that the apoptotic action demonstrated by PBOX-6 may be selective toward tumor cells.
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PBOX-6 Induces Phosphorylation of Bcl-2 in K562 and CEM Cells, Which Occurs in a Dose- and Time-Dependent Manner. Phosphorylation of Bcl-2 has been shown to result in its inactivation (Fan et al., 2000). To investigate the mechanism by which PBOX-6 overcomes Bcl-2-mediated resistance to apoptosis, we examined the effect of the compound on Bcl-2 phosphorylation. Cells were treated with either a range (0.1–10 µM) of PBOX-6 concentrations for 16 h or 10 µM PBOX-6 for various lengths of time (0–16 h). Phosphorylation of Bcl-2 is detected by the presence of a slower migrating band by SDS-PAGE. Results from Western blotting show that PBOX-6 induces a dose- and time-dependent phosphorylation of Bcl-2 in K562 cells (Fig. 4, A and B, respectively), CEM-Neo cells (Fig. 4, C and D, respectively) and CEM-Bcl-2 cells (Fig. 4, E and F, respectively). In the three cell lines examined, Bcl-2 phosphorylation first becomes apparent at 1 µM PBOX-6 and increases as drug concentration increases. In addition, Bcl-2 phosphorylation becomes visible after a 1-h treatment with PBOX-6 (10 µM) and increases gradually up to 16 h. Collectively, these results suggest that phosphorylation and inactivation of Bcl-2 may be part of the mechanism by which this novel compound overcomes Bcl-2-mediated resistance to apoptosis.
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Jurkat-AA/A Bcl-2 Cells Demonstrate Resistance against PBOX-6-Induced Apoptosis and Bcl-2 Phosphorylation When Compared with Parental Cells. To investigate further the role of Bcl-2 phosphorylation after PBOX-6 treatment, we assessed its effect on Jurkat clones stably expressing empty vector (Jurkat-Neo), wild-type Bcl-2 (Jurkat-WT Bcl-2), or a triple Bcl-2 mutant, whereby the phosphorylation sites serine 70, serine 87, and threonine 69 have been mutated to alanine (Jurkat-AA/A Bcl-2). It has been previously reported that Jurkat-AA/A Bcl-2 cells demonstrate increased resistance to paclitaxel (Taxol)-induced cell death compared with parental cells (Yamamoto et al., 1999). In agreement with this report, we found that Jurkat-Neo and Jurkat-WT Bcl-2 cells undergo >70% cell death after treatment with Taxol (1 µM) for 24 h, whereas significantly less death was induced in Jurkat-AA/A Bcl-2 cells (Fig. 5A). Treatment of Jurkat-Neo and Jurkat-WT Bcl-2 cells with PBOX-6 (10 µM) for 24 h induces approximately 75% cell death; however, the Jurkat-AA/A Bcl-2 cells display increased resistance against PBOX-6-induced apoptosis with >50% protection observed after 24-h treatment (Fig. 5B). This suggests that phosphorylation of Bcl-2 by PBOX-6 results in its inactivation. In addition, we assessed the ability of PBOX-6 to phosphorylate Bcl-2 in Jurkat cells expressing WT and mutant Bcl-2. It was found that PBOX-6 phosphorylates Bcl-2 in the Jurkat-Neo and Jurkat-WT Bcl-2 cells; however, only a faint band indicative of endogenous Bcl-2 phosphorylation was present in the mutant Jurkat-AA/A Bcl-2 cells (Fig. 5C). This suggests that PBOX-6-induced phosphorylation of Bcl-2 occurs within the unstructured loop on one or more of the mutated phosphorylation sites.
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PBOX-6 Induces a Dose- and Time-Dependent Phosphorylation of Bcl-XL in K562 Cells. Up-regulation of Bcl-XL has been implicated in the antiapoptotic properties of CML cells (Amarante-Mendes et al., 1998), and its phosphorylation is believed to result in its inactivation (Haldar et al., 1996). We assessed the effect of PBOX-6 on the phosphorylation status of Bcl-XL in K562 cells. Whole cell lysates were isolated from K562 cells after treatment with either a range (0.1–10 µM) of PBOX-6 concentrations for 16 h, or 10 µM PBOX-6 for various lengths of time (0–16 h), and the phosphorylation status of Bcl-XL was examined by Western blotting. Results reveal that PBOX-6 induces a dose- and time-dependent phosphorylation of Bcl-XL, as shown by the presence of a slower migrating band by SDS-PAGE (Fig. 6, A and B, respectively). The phosphorylation of Bcl-XL in K562 cells occurs at a later time than Bcl-2 phosphorylation. However, these results support the hypothesis that phosphorylation and inactivation of Bcl-XL may be part of the mechanism by which PBOX-6 induces apoptosis in chemotherapy-resistant CML cells.
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Expression Levels and Subcellular Localization of Bax Are Unaltered in K562 Cells after PBOX-6 Treatment. It has been suggested that Bcl-2 prevents apoptosis by inhibiting the translocation of Bax from the cytosol into the mitochondria where it can carry out its proapoptotic function (Nomura et al., 1999). The hypothesis that inactivation of Bcl-2 and Bcl-XL by phosphorylation may allow mitochondrial translocation of Bax was investigated. Levels of Bax expression were monitored in mitochondrial and cytosolic fractions from K562 cells prepared after treatment with either vehicle or PBOX-6 for 16 h. Equal amounts of protein were resolved by SDS-PAGE, and levels of Bax expression were determined by Western blotting. To determine the purity of each fraction, Bcl-2 was measured as a mitochondrial marker as shown previously (Gross et al., 1998; Khaled et al., 1999). The cytosolic enzyme, lactate dehydrogenase (LDH) was used as a marker of the cytosolic fractions and was expressed as a percentage of the total enzyme activity measured from whole cell extracts. Results from Western blotting reveal that Bax is present in the cytosolic and mitochondrial fractions of vehicle-treated cells, and treatment with PBOX-6 does not alter the distribution of Bax in these cells (Fig. 7A). The membrane was stripped and reprobed with Bcl-2, and results show the absence of Bcl-2 from the cytosolic fraction, indicating the purity of the fraction (Fig. 7B). Furthermore, results from the LDH assay indicates that the majority of LDH activity remains in the cytosolic fraction, whereas <1% was detected in the mitochondrial fraction, indicating that extensive contamination of the mitochondrial fraction with cytosolic components did not occur (Fig. 7C). These results suggest that Bax is present in both the cytosolic and mitochondrial fraction of K562 cells, and this cannot be accounted for by cross-contamination of subcellular fractions. However, the expression levels and subcellular localization of Bax remained unchanged after PBOX-6 treatment.
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Activation of JNK Occurs Upstream of Bcl-2 Phosphorylation in CEM-Neo and CEM-Bcl-2 Cells after PBOX-6 Treatment. It has been reported that the JNK mitogen-activated protein kinase phosphorylates Bcl-2 after Taxol-induced apoptosis in HL60 cells (Shiah et al., 2001). We have recently shown that activation of JNK is essential in the apoptotic pathway induced by PBOX-6 in K562 cells (Mc Gee et al., 2002a) To determine the sequence of events that occur during PBOX-6-induced apoptosis, JNK activity was assessed after PBOX-6 treatment of CEM cells. Using GST-c-Jun as a JNK substrate, a kinase assay was performed, and results show that PBOX-6 induces the transient activation of JNK in CEM-Neo and CEM-Bcl-2 cells (Fig. 8, A and B, respectively). JNK activation becomes visible after a 15-min treatment with PBOX-6 in the two cell lines. The early activation of JNK after PBOX-6 treatment of CEM cells is similar to data obtained previously using K562 cells (Mc Gee et al., 2002a) and suggests that JNK activation occurs upstream of Bcl-2 phosphorylation and therefore may be an important intermediate leading to inactivation of Bcl-2.
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JNK Inhibitors Dicoumarol and CEP-11004 Prevent PBOX-6-Induced Apoptosis in K562, CEM, and Jurkat Cells. The quinone reductase inhibitor dicoumarol specifically inhibits activation of JNK in response to a variety of stimuli (Cross et al., 1999; Krause et al., 2001). In agreement with these studies, we have previously shown that dicoumarol abolishes PBOX-6-induced JNK activation and apoptosis in K562 cells, outlining the importance of JNK in the signaling pathway induced (Mc Gee et al., 2002a). In the present study, we have found that dicoumarol blocks PBOX-6-induced apoptosis in CEM-Bcl-2 and Jurkat-WT Bcl-2 cells, demonstrating that JNK activation plays an equally important role during apoptosis in these cells (Fig. 9A). An alternative JNK inhibitor, CEP-11004, has been shown to inhibit JNK signaling by directly targeting and inhibiting members of the MLK family (Murakata et al., 2002). In agreement with this, using an in vitro kinase assay we have found that pretreatment of K562 cells with CEP-11004 (5 µM) completely inhibits PBOX-6-induced JNK activation (Fig. 9B). In addition, we have found that pretreatment of K562, CEM-Bcl-2, and Jurkat-WT Bcl-2 with CEP-11004 (5 µM) for 1 h before treatment with PBOX-6 (10 µM) for a further 16 h completely inhibits PBOX-6-induced apoptosis (Fig. 9C). The ability of the two JNK inhibitors to block PBOX-6-induced apoptosis in the three cell lines further emphasizes the importance of JNK activation during PBOX-6-induced apoptosis.
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Dicoumarol and CEP-11004 Prevent Bcl-2 Phosphorylation Induced by PBOX-6. To investigate whether JNK activation leads to Bcl-2 phosphorylation, the effect of dicoumarol and CEP-11004 on the phosphorylation status of Bcl-2 was assessed. Results show that pretreatment of K562, CEM-Bcl-2, and Jurkat-WT Bcl-2 cells with dicoumarol (200 µM) or CEP-11004 (5 µM) for 1 h before treatment with PBOX-6 (10 µM) for a further 16 h, completely blocks PBOX-6-induced phosphorylation of Bcl-2 (Fig. 10, A and B, respectively). These results suggest that phosphorylation of Bcl-2 occurs as a direct consequence of JNK activation in these cells.
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) has previously shown that overexpression of Bcl-2 in these cells confers resistance against apoptosis induced by etoposide, actinomycin D, and UV irradiation, and we have confirmed these findings. However, in contrast to these agents, we found that PBOX-6 induces apoptosis in CEM-Neo and CEM-Bcl-2 cells with similar potency. The induction of apoptosis, which occurs in a dose- and time-dependent manner, suggests that PBOX-6 can overcome Bcl-2-mediated resistance to apoptosis. The morphological criteria associated with apoptosis, which include cell shrinkage, chromatin condensation, nuclear fragmentation, and the appearance of apoptotic bodies, was visible in the two cell lines after PBOX-6 treatment. In direct contrast to data obtained using tumor cells, it was found that an equivalent concentration of PBOX-6 has no cytotoxic effect on normal PBMCs isolated from healthy volunteers. Induction of apoptosis in tumor cells was detected after a 4-h treatment with PBOX-6; however, it has no effect on normal PBMCs up to 48 h posttreatment. These data illustrate the ability of PBOX-6 to selectively induce apoptosis in tumor cells and highlight its potential in the treatment of cancer.
The mechanism by which antiapoptotic Bcl-2 proteins exert their effect remains uncertain, and a number of hypotheses have been proposed. These include dimer formation between opposing family members, prevention of Bax translocation, and inhibition of mitochondrial events. Post-translational modification of Bcl-2 proteins, such as proteolysis and phosphorylation, also control their function, although the functional significance of these modifications remains controversial. For example, the chemotherapeutic drug Taxol induces Bcl-2 phosphorylation and apoptosis in lymphoid cells and prostate cancer cells (Haldar et al., 1995, 1996), whereas phosphorylation of Bcl-2 by protein kinase C
enhances its antiapoptotic function, and it has been suggested that the role of Bcl-2 phosphorylation may depend on the phosphorylation site (Ruvolo et al., 1998).
CML cells express the chimeric oncogene bcr-abl and show elevated levels of Bcl-2 and Bcl-XL, which may be responsible for their antiapoptotic properties (Amarante-Mendes et al., 1998; Salomoni et al., 2000). We have previously reported that PBOX-6 induces apoptosis in the K562 CML cell line, in a pathway that bypasses the apoptotic suppressor Bcr-Abl (Mc Gee et al., 2001). In this study, we have found that PBOX-6 induces phosphorylation of Bcl-2 and apoptosis in K562, CEM-Neo, and CEM-Bcl-2 cells. Bcl-2 phosphorylation occurs in a dose- and time-dependent manner and this finding is consistent with others who report Bcl-2 inactivation by phosphorylation.
In addition to Bcl-2, Bcl-XL has been implicated in the antiapoptotic phenotype of Bcr-Abl-positive K562 cells. The Bcr-Abl tyrosine kinase inhibitor CGP57148 better known as STI571, causes down-regulation of Bcl-XL and apoptosis in K562 cells (Oetzel et al., 2000). In contrast to this, we found that PBOX-6 induces a dose- and time-dependent phosphorylation of Bcl-XL in K562 cells. Phosphorylation of Bcl-XL occurs at a later time than Bcl-2 phosphorylation in K562 cells and closely parallels the induction of apoptosis previously reported in these cells after PBOX-6 treatment (Mc Gee et al., 2001). In agreement with others, these findings support a role of Bcl-2 and Bcl-XL in the antiapoptotic properties of K562 cells and suggest that phosphorylation and inactivation of anti-apoptotic Bcl-2 proteins may be part of the apoptotic pathway induced by PBOX-6.
To further investigate the role of Bcl-2 phosphorylation after PBOX-6 treatment, we assessed its effect on Jurkat clones stably expressing empty vector, wild-type Bcl-2, or a triple Bcl-2 mutant whereby the primary phosphorylation sites Ser70, Ser87, and Th69 have been mutated to alanine. It has been found that whereas PBOX-6 potently induces apoptosis in Jurkat-Neo and Jurkat-WT Bcl-2 cells, the mutant Bcl-2-expressing cells demonstrate increased resistance against PBOX-6-induced apoptosis. This finding further suggests that phosphorylation of Bcl-2 after PBOX-6 treatment results in its inactivation and is in agreement with Yamamoto et al. (1999) who reported that Jurkat cells expressing the triple Bcl-2 mutant were more resistant to cell death induced by Taxol, which we have also verified. Furthermore, it was found that PBOX-6 phosphorylates Bcl-2 in Jurkat-WT Bcl-2 cells; however, Bcl-2 phosphorylation of the Bcl-2 triple mutant cells was greatly reduced. A faint band indicative of phosphorylated Bcl-2 was present in the Jurkat-Neo and mutant cells, which represents phosphorylation of endogenous Bcl-2. These data suggest that phosphorylation of Bcl-2 by PBOX-6 occurs within the unstructured loop region on one or more of the mutated phosphorylation sites. A small extent of cell death was detected in the Bcl-2 triple mutant cells after PBOX-6 treatment. This finding, which has also been reported by others (Yamamoto et al., 1999), may suggest that phosphorylation at Ser70, Ser87, and Th69 is not sufficient to antagonize all the antiapoptotic function of Bcl-2 and that PBOX-6 may induce modifications beyond phosphorylation. Collectively, these findings indicate that the phosphorylation and inactivation of Bcl-2 may be a critical upstream event leading to the induction of apoptosis by PBOX-6 in chemotherapy-resistant tumor cells.
A recent review suggests that Bcl-2 phosphorylation cannot accelerate cell death in apoptosis-resistant cell lines (Blagosklonny, 2001). In direct contrast to this, these results show that phosphorylation of Bcl-2 by PBOX-6 leads to the induction of apoptosis in a number of chemotherapy-resistant cell lines. Bcl-2 was phosphorylated on two residues in some instances, whereas we could only detect phosphorylation on one residue in other instances. This may be due to poorer resolution upon development of the Western blot, or the phosphorylation state of Bcl-2 may depend on the level of upstream kinase activation. However, these findings are consistent with others who report the phosphorylation of Bcl-2 on one or more residues. For example, up to five distinct bands were visible by Western blotting after Bcl-2 phosphorylation in COS cells (Maundrell et al., 1997). In addition, we did not detect any evidence of Bcl-2 degradation after phosphorylation by PBOX-6.
Bcl-2 normally resides in various subcellular locations such as endoplasmic reticulum and mitochondrial and nuclear membranes (Blagosklonny, 2001). From subcellular fractionation studies, we found that PBOX-6 phosphorylates mitochondrial Bcl-2. The unphosphorylated Bcl-2 present on Western blots from whole cell lysates may represent Bcl-2 at other subcellular locations that is not phosphorylated by PBOX-6. This hypothesis is supported by findings from IL-3-induced phosphorylation, where phosphorylation only affects a minor pool of soluble Bcl-2 and is not visible by immunoblotting (Blagosklonny, 2001).
One proposed mechanism of Bcl-2 function is the inhibition of Bax translocation from the cytosol to the mitochondria where is carries out it proapoptotic function (Gross et al., 1998; Nomura et al., 1999). We assessed the effect of PBOX-6 on the subcellular localization of Bax in K562 cells. In agreement with previous reports (Amarante-Mendes et al., 1998), it was found that Bax resides in both the cytosolic and mitochondrial fraction of viable cells. Levels of Bax expression in the two subcellular fractions remained unchanged after PBOX-6 treatment. In a manner similar to Fadeel et al. (1999) and others, the purity of the various fractions was confirmed by measuring the activity of the cytoplasmic enzyme LDH, as a cytosolic marker. In addition, Western blotting was used to determine levels of cross-contamination of subcellular fractions. Bcl-2 was detected in its phosphorylated form, in the mitochondrial fraction after PBOX-6 treatment but was absent from the cytosolic fraction. These results demonstrate that the antiapoptotic effects of Bcl-2 and Bcl-XL in K562 cells are not exerted through their inhibition of Bax translocation. Alternative mechanisms exist whereby antiapoptotic Bcl-2 and Bcl-XL exert their function such as their ability to dimerize with Bax and repress cell death (Yang et al., 1995). It has been shown that inactivation of Bcl-2 and Bcl-XL by phosphorylation, after Taxol treatment of PC3 prostate cancer cells, reduces the formation of Bax heterodimers and increases the formation of Bax-Bax homodimers, thus inducing apoptosis (Haldar et al., 1996). We found that Bax is present in the mitochondrial fraction of untreated K562 cells; therefore, it is possible that phosphorylation of Bcl-2 and Bcl-XL by PBOX-6 alters the dimerization properties of Bax at this location, thus promoting apoptosis. Further studies will be carried out to investigate this hypothesis.
The stress-activated kinase JNK has been identified as an upstream kinase responsible for phosphorylation of Bcl-2 in some instances (Fan et al., 2000). We have previously shown that JNK becomes activated in K562 cells after PBOX-6 treatment. Inhibition of JNK using the JNK-interacting protein-1 signaling inhibitor and dicoumarol abolished PBOX-6-induced activation of JNK and apoptosis, outlining an essential role of JNK in the apoptotic pathway induced by PBOX-6 in CML cells (Mc Gee et al., 2002a). In this study, we found that PBOX-6 induces the transient activation of JNK in CEM-Neo and CEM-Bcl-2, suggesting that a similar apoptotic pathway is triggered in these cells. The early activation of JNK, which occurs within 15 min, implicates it in the upstream pathway leading to Bcl-2 phosphorylation. In addition, the two JNK inhibitors dicoumarol and CEP-11004 inhibit Bcl-2 phosphorylation and apoptosis in K562, CEM, and Jurkat cells, providing further evidence that JNK activation occurs upstream of Bcl-2 phosphorylation. These findings are in agreement with others who report JNK as an upstream kinase responsible for the phosphorylation of Bcl-2 family members. For example, inhibition of JNK using antisense oligonucleotides prevented the phosphorylation of Bcl-2 and Bcl-XL in KB-3 human carcinoma cells after treatment with vinblastine (Barr and Bogoyevitch, 2001). In direct contrast to this, some stimuli that activate JNK such as DNA damaging agents, fail to phosphorylate Bcl-2 (Blagosklonny et al., 1997). These conflicting reports suggest that under certain cellular conditions substrate accessibility may be a key factor enabling JNK to phosphorylate Bcl-2. The indolocarbazole analog CEP-11004 inhibits JNK signaling by directly targeting and inhibiting members of the upstream MLK family. CEP-11004 has been shown to prevent JNK activation and apoptotic neuronal cell death in rat pheochromocytoma PC12 cells, primary cortical neurons and human SH-SY5Y cells after treatment with a variety of apoptotic stimuli (Murakata et al., 2002). In addition, CEP-11004 protects against lipopolysaccharide-induced activation of JNK and c-Jun in microglia cultivated from neonatal rats (Hidding et al., 2002). In agreement with these reports, we have found that CEP-11004 prevents JNK activation and apoptosis in K562 cells after PBOX-6 treatment, which correlates with results obtained previously using JNK-interacting protein-1 and dicoumarol (Mc Gee et al., 2002a). This finding suggests that members of the MLK family may be part of the upstream signaling pathway leading to JNK activation by PBOX-6 and further aids our understanding of the mechanism of action of this novel compound.
Identification of novel drug targets that may be exploited in therapeutic intervention is of high importance; however, it is limited due to an incomplete knowledge of the core cell death machinery (Baell and Huang, 2002). Therefore, understanding the mechanism of apoptosis has important implications in the prevention and treatment of cancer. Although significant advances have been made in recent years following the treatment of CML patients with the Abl tyrosine kinase inhibitor STI571, the development of drug resistance remains a problem and highlights the continued need for the development of novel drugs with different mechanisms of action. We have previously reported that the novel pyrrolo-1,5-benzoxazepine compound PBOX-6 induces apoptosis in Bcr-Abl-positive and Bcr-Abl-negative cells. In this study, we have identified Bcl-2 as a critical target of PBOX-6. Phosphorylation and inactivation of Bcl-2 abrogates its protective effect in a number of tumor cells after PBOX-6 treatment. Importantly, the noncytotoxic effect of PBOX-6 on normal PBMCs highlights its selectivity for tumor cells and its potential as a novel target-based drug for the treatment of cancer.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: JNK, c-Jun NH2-terminal kinase; MLK, mixed lineage kinase; CML, chronic myelogenous leukemia; PBOX-6, 7-[{dimethylcarbamoyl}oxy]-6-(2-naphthyl)pyrrolo-[2,1-d] (1,5)-benzoxazepine; PBMC, peripheral blood mononuclear cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; GST, glutathione S-transferase; DMSO, dimethyl sulfoxide; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis; LDH, dehydrogenase.
Address correspondence to: Dr. Daniela Zisterer, Department of Biochemistry, Trinity College, Dublin 2, Ireland. E-mail: dzistrer{at}tcd.ie
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-actin as a loading control (bottom). Mean arbitrary unit from densitometry analysis of three replicates is included. CEM-Neo (B) and CEM-Bcl-2 (C) cells were seeded at 3 x 105 cells/ml and either left untreated, UV-irradiated for 2 min and incubated at 37°C for a further 3 h, treated with etoposide (200 µM) for 24 h, actinomycin D (5 µM) for 24 h, or PBOX-6 (10 µM) for 24 h. The extent of apoptosis was determined by centrifuging an aliquot of cells (150 µl) onto a glass slide followed by RapiDiff staining. Values represent the mean ± S.E.M. of three separate experiments.
