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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
School of Biochemistry and Immunology, Trinity College, Dublin, Ireland (L.M.G., L.K., V.O., D.C.W., D.M.Z.); Dipartimento Farmaco Chimico Tecnologico, Universita'degli Studi di Siena, Italy (G.C.); and Institute of Molecular Medicine, St. James's Hospital and Trinity College, Dublin, Ireland (M.L.)
Received November 3, 2006; accepted December 29, 2006.
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Abstract |
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). The Bcr-Abl kinase promotes growth factor-independent cell growth and confers cellular resistance to DNA damaging drugs and irradiation (Sattler and Salgia, 1997; Skorski, 2002). It has been demonstrated that the Bcr-Abl kinase-mediated up-regulation of the antiapoptotic proteins Bcl-2 (Sanchez-Garcia and Martin-Zanca, 1997), Bcl-xL (de Groot et al., 2000), and, more recently, Mcl-1 (Aichberger et al., 2005) also contributed to Bcr-Abl-mediated cytotoxic drug resistance in CML cells. Furthermore, the tyrosine kinase activity of Bcr-Abl is known to regulate several other signaling/survival pathways, including those involving the modulation of Ras, Janus kinase/signal transducer and activator transcription factor, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3(OH)-kinase/Akt, and nuclear factor 
B (Hoover et al., 2001; Steelman et al., 2004).
Given that the constitutive activity of Bcr-Abl is central to the pathophysiology of CML, Bcr-Abl-targeted therapies, such as tyrosine kinase inhibitors (STI-571, PD173955), antisense oligonucleotides, inhibitors of Bcr-Abl translation (arsenic trioxide), compounds promoting the proteasomal degradation of Bcr-Abl (geldanamycin analogs), and immunomodulatory strategies targeted against Bcr-Abl have revolutionized the treatment of CML (Martiat et al., 1993; Bocchia et al., 1995; Cotter, 1995; Nimmanapalli and Bhalla, 2002).
STI-571 was the first Bcr-Abl tyrosine kinase inhibitor to enter clinical trials and is now the first line treatment for CML patients. However, despite high rates of hematological and cytogenetic response to STI-571 monotherapy, the emergence of resistance to STI-571 has led to the examination of combination therapies. Numerous studies have been conducted to date demonstrating the therapeutic efficacy of STI-571 in combination with an extensive range of anticancer agents, including DNA-damaging drugs and agents targeting signaling pathways downstream of Bcr-Abl (Kano et al., 2001; Yu et al., 2002).
Currently, little information is available concerning the effects of simultaneously causing microtubule disruption while inhibiting tyrosine kinase activity in CML cells. STI-571 was shown to exert an additive effect when used in combination with vincristine (a tubulin depolymerizer) in K562 cells (Kano et al., 2001). However, the exact mechanism of this interaction between vincristine and STI-571 was not further investigated. Previously, our group identified a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds that induce apoptosis in cancer cells derived from both solid tumor types and those from hematological malignancies, suggesting their potential as anticancer agents. Furthermore, PBOX-6, a representative proapoptotic compound, inhibited tumor growth in an aggressive murine model of mammary carcinoma (Greene et al., 2005). Tubulin was recently identified as the molecular target of the PBOX compounds (Mulligan et al., 2006). Both PBOX-6 and -15 caused a depolymerization of the microtubule network in human breast carcinoma-derived MCF-7 cells and inhibited the assembly of purified tubulin in vitro. As demonstrated with other microtubule-targeting agents (MTAs), we showed that activation of c-Jun NH2-terminal kinase (McGee et al., 2002), together with the phosphorylation and inactivation of the antiapoptotic proteins Bcl-2 and Bcl-xL are a prerequisite for PBOX-6-mediated apoptosis (McGhee et al., 2004). In addition, we demonstrate herein that the PBOX compounds induce the formation of polyploid cells, a characteristic shared with other MTAs (Verdoodt et al., 1999). Generally, in response to MTAs, cells enter mitosis, transiently arrest, and exit without undergoing cytokinesis. On existing mitosis, cells with defective microtubule spindles activate the G1 cell cycle checkpoint principally by inducing p21 and p53, preventing cell cycle progression to S phase. However, cells lacking p53, pRb, or the cyclin-dependent kinase inhibitors (CDKIs) p21 and p16 will enter S phase with a 
tetraploid (4N) DNA content, a process known as endoreplication, resulting in polyploidy (Khan and Wahl, 1998; Lanni and Jacks, 1998; Hong et al., 1999; Stewart et al., 1999). As with other MTAs, a significant proportion of K562 cells (
65%) remained resistant to PBOX-6, even at maximal concentrations; hence, we sought to determine whether cotreatment with STI-571 could potentiate PBOX-6-induced apoptosis.
Herein, we report that a pharmacologically achievable plasma concentration of STI-571 significantly enhanced PBOX-6-induced apoptosis in STI-571-sensitive and -resistant CML cells, compared with either drug alone. Similar results were observed when other lead MTAs where used in combination with STI-571 in CML cells. PBOX-6/STI-571-induced apoptosis was concomitant with a reduction in PBOX-6-induced G2M arrest, PARP cleavage, and down-regulation of Bcr-Abl, Bcl-xL, and Mcl-1. Together, these findings highlight the potential therapeutic efficacy in combining STI-571 with PBOX-6 as a novel antileukemic strategy.
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Materials and Methods |
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). CML cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS). HL-60 cells were grown in RPMI-1640 supplemented with 20% FBS. All media were supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Cells were maintained at 37°C in 5% CO2 in a humidified incubator.
Reagents. PBOX-6 and -15 were synthesized and described previously (Campiani et al., 1996) and dissolved in ethanol. Structures of the PBOX compounds were described by Mulligan et al. (2006). STI-571 was kindly provided by Witte-Maria Weber (Novartis Pharmaceuticals, Basel, Swizerland) and prepared as a 10 mM stock solution in sterile DMSO. Paclitaxel (former generic name, Taxol) and nocodazole were purchased from Sigma-Aldrich (Poole, Dorset, UK) and dissolved in sterile DMSO. All compounds once dissolved in the relevant solvent were stored at –20°C. RPMI 1640 medium was purchased from Biosciences Ltd. (Dublin, Ireland). L-glutamine, FBS, and penicillin/streptomycin were supplied from Sigma-Aldrich. Lymphoprep was obtained from Unitech (Dublin, UK). The anti-PARP mouse monoclonal antibody (mAb), the anti-c-Abl mAb, the anti-Bcl-xL mAb, and the anti-actin mAb were purchased from Merck Biosciences (Nottingham, UK). The anti-Bcl-2 mAb was obtained from Santa Cruz (Heidelberg, Germany). The enhanced chemiluminescence reagent was obtained from Amersham Biosciences (Buckinghamshire, UK). All other chemicals were obtained from Sigma-Aldrich.
Western Blot Analysis. After treatment, whole-cell pellets (1 x 107 cells) were washed in PBS, resuspended in 60 µl of PBS, lysed by the addition of 60 µl of 2x Laemmli buffer (1x= 30 mM Tris base, pH 6.8, 2% SDS, and 10% glycerol), and briefly sonicated. Homogenates were quantified by the Markwell protein assay, before the addition of reducing agent (50 mM dithiothreitol). Equal amounts of protein (50 µg) were boiled for 3 min, separated by SDS-polyacrylamide gel electrophoresis, and electroblotted to polyvinylidene difluoride membrane. The blots were stained in 0.1% Ponceau S (w/v) in 5% acetic acid (v/v) to ensure equal transfer. Membranes were then washed 2 x 2 min in Tris-buffered saline, pH 7.6, and 0.05% Tween 20 (TBS-T) and blocked for 1 h at room temperature (RT) in TBS-T containing 5% (w/v) dried milk (blocking buffer). After 1 h, the blots were then incubated overnight at 4°C in 1 µg/ml primary antibody diluted in 5% blocking solution. Blots were then washed 3 x 10 min in TBS-T and incubated for 1 h at RT in a 1:1000 dilution of horseradish peroxidase-conjugated secondary antibody. Blots were again washed 3 x 10 min in TBS-T and then developed by enhanced chemiluminescence.
Experimental Format. Logarithmically growing cells were seeded at 200,000/ml (K562), 300,000/ml (HL-60), and 500,000/ml (LAMA-84) in sterile plastic T-flasks. Cells were left untreated or treated with solvent control or designated drug/drug combination for 24 or 48 h. At the end of the incubation period, cells were harvested by centrifugation at 600g for 10 min at room temperature and prepared for subsequent analysis as detailed below.
Flow-Cytometric Analysis. The flow-cytometric evaluation of apoptosis (pre-G1 peak) and cell cycle analysis were performed as described previously (Greene et al., 2005). In brief, after treatment, cells were washed with ice-cold PBS and fixed in ice-cold 70% ethanol at 4°C for at least 1 h. Cells were then centrifuged at 800g for 10 min and resuspended in PBS containing 10 µg/ml RNase A and 100 µg/ml propidium iodide (PI). Following a 30-min incubation in the dark at 37°C, the PI fluorescence was measured on a linear scale using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). Data collection was gated to exclude cell debris and cell aggregates. Apoptotic cells are represented by a broad hypodiploid peak, which is easily discernible from the narrow peak of cells with diploid DNA content in the red fluorescence channel. All data were recorded and analyzed using the CellQuest software (Becton Dickinson).
3,4,5-Dimethylthiazol-2-yl-2,5-Diphenyl-Tetrazolium Bromide Assay. Parental K562 (K562S) and STI-571-resistant K562 (K562R) cells were treated with vehicle [0.05% DMSO (v/v)] and varying concentrations of STI-571 (0.1–5 µM). After 72 h, cell viability was assessed by the reduction of 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT). MTT was added to each well of the microtiter plate (final concentration of 1 mg/ml) and incubated in the dark for 1 h at 37°C. The purple formazan crystals were dissolved in DMSO, and the solution was vigorously mixed to dissolve the reacted dye. After 20 min, the plates were read on an automated microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at 595 nm. The blank solution (medium, MTT, and DMSO) was used to calibrate the spectrophotometer to zero absorbance. The relative cell viability (percent) related to control wells and was calculated by [A]test/[A]control x 100, where [A]test is the absorbance of the drug-treated cells, and [A]control is the absorbance of the vehicle control-treated cells. Dose-response curves were plotted, and IC50 values (concentration of drug resulting in 50% reduction in cell survival) were obtained using the commercial software package Prism (GraphPad Software Inc., San Diego, CA). Experiments were performed in triplicate on at least three separate occasions.
Normal Peripheral Blood Mononuclear Cells. Peripheral blood was obtained with informed consent from normal volunteers in syringes containing preservative-free heparin, diluted 1:1 with sterile PBS, and layered over a cushion of 15 ml of Lymphoprep in sterile 50-ml centrifuge tubes. After centrifugation for 30 min at 400g at RT, the interface layer, consisting of mononuclear cells, was extracted with a sterile Pasteur pipette and washed with sterile PBS. Cells were resuspended in RPMI 1640 medium containing 10% FBS at a density of 106 cells/ml. Experiments were approved by the St. James's Hospital and the Adelaide Meath and the National Children's Hospital Research Ethics Committee.
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). The DNA content of propidium iodide labeled cells was determined by flow cytometric analysis after 24 and 48 h (Figs. 1 and 2). Cells in the G0G1 and G2M phases of the cell cycle contain a diploid (2N) and 4N DNA content, respectively. Cells undergoing apoptosis typically show a pre-G1 or subdiploid peak due to nuclear fragmentation and display a <2N DNA content. Polyploid cells have a 4N DNA content. Polyploidy is a phenomenon whereby G1 checkpoint-defective cells endoreplicate following microtubule disruption. K562 cells are p53 null and consequently have a compromised G1 checkpoint. PBOX-6 alone at concentrations > 1 µM induced G2M cell-cycle arrest followed by late apoptosis (48 h; Figs. 1 and 2). However, 65% of PBOX-6 treated K562 cells failed to undergo apoptosis due to either a sustained G2M cell cycle arrest or polyploid formation in these cells. Following a 24-h treatment, STI-571 (250 nM) alone increased the percentage of cells in G0G1 to 44.9 ± 0.9% compared with 39.9 ± 0.4% observed in control cells. This finding is in agreement with previously published results demonstrating G0G1 cell cycle arrest in AML cells treated with STI-571 in the nanomolar range (Nishimura et al., 2003). STI-571 alone (250 nM) was minimally toxic to cells and induced apoptosis in approximately 5% of the cells after 48 h. Interestingly, combined exposure of PBOX-6 with STI-571 significantly increased the percentage of apoptotic cells by inhibiting a prolonged G2M cell cycle arrest (P = 0.001) and polyploidy (P = 0.02) induced by PBOX-6 (Fig. 2C). STI-571 did not affect early (24 h) G2M cell cycle arrest induced by PBOX-6 (Fig. 2A). Statistical analysis demonstrated that significantly more apoptosis was induced in cells treated with the STI-571/PBOX-6 combination than either drug alone (P < 0.01) (Fig. 1C). Furthermore, STI-571 also significantly enhanced the apoptotic potential of a second tubulin-targeting PBOX compound, PBOX-15, by impeding a prolonged G2M checkpoint (Fig. 3). Similar results were obtained with a third tubulin depolymerizer, vinblastine (data not shown).
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STI-571 Potentiates PBOX-6-Mediated Lethality in Bcr-Abl-Positive but Not Bcr-Abl-Negative Leukemia Cells. Next, we examined the effect of STI-571/PBOX-6 combinations on a second Bcr-Abl-positive cell line, LAMA-84 (Fig. 4). STI-571 (250 nM) alone was minimally toxic to the LAMA-84 cells and induced an increase in the percentage of cells in the G0G1 phase of the cell cycle. In more detail, after 24 h, 72.6 ± 2.8% of cells treated with STI-571 were in the G0G1 phase of the cell cycle compared with 61.2 ± 1.4% observed in control-treated cells. As previously reported, PBOX-6 (10 µM) alone induced apoptosis in LAMA-84 cells (Fig. 4, A and B). Here, we also demonstrate that PBOX-6 produced a significant amount of G2M cell cycle arrest and induced endoreplication following microtubule disruption in these cells (Fig. 4, C and D). Again, after 48 h, cotreatment with STI-571 (250 nM) significantly reduced PBOX-6 (10 µM)-mediated G2M arrest and polyploid formation with a concomitant increase in the percentage of apoptotic cells (Fig. 4).
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). STI-571 (250 nM) alone was not toxic to HL-60 cells, nor did it enhance the apoptotic efficacy of PBOX-6 in these cells. Finally, the administration of both agents either alone or in combination did not induce apoptosis in normal peripheral blood cells (Fig. 5B).
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). Given that STI-571 inhibited PBOX-6-induced polyploidy, we sought to investigate the effect of STI-571 on paclitaxel-induced polyploidy. Paclitaxel alone induced a dose-dependent increase in the number of polyploid cells (Fig. 6A). After a 48-h exposure to 10 µM Taxol, over 50% of the cells were polyploid. As anticipated, STI-571 (250 nM) significantly reduced the extent of Taxol-induced polyploidy with a concurrent increase in the percentage of apoptotic cells. It may be deduced that cotreatment with STI-571 may reverse resistance to Taxol, at least in part, in K562 CML cells.
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These findings prompted us to investigate the effects of exposing K562 CML cells to the tubulin depolymerizer, nocodazole (1 µM), in the presence and absence of STI-571 (250 nM). As shown in Fig. 6B, the apoptotic potential of nocodazole was significantly enhanced by STI-571-mediated inhibition of polyploidy. The formation of polyploidy in K562 cells following exposure to nocodazole was described previously by Verdoodt et al. (1999). A 10-fold increase in nocodazole (10 µM) strengthened the mitotic block induced by nocodazole and minimized mitotic slippage and polyploid formation. In this instance, cotreatment with STI-571 reduced G2M cell cycle arrest and enhanced nocodazole-induced apoptosis (data not shown).
Cotreatment of STI-571 with PBOX-6 in CML Cells Resulted in Enhanced PARP Cleavage and Promoted the Down-Regulation of the Antiapoptotic Proteins Bcr-Abl, Bcl-xL, and Mcl-1. Western blot analysis was used to determine the effects of combining STI-571 with PBOX-6 on the levels of the p210 Bcr-Abl protein and selected downstream signals in CML cells (Fig. 7). We have shown previously that PBOX-6-induced apoptosis was associated with a reduction in Bcr-Abl protein levels in LAMA-84, but not in K562 and KYO-1 CML cells, suggesting a Bcr-Abl-independent apoptotic pathway (McGee et al., 2001). As expected, after a 48-h treatment, PBOX-6 (10 µM)-induced apoptosis was associated with the down-regulation of the Bcr-Abl protein in LAMA-84 cells but not in K562 CML cells (Fig. 7, A and B). As shown here and elsewhere, STI-571 (250 nM) alone resulted in a minimum alteration in Bcr-Abl protein levels in both cell lines (Nimmanapalli et al., 2003). The combination of STI-571 with PBOX-6 resulted in a marked reduction in Bcr-Abl protein levels in K562 cells, whereas the expression of Bcr-Abl was undetectable in LAMA-84 cells treated for 48 h with the STI-571/PBOX-6 combination. Bcr-Abl-mediated cell survival has been linked with the increased expression of various antiapoptotic proteins, particularly the signal transducer and activator transcription factor-5-regulated Bcl-xL. We next sought to determine the effects of STI-571/PBOX-6 combinations on Bcl-xL and other members of the Bcl-2 family in LAMA-84 cells (Fig. 7C). Treatment with either drug alone resulted in a modest decrease in the levels of Bcl-xL; however, Bcl-xL was undetectable at 48 h in cells treated with both agents in combination. Likewise, cotreatment of LAMA-84 cells with STI-571 and PBOX-6 resulted in marked down-regulation in Mcl-1 protein levels at 48 h, whereas Mcl-1 remained unchanged in cells treated with either drug alone. We have previously shown that PBOX-6 induces the phosphorylation of Bcl-2 in K562 and CEM leukemia cells (McGee et al., 2004). Here, we demonstrate that following a 24-h treatment, PBOX-6 (10 µM) alone induced the phosphorylation of Bcl-2 in LAMA-84 cells. However, there is a marked reduction in the levels of PBOX-6-induced Bcl-2 phosphorylation by 48 h, suggesting that it is a transient event. Cotreatment with STI-571 did not alter PBOX-6-mediated Bcl-2 phosphorylation, nor did it modulate Bcl-2 protein levels. Finally, in accordance with the onset of apoptosis, STI-571/PBOX-6 combinations resulted in increased PARP cleavage at both 24 and 48 h compared with cells treated with single agents.
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Apoptotic Efficacy of PBOX-6 as a Single Agent and in Combination with STI-571 in STI-571-Resistant CML Cells. We next sought to determine whether cells resistant to STI-571 display cross-resistance to PBOX-6 and to determine whether coadministration with STI-571 enhances PBOX-6-induced lethality in these cells. In this experiment, parental K562 cells (K562S) and STI-571-resistant cells (K562R) that were derived from a multidrug-resistant cell line (Yanovich et al., 1989), where treated with vehicle, or PBOX-6 (0.1–10 µM) for 24 and 48 h (Fig. 8). The K562R cells display higher levels of the Bcr-Abl protein compared with parental K562 cells (Fig. 8A). As a result, these cells require higher concentrations of STI-571 to quench the kinase activity of the Bcr-Abl tyrosine kinase and ultimately induce cell death (Yu et al., 2002). As shown in Fig. 8B, K562R cells were 9-fold more resistant to STI-571 than K562S cells. On the other hand, K562R cells did not display any cross-resistance to PBOX-6 as determined by both a cell viability assay (Fig. 8B) and quantification of the pre-G1 peak, which is indicative of apoptosis (Fig. 8C). In addition, as observed in parental K562 cells, cotreatment with STI-571 significantly enhanced the apoptotic efficacy of PBOX-6 in K562R cells, compared with either drug alone (Fig. 8D). Again, the increased level of apoptosis in K562R cells treated with both PBOX-6 and STI-571 compared with either drug alone was attributed to a reduction in PBOX-6-induced G2M arrest by STI-571 (data not shown). These findings highlight the potential therapeutic benefit of the coadministration of STI-571 with PBOX-6 in STI-571-resistant cells that overexpress the Bcr-Abl protein.
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; McGee et al., 2001, 2004; Greene et al., 2005). In addition, PBOX-6 also demonstrated significant antitumor activity in vivo in an aggressive murine model of mammary carcinoma, further highlighting their potential as anticancer agents (Greene et al., 2005). However, despite the wide activity spectra displayed by PBOX-6 against various cancer cell types, a considerable proportion (65%) of the Bcr-Abl-positive K562 CML remained resistant to PBOX-induced apoptosis even at maximum concentrations (50 µM). K562 CML cells are inherently resistant to apoptosis induced by a variety of chemotherapeutic agents and growth factor deprivation, a characteristic attributed to the constitutive activation of the Bcr-Abl kinase. Hence, Bcr-Abl kinase activity is central to the pathophysiology of CML, and Bcr-Abl-targeted therapies such as the tyrosine kinase inhibitor STI-571 have revolutionized the treatment of CML. There are numerous studies to date demonstrating enhanced lethality in vitro and in vivo by combining STI-571 with other anticancer agents. For example, Kano et al. (2001), demonstrated that STI-571 produced additive cytotoxic effects when combined with hydroxyurea, cytarabine, homoharringtonine, doxorubicin, and etoposide in four Bcr-Abl-positive CML cell lines. However, although there is no disputing the potential therapeutic benefits of combining STI-571 with DNA-targeting agents in the treatment of CML, currently there are limited data supporting the simultaneous administration of STI-571 with microtubule-targeting agents.
Coadministration of STI-571 and the tubulin polymerizer, paclitaxel, have been shown to synergistically inhibit the growth of prostate (Kim et al., 2004), breast (Lev et al., 2005), ovarian (Apte et al., 2004), and gastric (Kim et al., 2005) carcinomas in vivo. In all studies, STI-571 enhanced the chemotherapeutic potential of paclitaxel by targeting the tyrosine kinase, platelet-derived growth factor receptor. Here, we demonstrate for the first time the potential antileukemic benefits incurred by targeting the Bcr-Abl tyrosine kinase with STI-571 while inhibiting the assembly of tubulin with a novel series of MTAs, the pyrrolo-1,5-benzoxazepines. Importantly, STI-571 also significantly enhanced the apoptotic efficacy of lead MTAs currently used within the clinic, including paclitaxel and vinblastine.
STI-571 enhancement of PBOX-6-induced lethality was associated with a reduction in PBOX-6-mediated G2M cell cycle arrest and diminished Bcr-Abl expression. It has been postulated that upon DNA damage, Bcr-Abl prolongs the activation of the G2M cell cycle checkpoint and in doing so allows the cell time to repair otherwise lethal perturbations (Skorski, 2002). Given that STI-571 did not significantly alter the initial G2M arrest in response to PBOX-6 but prevented a prolonged G2M arrest, it is tempting to speculate that Bcr-Abl may also contribute to the sustained G2M arrest observed in Bcr-Abl-positive CML cells in response to microtubule disruption. Moreover, a sustained G2M arrest was not observed in the Bcr-Abl-negative HL-60 cell line, following treatment with PBOX-6 for 24 or 48 h, and was reflected by increased levels of apoptotic cells. Additional studies revealed that HL-60 cells did transiently arrest in G2M (2–8 h) in response to PBOX-6 exposure (data not shown). STI-571 also inhibited a prolonged G2M cell cycle block induced by other MTAs, including vinblastine and nocodazole, demonstrating that this effect was not specific to the PBOX compounds (data not shown). Collectively, these findings support the speculation that the Bcr-Abl oncogene may contribute, at least in part, to a sustained G2M arrest in response to MTAs in CML cells. Down-regulation of the Bcr-Abl protein may allow the cells to progress through the cell cycle and in doing so activate the G1 cell cycle checkpoint and trigger the onset of apoptosis.
In addition to down-regulation of Bcr-Abl protein levels, the coadministration of STI-571 and PBOX-6 also resulted in a marked reduction of the levels of the antiapoptotic proteins Mcl-1 and Bcl-xL compared with the effects of either drug alone. It is noteworthy that Bcr-Abl lies upstream of the Stat family of transcription factors, which have been implicated in the regulation of Bcl-XL and Mcl-1 in leukemia cells (de Groot et al., 2000; Epling-Burnette et al., 2001; Aichberger et al., 2005). Therefore, it is likely that the observed down-regulation of Bcl-XL and Mcl-1 in STI-571/PBOX-6-treated cells was a direct consequence of Bcr-Abl down-regulation.
Another significant finding in this study was the observed inhibition of endoreplication and polyploidy induced by PBOX-6 and other MTAs by cotreatment with STI-571. MTAs principally function via the alteration of the microtubule dynamics during mitosis and subsequent activation of the mitotic spindle checkpoint. The mitotic spindle checkpoint delays the onset of anaphase until any defects in the spindle are corrected. However, cells can prematurely exit mitosis with defective spindles and activate the G1 cell cycle checkpoint, resulting in G1 cell cycle arrest, followed by apoptosis. Cells with a defective G1 checkpoint will fail to arrest and proceed to S phase with a 4N DNA content, resulting in polyploidy (Stewart et al., 1999). The G1 cell cycle proteins, p53 and pRb, and the CDKIs p21 and p16 have been shown to prevent endoreplication and polyploidy following aberrant mitotic exit of cells (Khan and Wahl, 1998; Lanni and Jacks, 1998; Hong et al., 1999; Stewart et al., 1999). In more detail, a p53-dependent block of DNA endoreplication subsequent to mitotic spindle inhibition was demonstrated by two independent groups using paired K562 (p53-deficient) and KS (p53-proficient) CML cell lines (Casenghi et al., 1999; Verdoodt et al., 1999). Additional analysis of the G1 phase CDK activities demonstrated that the induction of p21-inhibited endoreplication was mediated through direct cyclin E/CDK2 regulation (Stewart et al., 1999). Taken together, these results suggest that the G1S cell cycle checkpoint proteins prevent inappropriate S phase entry following mitotic slippage induced by prolonged MTA exposure. CDKIs have been shown previously to prevent MTA-induced endoreplication in human cancer cells (Motwani et al., 2000). Specifically, inhibition of CDKs by flavopiridol at clinically achievable nanomolar concentrations prevented endoreplication in human cancer cells (MDA-MB-468 and p21–/– HCT116 cells) defective in G1 checkpoint proteins (Motwani et al., 2000). Likewise, ectopic expression of the CDKI p21 in p53-deficient HIp21 cells also prevented endoreplication (Stewart et al., 1999).
The data presented in this study represent the first report to date demonstrating that a tyrosine kinase inhibitor can also prevent endoreplication following mitotic spindle disruption. Mazzacurati et al. (2004) demonstrated that STI-571 induces growth arrest in CML cells by activating the Chk2-Cdc25A-Cdk2 axis, a pathway complementary to p53 in the activation of the G1S cell cycle checkpoint. In this study, treatment with STI-571 alone induced an increase in the percentage of cells in the G1 phase of the cell cycle. It may be suggested that STI-571 may potentiate the inadequate pseudo-G1 arrest initiated by MTAs following mitotic slippage into G1 and inhibit endoreplication in p53 null K562 and LAMA-84 CML cells, perhaps through the Chk2-Cdc25A-Cdk2 pathway. In addition, overexpression of the antiapoptotic protein Bcl-xL has been shown to cooperate with loss of p53 to dramatically induce polyploidy by promoting the survival of cells with spindle damage (Minn et al., 1996). Therefore, it may be suggested that the reduction in the levels of the Bcl-xL protein in cells treated with both PBOX-6 and STI-571 may contribute to the reduction in PBOX-6-induced endoreplication. It is well documented that cellular polyploidy leads to genomic instability and ultimately promotes tumorigenesis (Fujiwara et al., 2005). These results highlight the potential clinical benefit in combining ST1–571 with MTAs to reduce genomic instability.
Finally, the noncytotoxic effects of STI-571/PBOX-6 combinations on Bcr-Abl-negative HL-60 leukemia cells and normal peripheral blood cells highlight its selectivity for Bcr-Abl-positive CML and its potential as an effective strategy in treating CML. In addition, given that PBOX-6/STI-571 combinations were equally effective at inducing apoptosis in K562-STI-571-resistant cells as that observed in the parental K562 cells, the use of PBOX-6/STI-571 combinations could also be explored in patients with CML resistant to STI-571. The K562-STI-571-resistant cells used in this study overexpress the Bcr-Abl oncogene. Although the ability of PBOX-6/STI-571 combinations to overcome this mechanism of resistance is clear, it would be worth determining whether PBOX-6/STI-571 combinations might circumvent other mechanisms of resistance, including cells expressing mutations within the Bcr-Abl kinase domain. Further studies are underway to determine whether this novel antileukemic strategy of simultaneously targeting tubulin and the Bcr-Abl oncogene can combat CML arising from the aberrant expression of mutant Bcr-Abl. In addition to inhibiting the kinase activity of Bcr-Abl, STI-571 also inhibits the tyrosine kinase activity of c-KIT and platelet-derived growth factor receptor (Druker, 2004); hence, the possible therapeutic benefits of PBOX-6/STI-571 combinations may be extended to other malignancies driven by these kinases.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CML, chronic myeloid leukemia; STI-571, imatinib mesylate; PBOX, pyrrolo-1,5-benzoxazepine; MTA, microtubule-targeting agent; CDKI, cyclin-dependent kinase inhibitor; 4N, tetraploid; PARP, poly(ADP-ribose) polymerase; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; mAb, monoclonal antibody; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline, pH 7.6/0.05% Tween 20; RT, room temperature; PI, propidium iodide; MTT, 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide; 2N, diploid; PD173955, pyrido[2,3-d]pyrimidine Src tyrosine kinase inhibitor.
Address correspondence to: Dr. Daniela Zisterer, School of Biochemistry and Immunology, Trinity College, Dublin 2, United Kingdom. E-mail: dzistrer{at}tcd.ie
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