Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells- the role of ATM-Chk2-Cdc25
J Cancer Res Clin Oncol (2008) 134:245–253
DOI 10.1007/s00432-007-0278-x
ORIGINAL PAPER
Bendamustine induces G2 cell cycle arrest and apoptosis in myeloma cells: the role of ATM-Chk2-Cdc25A
and ATM-p53-p21-pathways
Leander Gaul · Sonja Mandl-Weber · Philipp Baumann ·
Bertold Emmerich · Ralf Schmidmaier
Received: 21 March 2007 / Accepted: 4 July 2007/ Published online: 25 July 2007
? Springer-Verlag 2007
Abstract
Purpose Multiple myeloma is a fatal hematological dis-ease caused by malignant transformation of plasma cells. Bendamustine has been proven to be a potent alternative to melphalan in phase 3 studies, yet its molecular mode of action is still poorly understood.
Methods The four-myeloma cell lines NCI-H929, OPM-2, RPMI-8226, and U266 were cultured in vitro. Apoptosis was measured by X ow cytometry after annexin V FITC and propidium iodide staining. Cell cycle distribution of cells was determined by DNA staining with propidium iodide. Intracellular levels of (phosphorylated) proteins were deter-mined by western blot.
Results We show that bendamustine induces apoptosis with an IC50 of 35–65 g/ml and with cleavage of caspase 3. Incubation with 10–30 g/ml results in G2cell cycle arrest in all four-cell lines. The primary DNA-damage sig-naling kinases ATM and Chk2, but not ATR and Chk1, are activated. The Chk2 substrate Cdc25A phosphatase is degraded and Cdc2 is inhibited by inhibitory phosphoryla-tion of Tyr15 accompanied by increased cyclin B levels. Additionally, p53 activation occurs as phosphorylation of Ser15, the phosphorylation site for ATM. p53 promotes Cdc2 inhibition by upregulation of p21. Targeting of p38 MAPK by the selective inhibitor SB202190 signi W cantly increases bendamustine induced apoptosis. Additionally, SB202190 completely abrogates G2 cell cycle arrest.Conclusion Bendamustine induces ATM-Chk2-Cdc2-mediated G2 arrest and p53 mediated apoptosis. Inhibition of p38 MAPK augments apoptosis and abrogates G2 arrest and can be considered as a new therapeutic strategy in com-bination with bendamustine.
Keywords Multiple myeloma · Bendamustine ·
Cell cycle · Ataxia telangiectasia mutated protein · Checkpoint kinase 2
Introduction
Multiple myeloma (MM) is an incurable hematological dis-ease caused by malignant transformation of plasma cells. Over three decades neither a single agent nor any polyche-motherapy was proven to be superior to melphalan and prednisone (Myeloma Trialist’s 1998). Only dose escala-tion of melphalan, enabled by the technology of autologous stem cell transplantation to overcome haematotoxicity, sig-ni W cantly improves overall survival (Child et al. 2003; Attal et al. 1996). However, since high dose melphalan has evolved to be gold standard for all eligible patients (Pal-umbo et al. 2004), highly active genotoxic compounds for the treatment of post-transplant relapse are rare.
Bendamustine is a bifunctional agent that consists of a benzimidazol nucleus, which is linked to a nitrogen mus-tard moiety and therefore combines the features of alkylat-ing agents and purine analogs (Konstantinov et al. 2002). Due to this heterogeneity in structure and the fact that ben-damustine induced double strand breaks have been shown to be more stable (Strumberg et al. 1996), its interaction with DNA has to be considered more complex than of other cytotoxic agents. Bendamustine has been proven to be active in lymphoma (Heider and Niederle 2001; Herold
L. Gaul · S. Mandl-Weber · P. Baumann · B. Emmerich · R. Schmidmaier (&)
Department of Haematology and Oncology,
Klinikum der Universit?t München,
Medizinische Klinik Innenstadt,
Ziemssenstrasse 1, 80336 Munich, Germany
e-mail: ralf.schmidmaier@med.uni-muenchen.de
et al. 2006; Lissitchkov et al. 2006; Kath et al. 2001), mul-tiple myeloma, and breast cancer (von Minckwitz et al. 2005; Zulkowski et al. 2002; Ho V ken et al. 1998) and has been shown to be almost without cross-resistance to other alkylating agents like cyclophosphamide and melphalan (Leoni et al. 2003). According to a phase III study benda-mustine plus prednison revealed to be superior to standard mephalan/prednisone in terms of time to treatment failure, complete remission rate, and quality of life (Ponisch et al. 2006). Furthermore, bendamustine has been proven to be save and e V ective as salvage treatment after high dose mel-phalan (Knop et al. 2005). This is in accordance with the preclinical data regarding lack of cross resistance (Leoni et al. 2003; Strumberg et al 1996). Although the clinical e V ectiveness of bendamustine in the treatment of myeloma is clearly demonstrated, we must resume that its molecular mechanism of action is still poorly understood.
The aim of our study was to examine the in vitro toxicity of bendamustine in myeloma cell lines and to detect the involved pathways as a prerequisite for development of molecular targeted combination therapies.
Materials and methods
Cells
NCI-H929, U266, RPMI-8226, OPM-2 cell lines were obtained from the American Type Culture Collection (Rock-ville, USA), grown in RPMI 1640 medium (Boehringer, Ingelheim, Germany) containing 10% heat-inactivated fetal calf serum (Boehringer) in a humidi W ed atmosphere (37.0°C; 5% CO2), and seeded at a concentration of 1￡105cells/ml.
Reagents
We obtained annexin V/FITC-conjugated from BD Biosci-ence (San Jose, CA, USA), propidium iodide from Calbio-chem (Darmstadt, Germany). Bendamustine from Ribosepharm (Gr?fel W ng, Germany), ca V eine from Sigma–Aldrich (Taufkirchen, Germany), olomoucine, roscovitine, Chk2-inhibitor, p38-inhibitor SB202190 from Calbiochem (Darmstadt, Germany). P-ATM, P-ATR, P-cdc2, P-Chk1, P-Chk2, P-Cdc25C and P-p53-antibodies were obtained from Cell-signaling technology (Frankfurt am Main, Ger-many). Bad, bax, bcl-2, bcl-XL, Cyclin B and D2, p21, p53, XIAP, and actin-antibodies were requested from Santa Cruz (Santa Cruz, CA, USA)
Analysis of cell death and apoptosis by X ow cytometry
Myeloma cells were seeded in 6-well plates at a concentra-tion of 0.5￡105 cells/ml. After 48h myeloma cells were detached by pipetting vigorously and by using a cell scraper. Cells were stained with FITC-conjugated annexin V and propidium iodide. Brie X y, after two washes with washing bu V er (8g NaCl, 0.2g KCl, 1.44g Na2HPO4, 0.24g KH2PO4, 1l H2O, pH7.2), cells were resuspended in binding bu V er (10mM HEPES/NaOH, pH7.4, 140mM NaCl, 2.5mM CaCl2). A total of 100 l of this cell suspen-sion was incubated with 5 l annexin V-FITC and 10 l of 50 g/ml PI for 15minutes at room temperature in the dark. Cells were analyzed on a Coulter EPICS XL-MCL X ow cytometer (Beckman Coulter, Krefeld, Germany) within 30minutes.
Analysis of cell cycle
Cells were W xed overnight with 70% (w/v) ice-cold ethanol. After two washes with ice-cold phosphate-bu V ered saline (PBS), the W xed cells were resuspended in 1ml of PBS containing 40 g/ml propidium iodide (PI) and 500U/ml RNase A. Following incubation for 30min in the dark at room temperature, the cells were analyzed by X ow cytome-ter using the System II software. The PI X uorescence signal peak versus the integral was used to discriminate G2/M cells from G0/G1 doublets.
Immunoblot analysis
Cells were washed three times in ice-cold PBS and lysed in a bu V er containing 10mM Tris–HCl (pH 7.6), 137mM NaCl, 1mM Na3VO4, 10mM NaF, 10mM EDTA, 1% (v/v) Igepal CA-630 (NP-40) with the addition of 10 g/ml aprotinin, 10 g/ml leupeptin, and 1mM phen-ylmethylsulfonyl X uoride (PMSF). Protein concentration was adjusted using a colorimetric assay (Bio-Rad Protein Assay, Bio-Rad, Munich, Germany). Proteins were sub-jected to SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene di X uoride (PVDF) mem-brane (Millipore, Germany). The transfer bu V er contained 25mM Tris–HCl, 192mM glycine, 0.037 (w/v) SDS, and 20% (v/v) methanol. The membranes were blocked with PBS containing 5% dried milk and 0.05% Tween-20 (PBS/ Tween). After washing four times with PBS/Tween, the membranes were incubated with appropriate primary and secondary antibody and visualized by autoradiography using the ECL western blotting detection system (Amer-sham Pharmacia, Freiburg, Germany).
Proliferation assay
Cells were cultured in microplates after addition of the agent in a W nal volume of 100 l/well. 10 l/well WST-agent (Roche, Penzberg, Germany) was added for 1h. After shaking gently absorbance at 450nm was measured
using a microplate enzyme-linked immunosorbent assay reader to detect metabolically intact cells.Statistics
Kruskal–Wallis one-way analysis of variance on Ranks was used to determine the statistical signi W cance of treatment results. The pair wise multiple comparison procedure was performed according to Dunn’s method. Values of P <0.05were considered statistically signi W cant.
Results
Bendamustine induces apoptosis in myeloma cell lines Present clinical studies have demonstrated high in vivo e Y cacy of bendamustine in multiple myeloma (Ponisch et al. 2006). However, there are no published preclinical data available regarding the IC50 in vitro and the molecular mode of action. To determine the in vitro toxicity we incu-bated the MM cell lines NCI-H929, RPMI-8226, OPM-2,and U266 with increasing concentrations of bendamustine ranging from 1 to 100 g/ml over 48h and stained with annexin V (AV) and propidium iodide (PI). We found that concentrations of 10–30 g/ml result in loss of cell mem-brane asymmetry as a sign for early apoptosis (AV+) in 20–40% of the cells depending on the cell line (Fig.1a). NCI-H929 and OPM-2 are more sensitive with an IC50 of 35 g/ml in comparison to RPMI-8226 and U266 with an IC50 of 65 g/ml. Furthermore, cleavage of caspase 3could be demonstrated by western blot (Fig.1b) using an antibody against the large fragment (17/19) of activated caspase 3, showing that bendamustine induced cell death in myeloma cells is accompanied by activation of the apopto-sis cascade. Figure 1c illustrates that prolongation of the incubation period leads to a linear rise in apoptosis over 72h measured by binding of PI as a sign for cell permeabil-ity and late apoptosis. Since in vivo anti-myeloma activity of bendamustine has been already shown in large clinical phase 3 trials, in vitro experiments with primary MM cells from patients were consciously not performed.Bendamustine induces G2/M cell cycle arrest
Bendamustine is supposed to take action by crosslinking the DNA double strands leading to protein missynthesis during replication. Due to this mechanism we expected the drug to cause changes in the cell cycle. We analyzed the cell cycle distribution of myeloma cells by measuring the DNA content with PI after 1–100 g/ml bendamustine for 48h. For all four-cell lines an obvious G2/M-arrest at
Fig.1
Bendamustine induces apoptosis. a NCI-H929 (NCI), RPMI-8226 (RPMI), OPM-2 and U-266 myeloma cells were were treated with 1–100 g/ml (1, 3, 10, 30 and 100) bendamus-tine (benda) or control medium (Co ) over 48h. Apoptosis was determined by annexin V (aV )/propidium iodide (PI ) staining. b Intracellular levels of caspase 3 and cleaved caspase 3 were de-tected by western blotting using antibodies against caspase 3 and the 17/19 D cleavage products. Actin bands show equal loading. c. NCI-H929 (NCI) were treated with control medium alone or bendamustine 10/30/100 g/ml over 72h and PI+ apoptosis was monitored after 24, 48, and 72h
concentrations ranging from 3 to 30 g/ml could be observed (Fig.2a) that meets statistical signi W cance in U266, NCI-H929, and RPMI-8226 cells (P =0.005, 0.006,and 0.006 respectively). The increase of G2-cells was more prominent in NCI-H929 (32%) and RPMI-8226 (43%).High doses of 100- g/ml bendamustine overcame this G2/M arrest and resulted in a loss of DNA and a rising popula-tion of subG1-cells, a further sign of apoptosis.
Flow cytometry images of the two cell lines NCI-H929and U-266 are presented in Fig.2b. To exclude the so-called “mitotic catastrophe” as the pathogenetic mechanism for the e V ect, we counted cells and mitotic W gures by microscopy after Giemsa staining (not shown). The number of apoptotic bodies but not of mitotic W gures was increased with dose escalation. Furthermore, we analyzed cell proliferation after incubation with WST-1 over 72h and
Fig.
2Bendamustine induces G2/M-arrest. a NCI-H929 (NCI), RPMI-8226 (RPMI), OPM-2 and U-266 were treated with bendamustine (benda) 1–100 g/ml or medium (co ) over 48h and cell cycle distributon (percent cells) was analyzed af-ter PI-staining. b NCI-H929 and U-266 cells were incubated with control medium or 10 g/ml bendamustine over 48h. The FACS-images are shown for both cell lines, the G2-fraction is signalized as black area under the graph. c NCI-H929 cells (NCI) were incubated with con-trol medium (co ), 3, 10, and 30 g/ml bendamustine and seeded in 96-well plates. WST-agent was added after 0, 24, 48, and 72h and the turnover was measured by extinction at 440nm
measurement of the extinction at wavelength 450nm on days 0, 1, 2, and 3. Figure2c shows a time and concentra-tion dependent inhibition of proliferation without any sign for a “proliferation burst” in lower concentrations. Primary MM cells from patients arrest in vitro in G1 phase, even in the presence of bone marrow stromal cells. By supplement-ing growth factors und cytokines ex vivo survival can be improved, but a su Y ciently high proliferation rate is not achievable. Therefore the e V ects obtained with MM cell lines could not be proved in patient cells.
Bendamustine activates the ATM-Chk2-Cdc25A pathway
The key controller of the progression from G2 to mitosis is the Cdc2/CyclinB complex, the mitosis-promoting factor (MPF). It accumulates in an inactive form in late G2 and is regulated by nuclear import and export. This physiological cell cycle progression can be interrupted at several levels by apoptotic and antiproliferative stimuli.
We incubated myeloma cell lines with bendamustine in concentrations most potent to induce G2 arrest, 10 and 30 g/ml for NCI-H929, 30 and 60 g/ml for RPMI-8226 over 48h and blotted for the cell cycle checkpoint regula-tion proteins Cyclin B1, Cyclin E, Cyclin D, Cdk1 (Cdc2) and Cdk2. We found increased levels of Cyclin B at 10mg/ ml (NCI-H929) and 30 g/ml (RPMI-8226), whereas the level of Cyclin D, which is responsible for G1-S transition, was not altered (Fig.3a). Also, Cdk1(Cdc2), Cdk2, and Cyclin E levels remained unchanged (data not shown). As we presumed changes in G2/M transition and expected alterations in the activity of the enzymes, we incubated with antibodies against the phosphorylated isoforms of ATM, ATR, Chk1, Chk2, Cdc25c, Cdc2. Cdc2 is deacti-vated by inhibitory phosphorylation at Tyr15, the hallmark of G2-arrest (Fig.3a). Figure3b shows that ATM is acti-vated by phosphorylation of Ser1981, accompanied by acti-vation of Chk2 (Thr68) as a main target for ATM. In contrast, ATR was deactivated and Chk1 remained unchanged in NCI-H929 and could not be detected in RPMI-8226 (data not shown). Interestingly, the level of Cdc25C remained unchanged and its inhibitory phosphory-lation at Tyr162 was not altered (Fig.3c) even though it is known to be the major target for the two kinases Chk1 and Chk2 in mediating G2 arrest. Instead, we proved that Cdc25A is a mediator of the cascade. Figure3c shows that Cdc25A level is completely diminished which corresponds to proteolytic degradation of the phosphatase.
P53 and p21
As p53 is known to be involved in apoptosis and cell cycle arrest at several levels, we incubated the blots with p53 and P-p53 antibodies. Figure3d illustrates that p53 protein level is increased in NCI-H929 but not in RPMI-8226 cells. However, both cell lines exhibit strong activation which appears as phosphorylation at Ser15. The linkage of p53 activation to Cdc2 inhibition is mediated by p21, which is known to directly inhibit the MPF. P21 protein levels were increased in both cell lines, suggesting that p53 activation Fig.3
Cdc2 inactivation via ATM/Chk2/Cdc25A and p53/p21. NCI-H929 and RPMI-8226 cells were incubated with either control medium (co) or bendamustine (benda) 10/30 and 30/60 g/ml over 48h. Pro-tein levels were detected by western blotting using antibodies against a Cyclin B1, Cyclin D2, P-Cdc2 (Tyr15), b P-ATM (Ser1981), P-ATR (Ser 428), P-Chk2 (Thr 68), c P-Cdc25C (Ser 216), Cdc25A, d p-53, P-p53 (Ser15), and e bad, bax, bcl-2, bcl-X/L and XIAP. Actin blotting shows equal loading
by bendamustine is as well involved in the observed G2-arrest. The p53-target 14-3-3 , known to sequester MPF in the cytoplasm and by this to prevent initiation of mitosis, was not altered (data not shown). Levels of members of the pro- and antiapoptotic bcl-family like bax, bad, bcl2, bcl-x/l and XIAP remained unchanged as well (Fig.3e).
Inhibition of p38 -MAPK potentiates bendamustine induced apoptosis
To develop molecular targeted therapeutic options in com-bination with the highly anti-myeloma active compound bendamustine based upon the W ndings above regarding cell cycle control we tested small molecule inhibitors of central signaling enzymes in our in vitro model. In this context, Ca V eine is considered a potent ATM/ATR (Sarkaria et al. 1999) and Chk1/Chk2-inhibitor and Cdc2-activator and most potent in abrogating G2 checkpoint arrest (Bode and Dong 2006). We co-incubated cells with ca V eine (0.03–30mM) and bendamustine (10–30 g/ml) over 48h. No e V ect, neither pro- nor antiapoptotic could be observed. Also, a selective inhibitor of Chk2 kinase was not able to abrogate bendamustine-induced apoptosis. Similar experi-ments with the two Cdc2-inhibitors roscovitine and olo-moucine were also without rational W ndings (data not shown). An interesting role in cell cycle interaction has been frequently implicated for the family of MAPK. Espe-cially p38 has been reported to have important e V ects on proliferation and apoptosis, even though highly cell-type dependent. We preincubated MM cells for 30minutes with the p38-inhibitor SB202190 at a non-toxic concentration of 3/10 M and added bendamustine 30 g/ml for 48h. It resulted in a twofold and statistically signi W cant (P=0.014) increase in apoptosis (Fig.4a). This synergism was reproduced with RPMI-8226 (21%) and OPM-2 (14%) cells, yet the e V ect was remarkably most signi W cant in the two cell lines exhibiting the greatest G2 arrest.
P38 activation is known to arrest cells in G2 (Garner et al. 2002; Pedraza-Alva et al. 2006). Therefore we concluded that inhibition should be able to overcome G2 arrest. Cell cycle was analyzed as demonstrated in Fig.4b. As we expected, the inhibitor SB202190 totally abrogated benda-mustine-induced G2/M arrest (P=0.015). To verify the involvement of p38 in intracellular bendamustine e V ects, we detected p38 level and activation status by immunoblotting. Figure4b shows that p38 is expressed in the cells and acti-vated by phosphorylation when treated with bendamustine. Discussion
Anti-cancer agents like purine analogs and alkylating agents target the DNA and induce cross-linking of the dou-ble strands. This mechanism makes the cell vulnerable in the two cell cycle checkpoints G1 and G2. Many cytotoxic drugs have been shown to cause changes in checkpoint reg-ulation resulting in proliferation arrest (Damia and Brog-gini 2004; Bozko et al. 2005). This enables the cell to DNA repair in order to prevent cell death and in this line prolifer-ation arrest may mean drug resistance. However, above a certain threshold of pathway activation the cells commit programmed suicide, termed apoptosis. We show that ben-damustine induces apoptosis in MM cells with an IC50 of about 35–65 g/ml, and causes G2 arrest. To understand the importance of G2 arrest it is essential to illuminate the complex regulation mechanisms of this checkpoint. The progression from G2 to mitosis is mainly regulated by the mitosis-promoting factor (MPF), a complex of Cyclin B and Cdc2 (Nurse 1990). Its activity is a result of numerous Fig.
4Synergism of bendamustine and p38 MAPK inhibitor. NCI-H929 cells (NCI) were incubated either with control medium (co), ben-damustine (benda) 30 g/ml, 10 M SB202190 or both agents over 48h. a. Apoptosis was measured by AnnexinV (AV)/propidium iodid (PI)-staining. b G2/M-fraction was determined by cell cycle distribu-tion after PI-staining. c. P38 level and phosphorylation status was detected by western blotting. Actin control shows equal loading
pathways re X ecting the complexity of stimuli that occur during cell cycle progression and its inhibition a hallmark for G2-arrest. In our experiments Cdc2 is inhibited by phosphorylation at Tyr15 accompanied by an increased level of Cyclin B at low concentrations. The majority of cells arresting in late G2 maintain high levels of Cyclin B that is synthesized equally in G2 (Maity et al. 1996). The activity of the complex is re X ected by the activity of Cdc2, which is decreased with rising concentrations. But which pathway is responsible for apoptosis and the inhibition of CyclinB/Cdc2? The two protein kinases ATM (Ataxia-tel-angiectasia mutated kinase) and ATR (Ataxia-telangiecta-sia and Rad3-related kinase), both exhibiting homology to the PI3 kinase family, have been shown to play a major role in the primary signaling of DNA-damage (Abraham 2001). ATM phosphorylates and activates Chk2 (Matsuoka et al. 1998; Chaturvedi et al. 1999; Falk et al. 2001), whereas ATR phosphorylates Chk1 (Liu et al. 2000). Furthermore, ATM is able to activate the tumor suppressor p53 on sev-eral serine residues (Canman et al. 1998; Turenne et al. 2001; Miyakoda et al. 2002). The grade of activation has been shown to correlate with the amount of DNA strand breaks (Buscemi et al. 2004). Activation of ATR and Chk1 is caused by UV-damage (Tibbetts et al. 1999, O’Driscoll et al. 2003) and has proven to be the mechanism the purine analogon thioguanine mediates G2 arrest (Yan et al. 2004; Yamane et al. 2004), while ATM activation has frequently been associated with double strand breaks due to ionising radiation (IR) or radiomimetic agents but not to UV or alkylating agents (Canman et al. 1998). Chk1 and Chk2 can inactivate Cdc25C by inhibitory phosphorylation of Tyr162 (Chaturvedi et al. 1999; Sanchez et al. 1997) and directly activate p53 by phosphorylation at Ser20 (Hirao et al. 2000). In contrast, activation of p53 by ATM and ATR is mediated by phosphorylation at Ser15. Inactivated Cdc25C is no longer able to promote cell cycle progression by acti-vating the MPF. Activated p53 itself can inhibit MPF by regulation of p21 and 14-3-3 and force cells to undergo apoptosis due to its complex function as a transcription fac-tor. Our experiments demonstrate that ATM but not ATR is activated and this result is underlined by the further activa-tion of Chk2 but not of Chk1. The fact that these results are in contrast with studies denying a role for ATM in the action of alkylating agents points towards a more complex interaction with the DNA maybe due to the homology of the substance with purine analogs. As Cdc25c is not altered we conclude that this kinase is not essential for bendamus-tine induced G2 arrest, but the opposite can be demon-strated for Cdc25A. Cdc25A as an activator of cdk1(Cdc2) and cdk2 has been proven to regulate G1/S and G2/M tran-sition and to play a role in UV, IR and chemotherapeutic activated checkpoint pathways (Busino et al. 2004; Agner et al. 2005; Mailand et al. 2000). Our experiments show that Cdc25A is degraded which correlates with inhibition of MPF. As Chk1 is not activated and ATR even inhibited, we conclude that only Chk2 is able to activate Cdc25A thus causing its proteolytic degradation.
A second checkpoint pathway stimulated by bendamus-tine is based on the tumor suppressor p53. Phosphorylation of p53 at Ser15 points towards a direct activation by ATM. On the one hand, p53 additionally blocks cell cycle pro-gression via Cdc2/Cyclin
B inhibitor p21. On the other hand it initiates the apoptotic program, which explains the cyto-toxic potency of bendamustine. We inhibited the main kinase for G2 arrest Cdc2, with selective inhibitors to test whether this inhibition alters apoptosis. Interestingly none of them showed any e V ect, which means that inhibition of the MPF alone is not su Y cient to further enhance the apop-totic potential of bendamustine. This could be due to the relatively low potential of the inhibitors in comparison to the concurrence of stimuli.
Nonetheless we decided targeting G2 interacting path-ways as a promising option to further optimize and under-stand bendamustine impact on checkpoint activation. One of these is the p38 MAPK pathway. P38 has been shown to interfere with G2 checkpoint by inducing G2 arrest (Bula-vin et al. 2001; Garner et al. 2002; Pedraza-Alva et al. 2006). Concerning multiple myeloma, p38 inhibition increased the cytotoxicity of proteasome inhibitors (Hide-shima et al. 2004; Navas et al. 2006) and there is evidence that targeting the microenvironment, which is of major importance in the pathophysiology of the disease, by spe-ci W c inhibitors may become an interesting therapeutic option in the future (Hideshima et al. 2003; Nguyen et al. 2006; Wang et al. 2006). We investigated the ability of the p38 MAPK inhibitor SB202190 to act in synergism with bendamustine and found that, if added in a non-toxic dose, it was able to increase apoptosis by twofold. This e V ect has been investigated in three cell lines with similar results. In addition, we observed high levels of p38 in myeloma cells, which were further activated under treatment with benda-mustine suggesting the potential of p38 to initiate G2 arrest. This may be considered a regulatory activation upon treat-ment with DNA alkylating drugs in order to ensure cell via-bility during cell cycle arrest and DNA repair. We further demonstrated that the inhibitor was able to abrogate benda-mustine induced G2 arrest. The consequence is that cells which were arrested due to a certain DNA damage in order to allow repair mechanisms are forced to break the check-point with defective DNA and undergo apoptosis.
In summary bendamustine is a highly active anti-myeloma agent in vitro and in vivo. Myeloma cells respond to bendamustine induced DNA damage with apoptosis and ATM/Chk2/Cdc25A and ATM/p53/p21 mediated cell cycle arrest and p38 activation to enable DNA repair and cell survival. Targeting of p38 by SB202190 concomitantly
with bendamustine treatment synergistically induces apoto-sis in myeloma cells and herewith provides a good rationale for new therapeutic strategies in multiple myeloma. References
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