Activation of mitochondrial apoptotic pathway in mantle cell lymphoma: high sensitivity to mitoxantrone in cases with functional DNA-damage response genes

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Abstract

Mantle cell lymphoma (MCL) is a mature B-cell proliferation characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course, and poor response to chemotherapy. The majority of drugs currently used in the treatment of lymphoproliferative disorders induce cell death by triggering apoptosis, but few data concerning drug-induced apoptosis in MCL have been reported. We have analysed the mechanisms of drug-induced cell death in four cell lines with the t(11;14) and in primary cells from 10 patients with MCL. Mitoxantrone, a topoisomerase II inhibitor, induced a strong cytotoxic effect in three cell lines (JVM-2, REC-1, and Granta 519), and in primary MCL cells. This cytotoxic effect due to apoptosis induction was observed despite the presence of either p53 or ATM abnormalities. However, no cytotoxic effect was detected after incubation with DNA-damaging agents in the NCEB-1 cell line, carrying p53 and ATM alterations, despite the presence of functional mitochondrial machinery. These results support that mitoxantrone can be effective in the treatment of MCL but that this activity requires the integrity of functional DNA-damage response genes.

Introduction

Mantle cell lymphoma (MCL) is a lymphoproliferative disorder derived from a subset of naive pregerminal center cells with a mature B-cell phenotype and coexpression of CD5 (Swerdlow et al., 2001; Campo, 2003). MCL is characterized by the chromosomal translocation t(11;14)(q13;q32) that results in cyclin D1 overexpression (Bosch et al., 1994; Campo et al., 1999; Bertoni et al., 2004) with the consequent deregulation of cell cycle control at the G1–S checkpoint. In addition to classical MCL, a blastoid variant of the disease has been described, which is characterized by high mitotic rate and that is associated with INK4a/ARF deletions, p53 mutations, and complex karyotypes (Hernandez et al., 1996; Pinyol et al., 1997; Schlegelberger et al., 1999).

MCL is characterized by an aggressive clinical course and poor response to conventional therapy due to either rapid relapse after an initial response or primary resistance to drugs (Argatoff et al., 1997; Bosch et al., 1998). The DNA damage response pathway plays a crucial role in the pathogenesis and clinical behavior of tumoral cells. The ataxia-telangiectasia-mutated (ATM) gene codifies for a protein involved in p53 phosphorylation in response to DNA damage, thus promoting cell cycle arrest and apoptosis (Banin et al., 1998). ATM and p53 alterations are associated with drug resistance in lymphoproliferative disorders. ATM inactivation is commonly observed in MCL patients (Camacho et al., 2002) and p53 alterations are associated with blastoid variants (Hernandez et al., 1996; Fang et al., 2003). The mechanisms of resistance to chemotherapy in MCL are poorly understood. Experimental evidences demonstrate that most of the chemotherapeutic agents currently used exert their cytotoxic effects by inducing apoptosis. Moreover, recently it has been proposed that alterations in the apoptotic pathways may contribute to the development of MCL (Hofmann et al., 2001; Martinez et al., 2003).

The aim of this study was to analyse the mechanisms of drug-induced apoptosis in MCL. Towards this end, four cell lines carrying the t(11;14)(q13;q32), as well as primary MCL cells, were incubated in vitro with several drugs currently used in the treatment of lymphoproliferative disorders and drug-induced cell death was characterized.

Results

Cytotoxic effect of fludarabine, mafosfamide, and mitoxantrone on cell lines

JVM-2, REC-1, Granta 519, and NCEB-1 cell lines were incubated for 24 and 48 h with different concentrations of fludarabine (1–10 μg/ml), mafosfamide (1–10 μg/ml), and mitoxantrone (0.05–0.5 μg/ml), and the cytotoxic effect of these drugs was measured by annexin V-FITC/PI staining. The LD50 for mitoxantrone, fludarabine, and mafosfamide is shown in Table 1. Mitoxantrone induced the strongest cytotoxic effect in JVM-2, REC-1, and Granta 519 cell lines at concentrations lower than those achieved with the dose currently used in clinical practice (0.5 μg/ml). In contrast, pharmacological doses of fludarabine (1 μg/ml) and mafosfamide (1 μg/ml) were not able to induce a significant cytotoxicity on any of these cell lines. REC-1 was the unique cell line for which the LD50 for mafosfamide was less than 10 μg/ml whereas the LD50 for fludarabine was lower than 10 μg/ml only for Granta 519. No cytotoxic effect was observed after incubation of NCEB-1 cells with mitoxantrone, fludarabine, or mafosfamide, even when higher doses of these drugs were used (data not shown).

Table 1 LD50 for mitoxantrone (M), fludarabine (F), and mafosfamide (Mf) in MCL cell lines

To study the possible additive or synergistic effect of these drugs, cells from the four cell lines were incubated during 24 h with 0.25 μg/ml of mitoxantrone, in the presence or absence of 1 μg/ml of fludarabine and/or 1 μg/ml of mafosfamide. Although concentrations of fludarabine and mafosfamide corresponded to those used in the FCM regimen (fludarabine+cyclophosphamide+mitoxantrone) (Bellosillo et al., 1999), mitoxantrone concentration was reduced from 0.5 to 0.25 μg/ml due to the massive cytotoxic effect observed at 0.5 μg/ml. As shown in Granta 519 cell line (Figure 1), none of these combinations resulted in a significant increase of cytotoxicity to that observed with mitoxantrone alone. The same results were detected in JVM-2 and REC-1 cell lines (data not shown).

Figure 1
figure1

Cytotoxic effect of the combination of mitoxantrone with fludarabine and/or mafosfamide. Cells from Granta 519 cell line were incubated for 24 h in the absence of any factor (CT), with mitoxantrone alone (M; 0.25 μg/ml), and in combination with fludarabine (F; 1 μg/ml), and/or mafosfamide (Mf; 1 μg/ml). Cell viability was determined by annexin V binding as described in Materials and methods. Data are shown as the mean value ±s.d. of triplicate cultures

Drug-induced apoptosis on MCL cell lines

In order to determine whether the cytotoxic effect was due to induction of apoptosis, the role of caspases and the proteolytic cleavage of PARP were analysed. Cells from the four cell lines were incubated with 10 μg/ml fludarabine and 0.25 μg/ml mitoxantrone. The decrease on cell viability correlated with loss of ΔΨm, and with the detection of the active form of caspase-3 in Granta 519 (Figure 2a), JVM-2, and REC-1 cell lines. These results demonstrated that drug cytotoxicity was due to apoptosis induction. No changes in ΔΨm or activation of caspase-3 were observed in NCEB-1 cells (Figure 2b).

Figure 2
figure2

Activation of mitochondrial apoptotic pathway during drug-induced cell death. Cells from Granta 519 (a) and NCEB-1 (b) cell lines were incubated in the absence (CT) or presence of fludarabine (F; 10 μg/ml) or mitoxantrone (M; 0.25 μg/ml) for 24 hours. Cell viability was determined by annexin V binding. Changes in ΔΨm were assessed by staining with JC-1. Active form of caspase-3 was quantified by anti-active caspase-3 immunostaining. In mitoxantrone-treated cells, a shift in the signal of fluorescence 3 (630 nm) was observed owing to the incorporation of this drug. The percentage of positive cells is indicated in each panel and represents cells undergoing apoptosis as assessed by the different methods of analysis. Whole-cell lysates were obtained from 2 × 106 cells from Granta 519 (c) and NCEB-1 (d) in the same conditions, and analysed by Western blot. Blots were also probed with α-tubulin antibody as an internal control

To study the mechanisms leading to the activation of caspase-3, the proteolysis and subsequent activation of caspase-9 and -8 were analysed. Mitoxantrone and fludarabine induced processing of both proteases, as shown by the decrease of the procaspase-9 and -8 and the appearance of the intermediate cleavage product (43/41 kDa) of caspase-8. Activation of caspase-3 was also accompanied by proteolysis of PARP (Figure 2c). No activation of caspase-9, -8 or -3 or cleavage of PARP were detected in NCEB-1 cells (Figure 2d).

Apoptosis-related proteins in drug-induced cell death

Western blot analysis revealed that MCL cell lines expressed Bcl-2, Bax, Bak, Bcl-XL, and Mcl-1 proteins. After incubation with 10 μg/ml fludarabine and 0.25 μg/ml mitoxantrone, no changes in the overall protein levels of Bcl-2, Bax, and Bak were detected, whereas downregulation of Mcl-1 was observed in the sensitive cell lines. Furthermore, a minor decrease on Bcl-XL expression was also detected. We also determined the behavior of some IAP (Inhibitor of Apoptosis Proteins) family members. A downregulation of XIAP protein was observed after drug incubation of sensitive cell lines, whereas no changes in the survivin levels were detected. No downregulation of any Bcl-2 family member or IAP protein was observed in NCEB-1 cell line (Figure 3a).

Figure 3
figure3

Apoptosis-related proteins in drug-induced apoptosis. (a) Whole-cell lysates were obtained from 2 × 106 cells from Granta 519 and NCEB-1 cell lines incubated for 24 h in the absence (CT) or presence of fludarabine (F; 10 μg/ml) or mitoxantrone (M; 0.25 μg/ml), and analysed by Western blot. Blots were also probed with α-tubulin antibody as an internal control. Cell viability is indicated as the percentage of annexin V negative cells analysed by flow cytometry. (b) Cells from Granta 519 cell line were incubated for 24 h in the absence (CT) or presence of fludarabine (F; 10 μg/ml) or mitoxantrone (M; 0.25 μg/ml). Bax and Bak conformational changes were determined by staining with anti-Bax and anti-Bak antibodies and flow cytometry analysis, as described in Material and methods. The percentage of positive cells is indicated in each panel

Involvement of Bax and Bak proteins in drug-induced apoptosis was also analysed using anti-Bax and -Bak antibodies directed against the NH2-terminal region of both proteins. This region is occluded in unstressed intact cells and it is only available for binding to these antibodies following conformational changes of these proteins during the apoptotic process. An increase in the number of Bax and Bak-positive cells was detected in Granta 519 (Figure 3b), JVM-2, and REC-1 cells after incubation with 10 μg/ml fludarabine and 0.25 μg/ml mitoxantrone. No changes in Bax and Bak conformation were observed in NCEB-1 cell line after incubation with these drugs.

Preincubation of Granta 519 cells with 200 μM Z-VAD.fmk, a broad inhibitor of caspases, reversed drug-induced phosphatidilserine exposure, loss of ΔΨm, and caspase-3 activation, thus confirming the role of caspases in drug-induced cell death. In contrast, Bax and Bak conformational changes were observed despite inhibition of the caspase cascade, suggesting that these conformational changes occur upstream of the caspase activation or in a caspase-independent manner (data not shown).

Cell cycle analysis after drug exposure

The effect of mitoxantrone and fludarabine on cell cycle distribution and DNA content was analysed. A sub-G0/G1 subpopulation corresponding to apoptotic cells was detected in Granta 519 (Figure 4a), JVM-2, and REC-1 cell lines, and progressively increased after 48–72 h incubation. NCEB-1 cells showed a 4n DNA content in agreement with the nearly tetraploid karyotype observed by cytogenetic analysis. After incubation with mitoxantrone, NCEB-1 exhibited a marked arrest in the transition from S phase to G2–M without apoptotic peak in the sub G0/G1 region, even when cell cycle analysis was performed at longer periods of time (48 and 72 h). No changes in the cell cycle pattern were detected after incubation of NCEB-1 cells with fludarabine (Figure 4b).

Figure 4
figure4

DNA content analysis after drug exposure. Cells from Granta 519 (a) and NCEB-1 (b) were incubated for 24 h with medium alone and in the presence of fludarabine (F; 10 μg/ml) or mitoxantrone (M; 0.25 μg/ml). DNA content was quantified by PI staining and flow cytometry analysis as described in Material and methods. NCEB-1 cell line displays a 4n DNA content, according to the nearly tetraploid karyotype observed by cytogenetic analysis

Staurosporine-induced apoptosis in NCEB-1 cells

To ascertain if the apoptotic machinery was functional in NCEB-1 cell line, cells were incubated with staurosporine, an inducer of mitochondrial apoptotic pathway (Xue et al., 2003). After 24 h of drug exposure, a cytotoxic effect in NCEB-1 cells was observed. This effect was accompanied by loss of ΔΨm, activation of caspase-3, and conformational changes of Bax and Bak, demonstrating an efficient mitochondrial apoptotic machinery in this cell line (Figure 5).

Figure 5
figure5

Staurosporine-induced apoptosis in NCEB-1 cells. Flow cytometry analysis of cell viability by annexin V binding, loss of ΔΨm by JC-1 staining, active form of caspase-3, and conformational changes of Bax and Bak of a representative experiment. Cells from NCEB-1 cell line were incubated with medium alone (CT) or in the presence of staurosporine (STS; 5 μ M) for 24 h. The percentage of positive cells is indicated in each panel. All experiments were performed in triplicate

Drug-induced apoptosis in cells from MCL patients

Tumoral cells from 10 MCL patients were incubated with mitoxantrone, mafosfamide, and fludarabine at the previously mentioned concentrations. The characteristics of these patients are summarized in Table 2. The 17p deletion by FISH and consequently p53 mutations were detected in three cases. The deletion of ATM was detected in three additional patients. No cases lacking both p53 and ATM were found. Pharmacological concentrations of mitoxantrone induced the strongest cytotoxic effect, being the median LD50 for all the patients 0.37 μg/ml (range 0.10–0.76 μg/ml). A significant decrease in cell viability was also observed after incubation with mafosfamide 1 μg/ml (P=0.03), although this effect was due to the cytotoxicity observed in only three out of the 10 MCL patients. The median LD50 was 5.70 μg/ml (range 2.6–11.5 μg/ml). No cytotoxic effect was observed after incubation with fludarabine for 24 h, and only in one patient a significant cytotoxicity was detected after 48 h of incubation with high doses of this drug (5–10 μg/ml) (data not shown).

Table 2 Characteristics of MCL patients

The combination of pharmacological doses of mitoxantrone (0.5 μg/ml) and mafosfamide (1 μg/ml) had a significant additive effect (P=0.04) (Figure 6). However, using the method of Chou and Talalay (1984) this additive effect was only observed in cells from three MCL patients. No additive or synergistic effect was detected after the addition of fludarabine to this combination. The cytotoxic effect was accompanied by loss of ΔΨm, activation of caspase-9, -8, and -3, PARP proteolysis, and conformational changes of Bax and Bak. Furthermore, downregulation of Mcl-1, Bcl-XL, and XIAP was detected, whereas no changes in the overall protein levels of Bcl-2, Bax, and Bak were observed (Figure 7).

Figure 6
figure6

Cytotoxic effect of fludarabine, mitoxantrone, and mafosfamide alone or in combination in primary MCL cells. Lymphocytes from 10 MCL patients were incubated at 37°C for 24 h in the absence of any factor (CT), with pharmacological doses of fludarabine (F; 1 μg/ml), mitoxantrone (M; 0.5 μg/ml), and mafosfamide (Mf: 1 μg/ml), or with the combination of these drugs. Cell viability was determined by annexin V binding as described in Material and methods and is expressed as the percentage with respect to the viability of cells in medium alone at the beginning of the culture. Data are shown as the median value ± range

Figure 7
figure7

Drug-induced apoptosis in MCL primary cells. (a) Cells from a representative MCL patient were incubated with medium alone (CT) or in the presence of mitoxantrone (M; 0.5 μg/ml) for 24 h. Flow cytometric dot plots show cell viability as determined by annexin V binding, loss of ΔΨm by DiOC6 staining, quantification of active form of caspase-3, and Bax and Bak conformational changes, as described in Material and methods. In mitoxantrone-treated cells, a shift in the signal of fluorescence 3 (630 nm) was observed owing to the incorporation of this drug. The percentage of positive cells is indicated in each panel. (b) Whole-cell lysates were obtained from 2 × 106 cells from the same patient incubated in the presence or absence (CT) of mitoxantrone (M; 0.5 μg/ml) for 24 h and analysed by Western blot. Blots were also probed with α-tubulin as an internal control

Discussion

MCL is an aggressive lymphoproliferative disorder highly resistant to currently available therapy. In fact, only few patients achieve a complete response and in most cases the prognosis is very poor (Argatoff et al., 1997; Bosch et al., 1998). Although the current therapy is based on the combination of several drugs, no data regarding the effect of conventional cytotoxic drugs alone or in combination on MCL primary cells have previously been reported. In this study, the cytotoxic effect of different drugs used in the treatment of lymphoproliferative disorders has been analysed in cells from MCL patients, as well as in cell lines carrying the t(11;14)(q13;q32).

Our results demonstrate that mitoxantrone, an inhibitor of topoisomerase II, exerts the highest cytotoxic effect in three MCL cell lines (JVM-2, REC-1, and Granta 519) as well as in primary MCL cells from all patients tested. The LD50 in these cases was lower than that previously observed in primary CLL cells (Bellosillo et al., 1998).

Topoisomerase II inhibitors may act by interfering with the religation activity of the enzyme to DNA double-strand breaks. Several cell lines display natural resistance to mitoxantrone which is independent of drug transport and drug-target alterations (Bailly et al., 1997). Furthermore, low expression of topoisomerase II is one of the mechanisms of resistance to topoisomerase II inhibitors (De Isabella et al., 1991; Rasheed and Rubin, 2003). Thus, mitoxantrone rapidly induced apoptosis in sensitive cells, whereas it produced a G2–M block in resistant ones. In agreement with these results, cell cycle analysis of NCEB-1 cell line revealed a G2–M arrest in mitoxantrone- but not in fludarabine- or mafosfamide-treated cells. Recently, it has been described that topoisomerase II is one of the proliferation-related genes in MCL cells (Rosenwald et al., 2003) and that topoisomerase-IIα expression is a prognostic factor for clinical outcome in MCL (Schrader et al., 2004). In this regard, our results demonstrate a high cytotoxic effect in MCL cell lines and primary cells from blastic MCLs, accordingly to their proliferative index.

Fludarabine, alone or in combination, has demonstrated efficacy in the treatment of CLL and follicular lymphoma (Solal-Celigny et al., 1996; Flinn et al., 2000; Bosch et al., 2002). However, in our experience high doses of fludarabine were necessary to induce a minor cytotoxic effect on MCL cells in vitro. Recently, a case of a patient with MCL resistant to fludarabine has been reported, due to a deficient fludarabine uptake (Reiman et al., 2002). In this regard, a relationship between in vitro sensitivity to fludarabine and drug uptake via equilibrative transport system has also been described in primary CLL cells (Molina-Arcas et al., 2003).

In primary cells from three MCL patients, the combination of pharmacological doses of mitoxantrone and mafosfamide produced an additive cytotoxic effect. We had previously demonstrated that, in CLL cells, the combination of fludarabine with mafosfamide produced a significant synergistic effect, whereas the addition of mitoxantrone to this combination induced a significant increase in cytotoxicity only in previously treated CLL patients (Bellosillo et al., 1999). The mechanism of the additive effect between mafosfamide and mitoxantrone in B-cell lymphoproliferative disorders could be related to the primary DNA damage produced by mafosfamide and the inability of the cellular machinery to repair it due to the mitoxantrone-mediated inhibition of topoisomerase II.

The present study demonstrates that cytotoxic drugs exert their effect by activating the mitochondrial apoptotic pathway in MCL cells. Thus, these drugs induced loss of mitochondrial transmembrane potential and conformational changes of Bax and Bak that triggered caspase-9 activation and apoptosis. Activation of caspase-8 was also observed and it could be explained by the occurrence of a loop involving caspase-9 and -3 or due to the involvement of other caspases upstream of mitochondria (Slee et al., 1999). Inhibition of caspase activity using the pan-caspase inhibitor Z-VAD.fmk abolished the cleavage of caspase-3 and restored cell viability indicating that genotoxic-induced cell death in MCL cells depends on caspase activity. As previously described (Bellosillo et al., 2002), conformational changes of Bax and Bak were completely independent on caspase activation.

The Bcl-2 family of proteins plays a central role in controlling the apoptosis mitochondrial pathway (Cory and Adams, 2002; Cory et al., 2003). In primary MCL cells as well as in MCL cell lines, conformational changes of Bax and Bak were detected after drug treatment and it was also accompanied by downregulation of Mcl-1 and Bcl-XL. In this regard, downregulation of Mcl-1 has been described in CLL cells after several drug treatments (Bellosillo et al., 1999) and, recently, a downregulation of Bcl-XL after treatment of MCL cells with NF-κB inhibitors has also been reported (Pham et al., 2003). Furthermore, high expression of the antiapoptotic proteins Bcl-2, Mcl-1, and Bcl-XL in NHL has been associated with chemotherapy resistance (Khoury et al., 2003; Zhao et al., 2004). We have also detected a downregulation of XIAP, a member of the IAPs that inhibits the activity of caspases.

NCEB-1 was the unique cell line in which no cytotoxic effect was observed after incubation with genotoxic drugs, even when higher drug concentrations, longer incubation periods, and combination therapy were employed. In contrast, incubation of NCEB-1 cells with staurosporine induced the typical features of apoptosis, demonstrating that the mitochondrial apoptotic machinery is functional in NCEB-1 cells. The lack of response to genotoxic conventional drugs might be explained by the alteration of upstream regulators of Bcl-2 family proteins and/or in DNA damage response genes. In this regard, NCEB-1 cells showed a complex karyotype that included alterations in p53 and ATM. These anomalies have also been found in patients with CLL and MCL and are associated with therapy failure and shorter survival (Wattel et al., 1994; Dohner et al., 1995; Hernandez et al., 1996; Camacho et al., 2002).

Apoptosis induced by genotoxic drugs is accompanied by the stabilization of p53 after its phosphorylation by ATM (Banin et al., 1998; Stankovic et al., 2004). Since p53-mediated apoptosis is thought to underlie the cytotoxicity of most genotoxic drugs, and ATM mutations represent another potential mechanism of drug resistance, the simultaneous dysfunction of both p53 and ATM might explain the failure in the induction of apoptosis in the NCEB-1 cell line. In accordance with this, no apoptosis was observed by topoisomerase I and II agents in p53-null mouse embryonic fibroblast with no ATM function (Fedier et al., 2003). Recently, and in agreement with our results, an MCL cell line with alterations in both p53 and ATM genes was completely resistant to apoptotic cell death although was highly sensitive to in vitro radiation. In contrast, a MCL cell line with only p53 mutation underwent apoptosis (M’kacher et al., 2003).

Our results demonstrate that MCL cells have a functional mitochondrial apoptotic machinery and suggested that cells lacking both ATM and p53, but not cells lacking one of these proteins, are resistant to genotoxic apoptotic stimuli. Finally, these in vitro studies provide experimental support for including mitoxantrone in clinical trials.

Materials and methods

Cell lines

Cell lines carrying the t(11;14)(q13;q32) translocation were studied: Granta 519, NCEB-1, and REC-1 cell lines, all derived from MCL patients, and the JVM-2 cell line, derived from a B-prolymphocytic leukemia. Granta 519 and JVM-2 cell lines were purchased from the DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). NCEB-1 and REC-1 cell lines were kindly provided by Dr Niels S Andersen (Department of Hematology, Rigshospitalet, Copenhagen, Denmark) and Dr Christian Bastard (Department of Hematology, Centre Henri Becquerel, Rouen, France), respectively. p53 mutations were detected in NCEB-1 cell line (Bogner et al., 2003) and 11q alterations involving ATM in Granta 519 (Vorechovsky et al., 1997) and NCEB-1. No ATM protein expression was detected by Western blot in these two cell lines (data not shown).

Patients

Ten patients diagnosed with MCL according to the World Health Organization classification (Swerdlow et al., 2001) who had not received treatment for the previous 3 months were studied. Tumoral cells were obtained from peripheral blood in seven patients, splenic tissue in two, and lymph node in one. The percentage of malignant cells CD19+, CD5+, CD23−, and showing light chain restriction were analysed by flow cytometry. Cyclin D1 overexpression was demonstrated in all cases by immunohistochemistry. An informed consent was obtained from each patient in accordance with the Ethical Committee of the Hospital Clinic (Barcelona, Spain).

Isolation of MCL cells

Mononuclear cells from peripheral blood samples were isolated by Ficoll/Hypaque sedimentation (Seromed, Berlin, Germany). Tumoral cells were obtained after squirting lymph node or spleen biopsies with RPMI 1640 culture medium using a fine needle. Cells were either used directly or cryopreserved in liquid nitrogen in the presence of 10% dimethyl sulfoxide and 20% heat-inactivated fetal calf serum (Gibco BRL, Paisley, Scotland). Manipulation due to freezing/thawing did not influence cell response.

Cell culture

JVM-2, REC-1, Granta 519, and NCEB-1 cell lines (0.5 × 106/ml). and mononuclear cells from patients with MCL (1 to 5 × 106/ml) were cultured in RPMI 1640 culture medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine and 50 μg/ml penicillin–streptomycin, at 37°C in a humidified atmosphere containing 5% carbon dioxide. Cells were incubated for 24–48 h with fludarabine monophosphate (Schering; Berlin, Germany), mitoxantrone (Lederle Laboratories; Gosport, Hampshire, UK), mafosfamide, the active form of cyclophosphamide (ASTAMedica; Frankfurt, Germany), and staurosporine (Sigma Chemicals; St Louis, MO). When Z-VAD.fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) (Bachem; Bubendorf, Switzerland) was employed, cells were preincubated for 1 h prior to the addition of drugs.

Antibodies

Monoclonal and polyclonal antibodies against active caspase-3, and Bax (BD-Pharmingen, San Diego, CA, USA); caspase-8, Bak (Ab-1), p53 (Ab-2), ATM (Ab-3) and α-tubulin (Oncogene Research, Boston, MA, USA); caspase-9 (New England Biolabs Inc., Beverly, MA, USA); poly-adenosine diphosphate ribose polymerase (PARP) (Roche Diagnostics, Mannheim, Germany); Bcl-2 antibody (DAKO, Glostrup, Denmark); Mcl-1 (S-19) (Santa Cruz Biotechnology, CA); XIAP (Transduction Laboratories, Lexington, UK); survivin (Novus Biologicals, Littelton, CO, USA) and Bax (clone YTH-6A7) (Trevigen, Gaithersburg, MD, USA) were employed.

Cell viability by annexin V binding

Exposure of phosphatidylserine residues was quantified by surface annexin V staining as previously described (Bellosillo et al., 2002). Experiments were performed in triplicate. LD50 was defined for each drug as the concentration of drug required to reduce a 50% the cell viability.

Mitochondrial transmembrane potential (ΔΨm) and reactive oxygen species

Changes in ΔΨm were evaluated by staining with 1 nM 3,3′-diexyloxacarbocyanine iodide (DiOC6[3]) (Molecular Probes, Eugene, OR, USA) or with 1.5 mM 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethilbenzimidazolylcarbocyanine iodide (JC-1) (Molecular Probes). Reactive oxygen species (ROS) production was determined by staining with 2 μ M dihydroethidine (DHE) (Molecular Probes). Cells were incubated with dyes for 30 min at 37°C, washed, resuspended in PBS, and analysed by flow cytometry (Bellosillo et al., 2001). A total of 10 000 cells per sample were acquired in a FACScan flow cytometer. Experiments were performed in triplicate.

Detection of intracellular proteins

Cells were fixed and permeabilized as previously described (Bellosillo et al., 2002). Cells were stained with antibodies against the active form of caspase-3, Bax (0.25 μg/ml × 106 cells), and Bak (2.5 μg/ml × 106 cells) for 20 min at room temperature, followed by goat anti-rabbit-FITC (SuperTechs, Bethesda, MD, USA) or goat anti-mouse-FITC (DAKO), and analysed in a FACScan.

Western blot analysis was performed as previously described (Bellosillo et al., 2002). Antibody binding was detected using a chemiluminiscence (ECL) detection system (Amersham, Buckinghamshire, UK). Equal protein loading was confirmed with α-tubulin blots.

Cell cycle analysis

Cells were fixed in 80% ethanol for 5 min at 5°C, centrifuged, and washed twice in PBS. Cells were incubated for 15 min at room temperature in a citrate–phosphate buffer (1:24), centrifuged, resuspended in 0.25 ml of PBS containing PI (5 μg/ml) and Ribonuclease A (100 μg/ml) (Sigma Chemicals, St Louis, MO, USA), and incubated for 10 min in the dark (Nicoletti et al., 1991). The percentage of cells in G0/G1, S, G2–M, and the presence of a sub-G0/G1 peak were evaluated with ModFit LT (Verity Software House, Inc., Topsham, MA, USA).

Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) was performed on fixed cells from the NCEB-1 cell line and from 10 patients with MCL. The LSI p53 (17p13) SpectrumOrange-labeled and LSI ATM (11q22.3) SpectrumGreen-labeled probes (Vysis, Downers Grove, IL) were tested. Two different observers scored 200 nuclei. A true deletion was considered when more than 5% of nuclei showed one hybridization signal. This threshold was established using fixed material from 10 normal subjects.

p53 molecular studies

p53 mutational analysis was performed in nine MCL patients. Exons 4–8 were amplified by PCR. Single-stranded conformational polymorphism (SSCP) analysis and direct sequencing were performed as previously described (Pinyol et al., 2000).

Statistical analysis

Comparisons were performed by using the Mann–Whitney test as appropriate. A P-value <0.05 was considered to be statistically significant.

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Acknowledgements

This work was supported in part by Grants FIS 02/250, 03/0398, CICYT SAF 02/3261, and Red Estudio neoplasias Linfoides G03/179. AF had a fellowship from Hospital Clínic.

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Correspondence to Dolors Colomer.

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Keywords

  • apoptosis
  • lymphoma
  • mitoxantrone

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