Cell Death by Bortezomib-Induced Mitotic Catastrophe in Natural Killer Lymphoma Cells

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Cell death by bortezomib-induced mitotic catastrophe in natural killer lymphoma cells

Lijun Shen,1 Wing-Yan Au,2 Kai-Yau Wong,1 higher pharmacologic concentrations of bortezomib. Norio Shimizu,3 Junjiro Tsuchiyama,4 Hence, activating mitotic catastrophe by bortezomib Yok-Lam Kwong,2 Raymond H. Liang,2 may provide a novel therapeutic approach for treating and Gopesh Srivastava1 apoptosis-resistant NK-cell malignancies and other cancers. [Mol Cancer Ther 2008;7(12):3807–15] Departments of 1Pathology and 2Medicine, The University of Hong Kong, Queen Mary Hospital, Hong Kong, People’s Republic Introduction of China; 3Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and 4Department of Pathology, According to the WHO classification scheme, natural killer Kawasaki Medical School, Kurashiki, Okayama, Japan (NK)-cell neoplasms include extranodal NK/T-cell lymphoma (nasal type) and aggressive NK-cell leukemia (1). Theyare aggressive malignancies with poor treatment Abstract outcomes (2). The lymphoma cells are characteristically À The proteasome inhibitor bortezomib (PS-341/Velcade) is CD3 CD3q+CD56+ and are infected bythe EBV. NK-cell used for the treatment of relapsed and refractory multiple lymphomas show a geographic predilection, as they myeloma and mantle-cell lymphoma. We recently reported constitute 5% to 10% of all lymphomas in Asia and South its therapeutic potential against natural killer (NK)-cell America but are extremelyuncommon in the West. An neoplasms. Here, we investigated the molecular mecha- optimal treatment for NK-cell malignancies has yet to be nisms of bortezomib-induced cell death in NK lymphoma found (2). Radiotherapyof localized nasal NK-cell lym- cells. NK lymphoma cell lines (SNK-6 and NK-YS) and phomas maybe curative in patients with stage I disease. primary cultures of NK lymphomas treated with bortezo- For patients with stage II or more advanced disease, mib were examined for alterations in cell viability, however, treatment results are unsatisfactory. Despite the apoptosis, cellular senescence, and cell cycle status. availabilityof prognostic models (3) and accurate lympho- Bortezomib primarily induced mitochondrial apoptosis in ma load assessment byquantification of EBV DNA (4), NK-YS cells and in primary lymphoma cells at the same improvement in treatment results has not been obtained by concentration as reported in myeloma cells. Unexpectedly, conventional chemotherapy. Although allogeneic trans- SNK-6 cells required a significantly higher median inhibi- plantation mayrescue some patients, the rapid progression tory concentration of bortezomib (23 nmol/L) than NK-YS of chemorefractorydisease or the toxic side effects of high- and primary lymphoma cells (6-13 nmol/L). Apoptosis was dose chemotherapyoften preclude such an option. Thus, limited in SNK-6 cells due to the extensively delayed new therapeutic agents, preferablywithout dose-limiting turnover of Bcl-2 family members. These cells were killed effects, are urgentlywanted. To achieve meaningful clinical by bortezomib, albeit at higher pharmacologic concen- results, a more effective and less toxic agent will be critical trations, via mitotic catastrophe—a mitotic cell death to attain disease stabilization before transplantation. Re- associated with M-phase arrest, cyclin B1 accumulation, search on new drug development for this disease has been and increased CDC2/CDK1 activity. Our results suggest hampered due to the verylimited number of bona fide NK that, in addition to cell death by apoptosis at lower lymphoma cell lines that are available (5). Establishment of bortezomib concentrations, NK lymphoma cells resistant NK lymphoma cell lines has been very difficult over the to bortezomib-induced apoptosis can be killed via mitotic years. Similarly, successful development of NK lymphoma catastrophe, an alternative cell death mechanism, at primarycultures for the evaluation of therapeutics has been equallychallenging, as tumor biopsies are usuallysmall and contain significant necrosis. Tumorigenesis is generallyprevented bythe watchdog Received 7/9/08; revised 9/11/08; accepted 9/23/08. activityof tumor suppressors that trigger apoptosis. Grant support: Research Grants Council of Hong Kong Special Administrative Region, People’s Republic of China grant HKU 7627/06M Similarly, anticancer agents often work by inducing cancer (G. Srivastava and R.H. Liang). cells to undergo apoptosis (6). Cancer cells, however, may The costs of publication of this article were defrayed in part by the still escape apoptosis byenhancing the activityof onco- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to proteins or inactivating the tumor suppressors. These indicate this fact. mechanisms contribute to poor drug responses. Accumu- Requests for reprints: Gopesh Srivastava, Lymphoma Research Laboratory, lating evidence indicates that, if apoptosis is abnormally Department of Pathology, The University of Hong Kong, Queen Mary suppressed, cancer cells maybe eliminated byother Hospital, Pokfulam, Hong Kong, People’s Republic of China. Phone: 852-2855-4875; Fax: 852-2872-5197. mechanisms (7). Among these mechanisms is mitotic E-mail: [email protected] catastrophe, a type of cell death that involves abnormal Copyright C 2008 American Association for Cancer Research. mitosis. When assaulted bydrugs, cancer cells tend to doi:10.1158/1535-7163.MCT-08-0641 bypass the mitotic checkpoints and prematurely reenter the

Mol Cancer Ther 2008;7(12). December 2008

cell cycle, that is, they divide asymmetrically, leading to (BD Biosciences). The NK cells were purified from the cytogenetic chaos and mitotic catastrophe (8–10). New harvested cells with the NK Cell Negative Isolation Kit routes to successful cancer treatment maytherefore be (Dynal Biotech ASA) and maintained in the same medium discovered byinvestigating the factors that interrupt as described above. In the second patient with bone competent cell division. marrow involvement, mononuclear cells were isolated by Normal cellular activities are dependent on the orderly the Ficoll-Hypaque method from the patient’s bone degradation of obsolete cellular proteins bythe ubiquitin- marrow sample before anytreatment, and NK cells were proteasome system. The targeted proteins participate in a purified and maintained as described above. wide varietyof essential cellular activities, such as signal Antibodies and Reagents transduction, transcriptional regulation, stress response, Primaryantibodies against the following proteins were and cell cycle control (11). The process is tightly controlled used in this study: caspase-9, caspase-3, poly(ADP-ribose) bya network of coordinated enzymes: E 1 ubiquitin- polymerase, Mcl-1, Bcl-xL, Bcl-2, Bak, Bax, ubiquitin, cyclin 15 activating enzyme, E2 ubiquitin-conjugating enzyme, and B1, phospho-CDC2 (Tyr ), and CDC2 from Cell Signaling h E3 ubiquitin ligase. The ubiquitinated proteins are targeted Technology; -actin from Santa Cruz Biotechnology; Bax for proteolysis by the proteasome. Bortezomib (PS-341/ (clone 6A7) and Bak (clone TC-100) from Calbiochem; and Velcade) is a synthetic small molecule that specifically and MPM-2 from Upstate. Bortezomib was kindlyprovided by reversiblyinactivates the proteasome (12). It has been used Millennium Pharmaceuticals. Propidium iodide was to treat relapsed and refractorymultiple myelomaand obtained from Sigma, 4¶,6-diamidino-2-phenylindole (DAPI) mantle-cell lymphoma (11). We have described previously was from Roche Applied Science, and chloromethyl- constitutive activation of nuclear factor-nBinNK-cell X-rosamine (MitoTracker Red) was from Molecular Probes. lymphoma (13). Given the involvement of nuclear factor- Viable Cell Number Assay nB with tumorigenesis and the action of bortezomib against The viable cell number was measured with the AQueous nuclear factor-nB (11), we have recentlyinvestigated and One Solution MTS assay(Promega). Briefly,cells were reported its therapeutic potential for NK-cell neoplasms incubated in a 96-well plate at a densityof 2 Â 105 per well (14). In this study, we have examined the molecular in 100 AL culture medium. Theywere treated with various mechanisms of bortezomib-induced cell death in NK concentrations of bortezomib for 1 day. For each well, 20 AL lymphoma cell lines and primary lymphoma cells. Our MTS reagent was added and the plate was incubated at results suggest that, in addition to cell death byapoptosis at 37jC for 3 h. Absorbance was measured at 492 nm with the lower bortezomib concentrations, NK lymphoma cells that Vmax 96-well plate reader (Molecular Devices). are resistant to apoptosis via bortezomib can be killed via DNA Fragmentation Assay mitotic catastrophe, an alternative cell death mechanism, at The DNA fragmentation assaywas done as described higher pharmacologic concentrations of bortezomib. As the previously(17). Briefly,cells were harvested after 1 dayof apoptosis pathwayis often impaired in relapsed and exposure to bortezomib, washed once, and resuspended in chemorefractoryNK-cell malignancyand other cancers, 200 AL cold PBS. Theywere lysedbyadding 20 AL lysis activation of mitotic catastrophe bybortezomib may buffer [0.2 mol/L EDTA, 0.05 mol/L Tris-HCl (pH 8.0), and provide a novel therapeutic approach for the treatment of 5% Triton X-100] and 2 AL of 20 mg/mL proteinase K. apoptosis-resistant diseases. Thus, bortezomib has the Cellular proteins were digested at 55jC overnight. Cell potential to serve as a more generic antineoplastic agent debris was removed bycentrifugation at 12,000 Â g for than thought previously. 5 min and 200 AL supernatant was gentlymixed with 40 AL of 5 mol/L NaCl. Samples were vortexed gentlyfor 20 s to separate histones from DNA and kept at room temperature Materials and Methods to achieve complete histone disassociation. Cold absolute Approval of this studywas obtained from the Institutional ethanol (500 AL) was added 10 min later and the tubes were Review Board of the Universityof Hong Kong/Hospital incubated at -20jC overnight. DNA was recovered by Authority, Hong Kong West Cluster (institutional review centrifugation at 12,000 Â g for 10 min. Pellets were rinsed board reference no. UW 05-070 T/733). Informed consent brieflywith 70% ethanol, air dried, and then dissolved in was obtained in accordance with the Declaration of 30 AL Tris-EDTA (pH 7.6) with 100 Ag/mL RNase A. Helsinki. Samples were then loaded in a 1.5% agarose gel for NK lymphoma Cell Lines and Primary Cultures electrophoresis. Gel images were obtained with the NK lymphoma cell lines SNK-6 (15) and NK-YS (16) were GelDoc-It Imaging System (UVP). maintained in RPMI 1640 (Hyclone) supplemented with Flow Cytometry antibiotics, 10% heat-inactivated fetal bovine serum Flow cytometry was done on the FACSCalibur cytometer (Hyclone), and 100 units/mL recombinant human inter- (BD Biosciences). Data were collected with the CellQuest leukin-2 (PeproTech). Primarycultures were set up from software (BD Biosciences) and analyzed with the WinMDI fresh tumor specimens obtained from two nasal NK-cell version 2.8 software. For cell cycle analysis, cells were lymphoma patients. In the first patient, the biopsy was harvested, washed, and fixed with 70% cold ethanol for 2 h. obtained from an involved testis during relapse. The tumor Ethanol was removed bycentrifugation at 400 Â g for 5 min tissues were minced and strained through a cell strainer and the cell pellet was resuspended in PBS with 100 Ag/mL

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RNase A. An equal volume of 10 Ag/mL propidium iodide CDC2/CDK1Kinase Assay was added and the sample was mixed thoroughly. Samples The activityof CDC2 was measured with the CDC2/ were examined on the FL2 channel and 20,000 events were CDK1 kinase assaykit (Upstate) following the manufac- collected. The distribution of cells in G1-G0, S, and G2-M turer’s instructions. Briefly, cells were lysed on ice in phases was analyzed with the ModFit LT software (Verity the extraction buffer [50 mmol/L Tris-HCl (pH 7.4), Software House). To detect alteration in the mitochondrial 250 mmol/L NaCl, 1 mmol/L EDTA, 50 mmol/L membrane potential (DCm), cells were harvested, washed, NaF, 1 mmol/L DTT, 0.1% Triton X-100, 10 Amol/L and freshlystained with 100 nmol/L chloromethyl- leupeptin, 100 Ag/mL aprotinin, and 0.5 mmol/L phenyl- X-rosamine at 37jC for 30 min. Samples were examined methylsulfonyl fluoride]. Cell lysate was centrifuged at on the FL3 channel and 30,000 events were collected. 100,000 Â g for 1 h at 4jC and the supernatant was Western Blot Analysis transferred to a tube on ice. Protein concentration was Protein extracts were prepared bylysing2 Â 107 cells in measured bythe Bradford assay(Bio-Rad). An equal 1 mL cold radioimmunoprecipitation assaybuffer (Santa amount of protein was subjected to immunoprecipitation Cruz Biotechnology) and immediately heated at 100jC for using anti-CDC2 antibody(Cell Signaling Technology) 10 min. Samples were kept at room temperature for 30 min and protein G-Plus agarose (Santa Cruz Biotechnology). before centrifugation at 12,000 Â g for 10 min. Supernatant The purified protein was incubated in the reaction buffer was transferred into a new tube and the protein concen- with [g-32P]ATP (Amersham) and histone H1 for 10 min tration was measured with the Bradford assay(Bio-Rad) on at 30jC. Reaction products were mounted on phospho- the Lambda 3B spectrophotometer (Perkin-Elmer). An cellulose paper and radioactivitywas measured with a equal amount of protein lysate from each sample was liquid scintillation counter (LS5801; Beckman Instru- loaded in a 10% SDS-PAGE gel for electrophoresis and ments). Reactions without histone H1 were included as proteins were electrotransferred to a polyvinylidene fluo- the substrate background control. A reaction with recom- ride membrane (Amersham). The blotted membrane was binant CDK1/cyclin B (Cell Signaling Technology) was blocked with 5% Blotto milk (Santa Cruz Biotechnology) done as the positive control. An equal amount of CDC2 and incubated first with the primaryantibodyand then served as the reaction input standardization control. with horseradish peroxidase-conjugated secondaryanti- Mitotic Index body(Cell Signaling Technology).Each step was followed Mitotic index, which is the ratio of mitotic cells relative to bythree washes with TBS-Tween 20 solution (Bio-Rad). The the total number of cells, was calculated from 1,000 cells blots were visualized with an enhanced chemiluminescent stained with DAPI (Roche) for each sample. reagent (Amersham) according to the manufacturer’s Statistical Analysis instructions. Relative expression levels of the various A Student’s t test was used for statistical analysis and proteins were quantified bydensitometric analysisof the P < 0.05 was considered significant. Western blots with the Molecular Imaging Software (Kodak) using h-actin as the protein loading control. For Western blots showing multiple bands, densitometric Results analysis was done only for the full-length protein bands. SNK-6 Cells Were Less Sensitive to Bortezomib Than In situ Senescence-Associated B-Galactosidase NK-YS and Primary NK Lymphoma Cells Staining The cytotoxic effect of bortezomib was determined in the Senescence-associated h-galactosidase staining was done NK lymphoma cell lines (SNK-6 and NK-YS) and primary with the Senescent Cells Staining Kit (Sigma) according to lymphoma cells by measuring cell viability with the MTS the manufacturer’s instructions. Photographs were taken assay(Fig. 1A). Similar data from the NK-YS cell line with the Eclipse E800M microscope (Nikon). and primarylymphomacells have been reported byour Immunofluorescence group (14). The MTS assaywas repeated in this studyto After cytospin, cells were fixed with 70% cold ethanol and compare with the data from the SNK-6 cell line. Treated 4% paraformaldehyde. Autofluorescence from fixatives was with various concentrations of bortezomib, the lymphoma quenched by0.1% sodium borohydride.Cells were rinsed cells were killed in a dose-dependent manner. The median in TBS (pH 7.6; DAKO) three times and blocked with inhibitoryconcentration (IC 50) of SNK-6 cells was 9 ng/mL normal goat serum (DAKO). The primaryantibodywas (23 nmol/L), whereas the IC50 of NK-YS cells was 3 ng/mL applied on the slides and incubated at 4jC overnight in a (8 nmol/L), showing that the SNK-6 cells could tolerate humidified chamber. After washing, the ready-to-use higher concentrations of bortezomib than NK-YS cells. The biotinylated secondary antibody (Zymed) was applied sensitivityof primaryNK lymphomacells was similar to and incubated for 1 h at room temperature followed by that of NK-YS cell line. The IC50 of primarycultures was binding with streptavidin-FITC (1:33; DAKO). The cell 2.4 ng/mL (6 nmol/L) for the first patient and 5 ng/mL nucleus was counterstained with 1 Ag/mL DAPI (Roche) for (13 nmol/L) for the second. 1 min. The mounting medium Vectashield (Vector Labora- Apoptosis Was Limited in SNK-6 Cells Treated with tories) was applied to the slide with a coverslip to prevent Bortezomib the fluorescence signal from fading. Photographs were To identifythe mechanism of death, we examined the taken with an Eclipse E800M microscope (Nikon). apoptotic activityin both cell lines and compared the

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Figure 1. Bortezomib-induced cell death in NKlymphoma cell lines and primary NKlymphoma cells. A, SNK-6 and NK-YS cell lines and primary cultures of NKlymphomas from two patients were incubated with various concentrations of bortezomib ranging from 0.5 to 150 ng/mL, and a viable cell number was assessed by MTS assay. Relative absorbance is defined as a percentage of the absorbance reading relative to control. The background absorbance was not subtracted from the raw data shown. B, a gel image of DNA fragmentation assay of bortezomib-treated lymphoma cells. C, morphology of nucleus in the bortezomib- and mock-treated lymphoma cells. Fluorescent microscopy with the DAPI staining; original magnification, Â400. D, detection of depolarization in DCm by flow cytometry in bortezomib-treated NKlymphoma cell lines. Viable cells were gated from the cell population within the range M1 in the histograms. Mean of three independent assays; bars, SD (A and D).

expression of both proapoptotic and antiapoptotic mole- profiles of representative Bcl-2 familymembers that have cules associated with the mitochondrial pathway. In the antiapoptotic and proapoptotic activities. As expected, Bcl- DNA fragmentation assaythat distinguishes apoptosis xL levels decreased in the apoptosis-sensitive NK-YS cells. from other types of cell death, exposure to 15 ng/mL The expression levels, however, remained unchanged in bortezomib (a concentration used throughout the study SNK-6 cells (Fig. 2A). Additionally, antiapoptotic Bcl-2 unless otherwise specified) led to apoptosis in 24 h in the expression levels increased progressivelyin SNK-6 cells, NK-YS and the primarylymphoma cells. There was, whereas theyslightlydecreased in the NK-YS cells in however, no evidence of a DNA degradation ladder in which the band corresponding to the proapoptotic cleaved the SNK-6 cells (Fig. 1B). Consistent with the above Bcl-2 protein was clearlypresent at 24 h. Moreover, an findings, morphologic examination on NK-YS and primary increase in Mcl-1 expression levels was detected earlyafter lymphoma cells showed the typical features of apoptosis, bortezomib treatment in both cell lines, although its evident with significant DNA hyperchromicity (chromatin turnover differed significantlybetween them. Mcl-1 expres- condensation) and karyorrhexis (nuclear fragmentation). In sion increased steadilyin SNK-6 cells and remained high at the SNK-6 cells, however, the morphologic sign of 24 h, whereas Mcl-1 expression in NK-YS cells increased in apoptosis was not evident (Fig. 1C). These findings were the first 6 h but dropped later at 24 h (Fig. 2A). In addition to also supported bythe measurement of the DCm and these antiapoptotic molecules, we also examined the cleavage of caspases. With exposure to bortezomib, the expression and activation of proapoptotic molecules from DCm depolarized in NK-YS cells in 6 h, whereas it took the Bcl-2 family. With bortezomib, Bak expression levels 9 h in SNK-6 cells (Fig. 1D). Consistent with the DCm data, decreased slightlyin both cell lines (Fig. 2A) and its cleavages in caspase-9, caspase-3, and poly(ADP-ribose) conformational change, a hallmark of activation (18), polymerase were also limited in SNK-6 cells (Fig. 2A). coincided between them (Fig. 2B). In contrast, Bax expres- SNK-6 Cells Treated with Bortezomib Showed Antia- sion levels remained roughlythe same in both cell lines poptotic Bias (Fig. 2A). The Bax conformational change differed tempo- To better understand the diminished apoptotic response rallybetween them, with the active form (19) present at to bortezomib in SNK-6 cells, we examined the expression 6 h in NK-YS cells while appearing at 24 h in SNK-6 cells

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(Fig. 2B). To compare the apoptosis potentials of these two gated if bortezomib at higher concentrations might induce cell lines, we calculated the Bcl-2/Bax ratio. In SNK-6 cells, cellular senescence and mitotic catastrophe in the SNK-6 the Bcl-2/Bax value increased by30%, from 1.83 at 0 h to cell line. We examined both cell lines for senescence- 2.35 at 6 h, whereas it slightlydecreased by10%, from 1.85 to associated h-galactosidase activity, a hallmark of cellular 1.66, in NK-YS cells in the same time frame. Thus, there senescence (21). Unexpectedly, senescent cells remained appeared to be an overall bias toward antiapoptotic scarce in SNK-6 cells, whereas the number of senescent cell conditions in SNK-6 cells in response to bortezomib. increased at 6 h but dropped dramaticallyat 24 h due to Bortezomib Induced Mitotic Catastrophe in SNK-6 apoptosis in NK-YS cells (Fig. 2C). The morphologyof Cells SNK-6 cells after adding bortezomib was consistent with a Mitotic catastrophe is a form of cell death associated with drug effect on mitosis. The dying cells appeared in the cell abnormal mitosis and often accompanied bycellular culture from 12 h, with a significant accumulation of senescence (20). Based on the above findings, we investi- enlarged, distorted, flattened, spindle-shaped cells. Their

Figure 2. High pharmacologic concentrations of bortezomib-induced mitotic catastrophe in the SNK-6 cell line that was insensitive to apoptosis and cellular senescence. A, Western blots of the apoptosis-related proteins in the bortezomib-treated NKlymphoma cell lines. Relative expression levels of the indicated proteins were quantified by densitometric analysis of the Western blots. h-Actin was used as the protein loading control and the data are presented at the bottom of the Western blots. For Western blots showing multiple bands, densitometric analysis was done only for the full-length protein bands. B, active forms of Bak and Bax were examined with immunofluorescence in bortezomib-treated NKlymphoma cell lines using the conformation- specific monoclonal antibody TC-100 and 6A7 (both in green), respectively. Nuclei were counterstained with DAPI (blue). Fluorescent microscopy; original magnification, Â400. C, senescent cells (blue) were detected in both lymphoma cell lines after treatment with bortezomib. Original magnification, 200Â. D, morphologic analysis of bortezomib-induced mitotic catastrophe in SNK-6 cells. Various morphologic alterations were consistent with those of mitotic catastrophe (i-iii): the enlarged, flattened, spindle-shaped cells (long-tailed arrow); multinucleate cells (arrowhead); and plasma membrane blebbing in the dying cells (short-tailed arrow). Inverted phase-contrast microscopy; original magnification, Â800. Additionally, these enlarged, spindle- shaped dead cells (open arrow) were detected with MPM-2 antibody (green; iv-ix). Nuclei were counterstained with DAPI (blue). Fluorescent microscopy; original magnification, Â800.

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nuclear membranes were absent, the chromosomes spread 15 ng/mL) and subjected to cell cycle analysis (Fig. 3B). In erratically, and their plasma membrane was occasionally NK-YS cells, apoptosis was induced in both concentrations undulated and generated blebs around the cells (Fig. 2D, of bortezomib. The sub-G1 population from apoptotic cells i-iii). To identifyif theywere associated with mitosis, these increased progressivelyin a time-dependent manner, with bortezomib-treated SNK-6 cells were examined byimmu- a stable distribution of the cells in the G1 and G2-M phases. nofluorescence with the MPM-2 antibody, which recog- In contrast, SNK-6 cells survived in 5 ng/mL bortezomib, nizes the mitosis-specific phosphorylated proteins, thus and this concentration did not have anyeffect on the serving as a marker of mitotic cells (22). As expected, distribution of the cell cycle phases. When exposed to bortezomib induced an accumulation of MPM-2-positive 15 ng/mL bortezomib, however, SNK-6 cells displayed cells among the SNK-6 cells with the chromosomes in total G2-M arrest after 9 h, with the tetraploid G2-M cells disarray. This was in sharp contrast to the normal mitotic increasing and outnumbering the diploid G1 cells. Once cells from the control (Fig. 2D, iv-ix). again, the evidence for apoptosis was still lacking, as the Bortezomib-Induced Mitotic Catastrophe Was Asso- sub-G1 population from apoptotic cells remained consis- ciated with G2-M Arrest in SNK-6 Cells tentlylow in SNK-6 cells. Together, these findings indicated To quantifythe effect of bortezomib on mitosis, we a mitotic cell death associated with the G2-M arrest. compared the mitotic indices of SNK-6 and NK-YS cells Bortezomib-Induced Mitotic Catastrophe Caused after drug treatment. Bortezomib caused a significant Cyclin B1 Accumulation and Increased CDC2/CDK1 increase in the mitotic index in SNK-6 cells but not in Activity in SNK-6 Cells NK-YS cells (Fig. 3A). From the mitotic index data, we Because we have identified that bortezomib induced the f estimate that 25% of SNK-6 cells were killed through G2-M arrest in the progress of mitotic catastrophe, we mitotic catastrophe after drug treatment. To further explore next investigated whether the cyclin B1/CDC2 complex the effect of bortezomib on mitotic arrest, SNK-6 and NK- played a role in these events. As cyclin B1 is regulated by YS cells were treated with two concentrations (5 and the ubiquitin-proteasome system (23), we examined by

Figure 3. Mitotic index and cell cycle analysis of NKlymphoma cell lines treated with bortezomib. A, mitotic indices of the NK-cell lines treated with bortezomib. Col- umns, mean of three independent experiments; bars, SD. **, P < 0.001, versus control. B, examination of the cell cycle profiles by flow cytometry in the NKlym- phoma cell lines. The three-dimensional histograms were generated with the WinMDI software. Arrow, G2-M arrest in the SNK-6 cell line treated with 15 ng/mL bortezomib.

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Western blot analysis the amount of ubiquitin, cyclin B1, and CDC2 (CDK1) in the bortezomib-treated SNK-6 cells (Fig. 4A). With a steadyincrease in the ubiquitin levels, the cyclin B1 levels increased moderately and reached a plateau after 9 h. In the same time frame, however, the total CDC2 levels declined slightly, with the phospho- CDC2 (Tyr15) levels remaining stable. To achieve a better understanding of the cyclin B1/CDC2 complex activity in the progress of mitotic catastrophe, we measured the CDC2 kinase activityin the bortezomib-treated SNK-6 cells. A marginal increase in CDC2 kinase activitywas detected at the earlystage of mitotic catastrophe ( f9 h after adding bortezomib), and ultimately, a 2-fold increase in the kinase activitywas observed after 24 h (Fig. 4B). Discussion We have shown that low concentrations of bortezomib induce apoptosis in the NK-YS cell line and in primaryNK lymphoma cells. However, in SNK-6 cells, which were resistant to apoptosis at low drug concentrations, bortezomib induced mitotic catastrophe at higher pharmacologic concentrations. Mitotic catastrophe is a type of cell death induced bythe abnormal activations of cyclinB and CDC2, resulting in deficient cell cycle checkpoints (the DNA structure checkpoint and the spindle assemblycheckpoint; ref. 10). It is usuallytriggered bythe abnormal mitosis that culminates in aberrant chromosome segregations. Al- though its molecular mechanism needs further investiga- Figure 4. Expression and activity of cyclin B1 and CDC2 in the tion, some facts about this unique mitotic cell death have bortezomib-induced mitotic catastrophe in the SNK-6 cell line. A, Western been clearlyidentified. Unlike apoptosis, which is basically blot analysis of the ubiquitin, cyclin B1, and CDC2 proteins in the dependent on caspase activation (24), mitotic catastrophe bortezomib-treated SNK-6 cells. Relative expression levels of the various proteins were quantified by densitometric analysis of Western blots. h- maybe mediated in a caspase-dependent or caspase- Actin was used as the protein loading control and the data are presented at independent fashion (8). In some cases, mitotic catastrophe the bottom of the Western blots. B, CDC2 activity was examined in the shares the same signaling pathwaywith apoptosis. More bortezomib-treated SNK-6 cells by measuring its kinase activity on histone often, when apoptosis is deficient, a programmed cell death H1. Columns, mean of three independent experiments; bars, SD. **, P < 0.001, versus control. can still be executed through mitotic catastrophe in response to mitotic failure independent of apoptosis (10). From our observation of the bortezomib-induced cell death Mcl-1 protein might merelycompromise the bortezomib- in a lymphoma cell line (SNK-6) that was resistant to induced apoptosis in NK-cell lymphomas. apoptosis, we identified mitotic catastrophe with character- In the Western blot for caspase-3 of the bortezomib- istics of M-phase arrest, cyclin B1 accumulation, and treated NK lymphoma cell lines, an extra band of f20 kDa elevated CDC2/CDK1 activity. These bortezomib-induced was detected between the full-length and the cleaved alterations mayinterfere with mitosis, and as a result, caspase-3 (Fig. 2A). This band probablyrepresents the mitotic catastrophe is triggered without the typical apo- intermediate cleavage product of the sequential two-step ptosis activation. process in the caspase-3 activation. It has been reported that We examined SNK-6 cells for expression of antiapoptotic the caspase-3 precursor is first cleaved at Asp175/Ser176 Bcl-2 familymembers and found a global delayin the (between the large and the small subunits) to produce the degradation of Mcl-1, Bcl-xL, and Bcl-2 proteins and an p12 subunit and the p20 peptide (28). The p20 peptide is elevated Bcl-2/Bax ratio. This was accompanied with a then cleaved at Asp28/Ser29 to generate the mature p17 delayin the conformational activation of Bax. The subunit. With the caspase-3 antibodythat recognizes the alterations in the Bcl-2 familymembers represent an overall full length (f35 kDa) and the large subunit of casapase-3 antiapoptotic bias in SNK-6 cells. Recent studies have (f17 kDa), we detected this f20 kDa band on the Western shown that an accumulation in Mcl-1 protein accounts for blot. It most likelyrepresents the partial cleavage product the inhibition to apoptosis in bortezomib-treated cancer that results from cleavage at Asp175/Ser176, which occurs cells (25–27). Similarly, in our study, both NK lymphoma before cleavage at Asp28/Ser29 during processing of the cell lines showed Mcl-1 protein increases in response to prosegment. bortezomib treatment. Apoptosis, however, was still Cleavage of Bcl-2, which is a proteolysis product of active mediated in NK-YS cells, indicating that an increase in caspase-3 (29–32), generates a 23 kDa Bax-like fragment

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that was present in NK-YS but not in SNK-6 cells following One studyhas shown that mitotic arrest induced byMG132 24 h treatment with bortezomib. Bcl-2 cleavage at Asp34 is associated with the presence of relativelyhigh amounts removes the NH2-terminal BH4 domain, which is required of cyclin B1 but that MG132-arrested cells unexpectedly for apoptosis inhibition as it permits formation of a present a low CDC2 activity(36). There is also evidence heterodimer with Bax. The truncated form of Bcl-2 cannot that CDC2 inactivation, which drives the cell cycle out of bind to Bax and therefore releases Bax to form the mitosis, is triggered bya disassociation of cyclinB1 from proapoptotic homodimer. In addition, the Bax-like cleavage CDC2 rather than its degradation byproteasome (37). To fragment of Bcl-2 can induce apoptosis byinducing release clarifythese events, we examined CDC2 expression and of cytochrome c into the cytosol. Such processing therefore activation status in the SNK-6 cells treated with bortezo- promotes apoptosis in NK-YS cells via a positive amplifi- mib. In our study, CDC2 activity increased over 24 h with cation loop, from caspase-mediated Bcl-2 cleavage to bortezomib, supporting an association of cyclin B1 accu- increased mitochondrial cytochrome c release and further mulation with the maintenance of CDC2 activityduring activation of caspases. mitotic catastrophe. Previous studies have documented an increase in the Our present results provide an earlyindicator that tetraploid population in cancer cells treated with bortezo- bortezomib maybe an effective treatment for NK-cell mib (33). It was suggested that bortezomib might induce lymphomas. The IC50 of bortezomib in NK-YS cells, apoptosis through G2-M-phase arrest. Using the SNK-6 line patient-derived lymphoma cells (6-13 nmol/L), and in that is relativelyresistant to apoptosis, we have further SNK-6 cells (23 nmol/L) are clinicallyachievable, as the z characterized bortezomib-induced mitotic catastrophe. As IC50 for normal peripheral blood mononuclear cells is 100 illustrated in Fig. 5, cyclin B1 is degraded through the nmol/L (11). In the absence of preclinical studies, the ubiquitin-proteasome pathway(11, 23). Bortezomib inhibits present bortezomib administration schedules in lymphoma proteasome activityand stabilizes cyclinB1. As a result, patients have been derived from the management of cyclin B1 accumulates in the cells. CDC2 is a cyclin- multiple myeloma. The finding of an alternative cell death dependent kinase that controls entryand exit of mitosis. mechanism at higher pharmacologic concentrations and in Recent evidence shows that mitotic exit is reversibly different lineages of hematopoietic malignancies supports regulated byCDC2 kinase activity(34). Briefly,mitotic the utilityof exploring more intensive infusion schedules in entryis enforced byan increase in active CDC2, whereas refractorylymphomapatients. mitotic exit is triggered bya decrease. If cyclinB1 persists, In this study, we investigated an unusual mechanism of as in bortezomib-treated SNK-6 cells, it maymaintain CDC2 cell death, mitotic catastrophe, in a NK lymphoma cell line in an active form and in turn lead to mitotic arrest. that was resistant to apoptosis. In response to chemother- Interfering with mitosis, however, does not completely apy, cancer cells may protect themselves from destruction arrest the cell cycle in M phase. Indeed, mitotic checkpoint bysuppressing apoptosis. Under some circumstances, slippage occurs in the presence of an active checkpoint (35). however, these refractorycancer cells are prone to bypass This slippage then results in the observed cytogenetic chaos, the cell cycle checkpoints (6), leading to cell death via multinucleate cell formation, and mitotic catastrophe. mitotic catastrophe (38). We present evidence here that Earlier observations on the effects of cyclin B1 accumu- bortezomib has the unusual attribute to potentiate both lation induced byproteasome inhibitors are informative. apoptosis and mitotic catastrophe and maytherefore be

Figure 5. Schematic diagram of molecular events in the bortezomib-induced M-phase arrest and mitotic catastrophe. Normally, the active CDC2/cyclin- B1 complex plays an essential role in driving the cell cycle through the M phase (39). At the G2-M-phase transition, the inhibitory phosphoryl group (Tyr15) is removed from CDC2 by the phosphatase CDC25. This process is counteracted by the kinase Wee1. Active CDC2 is able to promote cyclin B1 phosphorylation, and phosphorylated cyclin B1 allows the whole complex to enter the nucleus. Nuclear translocation of CDC2/cyclin B1 triggers activation in various mitosis-associated molecules that drive cell cycles in progress. The anaphase- promoting complex/cyclosome (APC/C) complex forms during anaphase and acts as the ubiquitin E3 ligase that labels cyclin B1 with ubiquitins. Once these ubiquitin tags are recognized, cyclin B1 is degraded by proteasome, a critical step in driving the cell cycle out of M phase. Proteasome inhibitors, such as bortezomib, enable cyclin B1 stabilization and thus play a role in M-phase arrest as well as in mitotic catastrophe.

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Lijun Shen, Wing-Yan Au, Kai-Yau Wong, et al.

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