[CANCER RESEARCH 62, 1662–1668, March 15, 2002]

Significance of MAD2 Expression to Mitotic Checkpoint Control in OvarianCancer Cells1

Xianghong Wang, Dong-Yan Jin, Raymond W. M. Ng, Huichen Feng, Yong C. Wong, Annie L. M. Cheung, andSai W. Tsao2

Departments of Anatomy [X. W., H. F., Y. C. W., A. L. M. C., S. W. T.] and Biochemistry [D-Y. J., R. W. M. N.], Faculty of Medicine, University of Hong Kong, Hong Kong

ABSTRACT

Chromosome instability is a commonly observed feature in ovariancarcinoma. Mitotic checkpoint controls are thought to be essential foraccurate chromosomal segregation, and MAD2 is a key component of thischeckpoint. In this study, we investigated the competence of the mitoticcheckpoint and its relationship to the expression of MAD2 protein in sevenovarian cancer cell lines. We found that a significant number (43%, threeof seven cell lines) of the tested ovarian cancer cells failed to arrest in theG2-M phase of the cell cycle in response to microtubule disruption. Thisloss of mitotic checkpoint control was associated with reduced expressionof the MAD2 protein. To additionally understand the significance of theMAD2 to mitotic checkpoint control, we established an inducible expres-sion system in which MAD2 was induced by the addition of ponasteroneA. Notably, the induced expression of MAD2 in two checkpoint-defectiveovarian cancer cell lines led to the restoration of mitotic checkpointresponse to spindle-disrupting agents. Taken together, our findings sug-gest that the steady-state amount of MAD2 inside cells may represent amolecular switch for mitotic checkpoint control. This provides a novelinsight into the molecular basis of CIN in ovarian carcinoma and hasimplications for effective use of checkpoint-targeting drugs.

INTRODUCTION

Accurate chromosomal segregation is essential for cell survival andgenomic stability. The fact that the majority of human cancer cellsexhibit gains or losses of chromosomes suggests that CIN3 maycontribute to tumorigenesis (1). The mitotic checkpoint, also knownas the spindle assembly checkpoint, detects errors occurred in thespindle structure or in the alignment of the chromosomes on thespindle, and delays chromosome segregation and anaphase onset untilthe defects are corrected. Disruption of the spindle with microtubuletoxins such as nocodazole and colcemid arrests cells in mitosis, andthis arrest depends on a functional mitotic checkpoint (2, 3). Twomajor groups of mitotic checkpoint genes, budding uninhibited bybenomyl (BUB)1–3 and MAD1–3, have been identified in buddingyeast (4, 5). Mammalian homologues of the yeast mitotic checkpointproteins have also been characterized (6–9).

Kinetochores, which are linked to both chromosomes and micro-tubules, play an important role in generating the mitotic checkpointsignal. The function of kinetochores is to ensure that chromosomes arenot segregated until every one of them is aligned and attached to thespindle (2, 10). The majority of proteins associated with mitoticcheckpoint function have been shown to localize to kinetochoresunattached to the microtubules (6, 7, 11, 12). It has been proposed thatthe mitotic checkpoint proteins, especially MAD2, may be crucial for

generating the “wait” signal to prevent the onset of anaphase aftermicrotubule disruption (13–15). Several lines of evidence supportMAD2 as a key component of the mitotic checkpoint. Microinjectionof anti-MAD2 antibodies into HeLa cells abolished nocodazole-induced mitotic arrest and caused premature mitosis (6, 16). MAD2has also been shown to interact with other mitotic checkpoint proteinsincluding MAD1 and MAD3 (8, 17). In addition, MAD2 directlyinteracts with CDC20 and inhibits the anaphase-promoting complex(18–23). Furthermore, chromosome missegregation was observed inMAD2 knockout (MAD2�/�) mice (24), and deletion of one MAD2allele resulted in a defective mitotic checkpoint in both human coloncancer cells and murine primary embryonic fibroblasts (25).

Although the importance of MAD2 in mitotic checkpoint controlhas been established in yeast and mammalian cells, the significance ofMAD2 and the mitotic checkpoint to CIN in human cells and theirassociation with human tumorigenesis are incompletely understood.Human MAD1 has been identified as a cellular target of the humanT-cell leukemia virus type 1 oncoprotein Tax (8). Mitotic checkpointdefects have also been found frequently in human (6), colon (50%;Ref. 26), lung (44%; Ref. 27), and NPC (40%) cells (28). AlthoughMAD2 gene mutations are very rare in human bladder (29), breast (30,31), digestive tract (32), liver (29), and lung (27) cancers, aberrantlyreduced expression of MAD2 protein has been correlated with defec-tive mitotic checkpoint in breast cancer (6) and NPC (28) cells.Furthermore, mice with heterozygous deletion of MAD2 developedlung tumors at high rates after long latencies (25), suggesting that MAD2haplo-insufficiency might contribute to CIN and tumorigenesis.

Ovarian cancer is a leading cause of mortality among gynecologicalcancers. Notably, chromosomal aberrations were frequently found inovarian cancer (33–36). However, it is not known whether the mitoticcheckpoint is functional in ovarian cancer cells. Neither is it clearwhether and how defects in this checkpoint might cause CIN inovarian cancer. In the present study, we investigated the competenceof the mitotic checkpoint in seven human ovarian cancer cell lines.We showed that defects in the mitotic checkpoint are rather commonin ovarian cancer cells. To shed light on the mechanisms underlyingthe defects, we examined the expression of mitotic checkpoint pro-teins and demonstrated an association between MAD2 expression andcheckpoint response. The significance of MAD2 to the mitotic check-point control was additionally studied in an inducible MAD2-expres-sion system. The stable introduction of a MAD2-expressing plasmidinto two ovarian cancer cell lines with low basal levels of MAD2resulted in the restoration of the checkpoint response to microtubule-disrupting agents. Our findings implicate that reduction of MAD2expression may represent a critical event in the development of CINin ovarian cancer.

MATERIALS AND METHODS

Cell Lines and Cell Culture Conditions. Seven ovarian cancer cell lines,DOV13, SKOV3, OVCA3 (obtained from ATCC), Ovca420, Ovca429,Ovca432, and Ovca433 (37) were maintained in RPMI 1640 (Life Technolo-gies, Inc.) supplemented with 2 mM L-glutamine and 5% (v/v) FCS at 37°C.The cultures were grown for a maximum of 10 passages before retrieving freshcells from frozen stock. Ovca420, Ovca432, and Ovca433 cell lines were

Received 10/24/01; accepted 1/18/02.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.Corrected online July 19, 2018.

1 Supported in part by CRCG grants from the University of Hong Kong. D-Y. J. is aLeukemia and Lymphoma Society Scholar.

2 To whom requests for reprints should be addressed, at Department of Anatomy,University of Hong Kong, 1st Floor, Laboratory Block, 21 Sassoon Road, Pokfulam,Hong Kong. Phone: 852-2819-9228; Fax: 852-2817-0857; Email: gswtsao@hkucc.hku.hk.

3 The abbreviations used are: CIN, chromosome instability; MAD, mitotic arrestdeficient; NPC, nasopharyngeal carcinoma; BrdUrd, bromodeoxyuridine; Ab, antibody.

1662

established from freshly isolated ascites or tumor explants from patients withlate-stage ovarian adenocarcinomas with distinct characteristics (37). Severalindependent groups have also demonstrated the differential expression patternsof genes and proteins, such as BRCA1, BRCA2, HER-2, and epidermal growthfactor receptor in these cell lines (37, 38). Moreover, the independent originsof these lines were additionally verified by PCR analysis of polymorphicalleles. Using four sets of primers for four microsatellite markers on chromo-somes 3 (D3S162), 5 (D5S82), 17 (D17S855), and X (DXS981), we found thateach of the cell lines exhibited a distinct pattern of allelic polymorphism (datanot shown).

Mitotic Index. Cells were grown on Chamber slides and treated withnocodazole or Colcemid. The cells were then fixed in cold methanol/acetone(1:1) for 5 min and stained with 4�,6-diamidino-2-phenylindole. The presenceof condensed nuclear DNA was considered to indicate cells undergoing mi-tosis. To measure the mitotic index (percentage of viable cells arrested inmitosis), at least 500 cells were counted for each experiment using fluores-cence microcopy, and the data points represent the average results from threeindependent experiments.

BrdUrd Staining. Cells were grown on Chamber slides for 24 h and thenincubated with culture medium containing BrdUrd (10 �M) for 2 h at 37°C.The BrdUrd incorporation rate was examined by ABC method using a Vec-tastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, afterwashing with PBS, the cells were fixed in cold methanol/acetone (1:1) for 5min and then incubated with methanol containing 2% H2O2 for 10 min at roomtemperature. After washing three times with PBS, the cells were incubated inblocking solution (provided in the ABC kit) for 30 min at 37°C. The blockingsolution was drained and primary Ab (1:20; mouse anti-BrdUrd FormalinGrade; Roche Diagnostics, Mannheim, Germany) was added onto the mono-layers, and the cells were incubated at 37°C for 1 h. After washing three timeswith PBS, the cells were incubated with secondary Ab (biotinylated antimouseAb, provided in the ABC kit) for 30 min at 37°C, and visible brown color inthe positive cells was developed according to the protocol provided by themanufacturers. Then the slides were counterstained with hematoxylin, dehy-drated, and then mounted. The positive cells were identified as the presence ofbrown color in the cell nucleus. At least 500 cells were counted in eachexperiment, and the percentage of BrdUrd-positive cells was calculated andcompared with untreated controls. Each experiment was repeated at least threetimes, and the SE of the means was used as error bars.

Cell Cycle Analysis. Flow cytometric analysis was performed on an EP-ICS profile analyzer and analyzed using the ModFit LT2.0 software (Coulter)as described previously (28).

Western Blotting Analysis. Detailed experimental procedures were de-scribed previously. Briefly, cells were harvested in lysis buffer (28) and proteinconcentrations were determined. Approximately 30 �g of protein was sepa-rated on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, andincubated with antibodies against MAD1 (polyclonal, 1: 500) and MAD2(monoclonal, 1:500; Ref. 8), or actin (1:200; Roche). The relative amounts ofeach protein were quantitated as ratios to actin. Results represent the averageof three independent experiments.

Transfection. A 1.5-kb fragment containing the full-length human MAD2cDNA (Ref. 8; GenBank U) was cloned into the EcoRI and XbaI sites of anexpression vector pIND(SP1) driven by the ecdysone-inducible promoter(Invitrogen, Carlsbad, CA). The resulting construct pIND(SP1)-MAD2(conferring neomycin resistance) was then cotransfected with pVgRXR vector(conferring Zeocin resistance), which permits formation of a functionalheterodimeric ecdysone receptor in mammalian cells. Using the calcium-phosphate method, the stable pIND(SP1)-MAD2 and pVgRXR transfectantswere generated by selecting in both neomycin (150 �g/ml) and Zeocin (250�g/ml). Briefly, cells were plated at �50% confluency, and the cell culturemedium was changed to 5% DMEM containing 5% fetal bovine serum 1 hbefore transfection. DNA suspension (1.1 ml) was made [69 �l of 2 M CaCl2,550 �l of 2 � HBSS, 5 �g of each plasmid DNA, and appropriate amount ofH2O; HBSS [560 mM NaCl, 20 mM KCl, 3.0 mM Na2HPO4, 24 mM dextrose,and 100 mM HEPES (pH 7.02)] and added to the cell culture flask over night.Selective drugs were added 24 h later, and individual transfectants were visible�10 days after drug selection. Clones were then isolated and tested for MAD2expression. To induce MAD2 expression in the transfectants, 5 �M of ponas-terone A (Invitrogen) was added for 20 h. Two transfectants of Ovca432 andOvca433 cell lines were generated and studied in detail.

Confocal Immunofluorescence Microscopy. The cells were grown oncoverslips and treated with nocodazole (50 nM) for 24 h. Confocal immuno-fluorescence microscopy was performed on a Zeiss Axiophot system as de-scribed previously (8). Cells were then fixed in 4% paraformaldehyde, per-meabilized with methanol, and stained with anti-�-tubulin Ab (clone B-5-1-2;Sigma Chemical Co.).

RESULTS

Competence of Mitotic Checkpoint in Ovarian Cancer Cells.Defects in mitotic checkpoint are thought to contribute to CIN andtumorigenesis (14, 26). Deregulation of the mitotic checkpoint invarious cancers has been documented (6, 26–28). However, there isno information on the mitotic checkpoint control and its associationwith CIN in ovarian cancer. As a first step toward understanding themechanisms of CIN, we sought to assess the competence of mitoticcheckpoint control in seven ovarian cancer cell lines using nocodazoleand Colcemid, which inhibit spindle assembly by disrupting micro-tubules. On addition of nocodazole or Colcemid, three of the seven(43%) ovarian cancer cell lines, Ovca420, Ovca432, and Ovca433, didnot show a significant increase in the percentage of cells arrested atmitosis (Fig. 1, A and B, dotted lines), as measured by the number ofcells with condensed chromosomes, a characteristic of mitotic block(Fig. 1C). In addition, there were no significant differences in thepatterns of nuclear staining between nocodazole-treated cells and thecontrol, because both of them showed evidence of nuclear division(Fig. 1C, cells in anaphase or telophase are indicated by arrows,compare panel 6 to panel 5). These data suggest that nocodazoletreatment did not alter mitosis and cell cycle progression in these threecell lines. In contrast, four of the cell lines (Ovca429, Ovca3, Skov3,and Dov13) responded to these two agents with an increase in mitoticcells reaching a peak at �24 h after exposure (Fig. 1, A and B, solidlines). This response is similar to the reaction of HeLa cells, whichhave been shown to be mitotic-checkpoint competent (6, 27, 28). Flowcytometric analysis of Ovca420, Ovca432, and Ovca433 cell linesshowed a lack of G2-M phase arrest after treatment with nocodazoleor Colcemid for 18 h (Fig. 2A) in contrast to Ovca429, Ovca3, Skov3,Dov13, and HeLa cells, which showed a clear G2-M phase block (Fig.2A). However, a small percentage of cells with �4 N DNA contentwas observed in Ovca420, Ovca432, and Ovca433 cell lines at thistime point, and the percentage of �4 N cells increased with theextended incubation time (data not shown). These results indicate thatthree of seven (43%) ovarian cancer cell lines failed to arrest atmitosis in response to microtubule disruption.

To eliminate the possibility that the Ovca420, Ovca432, andOvca433 cells failed to accumulate in mitosis simply because theywere not actively cycling during drug treatment, we measured thefraction of cells in S phase by staining with BrdUrd. As shown in Fig.2B, after nocodazole treatment, Ovca420, Oca432, and Ovca433 celllines had a similar S phase index to the untreated controls, suggestingthat these cells did proceed through mitosis and enter the next S phasein the presence of nocodazole. In contrast, the Ovca429, Ovca3,Skov3, Dov13, and HeLa cells showed �80% reduction in BrdUrd-positive cells compared with the untreated control, reflecting a failureto reinitiate DNA synthesis because of the mitotic block.

To exclude the possibility that the failure of mitotic arrest inOvca420, 432, and 433 cell lines was simply because the microtubulesbecame resistant to nocodazole compared with the other four celllines, we studied the changes in microtubule structure in the presenceand absence of nocodazole. The seven cell lines were immunostainedfor �-tubulin, and the patterns were compared with those in HeLa andCNE3 cells, which have been shown previously to be competent anddefective, respectively, for mitotic checkpoint control (6, 27, 28). In

1663

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

all of the untreated cells, �-tubulin was observed in microtubulesarranged in long and slender fibers, which spread to the entire cyto-plasm. The microtubule organizing centers or centrosomes were alsoevident in some cells (Fig. 3, panels 1 and 3). In contrast, themicrotubule arrays in cells treated with nocodazole were largelydisrupted, and �-tubulin was found in the cell periphery (Fig. 3, panel4) as well as in the remaining microtubule organizing centers (Fig. 3,panels 2 and 4). Notably, microtubule disruption occurred in all of thecell lines. Generally the severity of microtubule disruption was notsignificantly different between cell lines that showed a mitotic arrestin response to nocodazole (i.e., HeLa, Ovca429, Ovca3, Skov3, andDov13) and the lines that failed to arrest (i.e., Ovca420, Ovca432, andOvca433). For instance, nocodazole induced mitotic arrest in Skov3cells (Figs. 1 and 2; Fig. 3, panel 2), whereas Ovca432 cells treatedwith nocodazole kept cycling (Figs. 1 and 2; Fig. 3, panel 4). How-ever, the microtubules in Ovca432 cells (Fig. 3, panel 4) appeared tobe even more severely disrupted than in the Skov3 cells (Fig. 3, panel2). These results suggest that microtubules in cells defective formitotic arrest are not resistant to nocodazole.

Collectively these results demonstrate that in the presence ofmicrotubule-interfering agents, three of seven (43%) ovarian cancercell lines progress through mitosis despite microtubule depolymeriza-tion and keep cycling into S phase, indicating a loss of mitoticcheckpoint.

Correlation of Defective Mitotic Checkpoint with Reduced Ex-pression of MAD2. The loss of mitotic checkpoint control in somecolorectal cancer cells has been shown previously to be associated

with the mutational inactivation of mitotic checkpoint protein BUB1(26). However, mutations of relevant checkpoint genes are extremelyrare in various cancers tested (27, 29–32). Instead, aberrantly reducedexpression of the MAD2 protein has been correlated with defectivemitotic checkpoint control in breast and NPC (6, 28). To additionallystudy the mechanisms underlying the defects in the mitotic checkpointresponse in ovarian cancer cells, we asked whether the expressionlevel of checkpoint proteins might be significantly different. As a firststep, we examined the expression of two key components of themitotic checkpoint, MAD1 and MAD2 (6, 8), by Western blot anal-ysis (Fig. 4). We found that the expression of MAD2 protein wassignificantly decreased (70–80% reduction) in Ovca420, Ovca432,and Ovca433 cell lines, which also exhibited a defective mitoticcheckpoint response (Fig. 4). The other four cell lines with competentmitotic checkpoint expressed MAD2 protein to �60% or more of thesteady-state amount in HeLa cells. However, there was no significantcorrelation between MAD1 expression and mitotic checkpoint com-petence in ovarian cancer cells as reported previously for NPC cells(28). These results suggest that decreased MAD2 expression maycontribute to the defective mitotic checkpoint control in Ovca420,Ovca432, and Ovca433 cells.

Effect of MAD2 Expression on Mitotic Checkpoint Control inOvca432 and Ovca433 Cells. The correlation of defective mitoticcheckpoint with reduced expression of MAD2 prompted us to addi-tionally investigate the roles of MAD2 in the checkpoint control. Tothis end, we sought to reintroduce MAD2 into the checkpoint-defec-tive ovarian cancer cell lines. One route to express MAD2 in mam-

Fig. 1. Mitotic indices of seven ovarian cancer cell lines. The cellswere treated with Colcemid (0.1 �g/ml; A) or nocodazole (50 nM; B)for the indicated times and analyzed at 6 h intervals. � � � � , highlightcells with defective mitotic checkpoint control. HeLa cell line was usedas a control with a normal mitotic checkpoint. C, representative resultsof fluorescence microscopic examination of HeLa (panels 1 and 2),Skov3 (panels 3 and 4), and Ovca433 (panels 5 and 6) before (panels1, 3, and 5), and after (panels 2, 4, and 6) treated with nocodazole (50nM) for 18 h. Accumulation of cells with condensed DNA indicates anormal spindle checkpoint in HeLa and Skov3 (panels 1–4) afterexposure to nocodazole. Photos were taken under �400 magnification.Arrows in Ovca433 cells indicate anaphase or telophase cells under-going nuclear division.

1664

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

malian cells is under the control of a constitutive promoter. However,we were concerned that constitutive and stable overexpression ofMAD2 might cause irreversible changes in cell physiology and cellcycle control. Thus, we constructed the human MAD2 expressionsystem using an inducible expression system in which MAD2 can beinduced transiently by a diffusible small molecule.

The ecdysone-inducible expression system is based on moltinginduction in the fruit fly and has been adapted for inducible expressionin mammalian cells (39). This system uses heterodimeric nuclearreceptors induced by a synthetic analogue of ecdysone (ponasteroneA) to activate the expression of the gene of interest. The aberrantreduction of MAD2 expression in checkpoint-defective cells (Ref. 6,28 and data shown above) raised the concept that the steady-stateamount of MAD2 inside the cells might determine the competence ofthe mitotic checkpoint. Thus, the MAD2 expression plasmid driven byan ecdysone-inducible promoter [pIND(SP1)-MAD2] and the plasmidexpressing the heterodimeric ecdysone receptor (pVgRXR) were co-transfected into Ovca432 and Ovca433 cell lines. Individual stabletransfectants were isolated by selecting in culture medium containingboth Zeocin (250 �g/ml) and G418 (150 �g/ml). Exogenous MAD2expression was induced by exposure to 5 �M ponasterone A for 20 h.As shown in Fig. 5, ponasterone A treatment resulted in a 2–4-foldincrease in MAD2 levels in two Ovca432-MAD2 clones (Clone 1 andClone 2) and two Ovca433-MAD2 clones (Clone 1 and Clone 2)compared with the untreated controls. The MAD2 expression levels inall of the transfectants were �50% compared with HeLa cells.

After ponasterone A treatment, there was no significant change inthe mitotic index (Fig. 6, A, �P), indicating that the levels of MAD2expression in these transfectants did not have any effect on mitoticcheckpoint control in the absence of spindle-disrupting agents. How-ever, when the cells were treated with both nocodazole (for 18 h) andponasterone A (Fig. 6A, �N�P), a significant increase in mitotic cellswas observed in all four of the clones compared with the cells treatedwith nocodazole alone (�N). This indicates that expression of MAD2enabled the cells to arrest at mitosis in the presence of microtubuletoxin. Among the four clones, Ovca432-MAD2-C1 showed the high-est MAD2 expression ratio (�3-fold increases after ponasterone Atreatment) compared with the untreated controls, and the MAD2expression levels in these cells were comparable with those in HeLacells (Fig. 5; �70%). As shown in Fig. 6A, the mitotic index profileof this cell line was also similar to HeLa cells, whereas the other threeclones showed partial mitotic arrest in the presence of nocodazole.This additionally supports the notion that levels of MAD2 expressionmay be one of the key determinants for mitotic checkpoint control inthese cells. Similar results were observed in cells treated with Colce-mid (data not shown).

Cell cycle studies by flow cytometry also confirmed these findings,because increased number of G2-M phase cells were observed in cellstreated with both nocodazole and ponasterone A (Fig. 6B, �N�P),whereas no significant changes were observed in the cells treated withnocodazole (�N) or ponasterone A (�P) alone. The fraction of Sphase cells was also decreased in the cells treated with both nocoda-zole or ponasterone A (Fig. 6C, �N�P) compared with the controland the cells treated with nocodazole (�N) or ponasterone A (�P)alone. Similar results were also observed when treated with Colcemid(data not shown). These results suggest that expression of MAD2restored mitotic checkpoint control, either fully or partially, in thepresence of microtubule inhibitors in ovarian cancer cells.

DISCUSSION

Ovarian cancer is the leading cause of death in female patients withgenital tract carcinomas, because most of the cases are presented at anadvanced stage. Although relatively little is known about the molec-ular basis of this cancer, CIN has been shown to be a common feature

Fig. 3. Nocodazole-induced microtubule disruption in ovarian cancer cell lines. Rep-resentative results are shown for Skov3 and Ovca432 cells. Cells were mock-treated(panels 1 and 3) and treated with 50 nM nocodazole for 24 h (panels 2 and 3). Photos weretaken under �126 magnification. Arrow indicates a cell arrested at metaphase. Similarresults were obtained for the other five ovarian cancer cell lines.

Fig. 2. Cell cycle distribution (A) and BrdUrd incorporation (B) in ovarian cancer celllines before and after Colcemid (0.1 �g/ml) or nocodazole (50 nM) treatment for 18 h. A,representative results showing the accumulation of G2-M phase cells in HeLa and Skov3cell lines after drug exposure in contrast to the lack of this accumulation in Ovca433 cells.B, significant reduction of BrdUrd incorporation was observed in HeLa, Ovca429, Ovca3,Skov3, and Dov13 but not in Ovca420, Ovca432, and Ovca433 cell lines compared withthe untreated control after exposure to nocodazole (50 nM; P � 0.001). Each data pointrepresents the average of three independent experiments; bars, �SE.

1665

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

(33–36). A recent study on 106 ovarian cancer patients using com-parative genomic hybridization demonstrated that 97% of the tumorsamples showed aberrant comparative genomic hybridization profile(40). In addition, the number and extent of chromosome changes weresignificantly increased in high-grade tumors compared with low-gradetumors. This indicates that CIN plays an important role in tumorigen-esis as well as tumor progression in ovarian cancer. In this study,using seven ovarian cancer cell lines, we identified a high frequencyof mitotic checkpoint defect (43%; three of seven cell lines). Ourfindings suggest that a defective mitotic checkpoint may contribute toCIN commonly observed in ovarian cancer cells. In addition, wefound that restoration of MAD2 expression in Ovca432 and Ovca433cells led to mitotic arrest in response to microtubule disruption. Ourdata point to a critical role for MAD2 in mitotic checkpoint control inhuman cancer cells.

Three salient points have emerged from our study. First, we dem-onstrated the frequent loss of mitotic checkpoint in ovarian cancercells. Second, we correlated the loss of checkpoint with the aberrantlyreduced expression of MAD2 protein. Third, we showed that themitotic checkpoint response might be restored if the dosage of MAD2was increased inside the cells.

Direct association between CIN and mitotic checkpoint defect has

been demonstrated in yeast cells (41); however, the mechanismsinvolving CIN in human cells are yet to be characterized. The mostconvincing evidence of the role of mitotic checkpoint defect in CIN inmammalian cells came from two recent studies in MAD2�/� mice(24), and in MAD2�/� human and mouse cells (25) showing thatdisruption of MAD2 expression resulted in CIN. In addition, theMAD2�/� mice developed lung tumors at high rates indicating thatdefects in mitotic checkpoint play an important role in tumorigenesis.In this study, we provide the first evidence for the high frequency ofmitotic checkpoint defect in ovarian cancer cells (Figs. 1 and 2).Because there is a strong correlation between frequency of chromo-somal changes and tumor grading in ovarian carcinoma (33, 40),which is important in determining patient survival, our data may proveuseful for diagnosis and patient selection. The front-line chemother-apeutic drug for the treatment of ovarian cancer is cisplatin, butcisplatin-resistant tumors occur at fairly high frequency. A betterunderstanding of the molecular basis of mitotic checkpoint control inovarian cancer cells may reveal novel strategies for more efficient useof checkpoint-targeting drugs such as Taxol. In this regard, onecritical issue is the differential response of mitotic checkpoint-competent versus mitotic checkpoint-defective cells to microtubuledisruption. It is noteworthy that all mammalian cells, irrespective oftheir competence in mitotic checkpoint control, will eventually adaptto microtubule-disrupting agents and exit mitosis through an as yetunknown mechanism (3, 42). However, if the dose of microtubuletoxins is sufficiently low, as in this study, or if the treatment issufficiently transient, checkpoint-competent cells arrested at mitosismay have a chance to repair their spindles and later proceed throughcell cycle. By contrast, if the microtubule challenge is persistent, thesecells will maintain a p53-dependent G1 arrest after adaptation andundergo apoptosis (42, 43). On the other hand, checkpoint-defectivecells do not arrest at mitosis in the presence of low-dose microtubuletoxins but lose chromosomes at a higher rate to induce apoptosis.Meanwhile, a prolonged exposure to microtubule toxins causespolyploidy in checkpoint-defective cells with no evidence of apopto-sis (7). Thus, the differential reaction to microtubule inhibitors mightbe exploited in selective killing of checkpoint-defective tumor cells.

MAD2 was first identified in screens for yeast mutants, which werehypersensitive to drugs that disrupt microtubules (4, 5). In mammalian

Fig. 4. Western blot analysis of MAD1 and MAD2expression in seven ovarian cancer cell lines. Protein (30�g) was analyzed, and the relative amounts of each proteinwere quantified as ratios to actin and then compared withHeLa cells. Results represent the average of three independ-ent experiments; bars, �SE.

Fig. 5. Forced expression of MAD2 restores mitotic checkpoint response in Ovca432and Ovca433 cells. Western blot analysis of MAD2 protein was performed in cellscotransfected with pIND(SP1)-MAD2 and pVgRXR plasmids. Increased MAD2 expres-sion was observed in cells treated with ponasterone A (5 �M) for 20 h (�P) compared withthe untreated control (�P). The relative MAD2 protein levels before and after ponasteroneA treatment were quantified as ratios to actin and compared with the level in HeLa cells.Results represent the average of three independent experiments; bars, �SE.

1666

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

cells, inactivation of MAD2 resulted in loss of mitotic checkpointcontrol and CIN (6, 24, 25). More recently, it has been reported thatsuppression of MAD2 protein by a carcinogenic compound tetrachlo-rodibenzo-p-dioxin is responsible for the inactivation of mitoticcheckpoint control in HeLa cells (44). Here we have shown thatdecreased MAD2 expression correlated to mitotic checkpoint defectin ovarian cancer cells (Figs. 1–4) and that restoration of MAD2expression induced mitotic arrest in response to microtubule disrup-tion (Figs. 5 and 6). Our findings support the model in which thesteady-state amount of MAD2 may serve as a molecular switch for themitotic checkpoint control in human cancer cells. This is consistentwith the recent finding that haplo-insufficiency of MAD2 led todevelopment of lung cancer in mice (25). Our model for mitoticcheckpoint control has useful implications for patient selection andtherapeutic intervention in ovarian cancer. Because mutations of themitotic checkpoint genes, including MAD2, are uncommon (26–28),analysis of the expression levels of MAD2 protein may allow identi-fication of mitotic checkpoint-defective tumors, thus facilitating theselection of patients for more effective chemotherapy.

REFERENCES

1. Lengauer, C., Kinzler, K. W., and Vogelstein, B. Genetic instabilities in humancancers. Nature (Lond.), 396: 643–649, 1998.

2. Rudner, A. D., and Murray, A. W. The spindle assembly checkpoint. Curr. Opin. CellBiol., 8: 773–780, 1996.

3. Wassmann, K., and Benezra, R. Mitotic checkpoints: from yeast to cancer. Curr.Opin. Genet. Dev., 11: 83–90, 2001.

4. Hoyt, M. A., Totis, L., and Roberts, B. T. S. cerevisiae genes required for cell cyclearrest in response to loss of microtubule function. Cell, 66: 507–517, 1991.

5. Li, R., and Murray, A. W. Feedback control of mitosis in budding yeast. Cell, 66:519–531, 1991.

6. Li, Y., and Benezra, R. Identification of a human mitotic checkpoint gene: hsMAD2.Science (Wash. DC), 274: 246–248, 1996.

7. Taylor, S. S., and McKeon, F. Kinetochore localization of murine Bub1 is required fornormal mitotic timing and checkpoint response to spindle damage. Cell, 89: 727–735,1997.

8. Jin, D. Y., Spencer, F., and Jeang, K. T. Human T cell leukemia virus type 1oncoprotein Tax targets the human mitotic checkpoint protein MAD1. Cell, 93:81–91, 1998.

9. Jin, D. Y., Kozak, C. A., Pangilinan, F., Spencer, F., Green, E. D., and Jeang, K. T.Mitotic checkpoint locus MAD1L1 maps to human chromosome 7p22 and mousechromosome 5. Genomics, 55: 363–364, 1999.

10. Amon, A. The spindle checkpoint. Curr. Opin. Genet. Dev., 9: 69–75, 1999.11. Waters, J. C., Chen, R. H., Murray, A. W., and Salmon, E. D. Localization of Mad2

to kinetochores depends on microtubule attachment, not tension. J. Cell Biol., 141:1181–1191, 1998.

12. Sharp-Baker, H., and Chen, R. H. Spindle checkpoint protein bub1 is required forkinetochore localization of mad1, mad2, bub3, and cenp-e, independently of its kinaseactivity. J. Cell Biol., 153: 1239–1250, 2001.

13. Pennisi, E. Cell division gatekeepers identified. Science (Wash. DC), 279: 477–478,1998.

14. Orr-Weaver, T. L., and Weinberg, R. A. A checkpoint on the road to cancer. Nature(Lond.), 392: 223–224, 1998.

15. Shah, J. V., and Cleveland, D. W. Waiting for anaphase: Mad2 and the spindleassembly checkpoint. Cell, 103: 997–1000, 2000.

16. Gorbsky, G. J., Chen, R. H., and Murray, A. W. Microinjection of antibody to Mad2protein into mammalian cells in mitosis induces premature anaphase. J. Cell Biol.,141: 1193–1205, 1998.

Fig. 6. Effect of MAD2 overexpression on mitotic checkpoint control in Ovca432 and Ovca433 cells. A, mitotic indices. B, cell cycle distribution. C, BrdUrd incorporation. �N,cells were treated with nocodazole (50 nM). �P, cells were treated with ponasterone A (50 �M). �N�P, cells were treated with nocodazole plus ponasterone A. Results represent theaverage of three independent experiments; bars, �SE.

1667

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

17. Chen, R. H., Brady, D. M., Smith, D., Murray, A. W., and Hardwick, K. G. Thespindle checkpoint of budding yeast depends on a tight complex between the Mad1and Mad2 proteins. Mol. Biol. Cell, 10: 2607–2618, 1999.

18. Hardwick, K. G., Johnston, R. C., Smith, D. L., and Murray, A. W. MAD3 encodesa novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p,and Mad2p. J. Cell Biol., 148: 871–882, 2000.

19. Fang, G., Yu, H., and Kirschner, M. W. The checkpoint protein MAD2 and themitotic regulator CDC20 form a ternary complex with the anaphase-promotingcomplex to control anaphase initiation. Genes Dev., 12: 1871–1883, 1998.

20. Hwang, L. H., Lau, L. F., Smith, D. L., Mistrot, C. A., Hardwick, K. G., Hwang, E. S.,Amon, A., and Murray, A. W. Budding yeast Cdc20: a target of the spindle check-point. Science (Wash. DC), 279: 1041–1044, 1998.

21. Kallio, M., Weinstein, J., Daum, J. R., Burke, D. J., and Gorbsky, G. J. Mammalianp55CDC mediates association of the spindle checkpoint protein Mad2 with thecyclosome/anaphase-promoting complex, and is involved in regulating anaphaseonset and late mitotic events. J. Cell Biol., 141: 1393–1406, 1998.

22. Kim, S. H., Lin, D. P., Matsumoto, S., Kitazono, A., and Matsumoto, T. Fission yeastSlp1: an effector of the Mad2-dependent spindle checkpoint. Science (Wash. DC),279: 1045–1047, 1998.

23. Wassmann, K., and Benezra, R. Mad2 transiently associates with an APC/p55Cdccomplex during mitosis. Proc. Natl. Acad. Sci. USA, 95: 11193–11198, 1998.

24. Dobles, M., Liberal, V., Scott, M. L., Benezra, R., and Sorger, P. K. Chromosomemissegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2.Cell, 101: 635–645, 2000.

25. Michel, L. S., Liberal, V., Chatterjee, A., Kirchwegger, R., Pasche, B., Gerald, W.,Dobles, M., Sorger, P. K., Murty, V. V., and Benezra, R. MAD2 haplo-insufficiencycauses premature anaphase and chromosome instability in mammalian cells. Nature(Lond.), 409: 355–359, 2001.

26. Cahill, D. P., Lengauer, C., Yu, J., Riggins, G. J., Willson, J. K., Markowitz, S. D.,Kinzler, K. W., and Vogelstein, B. Mutations of mitotic checkpoint genes in humancancers. Nature (Lond.), 392: 300–303, 1998.

27. Takahashi, T., Haruki, N., Nomoto, S., Masuda, A., Saji, S., Osada, H., andTakahashi, T. Identification of frequent impairment of the mitotic checkpoint andmolecular analysis of the mitotic checkpoint genes, hsMAD2 and p55CDC, in humanlung cancers. Oncogene, 18: 4295–4300, 1999.

28. Wang, X., Jin, D. Y., Wong, Y. C., Cheung, A. L., Chun, A. C., Lo, A. K., Liu, Y.,and Tsao, S. W. Correlation of defective mitotic checkpoint with aberrantly reducedexpression of MAD2 protein in nasopharyngeal carcinoma cells. Carcinogenesis(Lond.), 21: 2293–2297, 2000.

29. Hernando, E., Orlow, I., Liberal, V., Nohales, G., Benezra, R., and Cordon-Cardo, C.Molecular analyses of the mitotic checkpoint components hsMAD2, hBUB1 andhBUB3 in human cancer. Int. J. Cancer, 95: 223–227, 2001.

30. Percy, M. J., Myrie, K. A., Neeley, C. K., Azim, J. N., Ethier, S. P., and Petty, E. M.Expression and mutational analyses of the human MAD2L1 gene in breast cancercells. Genes Chromosomes Cancer, 29: 356–362, 2000.

31. Gemma, A., Hosoya, Y., Seike, M., Uematsu, K., Kurimoto, F., Hibino, S.,Yoshimura, A., Shibuya, M., Kudoh, S., and Emi, M. Genomic structure of the humanMAD2 gene and mutation analysis in human lung and breast cancers. Lung Cancer,32: 289–295, 2001.

32. Imai, Y., Shiratori, Y., Kato, N., Inoue, T., and Omata, M. Mutational inactivation ofmitotic checkpoint genes, hsMAD2 and hBUB1, is rare in sporadic digestive tractcancers. Jpn. J. Cancer Res., 90: 837–840, 1999.

33. Iwabuchi, H., Sakamoto, M., Sakunaga, H., Ma, Y. Y., Carcangiu, M. L., Pinkel, D.,Yang-Feng, T. L., and Gray, J. W. Genetic analysis of benign, low-grade, andhigh-grade ovarian tumors. Cancer Res., 55: 6172–6180, 1995.

34. Sonoda, G., Palazzo, J., du, M. S., Godwin, A. K., Feder, M., Yakushiji, M., andTesta, J. R. Comparative genomic hybridization detects frequent overrepresentationof chromosomal material from 3q26, 8q24, and 20q13 in human ovarian carcinomas.Genes Chromosomes Cancer, 20: 320–328, 1997.

35. Guan, X. Y., Sham, J. S., Tang, T. C., Fang, Y., Huo, K. K., and Yang, J. M. Isolationof a novel candidate oncogene within a frequently amplified region at 3q26 in ovariancancer. Cancer Res., 61: 3806–3809, 2001.

36. Wang, V. W., Bell, D. A., Berkowitz, R. S., and Mok, S. C. Whole genomeamplification and high-throughput allelotyping identified five distinct deletion re-gions on chromosomes 5 and 6 in microdissected early-stage ovarian tumors. CancerRes., 61: 4169–4174, 2001.

37. Rauh-Adelmann, C., Lau, K. M., Sabeti, N., Long, J. P., Mok, S. C., and Ho, S. M.Altered expression of BRCA1, BRCA2, and a newly identified BRCA2 exon 12deletion variant in malignant human ovarian, prostate, and breast cancer cell lines.Mol. Carcinog., 28: 236–246, 2000.

38. Xu, F., Yu, Y., Le, X., Boyer, C., Mills, G. B., and Bast, R. C. The outcome ofheregulin-induced activation of ovarian cancer cells depends on the relative levels ofHER-2 and HER-3 expression. Clin. Cancer Res., 5: 3653–3660, 1999.

39. No, D., Yao, T. P., and Evans, R. M. Ecdysone-inducible gene expression inmammalian cells and transgenic mice. Proc. Natl. Acad. Sci. USA, 93: 3346–3351,1996.

40. Kiechle, M., Jacobsen, A., Schwarz-Boeger, U., Hedderich, J., Pfisterer, J., andArnold, N. Comparative genomic hybridization detects genetic imbalances in primaryovarian carcinomas as correlated with grade of differentiation. Cancer (Phila.), 91:534–540, 2001.

41. Paulovich, A. G., Toczyski, D. P., and Hartwell, L. H. When checkpoints fail. Cell,88: 315–321, 1997.

42. Minn, A. J., Boise, L. H., and Thompson, C. B. Expression of Bcl-xL and loss of p53can cooperate to overcome a cell cycle checkpoint induced by mitotic spindledamage. Genes Dev., 10: 2621–2631, 1996.

43. Lanni, J. S., and Jacks, T. Characterization of the p53-dependent postmitotic check-point following spindle disruption. Mol. Cell. Biol., 18: 1055–1064, 1998.

44. Oikawa, K., Ohbayashi, T., Mimura, J., Iwata, R., Kameta, A., Evine, K., Iwaya, K.,Fujii-Kuriyama, Y., Kuroda, M., and Mukai, K. Dioxin suppresses the checkpointprotein, MAD2, by an aryl hydrocarbon receptor-independent pathway. Cancer Res.,61: 5707–5709, 2001.

1668

MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS

Correction

Correction: Significance of MAD2 Expressionto Mitotic Checkpoint Control in OvarianCancer Cells

In this article (Cancer Res 2002;62:1662–9), which was published in the March 15,2002, issue of Cancer Research (1), the authors did not disclose that portions of Fig. 1were previously published in an article in Carcinogenesis in 2000 (2). Several authorsfrom the Cancer Research article were authors on the Carcinogenesis article. Theauthors state that this oversight occurred because of mislabeling of images in theirlaboratory.

Specifically, 2 panels in Fig. 1 were incorrectly published in the Cancer Researcharticle. The same image (labeled as mitotic index of HeLa cells after treatmentwith nocodazole) was used for Panel 1 of Fig. 1C in the Cancer Research articleand the top right panel of Fig. 2B published in the Carcinogenesis article. Thesame image was used for Panel 2 in Fig. 1C (erroneously labeled as mitotic figuresof SKOV3 cells after treatment with nocodazole) in the Cancer Research articleand the middle right panel of Fig. 2B (labeled as mitotic index of SUNE1 aftertreatment with nocodazole) in the Carcinogenesis article.

The authors regret these errors.

References1. Wang X, Jin D-Y, Ng RW, Feng H, Wong YC, Cheung ALM, et al. Significance of MAD2

expression to mitotic checkpoint control in ovarian cancer cells. Cancer Res 2002;62:1662–9.2. Wang X, Jin D-Y, Wong YC, Cheung ALM, Chun ACS, Lo AKF, et al. Correlation of defective

mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngealcarcinoma cells. Carcinogenesis 2000;21:2293–7.

Published OnlineFirst March 20, 2012.doi: 10.1158/0008-5472.CAN-12-0583�2012 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 1905