[CANCER RESEARCH 62, 1662–1668, March 15, 2002] Significance of MAD2 Expression to Mitotic Checkpoint Control in Ovarian Cancer Cells1 Xianghong Wang, Dong-Yan Jin, Raymond W. M. Ng, Huichen Feng, Yong C. Wong, Annie L. M. Cheung, and Sai 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 generating the “wait” signal to prevent the onset of anaphase after microtubule disruption (13–15). Several lines of evidence support Chromosome instability is a commonly observed feature in ovarian MAD2 as a key component of the mitotic checkpoint. Microinjection carcinoma. Mitotic checkpoint controls are thought to be essential for of anti-MAD2 antibodies into HeLa cells abolished nocodazole- accurate chromosomal segregation, and MAD2 is a key component of this checkpoint. In this study, we investigated the competence of the mitotic induced mitotic arrest and caused premature mitosis (6, 16). MAD2 checkpoint and its relationship to the expression of MAD2 protein in seven has also been shown to interact with other mitotic checkpoint proteins ovarian cancer cell lines. We found that a significant number (43%, three including MAD1 and MAD3 (8, 17). In addition, MAD2 directly of seven cell lines) of the tested ovarian cancer cells failed to arrest in the interacts with CDC20 and inhibits the anaphase-promoting complex G2-M phase of the cell cycle in response to microtubule disruption. This (18–23). Furthermore, chromosome missegregation was observed in loss of mitotic checkpoint control was associated with reduced expression MAD2 knockout (MAD2Ϫ/Ϫ) mice (24), and deletion of one MAD2 of the MAD2 protein. To additionally understand the significance of the allele resulted in a defective mitotic checkpoint in both human colon MAD2 to mitotic checkpoint control, we established an inducible expres- cancer cells and murine primary embryonic fibroblasts (25). sion system in which MAD2 was induced by the addition of ponasterone Although the importance of MAD2 in mitotic checkpoint control A. Notably, the induced expression of MAD2 in two checkpoint-defective has been established in yeast and mammalian cells, the significance of ovarian cancer cell lines led to the restoration of mitotic checkpoint response to spindle-disrupting agents. Taken together, our findings sug- MAD2 and the mitotic checkpoint to CIN in human cells and their gest that the steady-state amount of MAD2 inside cells may represent a association with human tumorigenesis are incompletely understood. molecular switch for mitotic checkpoint control. This provides a novel Human MAD1 has been identified as a cellular target of the human insight into the molecular basis of CIN in ovarian carcinoma and has T-cell leukemia virus type 1 oncoprotein Tax (8). Mitotic checkpoint implications for effective use of checkpoint-targeting drugs. defects have also been found frequently in human (6), colon (50%; Ref. 26), lung (44%; Ref. 27), and NPC (40%) cells (28). Although MAD2 gene mutations are very rare in human bladder (29), breast (30, INTRODUCTION 31), digestive tract (32), liver (29), and lung (27) cancers, aberrantly Accurate chromosomal segregation is essential for cell survival and reduced expression of MAD2 protein has been correlated with defec- genomic stability. The fact that the majority of human cancer cells tive mitotic checkpoint in breast cancer (6) and NPC (28) cells. exhibit gains or losses of chromosomes suggests that CIN3 may Furthermore, mice with heterozygous deletion of MAD2 developed contribute to tumorigenesis (1). The mitotic checkpoint, also known lung tumors at high rates after long latencies (25), suggesting that MAD2 as the spindle assembly checkpoint, detects errors occurred in the haplo-insufficiency might contribute to CIN and tumorigenesis. spindle structure or in the alignment of the chromosomes on the Ovarian cancer is a leading cause of mortality among gynecological spindle, and delays chromosome segregation and anaphase onset until cancers. Notably, chromosomal aberrations were frequently found in the defects are corrected. Disruption of the spindle with microtubule ovarian cancer (33–36). However, it is not known whether the mitotic toxins such as nocodazole and colcemid arrests cells in mitosis, and checkpoint is functional in ovarian cancer cells. Neither is it clear this arrest depends on a functional mitotic checkpoint (2, 3). Two whether and how defects in this checkpoint might cause CIN in major groups of mitotic checkpoint genes, budding uninhibited by ovarian cancer. In the present study, we investigated the competence benomyl (BUB)1–3 and MAD1–3, have been identified in budding of the mitotic checkpoint in seven human ovarian cancer cell lines. yeast (4, 5). Mammalian homologues of the yeast mitotic checkpoint We showed that defects in the mitotic checkpoint are rather common proteins have also been characterized (6–9). in ovarian cancer cells. To shed light on the mechanisms underlying Kinetochores, which are linked to both chromosomes and micro- the defects, we examined the expression of mitotic checkpoint pro- tubules, play an important role in generating the mitotic checkpoint teins and demonstrated an association between MAD2 expression and signal. The function of kinetochores is to ensure that chromosomes are checkpoint response. The significance of MAD2 to the mitotic check- not segregated until every one of them is aligned and attached to the point control was additionally studied in an inducible MAD2-expres- spindle (2, 10). The majority of proteins associated with mitotic sion system. The stable introduction of a MAD2-expressing plasmid checkpoint function have been shown to localize to kinetochores into two ovarian cancer cell lines with low basal levels of MAD2 unattached to the microtubules (6, 7, 11, 12). It has been proposed that resulted in the restoration of the checkpoint response to microtubule- the mitotic checkpoint proteins, especially MAD2, may be crucial for disrupting agents. Our findings implicate that reduction of MAD2 expression may represent a critical event in the development of CIN Received 10/24/01; accepted 1/18/02. in ovarian cancer. 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. MATERIALS AND METHODS Corrected online July 19, 2018. 1 Supported in part by CRCG grants from the University of Hong Kong. D-Y. J. is a Leukemia and Lymphoma Society Scholar. Cell Lines and Cell Culture Conditions. Seven ovarian cancer cell lines, 2 To whom requests for reprints should be addressed, at Department of Anatomy, DOV13, SKOV3, OVCA3 (obtained from ATCC), Ovca420, Ovca429, University of Hong Kong, 1st Floor, Laboratory Block, 21 Sassoon Road, Pokfulam, Ovca432, and Ovca433 (37) were maintained in RPMI 1640 (Life Technolo- Hong Kong. Phone: 852-2819-9228; Fax: 852-2817-0857; Email: gswtsao@hkucc. gies, Inc.) supplemented with 2 mML-glutamine and 5% (v/v) FCS at 37°C. hku.hk. 3 The abbreviations used are: CIN, chromosome instability; MAD, mitotic arrest The cultures were grown for a maximum of 10 passages before retrieving fresh deficient; NPC, nasopharyngeal carcinoma; BrdUrd, bromodeoxyuridine; Ab, antibody. cells from frozen stock. Ovca420, Ovca432, and Ovca433 cell lines were 1662 MITOTIC CHECKPOINT CONTROL IN OVARIAN CANCER CELLS established from freshly isolated ascites or tumor explants from patients with Confocal Immunofluorescence Microscopy. The cells were grown on late-stage ovarian adenocarcinomas with distinct characteristics (37). Several coverslips and treated with nocodazole (50 nM) for 24 h. Confocal immuno- independent groups have also demonstrated the differential expression patterns fluorescence microscopy was performed on a Zeiss Axiophot system as de- of genes and proteins, such as BRCA1, BRCA2, HER-2, and epidermal growth scribed previously (8). Cells were then fixed in 4% paraformaldehyde, per- factor receptor in these cell lines (37, 38). Moreover, the independent origins meabilized with methanol, and stained with anti-␣-tubulin Ab (clone B-5-1-2; of these lines were additionally verified by PCR analysis of polymorphic Sigma Chemical Co.). alleles. Using four sets of primers for four microsatellite markers on chromo- somes 3 (D3S162), 5 (D5S82), 17 (D17S855), and X (DXS981), we found that each of the cell lines exhibited a distinct pattern of allelic polymorphism (data RESULTS not shown). Mitotic Index. Cells were grown on Chamber slides and treated with Competence of Mitotic Checkpoint in Ovarian Cancer Cells. nocodazole or Colcemid. The cells were then fixed in cold methanol/acetone Defects in mitotic checkpoint are thought to contribute to CIN and (1:1) for 5 min and stained with 4Ј,6-diamidino-2-phenylindole. The presence tumorigenesis (14, 26). Deregulation of the mitotic checkpoint in of condensed nuclear DNA was considered to indicate cells undergoing mi- various cancers has been documented (6, 26–28). However, there is tosis. To measure the mitotic index (percentage of viable cells arrested in no information on the mitotic checkpoint control and its association mitosis), at least 500 cells were counted for each experiment using fluores- with CIN in ovarian cancer. As a first step toward understanding the cence microcopy, and the data points represent the average results from three mechanisms