Correlation of defective mitotic checkpoint with aberrantly reduced expression of MAD2 protein in nasopharyngeal carcinoma cells

Xianghong Wang, Dong-Yan Jin1, Y.C. Wong, Annie L.M. Cheung, Abel C.S. Chun1, Angela K.F. Lo, Yu Liu and Sai Wah Tsao2

Department of Anatomy and
1 Institute of Molecular Biology, Faculty of Medicine, University of Hong Kong, Pok Fu Lam Road, Hong Kong


    Abstract
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 Abstract
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 References
 
Nasopharyngeal carcinoma (NPC) occurs with a high incidence in many countries in south-eastern Asia. Chromosomal abnormalities have been commonly found in NPC, but the underlying mechanism is not well understood. We determined mitotic indices, the staining pattern of nuclear DNA and cell cycle profiles of NPC cells in response to treatment with microtubule-disrupting agents, and found that the mitotic checkpoint was defective in two out of five (40%) of the tested NPC cell lines. We also observed that an aberrantly reduced expression of MAD2, one of the key components of mitotic checkpoint, correlated with the loss of checkpoint control. Our findings suggest that a defective mitotic checkpoint characterized by reduced expression of MAD2 contributes to chromosomal instability in NPC.

Abbreviations: NPC, nasopharyngeal carcinoma.


    Introduction
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 Abstract
 Introduction
 References
 
Nasopharyngeal carcinoma (NPC), although relatively rare among Caucasians, is common in Hong Kong, southern China and many other parts of south-eastern Asia (1). Although this disease is a significant cause of morbidity and mortality worldwide, the molecular basis of tumorigenesis in NPC is poorly understood. In contrast to many other human tumours, mutations of the p53 and Rb genes are not common events in NPC (2,3). However, chromosomal abnormalities, including numerical changes, chromosomal deletions and amplifications, have been frequently reported (47). It is generally accepted that nearly all solid tumours are genetically unstable. In most cases, chromosomal instability results in frequent gain and loss of chromosomes in human cancer cells. In some colon cancers, and in other cases, microsatellite instability reflects an elevated nucleotide mutation rate. In yeast, disruption of spindle checkpoint genes can lead to chromosomal instability since checkpoint-defective cells complete mitosis even in the presence of abnormal chromosomes (8). To date, however, little is known about the underlying mechanism of the mitotic checkpoint defects in human cancers. Since the identification of MAD (mitotic arrest deficient) and Bub (budding uninhibited by benzimidazole) gene families (9), attempts have been made to find mutations of mitotic checkpoint genes in human cancers with chromosomal instability. Only a small fraction of colorectal cancers have been observed to contain somatic mutations of the Bub1 gene (10). Consistent with this, somatic mutations of the MAD1 and MAD2 genes and of the CDC gene encoding p55 have also been found at a low frequency in lung cancer cell lines and primary tumour samples (11,12). It is possible that mutations and deletions of these genes may not be a frequent event and abnormalities at gene level may not be the major cause of chromosomal instability in most of these tumours. Nevertheless, cancer cells with chromosomal instability have been found to have a defective mitotic checkpoint more frequently (1012). In this regard, a previous report has correlated a reduced level of MAD2 protein expression with defective mitotic checkpoint control in breast cancer cell lines (13).

We have been studying the genetic and phenotypic alterations associated with NPC as well as the roles of these alterations in tumour development and progression (1416). To investigate whether mitotic checkpoint plays a role in the chromosomal instability commonly observed in NPC, we investigated the competence of the mitotic checkpoint and the possible cause of checkpoint defects in NPC cells. Using five NPC cell lines, we studied the mitotic index and cell cycle distribution in response to the microtubule-disrupting agents nocodazole and colcemid, and then investigated the expression of three key mitotic checkpoint proteins MAD1, MAD2 and p55CDC in these cell lines.

To evaluate the checkpoint function in response to microtubule disruption in NPC cells, five NPC cell lines CNE1, CNE2, CNE3, SUNE1 and 915 (16,17) and HeLa cells were treated with nocodazole and colcemid, two microtubule-disrupting agents that block spindle assembly through different mechanisms. Firstly, mitotic indices were determined to assess the percentage of cells arrested at mitosis at different time points up to 48 h after drug treatment. HeLa cells previously demonstrated to be checkpoint-competent (11,13) were used as positive control. We noted that HeLa cells responded to drug treatment by accumulating mitotic cells, with a peak at 18 h (Figure 1Go). Three NPC cell lines CNE1, 915 and SUNE1 (solid lines) behaved in a similar manner to HeLa, indicating an intact checkpoint function. By contrast, in CNE2 and CNE3 cells (dotted lines), there was either a complete lack of mitotic arrest or lack of clear peak in mitotic index after treatment with nocodazole and colcemid, suggesting a defect in checkpoint response. One simple explanation for the lack of mitotic arrest is that CNE2 and CNE3 cells could have been able to inactivate or expel nocodazole and colcemid. To rule out this possibility, we stained HeLa and CNE3 cells for {alpha}-tubulin by immunofluorescence in the presence and in the absence of nocodazole (Figure 2AGo). In untreated cells, {alpha}-tubulin was visualized in microtubules arranged in long fibres, which fill the entire cytoplasm (left panels). In cells treated with nocodazole, the microtubule arrays disappeared and {alpha}-tubulin was found in the cell periphery (right panels). These results suggest that in both cell lines, treatment with nocodazole results in the disruption of microtubules. Next we compared the staining patterns of nuclear DNA in treated and untreated cells (Figure 2BGo). This analysis revealed a significant difference between the two groups of cells. In the HeLa, CNE1, 915 and SUNE1 cells, which showed a peak in mitotic index in response to drug treatment, there was an accumulation of cells with condensed chromosomes, a characteristic of mitotic block. In the CNE2 and CNE3 cell lines, which showed much lower mitotic index after treatment, DNA condensation did not occur. Cell cycle analysis was then performed to verify the distribution of cells in different phases with or without treatment with nocodazole (Table IGo). A nearly complete G2/M block and a reduction of S phase cells in response to nocodazole treatment were documented for the CNE1, 915, SUNE1 and HeLa cell lines. An example of the original fluorescence activated cell sorting (FACS) trace for the HeLa cells is shown in Figure 2CGo. It can be seen that cells with 4N DNA content accumulated upon disruption of microtubules (compare right panel to the left). In sharp contrast, the cell cycle profiles of CNE2 and CNE3 cells did not change significantly after treatment with nocodazole. In addition, a lack of reduction in BrdU incorporation (data not shown) indicates that CNE2 and CNE3 cells continued to divide in spite of microtubule disruption. As shown in the original FACS tracing for the CNE3 cells (Figure 2CGo, panels 3 and 4), the ratio of cells with 2N and 4N DNA content remained constant after nocodazole treatment. However, the proportion of cells with >4N DNA content was higher, indicating that the mitotic checkpoint has been overridden. Results from the above assays consistently point to a defective mitotic checkpoint in CNE2 and CNE3 cells, i.e. in two of the five NPC cell lines tested (40%). This is comparable to data published on human lung cancers, in which four out of nine (44%) cell lines had a defective mitiotic checkpoint (11).



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Fig. 1. Mitotic indices of five NPC cell lines. HeLa cells were used as positive control. Cells were treated with nocodazole (50 nM) (A) or colcemid (1.0 µg/ml) (B) for the indicated times and harvested at 6 h intervals up to 48 h. The cells were then fixed in cold methanol/acetone (1:1) for 5 min and stained with 4',6-diamidino-2-phenylindole (DAPI). To measure the mitotic index (percentage of viable cells arrested in mitosis), >=500 cells were counted for one experiment using fluorescence microcopy. Each experiment was repeated at least twice. The dotted lines indicate the mitotic checkpoint defective cell lines.

 




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Fig. 2. Representative results of fluorescence microscopic and flow cytometric examination of HeLa, SUNE1 and CNE3 cell lines. (A) Anti-{alpha}-tubulin immunofluorescence staining. HeLa and CNE3 cells were treated with nocodazole as described in the legend of Figure 1Go and were stained with monoclonal anti-{alpha}-tubulin (clone B-5-1-2, Sigma). The photos were taken under 151x magnification on a confocal microscope (Zeiss). Experimental procedures for confocal immunofluorescence microscopy have been detailed elsewhere (18,28). (B) Propidium iodide staining. Cells were treated with nocodazole as in (A). Accumulation of cells with condensed chromosomes was observed in HeLa and SUNE1 cells, whereas a significant decrease in mitotic cells was seen in CNE3 cells. The photos were taken under 100x magnification on a fluorescence microscope. (C) DNA content of HeLa and CNE3 cells as analysed on an EPICS profile analyser (Beckman-Coulter).

 

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Table I. Analysis of cell cycle distribution after exposure to nocodazole
 
We provide the first evidence that the mitotic checkpoint is frequently defective in NPC cells. Our data indicate that defects in mitotic checkpoint could be one cause of the chromosomal instability commonly seen in NPC. Further characterization of the nature and molecular mechanisms of these checkpoint defects should provide important insights into the oncogenesis of NPC. As a first step towards this end, we searched for mutations in key components of the mitotic checkpoint such as the MAD1, MAD2 and p55CDC genes. No genetic alterations were found using this approach (data not shown), which generally agrees with the results from colon cancer cell lines (11).

We next determined the levels of expression of the mitotic checkpoint proteins MAD1, MAD2 and p55CDC in the five NPC cell lines. MAD1 and MAD2 are thought to be centrally involved in sensing chromosome positions and spindle attachment and transducing the signal to the cell cycle machinery (13,1820). There is evidence that MAD2 may be involved in signalling abnormalities in kinetochore–microtubule attachments (13). In addition, MAD2 forms a complex with MAD1 and seems to transmit its signal by associating with another protein, p55CDC (equivalent to yeast CDC20), which then activates the cyclin destruction machinery through the anaphase promoting complex (APC) (21,22). We have obtained highly specific antibodies against human MAD1, MAD2 and p55CDC (18). With these antibodies, we assessed the expression of MAD1, MAD2 and p55CDC proteins in the five NPC cell lines CNE1, CNE2, CNE3, SUNE and 915 by using western blot analysis (Figure 3Go). CNE1, SUNE1 and 915 cells expressed all three proteins to a similar level as seen in HeLa cells. However, CNE2 and CNE3 showed significantly lower expression of MAD2, both expressing 60–70% less MAD2 at steady state than in HeLa cells. The other two checkpoint proteins, MAD1 and p55CDC, were also differentially expressed in CNE2 and CNE3 cells. In contrast, expression of the tumour suppressor proteins p53, Rb and p21WAF1 was the same in all five cell lines (Figure 3Go). Collectively, our data suggest that aberrantly reduced expression of mitotic checkpoint protein MAD2 accompanies the checkpoint dysfunction in NPC cell lines CNE2 and CNE3.



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Fig. 3. Western blotting analysis of MAD1, MAD2, p55CDC, pRb, p53 and p21WAF1 proteins on NPC cell lines. Detailed experimental procedures were described previously (15). Briefly, the cells were harvested in lysis buffer and protein concentrations were determined. Approximately 50 µg proteins were separated on a 10% or 15% sodium dodecyl sulfate–polyacrylamide gel, transferred to nitrocellulose and incubated with antibodies against MAD1 (polyclonal, 1:500), MAD2 (monoclonal, 1:500), p55CDC (polyclonal, 1:500; Santa-Cruz, CA, USA), pRb (monoclonal, 1:1000; DAKO), p53 (monoclonal, 1:200; Oncogene, Cambridge, MA, USA), p21WAF1 (monoclonal, 1:1000; PharMingen, CA, USA) and actin (polyclonal, 1:500; Santa-Cruz, CA, USA). The MAD1 and MAD2 antibodies were detailed elsewhere (18). The relative amounts of each protein were quantified as ratios to actin. Results represent the average of three independent experiments and the error bars indicate SE.

 
In this study, we have shown for the first time that defects in mitotic checkpoint occur rather frequently (~40%) in NPC cells. This lends further support to the notion that interference with mitotic checkpoint function contributes significantly to the development of cancer (8). Chromosomal abnormalities are commonly found in NPC cells (47) and it has been shown that expression of the Epstein–Barr virus oncoprotein LMP-1, which is frequently associated with NPC, can influence cell cycle progression (23). The mitotic checkpoint is a surveillance system that ensures the proper attachment of all chromosomes to the spindle before the onset of anaphase. A premature onset as a result of a defective checkpoint would, presumably, lead to mis-segregation of chromosomes. Through this mechanism, checkpoint dysfunction may contribute to chromosomal instability and oncogenesis. The human MAD1 was initially identified as a binding partner and a cellular target of human T-cell leukemia virus type I oncoprotein Tax (18). Mutations of BUB1 and MAD1 have also been found in a small proportion of colon, lung and breast cancers (1012,24). However, mechanisms of aneuploidy and chromosomal deletion or amplification were not understood. Our finding that a mitotic checkpoint was affected provides an explanation for the chromosomal defects seen in NPC.

Previous cytogenetic analysis has documented significant chromosomal abnormalities in CNE2 cells (25). It is noteworthy that this NPC cell line has structurally abnormal chromosomes distinct from other NPC lines including CNE1. Two unique giant markers for CNE2 have been defined by chromosome banding and there are dramatic differences between CNE2 and CNE1 (25). These data suggest that checkpoint-defective CNE2 may have more severe chromosomal abnormalities than the other checkpoint-competent NPC lines. This further supports the concept that mitotic checkpoint dysfunction leads to chromosomal instability. In this regard, the five NPC cell lines merit a more extensive and systematic cytogenetic analysis.

Our evidence is also a first demonstration of the association between the steady-state amounts of MAD2 protein, which is thought to be one of the key proteins in regulating mitotic checkpoint (22), and the mitotic checkpoint competence. Thus, CNE2 and CNE3 cells showed lowest levels of MAD2 (Figure 3Go); this aberrantly reduced expression of MAD2 accompanied a defective mitotic checkpoint response to treatment with nocodazole and colcemid (Figures 1 and 2GoGo; Table IGo). In contrast, the checkpoint-competent cell lines showed comparable levels of this protein. However, the relative amount of p55CDC did not seem to affect the mitotic control as the protein was barely detectable in HeLa cells but a normal checkpoint response was observed. There was also no consistent correlation between the expression level of MAD1 and the mitotic checkpoint competence. Our results suggest that MAD2 expression may be crucial in regulating mitotic checkpoint in NPC cells. This hypothesis is supported by the evidence that neutralization of MAD2 protein by its antibody in HeLa cells resulted in the abolition of nocodazole-induced mitotic arrest (13). Recently, chromosome missegregation was observed in cells from MAD2 knockout mice (26). In addition, down-regulation of MAD2 by p21waf1 resulted in abnormal mitosis in human fibrosarcoma cells (27). In combination with our observations, the evidence so far strongly suggests the importance of MAD2 in controlling mitotic checkpoint. There is evidence that MAD2 transmits its signals by forming a complex with MAD1 and p55CDC (9). It is possible that the steady-state amount of MAD2, but not that of MAD1 or p55CDC, may represent the rate-limiting step in switching on/off the checkpoint. However, elucidation of the exact roles of MAD1, MAD2 and p55CDC in regulating the spindle checkpoint in NPC cells requires further investigation. In particular, it would be of great interest to understand whether genomic alterations in the MAD2 promoter region or changes occurring at other levels (transcriptional, post-transcriptional, translational or post-translational) could be responsible for the defects in the checkpoint response.

In summary, we have presented the first evidence for a defective mitotic checkpoint in NPC cells. In addition, we have demonstrated an association between reduced expression of MAD2 protein and loss of mitotic checkpoint in these cells. Our findings provide a novel mechanism to explain, at least in part, the chromosomal instability commonly seen in NPC. Since the mitotic checkpoint is targeted by front-line anti-cancer drugs such as taxol, the difference in the mitotic checkpoint protein expression may be informative in determining the difference between microsatellite instability and chromosomal instability in human tumours and facilitating the successful use of chemotherapy.


    Notes
 
2 To whom correspondence should be addressed Email: gswtsao{at}hkucc.hku.hk Back


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Received June 26, 2000; revised August 22, 2000; accepted August 28, 2000.