Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC.
Aneuploidy and the function of the spindle assembly checkpoint were examined using DNA flow cytometry and morphologic analysis with microtubule disrupting drugs. To explore the molecular basis, the authors examined the expression and alterations of the mitotic checkpoint gene, BUB1, using Northern hybridization and direct sequencing in 8 HCC cell lines and 50 HCC specimens. Furthermore, the authors examined the alterations of other mitotic checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, using direct sequencing in HCC cell lines with aneuploidy.
An impaired spindle assembly checkpoint was found in five (62.5%) of the eight aneuploid cell lines. Transcriptional expressions of the BUB1 gene appeared in all cell lines. While some polymorphic base changes were noted in BUB1, BUBR1, and CDC20, no mutations responsible for impairment of the mitotic checkpoint were found in either the HCC cell lines or HCC specimens, which suggests that these genes did not seem to be involved in tumor development in HCC.
Many systems protect genomic DNA from damage or alteration as a result of endogenous and exogenous injuries.1 A defect in one of these protective systems could increase mutation rates and cause genetic instability. Genetic instability is one of the characteristic features of various types of cancers and is thought to be important in terms of carcinogenesis and tumor growth. It can take two different forms: microsatellite instability (MI) and chromosomal instability (CI).2, 3 Microsatellite instability has been reported in hereditary nonpolyposis colorectal carcinoma and other sporadic cancers,4–8 and is largely the result of a defect of the mismatch repair genes with an increased mutation rate at the nucleotide level.9–11 In many other tumors, a CI leading to an abnormal number of chromosomes (aneuploidy) has been observed.12 Cahill et al.13 showed that human colon carcinoma cell lines with aneuploidy were consistently associated with the loss of a spindle assembly checkpoint that prevents the onset of anaphase and subsequent commitment to cellular division until the chromosomes are aligned properly on a bipolar spindle. Many genes whose products play a role in the spindle assembly checkpoint have been identified. In yeast, the budding uninhibited by benzimidazole (BUB) family genes BUB1, BUB2, BUB3, and mitotic arrest deficient (MAD) family genes MAD1, MAD2, and MAD3 were initially identified in screening for the checkpoint defect.14, 15 Additional work has shown that the yeast genes Mps1 and Cdc20/slp1+ also function in the spindle checkpoint pathway.16, 17 It has been reported that disrupting these genes can lead to CI.14–19 In addition, alterations in the expression or sequence of human spindle assembly checkpoint genes have been detected in some cancers. Li and Benezra20 have shown that the expression of human homologue of S. cerevisiae MAD2 gene decreases in breast carcinoma. Cahill et al.13 provided evidence for mutant alleles of human homologue of S. cerevisiae BUB1 (hBUB1) in colorectal carcinoma. They have also reported that mutations in the hBUB1 gene can function in a dominant-negative manner.
Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide.21 Hepatitis virus infection, alcohol consumption, and dietary exposure to toxins such as aflatoxin B1 are associated with the occurrence of HCC.22–26 However, genetic events in hepatic carcinogenesis are poorly understood. We previously investigated the incidence of MI and frameshift mutations of the TGFβRII, M6P/IGFIIR, hMSH3, hMSH6, and BAX genes in HCC.27 Only 4% of HCC cases showed MI, and no frameshift mutation was detected in these genes. Therefore, we conclude that it is unlikely for MI to be involved in the initiation and progression of HCC. Some reports showed that aneuploidy was observed frequently in HCC and liver cirrhosis and suggested that aneuploidy was involved in hepatocarcinogenesis.28–30 To investigate whether impairment of the spindle assembly checkpoint is associated with hepatocarcinogenesis and tumor growth, the function of the checkpoint in HCC was investigated in the current study. In addition, alteration in the human homologues of S. cerevisiae BUB1, BUBR1, BUB3, MAD2B, and CDC20 genes in HCC was examined.
MATERIALS AND METHODS
Cell Culture and DNA Ploidy Analysis
All HCC cell lines (Hep3B, HepG2, HLE, HT17, HuH-7, Li-7, Mahlavu, and PLC/PRF/5) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cells were harvested, fixed with ethanol and stained with propidium iodide (Sigma-Aldrich, Tokyo, Japan). Flow cytometric analyses were performed with a B-D FACScan flow cytometer (Becton-Dickinson, KY) configured with a 488 nm argon ion laser. Lymphocytes from healthy individuals were used as internal standards. A total of 10,000 events per sample were acquired. The median coefficient of variation (CV) of the G1 peaks of the lymphocyte standards was 1.8%. The DNA index (DI) was measured by the determination of the ratio of the DNA content of the aneuploid peak to the DNA content of the diploid peak. The DNA aneuploid population had a DI higher than 1.1 N in 10% or more of the nuclei.
For analysis of chromosome number, spread metaphase chromosomes were stained with Giemsa solution (Wako, Osaka, Japan) and counted by Cytovision (Applied Imaging Corp., CA).31
Cell Cycle Analysis
Approximately 0.1 × 106 cells were plated in 6 cm dishes. After 48 hours, nocodazole or colcemid was added to the media to a final concentration of 0.2 μg/mL or 1 μg/mL, respectively. Cells were harvested at 6 hour time intervals thereafter and then fixed with glutalaldehyde, stained with Hoechest 33258, and analyzed with fluorescence microscopy. At least 300 cells were counted for one measurement and each measurement was repeated at least twice.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from cell lines by lysing with Trizole reagent according to the manufacturer's instructions (Life Technologies, MD) and then resuspended in RNase-free water. Reverse transcription (RT) was performed at 30 °C for 10 minutes, 42 °C for 60 minutes, 95 °C for 10 minutes, and 5 °C for 5 minutes. Polymerase chain reaction (PCR) was then carried out with TaKaRa LA Taq (Takara, Shiga, Japan). The nucleotide sequences of the primer sets are listed in Table 1. Reaction mixtures were heated to 94 °C for 2 minutes, followed by 35 cycles of 94 °C for 30 seconds, 58 °C for 30 seconds, 72 °C for 4 minutes, and then 72 °C for 10 minutes for the final elongation.
Table 1. Primers for PCR Amplification
PCR: polymerase chain reaction.
A 20 μg amount of total RNA was fractionated on a 1% denaturing agarose gel and transferred to a Hybond-N+ membrane (Amersham Pharmacia Biotech, Buckinghamshire, England) in 20 × SSC buffer solution. Hybridization was performed in an Express Hyb Hybridization Solution (Clontech, CA) at 68 °C for 1 hour using BUB1 full-length RT-PCR products for probes labeled by random primers. After hybridization the blots were washed at room temperature in 2 × SSC, 0.05% sodium dodecyl sulfate for 40 minutes and at 50 °C in 0.1 × SSC, 0.1% SDS for 40 minutes, and exposed for 48 hours.
Patients and Tumor Samples
Fifty samples of histologically confirmed HCC from 50 patients (43 males and 7 females, aged 43–83 years) who had undergone hepatic resection were analyzed in the current study. Informed consent was obtained from all patients. Clinical and histologic details have been previously described.27 Genomic DNA was extracted from each sample as described by Miller et al.32
Each exon of the BUB1 gene was amplified by PCR with primers that were provided by D. P. Cahill, M.D., Ph.D.33 Polymerase chain reaction was carried out with Taq DNA polymerase (Takara). Reaction mixtures were heated to 94 °C for 2 minutes, followed by 35 cycles at 94 °C for 30 seconds, each annealing temperature for 30 seconds, 72 °C for 2 minutes, and then 72 °C for 10 minutes for the final elongation.
Reverse transcription PCR or PCR products were electrophoresed in an agarose gel and eluted from the isolated gel using Quantum PrepTM Freeze 'N Squeeze DNA Gel Extraction Spin Columns (BIO-RAD, CA). The products were sequenced with a Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Applied Biosystems, Courtaboeuf, France) and an ABI Prism 377 DNA sequencing system (Perkin-Elmer, Applied Biosystems) according to the manufacturer's recommendations.
DNA Ploidy Analysis of HCC Cell Lines
Aneuploidy was screened using DNA flow cytometry and defined by a DI higher than 1.0. All HCC cell lines examined in the current study exhibited aneuploidy. The DI values of Hep3B, HepG2, HLE, HT17, HuH-7, Li-7, Mahlavu, and PLC/PRF/5 were 1.69, 2.34, 1.50, 1.82, 1.55, 1.46, and 1.57, respectively (Fig. 1). A human colon carcinoma cell line, HCT116, was used as the diploid cell control. Furthermore, cytogenetic analysis confirmed the aneuploidy in all HCC cell lines (Fig. 2).
Spindle Assembly Checkpoint in HCC Cell Lines
By treatment with nocodazole or colcemid, which inhibits proper spindle assembly through different mechanisms, the function of the mitotic checkpoint was screened. Three cell lines (HLE, Mahlavu, and PLC/PRF/5) exhibited an accumulation of cells arrested at the M phase. However, other cell lines (Hep3B, HepG2, HT17, HuH-7, and Li-7) showed no accumulation at any time point, indicating that an impaired spindle assembly checkpoint was present in 62.5% of the HCC cell lines. These results were obtained using either nocodazole or colcemid (Fig. 3). HCT116 was used as the control for an intact checkpoint and SW480 for an abnormal checkpoint.
BUB1 Gene in HCC Cell Lines
Reverse transcription PCR was performed with total RNA isolated from cell lines with a checkpoint defect. All RT-PCR products exhibited one band of the expected size. No mutations were found by direct sequencing analysis in the BUB1 gene. Transcripts of the BUB1 gene were assessed using Northern hybridization. Transcriptional expressions of the BUB1 gene appeared in all cell lines, and no significant difference in the amount of transcript was detected between the normal and abnormal checkpoint cells (Fig. 4).
BUB1 Gene in HCC Specimens
Genetic DNA extracted from 50 HCC specimens was subjected to mutational analysis of the BUB1 gene, and 2 sequence variants were identified. One variant had serine (TCC) substituted for phenylalanine (TTC) at codon 375 in case 11, and the other had lysine (AAG) substituted for arginine (AGG) at codon 566 in case 28 (Fig. 5). Case 11 was a well-differentiated HCC in cirrhotic liver caused by hepatitis B virus, and case 28 was a moderately differentiated HCC in hepatitis C virus induced cirrhotic liver. Genomic DNA extracted from nontumorous liver tissue revealed the same variants in both cases.
BUBR1, BUB3, MAD2B, and CDC20 Genes in HCC Cell Lines
Reverse transcription PCR was performed with total RNA isolated from cell lines with a checkpoint defect. All RT-PCR products exhibited one band of the expected size. While some polymorphic base changes that have been previously reported were noted, no mutations were found in any of the HCC cell lines (Table 2).
Spindle assembly checkpoint is a surveillance mechanism that monitors the attachment of spindle microtubules to the kinetochores, thereby ensuring that the onset of anaphase occurs after the correct completion of metaphase. The current findings showed that an impaired checkpoint was present in five (62.5%) of eight aneuploidy HCC cell lines. To our knowledge, this is the first report to show that impairment of the spindle assembly checkpoint occurs in a significant proportion of HCC cell lines.
The data did not directly support the link between a defective spindle assembly checkpoint and aneuploidy. However, in vitro dysfunction of the spindle assembly checkpoint causes abnormalities of chromosome segregation.14–19 Furthermore, human colon carcinomas with dysfunction of the spindle checkpoint consistently exhibit aneuploidy.13 Various reports have indicated that a defect in the spindle checkpoint causes aneuploidy. Thus, the aneuploidy observed in the current five HCC cell lines was probably associated with the impairment of a checkpoint.
We found that some aneuploid HCC cell lines showed a normal spindle checkpoint. It has been reported that certain genes, except for the spindle checkpoint, are involved in aneuploidy in vitro. Zhou et al.34 showed that an overexpression of STK15/BTAK (aurora2), which encodes a centrosome associated kinase, showed both centrosome abnormality and aneuploidy in diploid cells. It is also noteworthy that Chial et al.35 reported that a change in the NDC1 dosage, required for a late step in spindle pole body duplication, could lead to aneuploidy in yeast. In addition, recent investigations have shown that adenomatous polyposis coli (APC) protein interacts with kinetochores and microtubles, and loss of APC sequence contributes to aneuploidy.26 These genes or other unknown mechanisms might partly contribute to aneuploidy in HCC and hepatocarcinogenesis, although impairment of spindle assembly checkpoint seems to play a critical role.
Using FACScan, we also examined whether cells with a dysfunctional spindle assembly checkpoint developed polyploidy in the presence of a microtubule-disrupting drug. Only a small fraction of cells exhibited polyploidy (data not shown). It was reported that a p53 and/or pRb-dependent postmitotic checkpoint induces G1 arrest in cells that have evaded mitotic arrest induced by an inhibitor of microtubule assembly.37, 38 These mechanisms probably support the spindle assembly checkpoint and also contribute to maintaining genomic stability.
Tylor and McKean39 reported that BUB1 is localized in the kinetochores and monitors proper spindle assembly. Ouyang et al.40 showed that BUB1 is closely linked to cell cycle proliferation. Furthermore, Cahill et al.13 showed that the mutational inactivation of this gene leads to a spindle checkpoint defect in colon carcinoma. Based on these reports, we first examined alterations in the BUB1 gene to explore the molecular basis of defects of the spindle assembly checkpoint in HCC. We found 2 sequence variants in 2 of 50 HCC specimens. Both variants were also found in nontumourous liver tissue. Accordingly, it is possible that these mutations were polymorphisms of the BUB1 gene. Myrie et al.41 reported that no mutations were found in breast carcinomas and Olesen et al.42 showed that no mutations and no loss of heterozygosity were found in bladder carcinomas. In lung carcinomas, one or no mutations have been detected.43, 44 The results of these and the current studies show that the BUB1 gene is rarely the target of genetic alterations.
Next, we examined other spindle checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, in five HCC cell lines. We noted several sequence variants, some of which indicated amino acid changes. However, they were reported previously in many cancers and normal tissues as germline variants, although their functions have not been shown.33, 41, 42, 44, 45 In colon carcinomas, Cahill et al.33 reported that a search in the MAD1L1, MAD2, BUB3, TTK (MPS1L1), and CDC20 genes did not identify mutations. In lung carcinomas, Takahashi et al.45 reported that mutations were not found in MAD2 and CDC20, and Nomoto et al.46 showed that 1 of 49 lung carcinomas exhibited a mutation in the MAD1 gene. A striking contrast between the frequency of this checkpoint defect and the mutational rate of these genes is obvious. Several reports have shown that other genes, such as p53,47, 48p38,49BRCA2,50 and cohesin,51 might be linked to the spindle assembly checkpoint. In addition, it appears that the yeast homologue of EB1, a human protein that binds APC, participates in another mitotic checkpoint that delays the final production of the two daughter cells in response to an incorrectly oriented spindle.52 These or unknown genes might contribute to this checkpoint defect.
Aneuploidy was detected in all HCC cell lines examined in the current study. Li et al.53 concluded that aneuploidy was the cause rather than a consequence of transformation, based on chromosome analysis of transformed cells. Duesberg et al.54 also showed that aneuploidy resulted in karyotypic and phenotypic heterogeneity of cancer cells. Chromosomal instability leading to aneuploidy may play a critical role in the carcinogenesis and progression of malignancy in many types of cancers. On the other hand, our previous study showed that MI did not occur frequently in HCC.27 Thus CI, rather than MI, may be important for hepatocarcinogenesis and tumor growth.
In conclusion, the current findings show that impairment of the spindle assembly checkpoint occurred with a high frequency in HCC with CI. The impairment of the spindle assembly checkpoint might be critical for CI in HCC and HCC development. However, other mechanisms that contribute to CI in HCC and the molecular basis of impairment of the mitotic checkpoint remain obscure. Further investigation will be required to understand this issue more fully.
The authors thank D. Cahill, M.D., Ph.D., The Johns Hopkins School of Medicine, for providing pBI–GFP–Bub and primer sequences.