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[Cancer Research 63, 8606-8613, December 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Epigenetic Inactivation of CHFR and Sensitivity to Microtubule Inhibitors in Gastric Cancer

Ayumi Satoh1, Minoru Toyota126, Fumio Itoh1, Yasushi Sasaki2, Hiromu Suzuki1, Kazuhiro Ogi2, Takefumi Kikuchi1, Hiroaki Mita2, Toshiharu Yamashita23, Takashi Kojima4, Masanobu Kusano5, Masahiro Fujita5, Masao Hosokawa5, Takao Endo1, Takashi Tokino2 and Kohzoh Imai1

1 First Department of Internal Medicine,
2 Department of Molecular Biology, Cancer Research Institute,
3 Department of Dermatology, and
4 Department of Pathology, Sapporo Medical University, Sapporo,
5 Keiyukai Sapporo Hospital, Sapporo, and
6 PREST, JST, Kawaguchi, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitotic checkpoints prevent errors in chromosome segregation that can lead to neoplasia. Therefore, it is notable that gastric cancers often show impaired checkpoint function. In the present study, we examined the functional consequences of epigenetic inactivation of the mitotic checkpoint gene CHFR in gastric cancers. CHFR expression was silenced by DNA methylation of the 5' region of the gene in 20% of the gastric cancer cell lines tested and in 39% of primary gastric cancers; expression could be restored by treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor. In addition, histones H3 and H4 were found to be deacetylated in cell lines showing aberrant methylation, indicating a role for histone deacetylation in the methylation-dependent gene silencing. Cells not expressing CHFR showed impaired checkpoint function, which led to nuclear localization of cyclin B1 after treatment with docetaxel or paclitaxel, two microtubule inhibitors. Apparently, the absence of CHFR is associated with sensitivity of cells to mitotic stress caused by microtubule inhibition, and restoration of CHFR expression by 5-aza-2'-deoxycytidine or adenoviral gene transfer restored the checkpoint. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in gastric cancer. Moreover, the aberrant methylation of CHFR appears to be a good molecular marker with which to predict the sensitivity of gastric cancers to microtubule inhibitors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although gastric cancers are the second most commonly diagnosed form of the disease (1 , 2) , much about the genetic and epigenetic abnormalities involved in gastric tumorigenesis remains unknown (3 , 4) . With respect to the latter, however, epigenetic changes such as DNA methylation are known to be involved in the inactivation of tumor suppressor genes (5 , 6) ; indeed, such aberrant methylation has been detected frequently in gastric cancer (7, 8, 9, 10) . In addition, DNA methylation of multiple CpG islands has been detected in a subset of gastric cancers that appears to have the CpG island methylator phenotype, which in most cases causes sporadic gastric cancers with microsatellite instability, followed by hMLH1 methylation (9) .

Many human cancers are sensitive to mitotic stress, a circumstance now being exploited in therapy (11) . Such sensitivity implies dysfunction of one or more cell cycle checkpoints. Some mitotic checkpoint genes (e.g., MAD2 and BUB1) are involved in preventing chromosome separation and entry into anaphase until the chromosomes are attached correctly to the chromosome segregation machinery (12 , 13) ; others (e.g., EB1) delay the final production of daughter cells until the spindle is oriented correctly (14) . Although impairment of molecules involved in mitotic checkpoints may be important in tumorigenesis, mutation of mitotic checkpoint genes is rarely detected, and the molecular mechanism underlying mitotic checkpoint defects remains unknown (15, 16, 17, 18) .

The silencing of another checkpoint gene, CHFR [checkpoint with fork head-associated (FHA) and ring finger], which delays chromosome condensation during prophase in response to mitotic stress caused by microtubule poisoning (19) , was noted recently in several human tumors (19, 20, 21) . CHFR contains a RING-finger domain that is critical for mitotic checkpoint activity and plays a role in ubiquitination of substrates such as polo-like kinase (PLK1) and CHFR itself (22 , 23) . CHFR also contains an FHA domain that is conserved in several checkpoint genes, including CHK2, RAD53, and MDC1 (24, 25, 26, 27, 28) . The FHA domain plays a role in recognizing phosphorylated proteins (29) , and deletion of the FHA domain has been shown to attenuate CHFR function (19) .

In the present study, we examined the methylation status of eight mitotic checkpoint genes in a panel of gastric cancer cell lines and primary gastric cancer specimens. Among them, only CHFR was specifically methylated in gastric cancers. This methylation was closely associated with loss of gene expression and with mitotic checkpoint dysfunction, which sensitized the affected cells to microtubule inhibitors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Line and Tissue.
Twenty gastric cancer cell lines were analyzed. Of these, 12 were obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan); the remaining 8 (HSC39, HSC40, HSC41, HSC42, HSC43, HSC44, HSC45, and SH-101) were described previously (30 , 31) . In addition, 61 primary gastric cancer specimens and 44 samples of stomach mucosa collected from areas adjacent to tumors were obtained from the Department of Surgery, Sapporo Keiyukai Hospital, after acquisition of informed consent from each patient. All of the cell lines were cultured in the appropriate medium. DNA was extracted using the phenol-chloroform method, whereas total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s instructions. To analyze restoration of CHFR expression, HSC44 and HSC45 cells were incubated for 72 h with 0.2 µM 5-aza-2'-deoxycytidine (5-aza-dC; Sigma, St. Louis, MO), a methyltransferase inhibitor, and/or for 24 h with various concentration of trichostatin A (TSA; 30 nM, 300 nM, or 3 mM), a histone deacetylase inhibitor (WAKO, Tokyo, Japan). The cells were then harvested, and total RNA was extracted for additional analyses.

Reverse Transcription-PCR.
Total RNA was prepared from samples of normal stomach and colon tissue, lymphocytes, and cancer cell lines, after which 5-µg samples were reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) to prepare first-strand cDNA. The primer sequences used were 5'-GGCGAGAGCGTTCCTCCAGTTG-3' (CHFRRT-F) and 5'-GCATGTCAGCGTCTCCTCCATCTTG-3' (CHFRRT-R). Controls consisted of RNA treated identically but without the addition of reverse transcriptase. The integrity of the cDNA was confirmed by amplifying glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously (32) . Samples (10 µl) of the amplified products were then subjected to 2.5% agarose gel electrophoresis and stained with ethidium bromide. Real-time PCR was carried out using SYBR Green sequence detection reagents (Applied Biosystems) in a solution containing l00 ng of cDNA, 25 µl of SYBR Green PCR Master Mix, and 2.5 pmol of each primer. The primers used were 5'-CCTCAACAACCTCGTGGAAGCATAC-3' (CHFRRT2-F) and 5'-TCCTGGCATCCATACTTTGCACATC-3' (CHFRRT2-R). The PCR cycling protocol included 1 cycle at 95°C for 5 min, and 40 cycles at 95°C for 30 s and 60°C for 1 min. Fluorescent signals were detected using an ABI 7000 Prism 7000 (Applied Biosystems), and the accumulation of PCR product was measured in real time as the increase in SYBR green fluorescence. Data were analyzed using ABI Prism 7000 SDS Software (Applied Biosystems). Standard curves relating initial template copy number to fluorescence and amplification cycle were generated using the amplified PCR product as a template, and were used to calculate mRNA copy number in each sample. Ratios of the intensities of the CHFR and GAPDH signals were used as a relative measure of the expression level of CHFR in each specimen.

Mutational Analysis.
Seventeen pairs of primers for individually amplifying and screening exons 2–18 of the CHFR gene were used for PCR with genomic DNA. Spanning the entire coding region of the gene, these primers are specified by GenBank (accession no. AC023047), and their sequences are available upon request. PCR was carried out in a 50-µl reaction mixture containing 1x PCR buffer (TaKaRa, Tokyo, Japan), 1 µM primers, 0.25 mM deoxynucleoside triphosphate mixture, and 1.0 unit of Hot Start Taq polymerase (TaKaRa). The amplified PCR products were electrophoresed in 1% Seaplaque gels, excised, purified using a PCR purification system (Promega), and sequenced. Primer sequences and PCR conditions used for mutational analysis are available upon request.

Bisulfite Treatment and Combined Bisulfite Restriction Analysis (COBRA).
Genomic DNA was initially treated with sodium bisulfite (Sigma) as described previously (33) . COBRA, a semiquantitative methylation assay, was carried out as described previously (34) . PCR was performed in a 50-µl volume containing 1x PCR buffer [67 mM Tris-HCl (pH 8.8), 16.6 mM (NH4)2SO4, 6.7 mM MgCl2, and 10 mM ß-mercaptoethanol], 0.25 mM deoxynucleoside triphosphate mixture, 0.5 µM each primer, and 1.0 unit of Hot Start Taq polymerase (TaKaRa). Touchdown PCR was then carried out as follows. After a hot start, the cycling protocol entailed 3 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s; 4 cycles of 94°C for 30 s, 53°C for 30 s, and 72°C for 30 s; 5 cycles of 94°C for 30 s, 51°C for 30 s, and 72°C for 30 s; and 26 cycles for 94°C for 30 s, 49°C for 30 s, and 72°C for 30 s. Primers were designed based on the nucleotide sequences obtained from GenBank (accession nos. CHFR, AC023047; BUB1, AC114776; BUB1B, AC025429; BUB3, AC012391; MAD2L1, AC025237; MAD2L2, HS330012; CENP-E, AC079919; and EB1, HS1085F17). The primers used for COBRA were: 5'-YGTTTATTAAGAGYGGTAGTTAAAG-3' (CHFRGM1-F) and 5'-AAA-ATCCTTAAAACTTCCAATCC-3' (CHFRGM1-R); 5'-GTTAATTTTTTGT-YGTYGTTATTAATG-3' (BUB1-F) and 5'-CCCCACCAAACRAACACTTAC-3' (BUB1-R); 5'-GTTTTAGAATGTTTTGGGYGAGA-3' (BUB1B-F) and 5'-CRCAACCTACCCTCRAAACTAC-3' (BUB1B-R); 5'-TTTTTTTTGAGTYGGAATAGGATGAT-3' (BUB3-F) and 5'-CCCTCAAAACACTCRCAAACTAAAC-3' (BUB3-R); 5'-GGAGAGTTGTAGYGTTATGGTTAGG -3' (MAD2L1-F) and 5'-AACAAATCCCCRCTACATAAAACTA-3' (MAD2L1-R); 5'-YGGTGTTTYGGTGGGTTTTAG-3' (MAD2L2-F) and 5'-CRACRAATCTCAACCCCTCC-3' (MAD2L2-R); 5'-GTTGAATTGGTTTTAGGAAAATGGT-3' (CENPE-F) and 5'-CCCCTCTCCTATTTAACAATAATCAC (CENPE-R); and 5'-AAAGGAGTYGGYGGAGGATG-3' (EB1-F) and 5'-ACTCACCRACTCTCRACAAAACC-3' (EB1-R). The PCR products were digested with a restriction endonuclease as follows: CHFR, NruI; BUB1, BstUI; BUB1B, AfaI; BUB3, MboI; EB1, HinfI; CENP-E, TaiI; MAD2L1, AfaI; and MAD2L2, AfaI. All of the restriction enzymes were purchased from TaKaRa except BstUI, which was from New England Biolabs. The resultant DNA fragments were subjected to 3% agarose gel electrophoresis and stained with ethidium bromide. The primer sequences, PCR conditions, and restriction enzymes used to examine other mitotic checkpoint genes are available upon request.

Bisulfite Sequence Analysis.
To sequence bisulfite-PCR products, fragments amplified using primers CHFRGM1-F and CHFRGM2-R (5'-TCCACCCTACCCACAAACAAC-3') were cloned into pCR4-TOPO vector using a TOPO-cloning kit (Invitrogen). The cycle sequencing reaction was then carried out using a BigDye terminator kit (Applied Biosystem), and the DNA was sequenced using an ABI 3100 automated sequencer (Applied Biosystem).

Chromatin Immunoprecipitation (ChIP) Analysis.
ChIP analysis was performed as described previously (35) . Briefly, cells were harvested, and their proteins cross-linked to DNA by incubation in 1.0% formaldehyde for 10 min at 37°C. The formaldehyde-fixed cells were allowed to settle on ice for 10 min and then spun down by brief centrifugation, after which the supernatant was carefully aspirated. The cells were then washed with ice-cold PBS containing protease inhibitors and resuspended in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), and protease inhibitor]. The nucleoprotein complexes were sonicated to reduce the sizes of the DNA fragments to 200-1000 bp and immunoprecipitated for 16 h at 4°C with rotation using anti-acetylated histone H3 or anti-acetylated histone H4 antibody (Upstate Biotechnologies, Lake Placid, NY). The resultant immune complexes were collected using protein A-agarose beads, after which the DNA was purified by phenol-chloroform extraction, precipitated with ethanol, and resuspended in distilled water. About 1:100 of the precipitated DNA was used for PCR, and 1:100 of the solution before adding antibody was used as an internal control for the quantitative accuracy of the DNA. PCR was performed in a solution containing 1x PCR buffer (TaKaRa), 1 µM primers, 0.25 mM deoxynucleoside triphosphate mixture, 10% DMSO, and 1.0 unit of Hot Start Taq polymerase (TaKaRa). The primer sequences used for the PCR reactions were 5'-CTCACCAAGAGCGGCAGCTAAAG-3' (CHFRCHIP-F) and 5'-ATTAGCGGGGCTCTCAGAATCCT-3' (CHFRCHIP-R). The amplified products were subjected to agarose gel electrophoresis, and the intensity of resultant bands was calculated using a Lane and Spot Analyzer (Atto, Tokyo, Japan).

Adenoviral Gene Transfer of CHFR.
Recombinant adenoviral vectors expressing CHFR (Ad-CHFR, amino acids 1–664), CHFR lacking the FHA domain (Ad-{Delta}FHA, amino acids 129–664) or bacterial lacZ (Ad-lacZ) were constructed using the replication-deficient E1-deleted Ad5 vector pJM17 (Microbix Biosystem, Toronto, Ontario, Canada). Expression of the corresponding genes was under the control of a human cytomegalovirus promoter/enhancer and a bovine growth hormone polyadenylation signal. To determine the relative efficiency of adenovirus-mediated gene transfer, cells were also infected with Ad-lacZ. For these experiments, the HSC44 human gastric cancer cell line was used because it showed relatively high efficiency for gene transfer.

Mitotic Index.
Approximately 5 x 104 cells were incubated overnight in 60-mm plates (37°C; 5% CO2), after which 1 µM docetaxel (Aventis Pharmaceuticals, Bridgewater, NJ) in culture medium was applied, and the cells were incubated for an additional 16 h. Thereafter, the cells were harvested with trypsin, incubated for 30 min at room temperature in 75 mM KCl, and spun down. After discarding the supernatant, the cells were resuspended in 5 ml of Carnoy’s fixative (3 parts methanol and 1 part glacial acetic acid) for 10 min at room temperature and spun down once again. The supernatant was then discarded, and the cells were resuspended in 100 µl of fixative, after which 10-µl aliquots of the cell suspension were dropped from a height of 10 cm onto glass slides and allowed to dry. After staining with propidium iodide, and washing with calcium- and magnesium-free PBS, a coverslip was placed above the cells, and the edges were sealed with clear nail polish. A confocal microscope (Zeiss) was then used to count at least 300 mitotic cells.

Immunofluorescent Staining.
To immunofluorescently stain cells for cyclin B1, cells fixed in 3.7% formalin solution for 10 min at 25°C were washed with PBS and then incubated overnight with rabbit anti-human cyclin B1 (Santa Cruz Biotechnology Inc.). The nuclei were stained with Hoechst33258 (WAKO).

Flow Cytometry Analysis.
Cells were treated with 1 µM docetaxel, 1 µM paclitaxel, or 50 µM VP16 for 16 h, or irradiated with UV at intensity of 8000 µJ/cm3. They were then harvested, fixed in 70% ethanol, incubated with 2 mg/ml RNase, and stained in 50 µg/ml of propidium iodide solution. Approximately 5 x 104 stained cells were analyzed using a Becton Dickinson FACScan flow cytometer (Braintree, MA).

Colony Formation Assays.
Cells (1 x 105) treated with 1 µM docetaxel for 24 h were washed with PBS, plated in drug-free medium, and incubated for 14 days. The resultant colonies were stained with crystal violet and counted using NIH Image software.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression and Mutation Analysis of CHFR.
To examine CHFR expression in gastric cancer cells, reverse transcription-PCR was carried out using cDNA prepared from various samples of normal stomach mucosa and from 20 gastric cancer cell lines (Fig. 1A)Citation . CHFR was expressed at readily detectable levels in the normal stomach mucosa and in 16 of the gastric cancer cell lines. Four cell lines (MKN74, KatoIII, HSC44, and HSC45) showed no expression at all, however. To test whether this silencing of CHFR expression was the result of gene mutation, we sequenced the entire coding region of the gene. Blast analysis of CHFR cDNA revealed the gene to contain 18 exons spanning ~50 kb on chromosome 12 (Fig. 1B)Citation . So using 17 sets of primers that respectively amplified exons 2–18, we carried out direct sequence analysis in all 20 of the cell lines, and discovered four base substitutions that were determined to be polymorphisms (Fig. 1C)Citation . Thus, inactivation of CHFR was caused by a mechanism other than mutation.



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  		Fig. 1. Expression and mutation analysis of CHFR in gastric cancer. A, analysis of CHFR expression in gastric cancer cell lines and normal stomach mucosa using RT-PCR. Controls consisted of carrying out PCR reactions in the absence of reverse transcription (RT-) and amplification of GAPDH to assess the quality of the cDNA. Cell lines are shown on the top. B, organization of the CHFR gene. Exons are indicated by vertical bars; arrows point to the start and stop codons. C, representative results of mutational analyses performed using 17 sets of primers that together span the entire coding region. In total, four base substitutions were found; arrows indicate their positions.

 
Aberrant CHFR Methylation.
CHFR has a typical CpG island in its 5' region (Fig. 2A)Citation , suggesting a role for DNA methylation in its silencing. Therefore, the methylation status of the CHFR 5' CpG island was examined in a panel of gastric cancer cell lines and primary tumors using COBRA, a semiquantitative methylation analysis, and aberrant methylation of CHFR was detected in all four of the gastric cancer cell lines in which the gene was silent (Fig. 2B)Citation . We next examined the methylation status of seven other mitotic checkpoint genes (BUB1, BUB1B, BUB3, MAD2L1, MAD2L2, CENP-E, and EB1) selected based on the presence of CpG islands in their 5' regions. COBRA was carried out using primers that covered the region around the transcription start site of each gene, but no methylation was detected in any of the genes tested in any of the cell lines (Fig. 2C)Citation . To evaluate the extent of the CHFR methylation, bisulfite sequencing was carried out in two methylated (KatoIII and HSC45) and two unmethylated (HSC43 and MKN28) cell lines (Fig. 2D)Citation . The CHFR CpG islands of the former were found to be densely methylated over the entire region analyzed, whereas those of the latter showed very little methylation.



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  		Fig. 2. Analysis of mitotic checkpoint gene methylation in gastric cancer. A, the CpG island of CHFR: CpG sites are indicated by vertical bars; exon 1 is shown as {blacksquare}; the arrow indicates the transcription start site. The primers used for COBRA analysis are shown by the arrowhead. The region analyzed by bisulfite-sequencing is indicated with a bar. B, bisulfite-PCR analysis of the CHFR CpG islands in a panel of gastric cancer cell lines. The extent of the methylation was determined by densitometry and is shown as percentages below the gels; M, methylated alleles. C, representative results of a COBRA of four mitotic checkpoint genes in a panel of gastric cancer cell lines. The methylation status of the indicated genes was examined by bisulphite-PCR using appropriate primers. As a positive control, DNA incubated with SssI-methylase (SssI-Mtase) was also analyzed. D, bisulfite-sequence analysis of the 5' prime region of CHFR. The PCR products were cloned into pCR4-TOPO, and at least 10 clones from each cell line were sequenced. Open and closed areas represent unmethylated and methylated CpG dinucleotides, respectively. Cell lines are shown below. The CpG sites in the region analyzed are indicated by vertical bars (top).

 
To examine the role of CHFR methylation in primary gastric cancers, we performed bisulfite-PCR analysis in a series of primary gastric cancer specimens. Aberrant methylation of CHFR was detected in 24 of 61 (39%) primary gastric cancers (Fig. 3A)Citation . No methylation was detected in samples of stomach tissue harvested from areas adjacent to the tumors, nor was it detected in gastric mucosa showing intestinal metaplasia, indicating this epigenetic change to be a cancer-specific event (data not shown). Bisulfite sequencing of the region around the transcription start site revealed that in the specimens shown by COBRA to be methylated, all of the CpG sites analyzed were densely methylated. Because the primary tumors we analyzed were not microdissected, unmethylated alleles derived from contaminating normal gastric mucosa were also present. Therefore, we used gastric cancer xenografts, which are relatively pure, to additionally examine the role of CHFR methylation in gene silencing in tumor tissues (Fig. 3, A–C)Citation . Four xenografts that showed no methylation of CHFR expressed its mRNA to various degrees. By contrast, CHFR was barely detectable in three xenografts that showed dense methylation. Thus, methylation-dependent silencing of CHFR is not a cell line-specific event.



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  		Fig. 3. Epigenetic inactivation of CHFR in primary gastric cancers and xenografts. A, bisulfite-PCR analysis of CHFR in primary gastric cancer and xenografts: N, normal stomach; T, gastric cancers. B, representative results of bisulfite-sequencing of CHFR in primary gastric cancer and xenografts. Specimens are shown on the left. Cases 114 and 116 showed completely unmethylated alleles derived from normal gastric mucosa. C, analysis of CHFR expression using real-time PCR. cDNA was prepared from gastric cancer xenografts, and PCR was performed using an ABI 7000. The bars show levels of CHFR expression normalized to that of GAPDH.

 
Histone Acetylation Status in Gastric Cancer Cell Lines Not Expressing CHFR.
To confirm the role of DNA methylation in silencing CHFR expression and to evaluate the role of the histone acetylation state, we tested whether interference with the activities of DNA methyltransferases and/or histone deacetylases might lead to reactivation of the silent genes. Two cell lines not expressing CHFR (HSC44 and HSC45) were treated with 5-aza-dC, a methyltransferase inhibitor and/or TSA, a histone deacetylase inhibitor (Fig. 4A)Citation . Expression of CHFR was partially restored by treating cells with 5-aza-dC, and TSA synergistically enhanced the effect of 5-aza-dC (Fig. 4A)Citation . By contrast, treatment with three concentrations (30 nM, 300 nM, and 3 mM) of TSA alone had no affect on gene expression (Fig. 4ACitation ; data not shown). Quantitative analysis using real-time PCR revealed that the level of CHFR mRNA expression in methylated cell lines treated with 5-aza-dC plus TSA is comparable with that in cell lines with no methylation (Fig. 4B)Citation .



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  		Fig. 4. Role of histone deacetylation in the methylation-dependent silencing of CHFR. A, effects of inhibiting methyltransferase and/or histone deacetylase on the expression of CHFR. The indicated cell lines (below) were treated with 0.2 µM 5-aza-dC and/or 300 nM TSA. B, quantitative reverse transcription-PCR analysis of CHFR in gastric cancer cell lines: 1, HSC44 Mock; 2, HSC44 cells with 300 nM TSA for 24 h; 3, with 0.2 µM 5-aza-dC for 72 h; 4, with 0.2 µM 5-aza-dC for 72 h and 300 nM TSA for 24 h; 5, with 2.0 µM 5-aza-dC for 72 h; 6, HSC45 mock; 7, HSC45 cells with TSA for 24 h; 8, with 0.2 µM 5-aza-dC for 72 h; 9, with 0.2 µM 5-aza-dC for 72 h and TSA for 24 h; 10, with 2.0 µM 5-aza-dC for 72 h; 11, SNU638; 12, NUGC3; 13, MKN28; 14, MKN45; 15, AZ521; 16, MKN7. C, acetylation status of histones H3 and H4 in a panel of gastric cancer cell lines. ChIP assays were carried out using antiacetylated histone H3 or H4 antibodies, after which ChIP-PCR analysis of CHFR was done. Band intensities were normalized to those of amplified DNA fragments from the 5' region of GAPDH. D, quantitative analysis of histone acetylation. The bars indicate the levels or H3/CHFR and H4/CHFR acetylation normalized to those of H3/GAPDH and H4/GAPDH. All values were determined by densitometry.

 
ChIP assays using antibodies against acetylated histone H3 and H4 were then carried out to determine whether methylation-dependent silencing of CHFR expression was associated with altered chromatin structure, namely hypoacetylation of histone (Fig. 4, C and D)Citation . Overall, the degree of acetylation of both histones correlated directly with gene expression and inversely with DNA methylation in the region around the transcription start site. Although the level of histone acetylation was increased by treatment with 5-aza-dC or TSA, expression of CHFR was restored only when cells were treated with 5-aza-dC (Fig. 4, A–D)Citation .

Mitotic Checkpoint Dysfunction Associated with Aberrant Methylation of CHFR.
The functional consequences of CHFR methylation in gastric cancer were evaluated by examining the mitotic index after treatment with a microtubule inhibitor (Fig. 5A)Citation .



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  		Fig. 5. Impaired mitotic checkpoint function in gastric cancer cell lines with CHFR methylation. A, representative sample of mitotic cells after treatment with microtubule inhibitor docetaxel. Cell lines were treated with docetaxel for 16 h and then stained with propidium iodide. B, correlation between CHFR expression and mitotic index. Cells were treated with docetaxel for 16 h. The level of CHFR mRNA expression normalized to that of GAPDH is shown on the Y-axis. The mitotic indexes of seven unmethylated cell lines and three methylated cell lines (HSC44, HSC45 and KatoIII), with or without 5-aza-dC pretreatment, are shown on the X-axis. C, expression of CHFR in HSC44 cells at the indicated times after infection with an adenoviral vector harboring CHFR. D, effect of adenovirus-mediated transfer of CHFR on the mitotic index. HSC44 cells were infected with Ad-LacZ, Ad-CHFR, or Ad-{Delta}FHACHFR at a multiplicity of infection of 50–200. Thirty-six h after infection, cells were treated with mock or 1 µM docetaxel for 16 h, and the mitotic index was determined. Introduction of CHFR reduced the mitotic index in HSC44 cells; {Delta}FHA-CHFR had a lesser effect.

 
After treatment with docetaxel, HSC44, HSC45, and KatoIII cells, which do not express CHFR, showed condensed chromosomes and high mitotic indexes (Fig. 5B)Citation , indicating impairment of the checkpoint regulating entry into mitosis. By contrast, the mitotic indexes of unmethylated cell lines were comparatively low; indeed, there was an inverse correlation between the level of CHFR expression and the mitotic index (Fig. 5B)Citation . The mitotic indexes of HSC44 and HSC45 cells were dramatically reduced when the cells were treated with 5-aza-dC, indicating that restoration of CHFR expression also restored the functionality of the checkpoint (86% to 2% for HSC44 and 80% to 10% for HSC45).

To additionally investigate the role of CHFR at the mitotic checkpoint, intact CHFR or {Delta}FHA-CHFR, which lacks the NH2 terminal of the gene (including the FHA domain), was introduced into HSC44 cells using an adenoviral vector (Fig. 5C)Citation . Introduction of CHFR dose-dependently reduced the mitotic index (Fig. 5D)Citation ; so did introduction of {Delta}FHA-CHFR, but to a lesser degree, indicating a role for the FHA domain in the checkpoint (Fig. 5D)Citation .

Cyclin B1 reportedly accumulates in the cytoplasm during the period between S phase and G2 phase, and then localizes in the nucleus during prophase (36) . The response of the CHFR checkpoint to microtubule poisoning was additionally characterized, therefore, by examining the intracellular distribution of cyclin B1. Consistent with earlier reports, cyclin B1 did not accumulate in most cells unless they were subjected to microtubule inhibition with docetaxel (Fig. 6)Citation . However, after treating CHFR-expressing MKN28 and SNU1 cells with docetaxel, cyclin B1 was localized mainly in the cytoplasm and did not enter nucleus because of the prophase checkpoint (Fig. 6)Citation . On the other hand, docetaxel induced both cytoplasmic accumulation and nuclear translocation of cyclin B1 in HSC44 and HSC45 cells, in which CHFR is silent (Fig. 6)Citation .



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  		Fig. 6. Distribution of cyclin B1 in gastric cancer cell lines with or without CHFR methylation. Gastric cancer cell lines were incubated for 16 h with mock or 1 µM docetaxel, fixed, and then incubated with anticyclin B antibody. Nuclei were stained with Hoechst33258. CHFR is unmethylated in MKN28 and SNU1 cells, but methylated in HSC44 and HSC45 cells.

 
Differential Sensitivity of Gastric Cancer Cells to Microtubule Inhibitors.
Cell cycle analyses were then carried out after treatment with docetaxel to investigate the role of CHFR in checkpoint function during mitotic stress. Forty-eight h after treatment with docetaxel, gastric cancer cell lines having a normal prophase checkpoint (MKN7 and AZ521) arrested at G2-M and were resistant to apoptosis (Fig. 7A)Citation . Cell lines that showed CHFR methylation (HSC44 and HSC45), by contrast, underwent apoptosis after being treated with docetaxel (Fig. 7A)Citation , and similar results were obtained when cells were treated with another microtubule inhibitor, paclitaxel. Statistical analyses showed that when treated with docetaxel or paclitaxel, the incidence of apoptosis was significantly (P < 0.001 and P < 0.005) lower among cells expressing CHFR than among those in which the gene is silent. In addition, introduction of CHFR using an adenoviral vector restored the cell cycle checkpoint, which led to a decrease in the number of apoptotic cells after treatment with docetaxel or paclitaxel (Fig. 7, A and B)Citation . There was no correlation between CHFR methylation and sensitivity to another chemotherapeutic agent (VP16) or to UV (P = 0.644 and P = 0.156), and introduction of CHFR failed to decrease sensitivity to these drugs (Fig. 7B)Citation . These results suggest that methylation of CHFR correlates specifically with sensitivity to microtubule inhibitors.



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  		Fig. 7. Differential susceptibility of gastric cancer cells to microtubule inhibitors. A, flow cytometric analysis of methylated (HSC44 and HSC45) and unmethylated (MKN7 and SNU1) cell lines treated with mock or docetaxel. HSC44 cells infected with Ad-CHFR were also examined. Percentages of sub-G1 cells are shown at the top. B, effects of docetaxel, paclitaxel, VP16, and UV on the incidence of apoptosis in a panel of methylated and unmethylated gastric cancer cell lines. The bars indicate the percentages of apoptotic cells.

 
That conclusion was confirmed by subsequent clonogenic assays, which provide a more stringent test of chemosensitivity. Fourteen days after exposure to docetaxel for 24 h, the incidence of cell death was markedly higher among the methylated cell lines than among the unmethylated ones (Fig. 8, A and B)Citation .



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  		Fig. 8. Loss of CHFR expression is associated with decreased colony formation after treatment with a microtubule inhibitor. A, assays of colony formation by gastric cancer cell lines with or without CHFR methylation. Cells were treated with 1 µM docetaxel for 24 h, plated, and stained after 14 days. B, quantitative analysis of colony formation. Bars indicate the number of colonies counted.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although mutation of mitotic checkpoint genes is rare in gastric cancers (37 , 38) , the present study indicates that epigenetic inactivation of the checkpoint gene CHFR occurs frequently in gastric cancers. Moreover, this effect appears to be unique to CHFR, because other mitotic checkpoint genes (BUB1, BUB1B, BUB3, MAD2L1, MAD2L2, CENP-E, and EB1) are not inactivated, although they contain CpG islands in their 5' regions. Several lines of evidence suggest that gene methylation occurs during aging (39) . However, the fact that methylation of the region around the transcription start site of CHFR was not detected in the gastric mucosa adjacent to tumors indicates that this event is not age-related but is a cancer-specific phenomenon. That treatment with a methyltransferase inhibitor, 5-aza-dC, restored expression of CHFR indicates the transcriptional silence was caused by the DNA methylation and not by the absence of a transcription factor or by gene mutation. Finally, our ChIP analysis showed that histone deacetylation also plays a role in the methylation-dependent silencing of CHFR, which is consistent with recent reports showing histone deacetylation to be involved in the methylation-dependent silencing of various genes (10 , 40, 41, 42) . The role of DNA methylation in gene silencing appears dominant over histone acetylation, because treating methylated cell lines with various concentrations of TSA failed to restore gene expression. Re-expression of CHFR was only observed when DNA methylation was inhibited. Clarification of the role of histone modification in the silencing of CHFR awaits further study.

CHFR was initially cloned as a human homologue of Dma1, which functions in a mitotic checkpoint in yeast, delaying entry into M phase when there is mitotic stress (43) . In fact, whereas gastric cancer cell lines expressing CHFR did not enter mitosis when treated with a microtubule inhibitor, cell lines not expressing CHFR did so and, consequently, showed high mitotic indexes. Re-expression of CHFR induced by treatment with 5-aza-dC or introduction of CHFR by adenoviral gene transfer reduced the number of mitotic cells significantly, strongly suggesting that CHFR inactivation impairs the function of the mitotic checkpoint in gastric cancer.

The molecular mechanism by which CHFR regulates the mitotic checkpoint remains unclear, although several lines of evidence suggest that maintenance of the cytoplasmic localization of cyclin B1 during mitotic stress is a crucial component. Notably, introduction of {Delta}FHA-CHFR, which lacks the FHA domain known to interact with phosphorylated proteins (25 , 29) , had less effect on the mitotic checkpoint than intact CHFR. In that regard, phosphorylation of serine residues in the cyclin B1 nuclear localization signal is reportedly necessary for transport of cyclin B1 to the nucleus (44) . That this phosphorylation is regulated by PLK1, which is a target for CHFR-dependent ubiquitination (23) , suggests that CHFR may regulate the localization of cyclin B1 during mitotic stress through degradation of PLK1. It remains unclear whether the FHA domain of CHFR is necessary for recognition of PLK1 as a substrate for ubiquitination, and further study will be necessary to determine whether there are any other proteins that associate with CHFR via its FHA domain. In addition, the fact that introduction of {Delta}FHA-CHFR reduced mitotic entry, albeit to a lesser degree than full-length CHFR, suggests that domains other than FHA also play roles in the function of the checkpoint.

Cell cycle checkpoint dysfunction is often associated with sensitivity to chemotherapeutic agents (11 , 32) . In the present study, microtubule inhibitors (docetaxel and paclitaxel) induced apoptosis in gastric cancers with CHFR methylation; this is also seen in a subset of colorectal cancer cells (19 , 22) and may indicate that CHFR methylation is a good molecular marker of tumors that will respond to microtubule inhibitors, and is, thus, of significant clinical importance. In fact, aberrant methylation of checkpoint and DNA repair genes has been shown previously to be indicative of sensitivity to other chemotherapeutic agents, e.g., O6-methylguanine-DNA methyltransferase (MGMT) methylation indicates sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (45) , 14–3-3{varsigma} methylation indicates sensitivity to adriamycin (32) , and FANCF methylation indicates sensitivity to cisplatin (46) . In addition, cells that express CHFR could be sensitized to microtubule inhibitors by impairing CHFR function using a gene knockdown technique such as RNA interference. Although the relationship between CHFR expression and mitotic index in primary tumors remains unknown, it is plausible that within primary gastric tumors, cells with CHFR methylation would be especially sensitive to microtubule inhibitors. This means it may be possible to select patients with indications for microtubule inhibitors based on their CHFR methylation status. Further study will be necessary to clarify the extent to which CHFR methylation status correlates with the response to microtubule inhibitors in gastric cancer patients.

In summary, our data show that CHFR gene expression is frequently silenced by DNA methylation in gastric cancer. Gastric cancer cells not expressing CHFR lack a mitotic checkpoint and are highly susceptible to microtubule inhibitors. This suggests that DNA methylation of CHFR may be a useful molecular marker with which to predict the responsiveness of gastric cancers to treatment with microtubule inhibitors.


   ACKNOWLEDGMENTS
 
We thank Dr. William F. Goldman for editing the manuscript.


   FOOTNOTES
 
Grant support: Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (M. T., F. I., T. T. and K. I.). M. T. is a researcher supported by The Akiyama Foundation. A. S. is a research fellow from the Japanese Society for the Promotion of Science.

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.

Requests for reprints: Minoru Toyota, First Department of Internal Medicine, Sapporo Medical University, S-1, W-16, Chuo-ku, Sapporo, 060-8543, Japan. Phone: 81-11-611-2111, extension 3211; Fax: 81-11-611-2282; E-mail: mtoyota{at}sapmed.ac.jp

Received 1/21/03. Revised 9/18/03. Accepted 10/15/03.


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 RESULTS
 DISCUSSION
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