Abstract. Background: Aberrant methylation of the CHFR
gene associated with gene silencing has been reported in several
primary tumors. In order to define the role of CHFR in the
tumorigenic pathway of the colorectum, the methylation of
CHFR was examined in tumors from colorectal cancer patients.
Materials and Methods: Ninety-eight colorectal cancer patients
were examined using a methylation-specific PCR (MSP) for
CHFR CpG island in primary tumors. Results: An aberrant
methylation of the CHFR gene was detected in 25 out of 98
(26%) primary colorectal cancers. No methylation was detected
in the corresponding normal tissue specimens. This finding
suggested that an aberrant methylation of the CHFR gene
occurs frequently in colorectal cancers. After a methylation
analysis of all samples, the clinicopathological data were
correlated with these results. A significant difference was found
in the tumor (p=0.035), thus, indicating that in early colorectal
cancer the CHFR gene was more frequently methylated than in
advanced cases. Conclusion: These findings suggest that CHFR
might act as a tumor suppressor in at least some colorectal
cancers and that CHFR methylation might, therefore, be a
particular phenomenon of early colorectal cancer.
A series of genetic alterations in both dominant oncogenes
and tumor suppressor genes are involved in the pathogenesis
of human colorectal cancer. The activation of oncogenes,
such as the ras gene, and the inactivation of tumor suppressor
genes, such as the APC and p53 genes, have been identified in
colorectal cancer (1-3). In addition, several other genes have
also been found to be related to the pathogenesis of
colorectal cancer (4, 5). An investigation of the genetic
changes is, therefore, important in clarifying the tumorigenic
pathway of colorectal cancer (6).
In recent years, the CHFR (checkpoint with FHA and
RING finger) gene has been reported as a new mitotic
checkpoint gene. CHFR protein delayed chromosome
condensation and entry into metaphase in response to the
mitotic stress induced by a microtubule inhibitor (7).
However, cancer cell lines lacking CHFR entered into
metaphase without any delay after treatment with a
microtubular inhibitor, thus, suggesting the function of CHFR
as a mitotic checkpoint. Recently, the aberrant methylation
of the CHFR gene associated with gene silencing has been
reported in several primary tumors (8-12). These results
prompted us to examine the methylation status of the CHFR
gene in surgically removed colorectal cancers.
In the present study, the methylation status and gene
expression of CHFR in several cancer cell lines were first
examined using methylation-specific PCR (MSP) and
reverse transcription-PCR (RT-PCR), respectively. The
methylation status of the CHFR gene in primary tumors and
corresponding normal tissues derived from 98 patients with
colorectal cancer was then examined and the correlation
between the methylation status and the clinicopathological
findings was evaluated.
Materials and Methods
Sample collection and DNA preparation. An esophageal cancer cell
line (NUEC3) and a gastric cancer cell line (NUGC3) were
established in our laboratory. Three colorectal cancer cell lines
(DLD1, SW480 and SW1116) were obtained from the American
Type Culture Collection (Manassas, VA, USA). They were grown
in RPMI 1640 supplemented with 10% fetal bovine serum and
incubated in 5% CO2 at 37ÆC.
Ninety-eight primary tumor and corresponding normal tissue
specimens were collected consecutively at Nagoya University
Hospital, Japan, from colorectal cancer patients during colorectal
surgery. All tissue specimens were confirmed histologically. Written
informed consent, as required by the Institutional Review Board,
was obtained from all patients. Collected samples were stored
immediately at –80ÆC until analysis. DNA was prepared as
Abbreviations: MSP, methylation-specific PCR; RT-PCR, reverse
Correspondence to: Kenji Hibi, Gastroenterological Surgery,
Nagoya University Graduate School of Medicine, 65 Tsurumai-
cho, Showa-ku, Nagoya 466-8560, Japan. Tel: +81527442245, Fax:
+81527442255, e-mail: firstname.lastname@example.org
Key Words: CHFR, methylation-specific PCR, colorectal cancer.
ANTICANCER RESEARCH 26: 4267-4270 (2006)
Aberrant Methylation of the CHFR Gene is Frequently
Detected in Non-invasive Colorectal Cancer
YUKI MORIOKA, KENJI HIBI, MITSURU SAKAI, MASAHIKO KOIKE, MICHITAKA FUJIWARA,
YASUHIRO KODERA, KATSUKI ITO and AKIMASA NAKAO
Gastroenterological Surgery, Nagoya University Graduate School of Medicine, Showa-ku, Nagoya 466-8560, Japan
described elsewhere (13). The clinicopathological characteristics of
the patients enrolled in the study are shown in Table I.
Sodium bisulfite modification. One Ìg of the genomic DNA
extracted from the tumor and corresponding normal colorectal
tissue specimens was subjected to bisulfite treatment, as described
previously (14). Briefly, alkali-denatured DNA was modified by 2.1
M sodium bisulfite / 0.5 mM hydroquinone at pH 5.0. The bisulfite-
reacted DNA was then treated by NaOH, purified using the
Wizard DNA Clean-Up System (Promega, Madison, WI, USA),
precipitated with ethanol and resuspended in distilled water.
MSP. The bisulfite-treated DNA was amplified with MSP. The
primer sequences of CHFR for the unmethylated reaction were:
CHFR UMS (sense), 5’-GTTTTAATATAATATGGTGTTGATT-
3’, and CHFR UMAS (antisense), 5’-AAAACAAC AACTAA
AACAAAACCA-3’, which amplify a 144-base pair product. Primer
sequences of CHFR for the methylated reaction were: CHFR MS
(sense), 5’-TTTTAATATAATATGGC GTCGATC-3’, and CHFR
MAS (antisense), 5’-AACGACAA CTAAAACGAAACCG-3’,
which amplify a 141-base pair product. The PCR amplification of
modified DNA samples consisted of 1 cycle at 95ÆC for 5 min, 1
cycle at 78ÆC for 10 min, 37 cycles of denaturing at 95ÆC for 30 sec,
1 min of annealing at 57ÆC, 1 min of extension at 72ÆC and a final
extension step of 10 min at 72ÆC. Modified DNAs obtained from
NUEC3 and DLD1 were used as positive controls for unmethylated
and methylated alleles, respectively. The controls without DNA were
included in each assay. Ten Ìl of each PCR product was loaded
directly onto non-denaturing 10% polyacrylamide gel, stained with
ethidium bromide, and were then visualized under UV illumination.
Each MSP was repeated at least twice.
RT-PCR. The expression of the CHFR gene was analyzed using
RT-PCR. Total RNA was extracted from various cancer cell lines
with ISOGEN®(Nippon Gene, Tokyo, Japan) following the
manufacturer’s instructions. First strand cDNA was generated from
RNA, as described elsewhere (15). cDNA was then amplified using
a primer set that was specific for the CHFR gene. The primer
sequences were: CHFR S (sense), 5’-GGCGAGAGCGTTCCT
CCAGTTG-3’, and CHFR AS (antisense), 5’-GCATGTCAGCG
TCTCCTCCATCTTG-3’. The PCR amplification consisted of 1
cycle at 94ÆC for 2 min, 30 cycles at 94ÆC for 30 sec, 55ÆC for 30
sec and 72ÆC for 30 sec, and 1 cycle at 72ÆC for 5 min. The
expression of ‚-actin was used as a control to confirm the success
of the RT reaction. The PCR products were visualized on 1.3%
agarose gel stained with ethidium bromide.
Statistical analysis. The associations between CHFR promoter
methylation and clinicopathological parameters were analyzed
using Fisher’s exact tests or Student’s t-tests. A p-value <0.05
indicated statistical significance.
We first examined the methylation status of CHFR promoter
in 1 esophageal, 1 gastric and 3 colorectal cancer cell lines
using the MSP technique (Figure 1). The aberrant
ANTICANCER RESEARCH 26: 4267-4270 (2006)
Table I. Clinicopathological features and CHFR promoter methylation in
Maximal size (cm) 985.1±1.43
Tumor extent <mt4
II, III, IV
2TNM stage 0.1211
1Fisher’s exact test; 2Student’s t-test; 3mean±S.D; 4mt, muscular tunic.
Figure 1. Methylation-specific PCR analysis for the methylation status of
CHFR promoter in human cancer cell lines. The presence of a visible PCR
product in lane U indicates the existence of unmethylated genes; the
presence of a product in lane M indicates the existence of methylated
genes. The aberrant methylation of CHFR was detected in 1 colorectal
cancer cell line (DLD1).
Figure 2. An analysis of the CHFR expression by RT-PCR. DLD1, which
showed a methylated allele in an MSP analysis, lacked any CHFR
expression, whereas CHFR was expressed in all other cell lines with non-
methylated CHFR promoter. The expression of ‚-actin was used as a
control to confirm the success of the RT reaction.
methylation of CHFR was detected in 1 colorectal cancer cell
line (DLD1). In order to confirm the status of the CHFR
gene according to its methylation pattern, CHFR expression
in these cell lines was examined using RT-PCR (Figure 2).
As expected, DLD1 which was the only cell line to
demonstrate methylation of the CHFR promoter, lacked
CHFR expression, whereas CHFR was expressed in all other
cell lines without methylation of the CHFR promoter.
The methylation status of the CHFR promoter was then
examined in primary colorectal cancer samples (Figure 3).
An aberrant methylation of the CHFR gene was detected in
25 out of 98 (26%) primary colon cancers. As a control, the
corresponding normal tissues of these same patients was
screened for CHFR methylation, and no methylation was
detected. Our results suggested that the aberrant
methylation of the CHFR gene was frequently observed in
After a methylation analysis of all samples, the
clinicopathological data were correlated with these results.
No significant correlations were observed between the
presentations of abnormal methylation in colorectal cancers
and patient gender, age or tumor maximal size, TNM stage
and lymph node metastasis (Table I). A significant
difference was observed in the tumor (p=0.035), indicating
that early colorectal cancers were more frequently
methylated than advanced ones.
Colorectal cancer is one of the most aggressive cancers and
occurs at a high incidence in most countries (16). In order
to remove this fatal cancer from patients, surgical
operations and subsequent chemotherapy and radiotherapy
are performed. For this purpose, it is important to identify
the occurrence of genetic alterations as a new parameter for
the estimation of the malignancy of the cancer.
CHFR, a mitotic checkpoint gene, was recently cloned
and localized to chromosome 12q24. It has been reported
that the CHFR protein delays chromosome condensation
and entry into metaphase in response to the mitotic stress
induced by microtubule inhibitors, such as nucodasole or
taxol (7). The CHFR protein contains three separate
domains, a forkhead-associated (FHA), a ring finger (RF)
and a cystein-rich (CR) domain. The FHA domain is
conserved in several checkpoint genes, including CHK2,
RAD53 and MDC1 (17-21). Based on a mutagenesis
analysis, both the FHA and CR domains are required for
CHFR’s checkpoint function (7). The RF domain is also
critical for the mitotic checkpoint activity, while also plays
a role in the ubiquitination of substrates, such as polo-like
kinase and CHFR itself (22).
In the present study, the frequent methylation of CHFR
in colorectal cancer was observed, whereas the same
methylation was not detected in corresponding normal
tissue specimens. The methylation status of CHFR in
colorectal cancer patients was also compared with their
clinicopathological features and demonstrated that CHFR
in early colorectal cancer patients was more frequently
methylated than in advanced cases. Therefore, CHFR
methylation could be used as a tumor marker in clinical
samples, such as serum and stool, for the early detection of
digestive tract cancers (23, 24).
Our findings suggest that CHFR may play a role in the
carcinogenic pathway in some patients with colorectal
cancers, and that tumor formation in the colorectum may
be controlled by inducing the expression of silenced CHFR
using demethylating reagents. This study provides solid
evidence that can be used in further studies on the
molecular mechanism of CHFR in colorectal cancers.
1Bos JL, Fearon ER, Hamilton SR, Verlaan-de Vries M, van
Boom JH, van der Eb AJ and Vogelstein B: Prevalence of ras
gene mutations in human colorectal cancers. Nature 327: 293-
Morioka et al: CHFR Methylation in Colon Cancer
Figure 3. The representative MSP of CHFR promoter in primary colorectal cancers. Modified DNAs obtained from DLD1 and NUEC3 were used as
positive controls for the methylated and unmethylated alleles, respectively. Cases 80 and 81 exhibited methylation.
2 Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A, Download full-text
Koyama K, Utsunomiya J, Baba S, Hedge P, Markham A,
Krush AJ, Petersen G, Hamilton SR, Nilbert MC, Levy DB,
Bryan TM, Preesinger AC, Smith KJ, Su LK, Kinzler KW and
Vogelsteen B: Mutations of chromosome 5q21 genes in FAP
and colorectal cancer patients. Science 253: 665-669, 1991.
Baker SJ, Markowitz S, Fearon ER, Willson JK and Vogelstein
B: Suppression of human colorectal carcinoma cell growth by
wild-type p53. Science 249: 912-915, 1990.
Hibi K, Nakamura H, Hirai A, Fujikake Y, Kasai Y, Akiyama S,
Ito K and Takagi H: Loss of H19 imprinting in esophageal
cancer. Cancer Res 56: 480-482, 1996.
Hibi K, Taguchi M, Nakamura H, Hirai A, Fujikake Y, Matsui
T, Kasai Y, Akiyama S, Ito K and Takagi H: Alternative
splicing of the FHIT gene in colorectal cancer. Jpn J Cancer
Res 88: 385-388, 1997.
Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger
AC, Leppert M, Nakamura Y, White R, Smits AM and Bos JL:
Genetic alterations during colorectal-tumor development. N
Engl J Med 319: 525-532, 1988.
Scolnick DM and Halazonetis TD: CHFR defines a mitotic
stress checkpoint that delays entry into metaphase. Nature 406:
Toyota M, Sasaki Y, Satoh A, Ogi K, Kikuchi T, Suzuki H, Mita
H, Tanaka N, Itoh F, Issa JP, Jair KW, Schuebel KE, Imai K
and Tokino T: Epigenetic inactivation of CHFR in human
tumors. Proc Natl Acad Sci USA 100: 7818-7823, 2003.
Satoh A, Toyota M, Itoh F, Sasaki Y, Suzuki H, Ogi K, Kikuchi
T, Mita H, Yamashita T, Kojima T, Kusano M, Fujita M,
Hosokawa M, Endo T, Tokino T and Imai K: Epigenetic
inactivation of CHFR and sensitivity to microtubule inhibitors
in gastric cancer. Cancer Res 63: 8606-8613, 2003.
10 Mizuno K, Osada H, Konishi H, Tatematsu Y, Yatabe Y,
Mitsudomi T, Fujii Y and Takahashi T: Aberrant
hypermethylation of the CHFR prophase checkpoint gene in
human lung cancers. Oncogene 21: 2328-2333, 2002.
11 Shibata Y, Haruki N, Kuwabara Y, Ishiguro H, Shinoda N, Sato
A, Kimura M, Koyama H, Toyama T, Nishiwaki T, Kudo J,
Terashita Y, Konishi S, Sugiura H and Fujii Y: CHFR expression
is downregulated by CpG island hypermethylation in esophageal
cancer. Carcinogenesis 23: 1695-1699, 2002.
12 Corn PG, Summers MK, Fogt F, Virmani AK, Gazdar AF,
Halazonetis TD and El-Deiry WS: Frequent hypermethylation of
the 5' CpG island of the mitotic stress checkpoint gene CHFR in
colorectal and non-small cell lung cancer. Carcinogenesis 24: 47-
13 Hibi K, Nakayama H, Koike M, Kasai Y, Ito K, Akiyama S and
Nakao A: Colorectal cancers with both p16 and p14 methylation
show invasive characteristics. Jpn J Cancer Res 93: 883-887, 2002.
14 Hibi K, Taguchi M, Nakayama H, Takase T, Kasai Y, Ito K,
Akiyama S and Nakao A: Molecular detection of p16 promoter
methylation in the serum of patients with esophageal squamous
cell carcinoma. Clin Cancer Res 7: 135-138, 2001.
15 Hibi K, Takahashi T, Sekido Y, Ueda R, Hida T, Ariyoshi Y,
Takagi H and Takahashi T: Coexpression of the stem cell factor
and the c-kit genes in small-cell lung cancer. Oncogene 6: 2291-
16 Greenlee RT, Murray T, Bolden S and Wingo PA: Cancer
statistics, 2000. CA Cancer J Clin 50: 7-33, 2000.
17 Sun Z, Hsiao J, Fay DS and Stern DF: Rad53 FHA domain
associated with phosphorylated Rad9 in the DNA damage
checkpoint. Science 281: 272-274, 1998.
18 Stewart GS, Wang B, Bignell CR, Taylor AM and Elledge SJ:
MDC1 is a mediator of the mammalian DNA damage
checkpoint. Nature 421: 961-966, 2003.
19 Lou Z, Minter-Dykhouse K, Wu X and Chen J: MDC1 is
coupled to activated CHK2 in mammalian DNA damage
response pathways. Nature 421: 957-961, 2003.
20 Li J, Williams BL, Haire LF, Goldberg M, Wilker E, Durocher
D, Yaffe MB, Jackson SP and Smerdon SJ: Structural and
functional versatility of the FHA domain in DNA-damage
signaling by the tumor suppressor kinase Chk2. Mol Cell 9:
21 Goldberg M, Stucki M, Falck J, D'Amours D, Rahman D, Pappin
D, Bartek J and Jackson SP: MDC1 is required for the intra-S-
phase DNA damage checkpoint. Nature 421: 952-956, 2003.
22 Kang D, Chen J, Wong J and Fang G: The checkpoint protein
CHFR is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at
the G2 to M transition. J Cell Biol 156: 249-259, 2002.
23 Hibi K, Robinson CR, Booker S, Wu L, Hamilton SR,
Sidransky D and Jen J: Molecular detection of genetic
alterations in the serum of colorectal cancer patients. Cancer
Res 58: 1405-1407, 1998.
24 Nakayama H, Hibi K, Taguchi M, Takase T, Yamazaki T, Kasai
Y, Ito K, Akiyama S and Nakao A: Molecular detection of p16
promoter methylation in the serum of colorectal cancer
patients. Cancer Lett 188: 115-119, 2002.
Received July 31, 2006
Revised October 20, 2006
Accepted October 25, 2006
ANTICANCER RESEARCH 26: 4267-4270 (2006)