Yonago Acta medica 2009;52:85–90

Allele-Specific Expression Analysis of PEG1/MEST
in Head and Neck Squamous Cell Carcinomas
Hideyuki Kataoka, Seiji Nakano*, Yasuomi Kunimoto, Naoko Sugie*, Mitsuhiko Osaki*,Narikazu Uzawa†, Mitsuaki A. Yoshida‡, Mitsuo Oshimura* and Hiroya Kitano
Division of Otolaryngology, Head and Neck Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, *Divisionof Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative
Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago 683-8503,†Maxillofacial Surgery, Maxillofacial Reconstruction and Function, Division of Maxillofacial andNeck Reconstruction, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku 113-8510
and ‡Biodosimetry Section, Department of Radiation Dosimetry, Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences, Chiba 263-8555 Japan

Genomic imprinting is an epigenetic feature that plays a significant role in carcinogenesis. In this study, we examined the expression status of an imprinted gene, paternally expressed gene 1/mesoderm-specific transcript (PEG1/MEST), in 38 cases of head and necksquamous cell carcinomas (HNSCCs) and in 17 oral squamous cancer cell lines. Loss ofimprinting (LOI) of PEG1/MEST was found in 8 of 10 (80%) in tumor specimens, and 6 of
10 (60%) informative cases even in the extracted normal tissue specimens. As for the oral
squamous cancer cell lines, LOI was detected in 5 of 8 (62.5%) informative cases in PEG1/
MEST. Thus, these data showed that abnormal expression of PEG1/MEST was found at ahigh frequency in the tumor, the extracted normal tissue specimens and the oral squamouscancer cell lines. PEG1/MEST LOI in extracted normal tissue specimens may have a potential individual cancer risk for HNSCC.
Key words: head and neck squamous cell carcinoma; loss of imprinting; PEG1/MESTHead and neck cancers are almost always squamouscell types arising in the oral cavity, tongue, pharynx
and larynx. Understanding the molecular mechanisms involved in tumor development and progression has enabled the design of new biological approaches. This study focuses on molecular studies
on the roles of genomic imprinting. Almost all imprinted genes identified to date can be classified as
regulators of embryonic growth, placental growthor adult metabolism (Morison et al., 2005). Monoallelic expression of either the maternal or paternal

copy occurs while the other copy is silenced in normal somatic tissue specimens. Imprinted genes areoften clustered in chromosomal domains and arethought to be coordinately regulated by imprinting
control centers. More than 60 types of imprinted
genes have been identified in humans (Delaval andFeil, 2004).
Mutations that affect the epigenetic states ofimprinted domains underlie a number of humandiseases (Walter and Paulsen, 2003). Loss of imprinting (LOI) is one of the most frequent alterations

Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; HNSCC, head and neck squamous cell carcinoma; LOI, loss ofimprinting; PEG1/MEST, paternally expressed gene 1/mesoderm-specific transcript; RFLP, restriction fragment length polymorphism; RT, reverse transcriptase; SNP, single nucleotide polymorphism

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H. Kataoka et al.

tion and matched with extracted normal tissue
specimens from 38 patients, were analyzed in thisstudy. They consisted of 16 oral cancers, 4 oropharyngeal cancers, 5 hypopharyngeal cancers and13 laryngeal cancers. All tissue specimens wereobtained during surgery at the Clinical Department of Otolaryngology, Head and Neck Surgery,Tottori University Hospital. Each resected tumor
was carefully trimmed to remove normal tissue.
Then extracted normal tissue was selected as far
from the cancerous areas as possible. We usually
picked extracted normal tissue from the muscle,
which was removed when neck dissections were
performed. Informed consent to participate in thisstudy was obtained from each patient. The institutional review board of Tottori University approved
this study (approval number 746). All tissue specimens were stored at –80°C until analysis.

found in cancers and involves abnormal activationof a normally silent allele. Jaenisch and colleagues
have demonstrated that global LOI leads to tumor
formation in chimeric mice. All the tumors in themice were derived from non-imprinted cells. Most
importantly, tumors were not seen in the offspring of
the chimaeras, as imprinting is reset during gametogenesis (Goymer, 2005).
Imprinting is therefore an epigenetic tumorsuppressing phenomenon. When imprinting is lost,
cells are immortalized through the inappropriate
regulation of both tumor suppressors and oncogenes(Holm et al., 2005). The genes exhibiting LOI in
human cancer leading to biallelic expression has
been reported in a variety of tumors including headand neck squamous cell carcinomas (HNSCCs)(Schofield et al., 2001; Feinberg and Tycko, 2004).
Previous studies regarding imprinted genes in
HNSCC suggested that LOI of IGF2, H19 andp57KIP2 loci play an important role in oncogenesis(el-Naggar et al., 1999; Lai et al., 2000; Rainho et al.,2001). However, the involvement of other imprinted
genes is still poorly understood in HNSCC.
Paternally expressed gene 1/mesodermspecific transcript (PEG1/MEST) is a paternallyexpressed gene located on human chromosome
7q32 (Kobayashi et al., 1997). PEG1/MEST LOI
has been reported in lung cancers (Suda et al., 2003;
Nakanishi et al., 2004), breast cancers (Pedersen etal., 1999, 2002) and colorectal cancers (Nishihara etal., 2000). In these previous studies, it was postulated
that the disruption of PEG1/MEST might be implicated in the etiology of these cancers. In this study,
we examined the imprinting status of PEG1/MESTin HNSCC specimens and oral squamous cancer celllines with special attention to the role of PEG1/MEST
LOI on oncogenesis in HNSCC tissue specimens andseveral oral squamous cancer cell lines.

Cell lines
Seventeen oral squamous cancer cell lines wereanalyzed. HSC-2, HSC-3 and HSC-4 were established and provided by Uzawa and associates(Uzawa et al., 1995). HO-1-N-1, HO-1-u-1, KON,
KOSC-2 cl3-43, OSC-19, OSC-20, SCC-4 andSKN-3 were obtained from the Japanese Collectionof Research Bioresources (Osaka, Japan). SAS
were obtained from the Cell Resource Center for
Biomedical Research, Tohoku University (Sendai,
Japan). Eight oral squamous cancer cell lines(HSC-2, HSC-3, HSC-4, HSC-5, HSC-6, HSC7, KON and SCCKN) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% fetal bovine serum. Two (HO-1-N-1 and HO-1-u-1)
were grown in DMEM-F12 medium (1:1 mix) with
10% fetal bovine serum. Two (OSC-19 and OSC20) were grown in DMEM-F12 medium (1:1 mix)
without serum. SCC-4 was grown in DMEM-F12
medium (1:1 mix) with 10% fetal bovine serum and0.4 µg/mL hydrocortisone. Four (KOSC-2 cl3-43,
SAT, SAS and SKN-3) were grown in RPMI 1640
medium with 10% fetal bovine serum. These cells
were cultured and growing cells with 70% confluence were harvested for analyses.

Materials and Methods
Tissue samples
HNSCCs, diagnosed by histopathologic examina86

PEG1/MEST in HNSCC

Fig. 1.
A: A schematic of the AflIII polymorphism of human PEG1/MEST is shown. The arrow indicates the site of the polymorphism. PCR products contain this polymorphic site. A-a represents the undigested product (792 bp) and A-b represents
product digested with AflIII (383 and 409 bp). PEG1/MEST, paternally expressed gene 1/mesoderm-specific transcript.
B: Heterozygosity called informative is necessary for allele-specific analysis.

DNA and RNA sample preparation

Allele-specific expression analysis

Genomic DNA was isolated using a Puregene
DNA Isolation Kit (Gentra Systems, Minneapolis,
MN). Total RNA was isolated using an RNeasy
Kit (QIAGEN, Valencia, CA) according to the
manufacturer’s instructions. Total RNA wastreated with RNase-free DNase I (Takara Bio,
Shiga, Japan) to remove any DNA contamination.
First-strand cDNA synthesis was carried out withor without SuperScript III Reverse Transcriptase(RT+ or –) (Invitrogen, Carlsbad, CA) by using anoligo (dT)15 primer (Roche, Basel, Switzerland).
The sequences of the primers were 5-CACTGATGCAGAAAGACGTTC-3 and 5-CAGCACCATTTGCTCATAGG-3.

Allele-specific expression analysis with RT-PCR
and digestion by restriction enzymes was performed under the same conditions as those used todetect genomic polymorphisms. To confirm theresults of restriction fragment length polymorphism(RFLP), we also performed a method for allelespecific expression analysis of imprinted genes byreal-time PCR with Cycling Probe Technology(Takara Bio). In this method, one end of the probe
is labeled with a fluorescent substance and the otherend is labeled with a quencher, which quenches thefluorescence emitted from the substance. RNaseH
specificially cuts the RNA region of this probe,
resulting in emission of strong fluorescence, but it
does not cut the RNA probe region including any
mismatches.

Identification of polymorphisms
Imprinting analysis for PEG1/MEST was carried out by single nucleotide polymorphism (SNP)
analysis for informative heterozygotes at the G/A(exon 12; rs10863) polymorphism. An informative
DNA polymorphism at an AflIII site was identified
as the AflIII-digestible (ACACGT) or indigestible(ACACAT) sequence. PCR was performed for 30
cycles including denaturation at 95˚C for 30 s, annealing at 62˚C for 30 s and extension at 72˚C for 30 s.

Results
We analyzed 38 primary HNSCCs for the LOI in
PEG1/MEST. First of all we identified genomic
polymorphisms using RFLP analysis. Informative
cases were then further analyzed to determine theirallele-specific expression. As shown in Figs. 1 and
2, we distinguished the 2 alleles and determined
whether a case is informative or not. This polymorphism (G/A; rs10863) results in the presence87

H. Kataoka et al.

		Genome(bp)

Extracted specimen
Normal
Tumor

+

–

+

Genome–(bp)

Extracted specimen
Normal
Tumor

+

–

+

–

792

792

409
383

409
383

Imprinted

LOI

LOI

Case 4

LOI

Case 1

Fig. 2. Allele-specific expression analysis of PEG1/MEST in head and neck squamous cell carcinoma tissue specimens. The
allele-specific expressions of PEG1/MEST were assessed by restriction fragment length polymorphism analysis. Representative results of PEG1/MEST are shown. +, presence of reverse transcriptase (RT); –, absence of RT; LOI, loss of imprinting;
PEG1/MEST, paternally expressed gene 1/mesoderm-specific transcript.

or absence of an AflIII site. The 3 different bands(792, 409 and 383 bp) after AflIII digestion of the
genomic PCR fragment indicated an informative
heterozygous case. Next, to determine the expression status of PEG1/MEST, the RT-PCR products
are digested with AflIII to determine whether one
or both copies are expressed. The presence of 3
bands indicates biallelic expression, that is, PEG1/
MEST LOI. On the other hand, a single band of
792 bp or 2 bands of 409 bp and 383 bp showedmonoallelic expression, indicating the normal imprinting status of PEG1/MEST. Among the cases
screened, we observed 10 informative cases of
PEG1/MEST. PEG1/MEST LOI was found in 8
informative cases (80%), and in 6 cases (60%) it
was detected even in the normal tissue specimens(Table 1), regardless of tumor location or histology.

These data suggest that PEG1/MEST LOI occurs
in HNSCC specimens, suggesting its fundamentalrole in HNSCC oncogenesis.
Next, we analyzed the imprinting status of
PEG1/MEST in 17 oral squamous cancer cell lines
to substantiate the results from the tissue specimens.
The results for the histopathologic types of theseoral squamous cancer cell lines are summarized inTable 2. PEG1/MEST LOI was observed in 5 of 8
informative cases (62.5%). The results from the celllines are summarized in Table 2, which were consistent with the high frequency of LOI at the PEG1/
MEST loci detected in the HNSCC tissue specimens. Thus, abnormal expression of PEG1/MEST
was found at a high frequency in the cancer tissue
specimens, the extracted normal tissue specimens
and the oral squamous cancer cell lines.

Table 1. Summary of PEG1/MEST gene expression profiles in informative cases of tissue specimens of head and neck squamous cell carcinoma
CaseType of cancer
Histological classification
number			
1
2
3
4
5
6
7
8
9
10

Tongue cancer
Laryngeal cancer
Laryngeal cancer
Hypopharyngeal cancer
Tongue cancer
Tongue cancer
Tongue cancer
Laryngeal cancer
Oropharyngeal cancer
Laryngeal cancer

Moderately differentiatedModerately differentiatedWell differentiatedPoorly differentiatedWell differentiatedModerately differentiatedModerately differentiatedModerately differentiatedModerately differentiatedPoorly differentiated

NormalLOI
LOI
LOI
Imprinted
Imprinted
Imprinted
Imprinted
LOI
LOI
LOI

LOI, loss of imprinting; PEG1/MEST, paternally expressed gene 1/mesoderm-specific transcript.

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PEG1/MEST
Tumor
LOI
LOI
LOI
LOI
Imprinted
LOI
LOI
LOI
LOI
Imprinted

PEG1/MEST in HNSCC

Table 2. Summary of allele-specific expressionof PEG1/MEST in 17 oral squamous cancer celllines

Discussion

Cell line
NumberNameSeveral lines of evidence suggest that the disruption of imprinting mechanisms play a critical rolein oncogenesis including HNSCC, but none haveexamined PEG1/MEST in HNSCC. The data frommice studies of Mest have suggested a possible
role of Mest as a regulator of embryonic growth.
Mest paternal knockouts have been shown to resultin an imprinted phenotype characterized by fetal
and placental growth retardation (Lefebvre et al.,1998). In our report, we provide the first evidence
regarding the presence of biallelic expression of
PEG1/MEST in HNSCC. PEG1/MEST LOI was
identified 8 out of 10 cases in HNSCC tissue specimens and 5 out of 8 cases in oral cancer cell lines.
Interestingly, PEG1/MEST LOI was observed in
the extracted normal tissue surrounding the tumor
specimens.
IGF2 and PEG1/MEST are known as imprinted genes important in fetal growth. The correlation between IGF2 LOI in normal cells andcolorectal cancer has been well documented (Cuiet al., 2003; Nakano et al., 2006). IGF2 LOI has
been found in 10% of normal individuals (Sakatani
et al., 2001) and it appears to be 5 times morecommon in patients with a family history of colon
carcinoma, and 21 times more common in patientswith a personal history of colorectal neoplasia (Cuiet al., 2003). Moreover, a mouse model with Igf2
LOI suggests that mice with abnormal imprinting
acquired twice as many intestinal adenomas as
those whose imprinting was normal (Sakatani etal., 2005). These studies indicated that IGF2 LOImight be a valuable predictive marker of an individual’s risk for colorectal cancer.
PEG1/MEST LOI in both extracted normal
and tumor tissue specimens suggests that the detection of PEG1/MEST LOI in extracted normal tissue
specimens may also pose a potential cancer risk inindividuals for HNSCC. IGF2 LOI has been foundin 10% of normal individuals. However, there are
no reports indicating the incidence of PEG1/MEST
LOI in normal individuals. It will be necessary to

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

HSC-2
HSC-3
HSC-4
HSC-5
HSC-6
HSC-7
HO-1-N-1
HO-1-u-1
KON
KOSC-2 cl3-43
OSC-19OSC-20
SAS
SAT
SCCKN
SCC-2
SKN-3

Frequency of LOI

Type of cancer

PEG1/MEST

Oral cavity
TongueTonguePoorly differentiatedTongueTongueBucchal mucosa
Mouth floor
Oral floor
Oral floor
TongueTongueTongueOral cavity
Oral cavity
TongueOral cavity

Imprinted
Imprinted
*
*
*
LOI
LOI
*
*
Imprinted
LOI
*
LOI
*
*
*
LOI

5/8 (62.5%)* Non-informative case.
LOI, loss of imprinting; PEG1/MEST, paternally expressedgene 1/mesoderm-specific transcript.

clarify the incidence of PEG1/MEST LOI in normal individuals in order to elucidate its predictive
value for an individual’s cancer risk for HNSCC.
References
1 Cui H, Cruz-Correa M, Giardiello FM, Hutcheon DF,
Kafonek DR, Brandenburg S, et al. Loss of IGF2 imprinting: a potential marker of colorectal cancer risk.
Science 2003; 299:1753–1755.
2 Delaval K, Feil R. Epigenetic regulation of mammalian
genomic imprinting. Curr Opin Genet Dev 2004;14:
188–195.
3 el-Naggar AK, Lai S, Tucker SA, Clayman GL,
Goepfert H, Hong WK, et al. Frequent loss of imprinting at the IGF2 and H19 genes in head and necksquamous carcinoma. Oncogene 1999;18:7063–7069.
4 Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev Cancer 2004;4:143–153.
5 Goymer P. Cancer epigenetics: dangerous unmarked
genes. Nature Reviews Genetics 2005; 6:878-878.
6 Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y,
Rideout WM 3rd, Jaenisch R. Global loss of imprinting
leads to widespread tumorigenesis in adult mice. Cancer Cell 2005; 8:275-285.

89

H. Kataoka et al.

7 Kobayashi S, Kohda T, Miyoshi N, Kuroiwa Y, Aisaka K,
Tsutsumi O, et al. Human PEG1/MEST, an imprinted
gene on chromosome 7. Hum Mol Genet 1997;6:781–
786.
8 Lai S, Goepfert H, Gillenwater AM, Luna MA,
El-Naggar AK. Loss of imprinting and genetic alterations of the cyclin-dependent kinase inhibitor p57KIP2
gene in head and neck squamous cell carcinoma. Clin
Cancer Res 2000;6:3172–3176.
9 Lefebvre L, Viville S, Barton SC, Ishino F, Keverne EB,
Surani MA. Abnormal maternal behaviour and growth
retardation associated with loss of the imprinted gene
Mest. Nat Genet 1998;20:163–169.
10 Morison IM, Ramsay JP, Spencer HG. A census of
mammalian imprinting. Trends Genet 2005; 21:457–
465.
11 Nakanishi H, Suda T, Katoh M, Watanabe A, Igishi T,
Kodani M, et al. Loss of imprinting of PEG1/MEST inlung cancer cell lines. Oncol Rep 2004;12:1273–1278.
12 Nakano S, Murakami K, Meguro M, Soejima H,
Higashimoto K, Urano T, et al. Expression profile of
LIT1/KCNQ1OT1 and epigenetic status at the KvDMR1
in colorectal cancers. Cancer Sci 2006;97:1147–1154.
13 Nishihara S, Hayashida T, Mitsuya K, Schulz TC,
Ikeguchi M, Kaibara N, et al. Multipoint imprinting
analysis in sporadic colorectal cancers with and without
microsatellite instability. Int J Oncol 2000;17:317–322.
14 Pedersen IS, Dervan PA, Broderick D, Harrison M,
Miller N, Delany E, et al. Frequent loss of imprintingof PEG1/MEST in invasive breast cancer. Cancer Res
1999;59:5449–5451.
15 Pedersen IS, Dervan P, McGoldrick A, Harrison M,
Ponchel F, Speirs V, et al. Promoter switch: a novel
mechanism causing biallelic PEG1/MEST expression ininvasive breast cancer. Hum Mol Genet 2002;11:1449–
1453.
16 Rainho CA, Kowalski LP, Rogatto SR. Loss of imprinting and loss of heterozygosity on 11p15.5 in head andneck squamous cell carcinomas. Head Neck 2001;23:
851–859.
17 Sakatani T, Kaneda A, Iacobuzio-Donahue CA, Carter
MG, de Boom Witzel S, Okano H, et al. Loss of imprinting of Igf2 alters intestinal maturation and tumorigenesis in mice. Science 2005;307:1976–1978.
18 Sakatani T, Wei M, Katoh M, Okita C, Wada D,
Mitsuya K, et al. Epigenetic heterogeneity at imprinted
loci in normal populations. Biochem Biophys Res
Commun 2001;283:1124–1130.
19 Schofield PN, Joyce JA, Lam WK, Grandjean V,
Ferguson-Smith A, Reik W, et al. Genomic imprinting
and cancer: new paradigms in the genetics of neoplasia.
Toxicol Lett 2001;120:151–160.
20 Suda T, Katoh M, Hiratsuka M, Fujiwara M, Irizawa Y,
Oshimura M. Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene. Int J
Mol Med 2003;12:243–246.
21 Uzawa N, Yoshida MA, Oshimura M, Ikeuchi T. Suppression of tumorigenicity in three different cell lines ofhuman oral squamous cell carcinoma by introduction ofchromosome 3p via microcell-mediated chromosometransfer. Oncogene 1995;11:1997–2004.
22 Walter J, Paulsen M. Imprinting and disease. Semin
Cell Dev Biol 2003;14:101–110.
Received March 25, 2009; accepted May 20, 2009
Corresponding author: Hideyuki Kataoka, MD

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