Genetic effects of X-ray and carbon ion irradiation in head and neck carcinoma cell lines.

Abstract

The effects of X-ray and carbon ion irradiation on DNA and genes in head and neck carcinoma cells were examined. Four head and neck cancer cell lines (squamous cell carcinoma, salivary gland cancer, malignant melanoma, normal keratinocyte) were treated with 1, 4, and 7 GyE of carbon ion, or 1, 4, and 8 Gy of X-ray, respectively. DNA and RNA in the treated cells were extracted and purified. PCR-LOH (polymerase chain reaction-loss of heterozygosity) analysis with 6 microsatellite regions on chromosome 17 was performed to determine DNA structural damage, and then microarray analysis was performed to reveal changes in gene expression. PCR-LOH analysis detected high LOH in cells treated by radiation, indicating that most of the damage by X-ray occurred in the target region on one of the homologous chromosomes. However, carbon ion caused homo-deletion, which means deletion of the counterparts in both homologous chromosomes.

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Title
Geneti c effects of x-ray and carbon i on i rradi ati on
i n head and neck carci nom a cel l l i nes
Author(s)
Yam am oto, N ; Ikeda, C; Yakushi j i , T; N om ura, T;
Katakura, A; Shi bahara, T; M i zoe, J E
J ournalBul l eti n of Tokyo Dental Col l ege, 48(4): 177-185
URL http: //hdl . handl e. net/10130/418
Right

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Original Article
177
Bull Tokyo Dent Coll (2007) 48(4): 177–185
Genetic Effects of X-Ray and Carbon Ion Irradiation in
Head and Neck Carcinoma Cell Lines
Nobuharu Yamamoto, Chihaya Ikeda, Takashi Yakushiji, Takeshi Nomura,
Akira Katakura, Takahiko Shibahara and Jun-etsu Mizoe*
Department of Oral and Maxillofacial Surgery, Tokyo Dental College,
1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan
* Hospital, Research Center for Charged Particle Therapy National Institute of
Radiological Sciences,
4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan
Received 1 June 2007/Accepted for publication 17 December, 2007
Abstract
The effects of X-ray and carbon ion irradiation on DNA and genes in head and neck
carcinoma cells were examined. Four head and neck cancer cell lines (squamous cell
carcinoma, salivary gland cancer, malignant melanoma, normal keratinocyte) were treated
with 1, 4, and 7 GyE of carbon ion, or 1, 4, and 8 Gy of X-ray, respectively. DNA and RNA
in the treated cells were extracted and purified. PCR-LOH (polymerase chain reaction-
loss of heterozygosity) analysis with 6 microsatellite regions on chromosome 17 was
performed to determine DNA structural damage, and then microarray analysis was per-
formed to reveal changes in gene expression. PCR-LOH analysis detected high LOH in
cells treated by radiation, indicating that most of the damage by X-ray occurred in the target
region on one of the homologous chromosomes. However, carbon ion caused homo-
deletion, which means deletion of the counterparts in both homologous chromosomes.
Key words: Head and neck carcinoma—Loss of heterozygosity (LOH)—
X-ray irradiation—Carbon ion irradiation
Introduction
Head and neck cancer is one of the most
common malignancies worldwide. In the Far
East Asia and India, in particular, the inci-
dence is much higher, with up to 40% of
malignancies occurring in the head and neck
regions
Human cancers result from the accumulation
of genetic alterations at specific chromosomal
regions, involving a multistep process
2).
19,30,31),
and much evidence indicates that there are
a number of tumor suppressor genes (TSGs)
involved in carcinogenesis. On the other hand,
the treatment of head and neck tumor is very
difficult, because this region is involved in
many important functions such as articula-
tion, mastication, and swallowing. These func-
tions are closely connected with the patient’s
personality and self-confidence. Carbon ion
radiotherapy, one of the new conservative
radiotherapies, is focused on from this point

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view. Therefore, it is important to investigate
the mechanism of the effects of carbon ions on
DNA structure and gene expression. Although
conventional X-ray treatment is an effective
modality for a wide variety of human cancers,
in certain cases it continues to provide poor
results.
To obtain an improved therapeutic effect,
dose escalation is essential, but this increases
the risk of oral toxicity. High linear energy
transfer (LET) radiotherapy with heavy ions,
such as neon and carbon ions, provides
superb biologic effects and has excellent dose-
localizing properties
charged particles can severely damage the
tumor, with fewer effects on normal tissue.
Beam modulation by bolus absorbers and
collimator blocks allows precise beam penetra-
tion and sharp lateral edges in three dimen-
sions. The resulting isodose distribution can
be made to conform closely to the target vol-
ume, allowing a high dose to the tumor, with
minimal irradiation of surrounding normal
tissues.
Carbon ion beams emit high LET radiation
characterized by higher relative biological effec-
tiveness (RBE) than low LET radiation such
as X-rays. The efficacy of carbon ion therapy
has been demonstrated in clinical trials at the
National Institute of Radiological Sciences
(NIRS), Chiba, Japan, since 1994
bon ions were selected for clinical trials,
because they have the biologic characteristics
of high LET, with 78KeV/?m at the distal
end of the spread-out Bragg peak (SOBP),
and because they show good dose-localizing
properties compared with heavier ions. These
advantages have been shown in various can-
cers
clinical trials have shown extremely favorable
therapeutic results in the treatment of head
and neck cancers (including oral cancers)
that were otherwise intractable with conven-
tional photon radiation
radiotherapy with heavy charged particles is
significantly effective in the therapy of head
and neck cancers. However, severe adverse
effects such as refractory ulceration at the adja-
cent normal tissues have also been reported.
4,6,14,15,17,23). These high LET-
16,27,29,40). Car-
3,16,27,28,33,36). Preliminary results of phase II
16,27). As stated above,
A suitable treatment strategy is certainly neces-
sary to reduce injury to surrounding normal
tissues.
Although several studies have focused on
the biologic effects of carbon ions, few have
attempted to understand the molecular basis
of carbon ion therapy. There is an urgent need
to elucidate the molecular mechanisms and
processes underlying carbon ion irradiation.
In recent years, a cDNA microarray system
has been used widely for comprehensive
gene expression analysis
technology of high-density cDNA microarray
provides the ability to analyze comparatively
the mRNA expression of thousands of genes
in parallel.
In the present study, DNA structural muta-
tions were examined by PCR-LOH (poly-
merase chain reaction-loss of heterozygosity)
analysis. The effects of carbon ions on carci-
noma cells are discussed in comparison with
X-ray.
7,9,39). The emerging
Materials and Methods
1. Cell line and cell culture conditions
The following head and neck carcinoma-
derived cell lines were used for this study:
Ca9-22 (derived from oral squamous cell
carcinoma: OSCC), HSG (from salivary gland
tumor), G361 (from malignant melanoma),
and HaCaT (from normal human squamous
cells) (Human Science Research Resources
Bank, Osaka, Japan). All cell lines were grown
in RPMI-1640 medium supplemented with
10% fetal bovine serum and 50units/ml
penicillin and streptomycin. All cultures were
grown at 37°C in a humidified atmosphere
of 5% carbon dioxide for routine growth.
Transfer to fresh medium was performed
when confluence was ?90%.
2. Radiation treatment
The cell lines were treated with different
doses (1, 4, and 8Gy) of X-ray and also with
different doses (1, 4, and 7GyE) of carbon
ion beam. All procedures of X-ray and carbon
ion irradiation were carried out at the NIRS.
Yamamoto N et al.

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Briefly, a 290-MeV/nucleon carbon ion beam
with 6-cm SOBP was used through on experi-
mental port. Cells plated in 75cm
flasks (Corning Inc., Corning, NY) were
irradiated at the distal end of the SOBP
(LET?75keV/?m). Structural damage was
determined using DNA extracted at 1, 24,
and 48h after irradiation.
2 plastic
3. Clonogenic survival assay of Ca9-22
Cell survival was measured using a clono-
genic survival assay. After exposure to various
doses of either carbon ion beams or X-rays,
cells were seeded into 60-mm tissue culture
dishes and cultured for approximately 14 days
to allow colonies to form. The colonies were
stained with a solution of crystal violet
(Sigma) and counted. The survival fraction
at each dose was determined as a ratio of
plating efficiencies for irradiated and non-
irradiated cells. These experiments were per-
formed once.
4. DNA preparation
Genomic DNAs were isolated by the standard
method using phenol-chloroform extraction
and refined, washed and precipitated with
ethanol
DNA were estimated by spectrophotometric
method and kept frozen at ?80°C. From each
DNA sample, 50ng/?l was used as a template
for the PCR amplification procedure.
25,26). The concentrations of extracted
5. DNA analysis on microsatellite loci
We selected 6 highly informative micro-
satellite markers (D17S261, D17S1176, TP53,
D17S250, D17S1320, and D17S1329) on
chromosome 17 (Table 1). All primers were
obtained from Research Genetics (Huntsville,
AL). PCR amplification was performed in a
total reaction volume of 20?l, as described
previously
tained 250ng sample DNA, 20pmol each
primer, 10mM Tris-HCl (pH8.3), 50mM KCl,
3.0mM MgCl2, 2mM dNTP, and 0.5 unit
Taq DNA polymerase (Perkin-Elmer Cetus,
Norwalk, CT). PCR was performed with 26 to
30 cycles of denaturation at 94°C for 1min,
annealing at 52 to 58°C for 1min, and exten-
sion at 72°C for 1min using a DNA Thermal
Cycler (Perkin-Elmer Cetus, Norwalk, CT).
After dilution with an adequate volume of
formamide-dye mixture (95% formamide,
20mM EDTA, 0.05% bromophenol blue, and
0.05% xylene cyanol), the PCR products were
heat-denatured (98°C, 5min.), chilled on ice,
and electrophoresed on 6% urea-formamide-
polyacrylamide gel at 3W for 2 to 3h, depend-
ing on fragment size. Silver staining of the
gels was performed using the DNA Silver
Staining Kit (Amersham Pharmacia Biotech
AB. Sweden). To ensure reproducibility in each
case with LOH or microsatellite instability
(MSI), all tests were performed under the
21). Each PCR reaction mixture con-
Carbon Ion-Enhanced Genes in Oral Cancer
Table 1Sequence of primers used for PCR-LOH analysis
MarkersLocations
Size of PCR
products (bp)
Sequence of primers
D17S26117p12-11.1157–171
5?-CAGGTTCTGTCATAGGACTA-3?
5?-TTCTGGAAACCTACTCCTGA-3?
5?-ACTTCATATACATATCACGTGC-3?
5?-TCAATGGAGAATTACGATAGTG-3?
5?-TTGCCTCTTTCCTAGCACTG-3?
5?-CCAAGACTTAGTACCTGAAG-3?
5?-GGAAGAATCAAATAGACAAT-3?
5?-GCTGGCCATATATATATTTAAACC-3?
5?-ACTTTCCAGAAAATCTCTGCTC-3?
5?-CCACGTCTTTTCTGTGTTCC-3?
5?-GACTCTGAAGGTAAAGAGCAA-3?
5?-CTCCCCTGCCTTGGGAGTAG-3?
D17S117617p13.1 95–109
TP5317p13.1103
D17S25017q11.2-12151–169
D17S132017q21180
D17S132917q21170

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same conditions.
6. Assessment of LOH and MSI
LOH in the tumor DNA samples was assessed
by scanning densitometry and analyzed with
National Institute of Health (NIH) software
(Image version 1.62, Dr. W. Rasband, NIH,
Bethesda, MD, USA). The intensities of the
signals in tumor DNA were compared with
those of the corresponding normal DNA. A
reduction in signal intensity of more than 50%
was required for LOH. Commonly deleted
regions were defined by considering the loci
most frequently showing LOH, together with
multiple interstitial deletions. Microsatellite
instability (MSI) for DNA samples was also
assessed as positive in cases with additional
bands in the tumor sample that were not
observed in the corresponding normal sample
or in cases with a band shift in the tumor
sample that contrasted with those of the cor-
responding normal bands.
Results
1. Analysis of allelic loss
Structural DNA changes occurring on
chromosome 17 after X-ray and carbon ion
irradiation of cell lines derived from malig-
nant tumors in head and neck were analyzed
using PCR-LOH assay. Typical results of elec-
trophoresis are shown in Fig. 1. Deletion
(DLT), LOH, ROH, and NI signify homo-
deletion, heterodeletion, retention of hetero-
zygosity, and not informative, respectively. A
deletion map was created covering both kinds
of beam (X-ray and carbon ion), 3 different
doses (1 Gy/GyE, 4 Gy/GyE, 8 Gy/7 GyE), 4
cell lines, and 3 different DNA-extracted times
(1, 24, and 48h after irradiation) (Fig. 2).
PCR-LOH analysis revealed high LOH, such
as in Ca9-22, HSG and G361, when they
were treated with X-ray. However, in normal
keratinocyte cell line, HaCaT, only two cases
of DNA mutations (DLT or MSI) were found.
Yamamoto N et al.
Fig. 1 Typical patterns of electrophoresis
Microsatellite polymorphism analysis in cell lines. Carbon ion irradiated-doses are shown at top, and locus
symbols at bottom. Paired control (C) and tumor (T) cell lines demonstrating deletion of both alleles
(DLT), loss of upper allele (LOH), retained heterozygosity (ROH) and not-informative (NI), respectively.
DLT: deletion (homozygosity)
LOH: loss of heterozygosity
ROH: retain of heterozygosity
NI: not informative
C: control
DLT LOH ROHNI
C 4GyEC 7GyEC 1GyEC 4GyE
HSG (24h)
D17S1176
(17p13.1)
G361 (48h)
TP53
(17p13.1)
Ca9-22 (48h)
D17S1320
(17q21)
HaCaT (24h)
D17S250
(17q11.2-12)
???
???
???

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181
In contrast, after carbon ion irradiation, DLT
occurred at many region regardless of type of
cell line. However, LOH was detected at only
one locus.
2. Survival rates
The survival rates for Ca9-22 cell exposed
to carbon ion beams or X-rays are shown in
Fig. 3. Each curve represents one experiment.
In Ca9-22 cells, there was a significant differ-
ence in survival curves for carbon ion beams
and X-rays. The survival curve for Ca9-22 cells
irradiated with carbon ion beams showed a
steep curve, whereas X-ray-irradiated Ca9-22
cells showed a gentle curve.
Discussion
Radiotherapy, an inevitable component of
modern cancer management, is a major treat-
ment modality that can potentially provide a
cure for patients with OSCC
34). The success or
Carbon Ion-Enhanced Genes in Oral Cancer
Fig. 2
Doses and beams are shown at top and locus symbols and DNA-extracted times on left.
Deletion mapping of chromosome 17 in 4 head and neck cancer cell lines
D17S261
D17S1176
1h
TP53
D17S250
D17S1320
D17S1329
D17S261
D17S1176
24h
TP53
D17S250
D17S1320
D17S1329
D17S261
D17S1176
48h
TP53
D17S250
D17S1320
D17S1329
Ca9-22
1GY
X
4GY
X
8 7
C C X C
HSG
1GY
X
4GY
X
8 7
C C X C
G361
1GY
X
4GY
X
8 7
C C X C
HaCaT
1GY
X
4GY
X
8 7
CC X C
X: X-ray
C: Carbon ion
DLT
LOH
MSI
ROH
NI
ˆ
¯
˜
˜
˝
˜
˜
¯
ˆ
¯
˜
˜
˝
˜
˜
ˆ
˜
˜
˝
˜
˜
Fig. 3Survival curves of Ca9-22 cells exposed to carbon
ion beams or X-rays
Each point represents value of one experiment.
Carbon
X-ray
1
0.1
0.01
0.001
02468 10
Dose (Gy)
Surviving rate

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182
failure of radiotherapy can be affected by the
radiosensitivity of the tumor target and the
limits imposed on treatment by the radio-
sensitivity of normal tissues. Recently, several
studies using microarrays technique have
successfully identified and classified a set of
human genes that are radiosensitive to X-ray
irradiation
Modern curative radiotherapy requires
higher doses to tumors and minimal irradia-
tion to the surrounding normal tissues. Car-
bon ions produce increased density of local
energy deposition with high LET components,
resulting in radiobiologic advantages. It is an
area of active investigation to elucidate the
mechanisms underlying the increased biologic
effectiveness of dense irradiation. Several
studies have evaluated the correlation between
tumor responses to carbon ion irradiation and
the expression status of known genes
Irradiation with high LET carbon ion beams
caused glioma cells with either the wild-type
or mutant p53 gene to fail to proliferate and
apoptosis, more effectively than X-rays
addition, the effects of carbon ion beams
are reduced by G1 arrest, which is indepen-
dent of p21 expression
has focused on the gene expression profiles
of head and neck carcinoma cells exposed
1,8,10,12,18,32).
11,13,38,41).
15). In
15). To date, no report
to X-ray and carbon ion beam irradiation
simultaneously.
Gene expression profiling using high-density
microarrays is an excellent tool to identify
novel candidate biomarkers in human cancers
associated with regulation of important cancer-
related cellular events, such as cell growth
regulation and apoptosis. Indeed, several
studies have successfully used microarrays to
identify and classify a set of human genes in
response to ionizing radiation
light gene expression changes in OSCC cells
exposed to carbon ion beams, we used a
high-throughput gene chip containing 54,675
oligonucleotide-based probe sets to analyze
change in gene expression after carbon ion
irradiation. It has been demonstrated that
gene expressions are dramatically changed
between 1 to 72h after irradiation
In particular, changes in gene expression
profiles at 3 or 4h postirradiation have been
identified in keratinocytes
vein endothelial cells
In the current study, structural DNA
changes occurring on chromosome 17 after
X-ray and carbon ion irradiation of cell popu-
lations derived from malignant tumors in the
head and neck were analyzed using PCR-LOH
assay. After X-ray irradiation, a larger amount
8,10,12,18). To high-
5,22,24,35,37).
20) and in umbilical
25).
Yamamoto N et al.
Table 2 Genetic expressions in the carbon-irradiated OSCC cell line
FunctionsGenes
ACTB, ADRB2, AKAP12, BRF2, CLK1, COTL1, EMP1,
FST, H3F3B, INHBA, INHBB, IRF1, JUN, KLF2,
MAPK3, MAPK8, MYC, ODC1, POLR2A, POLR2F,
POLR2L, PTHLH, PTN, SFRS12, SFRS2, SFRS6,
SNAPC1, SNAPC2, SNAPC3, SNAPC4, SPN, SRPK1,
TBP, TFRC, VIL2, ATP2B1, BCAR1, BCAR3, CASP3,
CLTC, CXCL2, CXCL3, DACH1, EHD1, FGF5, IGF1R,
IGF2, IL18, IL8RB, INSR, IRS1, IRS2, ITPR1, JAK2,
NEDD4, NEDD9, NPM1, NRG1, NUP98, PTGS2,
PTPN12, RAPGEF2, RELA, SCN2A1, SNAP29, SOCS1,
STAT1, SYNCRIP, TNFAIP3, CBLB, CSF1R, DTR,
DUSP4, EIF3S1, EIF3S3, EIF3S6, EIF3S7, EIF3S8,
EIF3S9, GLIPR1, GRB2, IL11, IL11RA, IL6ST, JAK1,
MAPK14, MAPK3, MYOD1, NONO, NP, PML, PTPRE,
SARA1, SFPQ, SPHK1, SPRY2, TNFAIP3, TOP1, TP53,
TRAF2, TYK2, VAV1
Gene expression
Cancer
Cell growth and proliferation
Cell death
Cell compromise
DNA replication
Recombination and repair
Carbohydrate metabolism
Cell morphology
Cellular movement
Cell cycle
Cell development
Immune and lymphatic system development and
function
Hematologic system development and function
Protein synthesis

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of LOH was detected rather than DLT. At
high doses, however, it was found that LOH
tended to decrease. In addition, after carbon
ion irradiation, LOH was detected only in one
location, whereas all other DNA impairments
were marked by the presence of DLT. These
results indicated that most of the damage by
X-ray occurred in the target region on one of
the homologous chromosomes in carcinoma
cells. Carbon ion beam caused homo-deletion
(DLT), which means deletion of the counter-
parts in both homologous chromosomes.
We selected the time point of 4h to monitor
the early response of OSCC cells to irradia-
tion, and identified 98 genes that were modu-
lated by carbon ion irradiation at all doses in
each of the OSCC-derived cell lines, Ca9-22 by
using microarray analysis.
In conclusion, this comprehensive gene
expression analysis provided an interesting
approach to effectively identifying candidate
genes involved in cellular radioresistance.
These genes may help to disclose the molecu-
lar mechanisms of radioresistance in head
and neck carcinoma, and could serve as radio-
therapeutic molecular markers for choice of
the appropriate radiotherapy in this disease.
Acknowledgements
This work was supported by a Research
Grant from the Ministry of Education, Science
and Culture, Japan (No.15592135).
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Reprint requests to:
Prof. Takahiko Shibahara
Department of Oral and
Maxillofacial Surgery,
Tokyo Dental College,
1-2-2 Masago, Mihama-ku,
Chiba 261-8502, Japan
E-mail: shibahara@tdc.ac.jp
Carbon Ion-Enhanced Genes in Oral Cancer