Infrequent mutation of lysophosphatidic Acid receptor-1 gene in hamster pancreatic duct adenocarcinomas and established cell lines.
ABSTRACT To evaluate the involvement of lysophosphatidic acid receptor-1 (LPA1) gene alteration in pancreatic carcinogenesis, we investigated mutations in the LPA1 gene in hamster pancreatic duct adenocarcinomas (PDAs) and established cell lines. Female Syrian golden hamsters received 30 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP) followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with DL-ethionine and then L-methionine and a further administration of 20 mg/kg BOP. A total of 10 PDAs obtained 10 weeks after beginning the experiment and three cell lines established from subcutaneously transplantable PDAs in syngeneic hamsters were examined for mutations using reverse transcription-polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP) analysis. A mutation was detected in only one PDA (1/10, 10%) in the form of a GGA to GTA (Gly to Val) transversion at codon 355, and no mutations were detected in the three cell lines. These results suggest that the LPA1 gene mutation may play roles in a limited fraction of BOP-induced pancreatic duct carcinogenesis in hamsters.
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ABSTRACT: Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA(1)), LPA(2), LPA(3), LPA(4), LPA(5) and LPA(6). These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models.Journal of Toxicologic Pathology 09/2011; 24(3):143-8. · 0.34 Impact Factor
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ABSTRACT: Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, frequent mutations of the LPA receptor-1 (LPA1) gene were detected in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). In this study, to evaluate the involvement of other LPA receptor gene alterations during lung carcinogenesis, we investigated mutations of the LPA2, LPA3, LPA4 and LPA5 genes in lung adenocarcinomas induced by BHP in rats. Fifteen male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks, and 15 adenocarcinomas were obtained. Genomic DNAs were extracted from frozen tissues, and the LPA2, LPA3, LPA4 and LPA5 genes were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No mutations of LPA2, LPA3, LPA4 and LPA5 were detected in the 15 adenocarcinomas. These results suggest that alterations due to LPA2, LPA3, LPA4 and LPA5 gene mutations might not be involved in the development of lung adenocarcinomas induced by BHP in rats.Journal of Toxicologic Pathology 03/2010; 23(1):63-6. · 0.34 Impact Factor
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ABSTRACT: In example of OSC, BrCN, O3 molecules, we show that the fitting method suggested in our previous work is applicable for retrieving interatomic separations of molecules with two structural parameters from high-order harmonics generated from these molecules in interaction with intense ultra-short infrared laser pulses. For linear molecules such as OSC, BrCN we find that the accurate structural information can be obtained using a small set of harmonics of only one alignment angle. However, for the planar molecule, O3, we need high harmonics of at least two alignment angles to retrieve two structural parameters of the molecule. We show that for both linear and planar molecules, the interatomic separations can also be retrieved from harmonics generated by isotropically oriented molecules. To develop the method, we also apply the genetic algorithm in the fitting procedure that really reduces time consuming compared to the direct scanning method.Cancer Letters - CANCER LETT. 01/2011; 964(1):12-17.
J Toxicol Pathol 2009; 22: 89–92
Infrequent Mutation of Lysophosphatidic Acid Receptor-1
Gene in Hamster Pancreatic Duct Adenocarcinomas and
Established Cell Lines
Toshifumi Tsujiuchi1, Mami Furukawa1, Yumi Obo1, Ayako Yamasaki1,
Mayuko Hotta1, Chie Kusunoki1, Naoko Suyama1, Toshio Mori2,
Kanya Honoki3, and Nobuyuki Fukushima4
1Laboratory of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering,
Kinki University, 3–4–1 Kowakae, Higashiosaka, Osaka 577-8502, Japan
2RI Center, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
3Department of Orthopedic Surgery, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan
4Laboratory of Molecular Neurobiology, Department of Life Science, Faculty of Science and Technology, Kinki
University, 3–4–1 Kowakae, Higashiosaka, Osaka 577-8502, Japan
Abstract: To evaluate the involvement of lysophosphatidic acid receptor-1 (LPA1) gene alteration in pancreatic
carcinogenesis, we investigated mutations in the LPA1 gene in hamster pancreatic duct adenocarcinomas (PDAs) and
established cell lines. Female Syrian golden hamsters received 30 mg/kg of N-nitrosobis(2-oxopropyl)amine (BOP)
followed by repeated exposure to an augmentation pressure regimen consisting of a choline-deficient diet combined with
DL-ethionine and then L-methionine and a further administration of 20 mg/kg BOP. A total of 10 PDAs obtained 10
weeks after beginning the experiment and three cell lines established from subcutaneously transplantable PDAs in
syngeneic hamsters were examined for mutations using reverse transcription-polymerase chain reaction-single strand
conformation polymorphism (RT-PCR-SSCP) analysis. A mutation was detected in only one PDA (1/10, 10%) in the
form of a GGA to GTA (Gly to Val) transversion at codon 355, and no mutations were detected in the three cell lines.
These results suggest that the LPA1 gene mutation may play roles in a limited fraction of BOP-induced pancreatic duct
carcinogenesis in hamsters. (J Toxicol Pathol 2009; 22: 89–92)
Key words: pancreatic duct adenocaricinoma, LPA1, mutation, hamster, nitrosamine
Pancreatic duct adenocarcinomas (PDAs) have one of
the lowest cure rates among human malignancies1. It is
important to understand the molecular mechanisms
underlying pancreatic carcinogenesis. However, at present,
very little information about rate-limiting molecular events
is available. Experimental models suitable for investigation
of human PDA development have been established in
hamsters using the carcinogen N-nitrosobis(2-
oxopropyl)amine (BOP)2, and to facilitate studies on the
underlying mechanisms, a rapid production approach has
previously been developed3,4. Indeed, we have reported
several genetic and epigenetic changes in this model. For
example, Ki-ras mutations are frequently found in the early
stages of pancreatic ductal carcinogenesis, but infrequent
mutation of Smad4 gene is detected in PDAs5,6. In addition,
we have provided aberrant DNA methylation in tumor
suppressor genes, such as p16, E-cadherin, Tslc1 and
Lysophosphatidic acid (LPA) is a bioactive mediator
that induces diverse cellular effects, including regulation of
cell proliferation, differentiation, transcellular migration,
morphogenesis and protection from apoptosis11–16. LPA can
induce cell proliferation, migration, invasion and production
of angiogenic factors in human ovarian cancer cell lines,
suggesting that LPA may play an important role in the
development of tumor cells12,13,15–19. LPA also interacts with
at least five G protein-coupled transmembrane receptors,
lysophosphatidic acid receptor-1 (LPA1), LPA2, LPA3,
LPA4 and LPA518–21. LPA1 is ubiquitously expressed in
normal tissues, but the expressions of other LPA receptor
subtypes are relatively restricted, suggesting that these
receptors may have different biological functions regarding
LPA7,18,19. Recently, aberrant expressions of LPA1 have
been reported in human and rat tumors, demonstrating that
Received: 11 November 2008, Accepted: 9 December 2008
Mailing address: Toshifumi Tsujiuchi, Laboratory of Cancer Biology
and Bioinformatics, Department of Life Science, Faculty of Science
and Engineering, Kinki University, 3–4–1 Kowakae, Higashiosaka,
Osaka 577-8502, Japan
TEL: 81-6-6721-2332 FAX: 81-6-6730-5880
90LPA1 Mutation in Hamster Pancreatic Tumors
alteration of LPA1 expression might be important in
malignant transformation of tumor cells as well as LPA per
se12,13,16,17,22,23. Moreover, we have reported that loss of
LPA1 expression is due to its aberrant DNA methylation in
rat tumor cell lines24.
Recently, we reported frequent mutations of the LPA1
gene in rat hepatocellular carcinomas (HCCs) induced by N-
nitrosodiethylamine and a choline-deficient L-amino acid
defined diet25. In the present study, to assess the
involvement of the LPA1 gene in pancreatic carcinogenesis,
we investigated mutations of the LPA1 gene in hamster
PDAs induced by BOP and three established cell lines.
A total of 12 female Syrian golden hamsters, weighing
approximately 100 g each, were used in the present study
(Japan SLC Inc., Shizuoka, Japan). PDAs were induced in
10 animals according to the rapid production model3,4.
Briefly, BOP (30 mg/kg body weight) (Nacalai Tesque, Inc.,
Kyoto, Japan) was given subcutaneously as the initiation,
followed by two cycles of augmentation pressure consisting
of choline-deficient diet administration and ethionine-
methionine-BOP injection. To obtain normal control
tissues, including the pancreas, the remaining 2 animals were
untreated and maintained free from carcinogen exposure
throughout the experimental period. All hamsters were
sacrificed under light ether anesthesia 10 wk after the
beginning of the experiment, and their pancreases were
immediately excised. Macroscopically apparent nodules
were dissected from the surrounding tissue and frozen in
liquid nitrogen. Portions of the nodules were also fixed in
10% neutrally buffered formalin at 4°C, routinely processed
for embedding in paraffin, sectioned and stained with
hematoxylin and eosin for histological examination.
The details of establishment of the three cell lines,
HPD-1NR, HPD-2NR and HPD-3NR, have been reported
previously26. Frozen cell lines were cultured in Dulbecco’s
modified Eagle’s medium (Nissui Pharmaceutical Co., Ltd.,
Tokyo, Japan) containing 10% fetal bovine serum (Flow
Laboratories, McLean, VA, USA), 2 mM L-glutamine, 100
U/ml penicillin and 100 mg/ml streptomycin sulfate.
Total RNA was prepared from frozen normal liver
tissue using an ISOGEN kit (Nippon Gene, Inc. Toyama,
Japan), and first-strand cDNA was synthesized from 0.5 μg
aliquots with Ready-To-Go Your-Prime First-Strand Beads
(GE Healthcare UK Ltd., Buckinghamshire, England). To
determine the sequences of the open reading frame (ORF)
and 5’ upstream and 3’ downstream regions, PCR
amplifications were performed with primer sets based on the
rat LPA1 cDNA sequence (NCBI accession number
NM_053936) as described previously8–10. The amplified
products were separated on 1% NuSieve agarose gels (BMA,
Rockland, ME, USA) containing 0.05 μg/ml ethidium
bromide, extracted and directly sequenced with a BigDye
Terminator v3.1 Cycle Sequencing Ready Reaction Kit
(Applied Biosystems Japan Ltd., Tokyo, Japan) and an ABI
PRISM 310 genetic analyzer (Applied Biosystems Japan
Total RNA was prepared from the 10 frozen PDA
samples, 3 cell lines and 2 normal pancreases using an
ISOGEN kit (Nippon Gene, Inc.), and then the first-strand
cDNA was synthesized from 0.2 μg aliquots with Ready-To-
Go Your-Prime First-Strand Beads (GE Healthcare UK
Ltd.). To eliminate possible false positives caused by
residual genomic DNA, all samples were treated with
RT-PCR-SSCP analysis was performed with the
primers listed in Table 1. All were designed from the
hamster LPA1 cDNA sequence obtained in the above
analysis. Briefly, PCR for SSCP was performed in 10 μl of
reaction mixture containing 1 μM of each primer, 200 μM of
each dNTP, 1×PCR buffer (Applied Biosystems Japan Ltd.),
2.5 units of Ampli Taq (Applied Biosystems Japan Ltd.) and
0.5 μl of synthesized cDNA mixture under the following
reaction conditions: primary denaturation for 2 min at 95°C;
36 cycles of 15 s denaturation at 95°C, 15 s annealing at 58–
64°C and 1 min extension at 72°C; and a final extension for
5 min at 72°C. PCR products were diluted with 10 μl of
loading solution containing 90% formide, 20 mM EDTA and
0.05% xylene cyanol and bromophenol blue. Aliquots
containing 6 μl of diluted products were electrophoresed on
polyacrylamide gel using a GeneGel Excel 12.5/24 kit (GE
Healthcare UK Ltd.) at 5, 10, 15 and 20°C for 90 min at 15
W with a GenePhor Electrophoresis Unit (GE Healthcare
UK Ltd.). After electrophoresis, the gels were stained with a
DNA Silver Staining kit (GE Healthcare UK Ltd.).
Following RT-PCR-SSCP analysis, the DNA fragment
from the abnormal shift band in the gel was extracted and
reamplified. The obtained PCR product was directly
sequenced using a BigDye Terminator v3.1 Cycle
Sequencing Ready Reaction Kit (Applied Biosystems Japan
Ltd.) and an ABI PRISM 310 genetic analyzer (Applied
Biosystems Japan Ltd.). To confirm the results, PCR
amplification was repeated using the same samples, and each
PCR product was sequenced with the forward and reverse
primers at least twice.
Nodules developed in all 10 hamsters treated with BOP.
These lesions were evaluated according to the diagnostic
criteria described previously3,4. All nodules obtained were
histologically well-differentiated PDAs. Since there is no
genetic information available about the hamster LPA1 gene,
we first identified the ORF of the hamster LPA1 gene cDNA
sequence (GenBank accession number AB257088). Based
Table 1. Primers Used for RT-PCR-SSCP Analysis
cDNA location PrimersAnnealing
nt -20 - 315 1F: 5’-TTTCAGACTACAGCACCGTC-3’
nt 260 - 57362
nt 533 - 85264
Tsujiuchi, Furukawa, Obo et al.
on this sequence, primers for the RT-PCR-SSCP analysis
were designed (Table 1). The amplified PCR products with
these primer sets indicated a clear single band in 1% agarose
gel. Homozygous deletion was not found. No changes of
LPA1 gene expression were found any of the PDAs
compared with normal pancreatic tissues (data not shown).
Representative results of the RT-PCR-SSCP and
sequencing analyses are shown in Fig.1 (A) and (B),
respectively. One out of 10 PDAs (10% incidence) produced
an abnormal band shift using the primer set of 4F-4R.
Sequence analysis revealed the mutation to be a GGA to
GTA (Gly to Val) transversion at codon 355. Although this
codon is located in the intracellular domain of the carboxyl-
terminal end, which is important in activation of several
biological signaling pathways11,27, it is unclear whether this
mutation can affect LPA1 function. Normal sized PCR
products amplified from 1F-1R, 2F-2R and 3F-3R produced
no mutations (data not shown).
In human tumors, aberrant expression levels have been
reported for the LPA1 gene12,16,17,22, whereas there have been
no reports of LPA1 mutations. Although the reported
expression level of LPA1 has varied, no consistent change
between normal and transformed epithelial ovarian cancer
cells has been found in ovarian cancer cells12,16,17. By
contrast, reduced expression of LPA1 gene has been
detected in human colorectal cancers, suggesting that
reduction of LPA1 expression may occur during malignant
transformation22. Previously, we reported that the promoter
region of the LPA1 gene is highly methylated in rat tumor
cell lines, correlating with loss of LPA1 expression24. In our
recent study, we detected frequent mutations of LPA1 gene
in HCCs induced by N-nitrosodiethylamine and a choline
deficient L-amino acid defined diet (46.7% and 41.7%
incidences, respectively)25. Therefore, if LPA1 gene acts as
a tumor suppressor gene, mutation of the LPA1 gene may
play important roles in inactivating LPA1 during
G/C to A/T transition is considered a common mutation
induced by ethylating N-nitroso compounds28. However, in
the present study, one mutation of LPA1 was a G/C to T/A
transversion. By contrast, the Ki-ras mutations were all G/C
to A/T transitions at codon 125. Therefore, it seems that the
Ki-ras mutations were caused by BOP per se and that the
LPA1 mutation may have been due to some other factors,
such as DNA damage caused by chronic oxidative stress,
acting during pancreatic carcinogenesis as a result of BOP.
In conclusion, we found infrequent mutation of the
LPA1 gene in hamster PDAs induced by BOP, suggesting
that LPA1 gene mutation may play roles in a limited fraction
of BOP-induced pancreatic duct carcinogenesis in hamsters.
The observed mutation frequency was markedly lower than
that of rat liver tumor cases. It seems this discrepancy may
be due to organ or species differences. To better understand
the involvement of LPA receptors in pancreatic duct
carcinogenesis, alterations of other receptors, such as LPA2
and LPA3, should be further studied.
Acknowledgments: This study was supported in part by the
Foundation for Promotion of Cancer Research in Japan, a
Grant-in-Aid (20591765) for Scientific Research from the
Ministry of Education, Culture, Sports, Science and
Technology of Japan and by grants (RK17-027, RK-027)
from the Faculty of Science and Engineering, Kinki
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Fig. 1. Mutation of the LPA1 gene in hamster PDAs and established
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