Download Transgenic rats carrying copies of the human c-Ha

Carcinogenesis vol.21 no.7 pp.1391–1396, 2000
Transgenic rats carrying copies of the human c-Ha-ras
proto-oncogene exhibit enhanced susceptibility to
N-butyl-N-(4-hydroxybutyl)nitrosamine bladder carcinogenesis
Tomonori Ota1,5,6, Makoto Asamoto1,
Hiroyasu Toriyama-Baba1, Fumi Yamamoto1,
Yoichiro Matsuoka1, Takahiro Ochiya2, Takao Sekiya3,
Masaaki Terada4, Hideyuki Akaza5 and
Hiroyuki Tsuda1,7
1Experimental
Pathology and Chemotherapy Division, 2Section for Studies
on Metastasis, 3Oncogene Division and 4Director, National Cancer Center
Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo 104-0045, 5Department
of Urology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1,
Tennodai, Tsukuba City, Ibaraki 305-8575, and 6Department of Urology,
Tokyo Metropolitan Komagome Hospital, 3-18-22 Hon-Komagome,
Bunkyo-ku, Tokyo 113-8677, Japan
7To
whom correspondence should be addressed
Email: [email protected]
We have established a transgenic rat line carrying three
copies of the human c-Ha-ras proto-oncogene with its own
original promoter region, Jcl/SD-TgN(HrasGen)128Ncc
(Hras128) rat. c-Ha-ras protein from expression of transduced and endogenous c-Ha-ras genes could be detected in
the bladder epithelium of untreated transgenic rats. To
examine their susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis, male transgenic and wild-type littermates were treated
with 0.05% BBN in their drinking water for 10 weeks and
then killed at week 20. The numbers and volumes of total
macroscopic bladder tumors including both transitional
cell papillomas and carcinomas (TCC) per rat were much
greater in Hras128 rats than in their wild-type counterparts. The numbers of carcinomas per rat were also
significantly greater in Hras128 rats. Two cases of TCC
exhibiting invasion of the bladder muscle layer, which
is extremely rare in the wild-type animals under the
experimental conditions used, were also observed in
Hras128 rats. The GGC→GAC mutations at codon 12 of
the transgene were observed in only two TCC out of 21
bladder tumors (9.5%), assessed by RFLP analysis and
direct sequencing. SSCP analysis did not show any endogenous c-Ha-ras gene mutations. One of 25 tumors (4.0%) in
wild-type rats had an endogenous c-Ha-ras gene mutation
at codon 12 that was detected (GGA→GAA) by singlestrand conformation polymorphism and direct sequencing.
These results indicate that the Hras128 rat is highly
susceptible to BBN carcinogenesis and may be utilized as
a rat model for analysis of bladder tumor development.
The mutation findings indicate that the enhanced tumor
development is not primarily due to mutations occurring
in the transgene.
Introduction
Various kinds of transgenic and knockout mice have proved
to be good animal models for analysis of gene functions. In
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; Hras128, Jcl/
SD-TgN(HrasGen)128Ncc; RFLP, restriction fragment length polymorphism;
SSCP, single-strand conformation polymorphism; TCC, transitional cell carcinoma.
© Oxford University Press
the chemical carcinogenesis field, transgenic mice carrying
human c-Ha-ras proto-oncogenes (rasH2 mice) have been
shown to be highly susceptible to various carcinogens (1–6).
However, rats have been more frequently used for analysis of
events occurring during tumor development. There are many
more basic experimental data available for rats than mice
regarding chemical carcinogenesis, and a variety of preneoplastic lesions, including liver enzyme-altered foci, for
example, are available in rats as markers (7–13). In the urinary
bladder, carcinomas in human and animals are classified into
two categories, superficial and invasive. Those induced by
N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats are
mostly of the superficial type, while the invasive type is more
frequent in mice. In the latter, ~50% of carcinomas are
squamous cell carcinomas, whereas in rats most are transitional
cell carcinomas (TCCs) (14). In the human bladder, ~70% of
TCCs are classified as superficial type at the onset. They are
associated with frequent recurrence, and ~10% progress to
exhibit invasion of the muscle layer (15). The reasons for such
recurrence and invasive characteristics have not yet been
elucidated in detail. Therefore, it is important to establish
appropriate animal models featuring highly malignant superficial type carcinomas as observed in human cases.
The ras genes, which are involved in the regulation of cell
proliferation and differentiation, are frequently activated by
somatic point mutations in a variety of human and experimental
animal cancers (16,17). Activation of Ha-ras genes has been
reported in 5–18% of human bladder cancer cases (18–20),
while overexpression without any mutation may be associated
with change from superficial to invasive phenotype (21).
Recently, we generated transgenic rats carrying three copies
of the human c-Ha-ras proto-oncogene [Jcl/SD-TgN
(HrasGen)128Ncc (Hras128) rats] and found females to be
highly susceptible to mammary carcinogenesis, mutations of
the transgene not playing a major role (22). In the present
paper, we report that the rats are also highly susceptible to
BBN-induced urinary bladder carcinogenesis, again without
frequent gene mutation.
Materials and methods
Transgenic rats
The Hras128 rat line (22) was generated by injecting copies of the human
c-Ha-ras proto-oncogene into pronuclei of fertilized rat oocytes from Sprague–
Dawley rats obtained from Clea Japan, Inc. (Tokyo, Japan). Sexually mature
Sprague–Dawley female rats were induced to superovulate using pregnant
mare’s serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG).
Fertilized eggs were collected and microinjected with BamHI fragments of
the human c-Ha-ras proto-oncogene with its own promoter region. After
microinjection, embryos at the two-cell stage were transplanted into foster
females mated with vasectomized males. The DNA construct utilized for the
transgenic rats has been described previously (23). In order to determine the
presence of the transgene, DNA samples from the tail were isolated by the
proteinase K phenol–chloroform method. PCR reactions were performed using
Ampli Taq Gold (Perkin Elmer, NJ) and human c-Ha-ras exon 2 specific
primers (hHras2F, 5⬘-AGCCCTGTCCTCCTGCAGGAT-3⬘ and hHras2R,
5⬘-GGCCAGCCTCACGGGGTTCA-3⬘) which amplify a 218 kb fragment of
the human c-H-ras proto-oncogene at an annealing temperature of 63°C with
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T.Ota et al.
35 cycles. DNAs were digested with HindIII, XhoI, XbaI, EcoRI or SacI and
Southern blots were probed with a 32P-labeled SacI fragment of the human
c-Ha-ras proto-oncogene. The number of copies of the transgene was
determined from the restriction enzyme cleaving pattern and the levels of
hybridization signals in 10 µg of genomic DNA, in comparison with signals
for known human c-Ha-ras proto-oncogene amounts. Signals were measured
with a BAS2000 image analyzer (Fuji Film Co., Tokyo, Japan). Subsequently,
mating was carried out between transgenic and wild-type littermates, resulting
in transgenic offspring according to Mendelian genetics. mRNA expression
from the transgene has been detected in all organs examined, including the
urinary bladder, using northern blotting (22). The human c-Ha-ras protooncogene mRNA specific oligonucleotide probe (5⬘-GGGGTTCCGGTGGCATTTGG-3⬘) for northern blotting was labeled using [γ-32P]ATP and T4
polynucleotide kinase (TaKaRa, Otsu, Japan).
Bladder carcinogenesis study
Twenty transgenic and 15 wild-type littermate male rats of the Hras128 line
were treated with 0.05% BBN (Tokyo Chemical Industry Co., Tokyo, Japan)
in their drinking water for 10 weeks starting at 6 weeks of age. Intake of
basal diet (Oriental Yeast Co., Tokyo, Japan) and drinking water during the
carcinogen administration period was recorded. Ten weeks after the end of
BBN treatment, the rats were killed and their bladders were removed for
histological evaluation and mutation analysis of the transduced human
c-Ha-ras proto-oncogene and the endogenous rat c-Ha-ras gene in tumors.
The bladders were fixed in ice-cold acetone and processed for paraffin
embedding for hematoxylin and eosin staining and mutation analysis. The
diameters of neoplastic lesions were measured to allow volumes to be calculated.
Immunoblotting
For the purpose of analyzing the transduced protein expression in the bladder,
mucosal epithelium from normal and transgenic and wild rats was scraped
with a clean slide glass then solubilized in 0.5 ml of 50 mM Tris–HCl pH
7.5, 6 M urea, 1% SDS, 5 mM sodium EDTA, 1 mM DTT, 10 µg/ml leupeptin,
1 mM PMSF. Proteins (10 µg) were separated by SDS–PAGE on 20% gels
in 0.1% SDS. After electrophoresis, the proteins were transferred to polyvinilidene difluoride membrane and probed with a mouse monoclonal antibody
specific to c-Ha-ras protein (Calbiochem, San Diego, CA). The bound antibody
was then detected by a horseradish peroxidase conjugated goat anti-mouse
IgG1 antibody (Southern Biotechnology Associates, Inc., Birmingham, AL)
and an enhanced chemiluminescence system (Amersham Pharmacia Biotech,
Buckinghamahire, UK). Data for density were analyzed with NIH Image
software.
Mutation analysis
DNAs from paraffin sections (20 µm) of bladder tumors were isolated with
DEXPAT® (TaKaRa) and amplified by PCR. For detection of mutations of
the transgene, DNA was analyzed by the PCR–restriction fragment length
polymorphism (RFLP) method, and for the endogenous c-Ha-ras gene,
PCR–single-strand conformation polymorphism analysis (SSCP) of codon 12
in the transgene was performed to detect the presence of an MspI-digestionresistant band. A 167 bp fragment amplified with primers hHras1F
(5⬘-GCAGGCCCCTGAGGAGCGAT-3⬘) and hHras1RN (5⬘-AGCAGCTGCTGGCACCTGGA-3⬘) at an annealing temperature of 60°C with 35 cycles
could clearly be distinguished from the 117 and 50 bp fragments resulting
from cleavage of the wild-type sequence (GGC). Similarly, detection of
mutations in codon 61 revealed the presence of an AlwNI-resistant band. An
AlwNI site was generated by use of a mismatched primer (H61/2A2,
5⬘-CGCATGGCGCTGTACAGCTC-3⬘). For the first round PCR, 218 bp
fragments of exon 2 were amplified by primers hHras2F and hHras2R at an
annealing temperature of 63°C with 35 cycles. They were then diluted 100
times with distilled water and used as templates for the second round PCR
using primers hHras2F and H61/2A2 at an annealing temperature of 60°C
with 35 cycles. The wild-type sequence (CAG) is cleaved into 93 and 17 bp
by AlwNI, while fragments with mutations in codon 61 remain undigested,
giving a 110 bp band. Digested samples of each reaction were electrophoresed
on 2 or 4% agarose gels. When an enzyme-resistant band could be visualized,
the responsible fragments were amplified using hHras1F and hHras1R
(5⬘-GAGCTCACCTCTATAGTGGG-3⬘) for codon 12 and hHras2F and H61/
2A2 for codon 61, and then directly sequenced using 32P-end-labeled primers,
hHras1F for codon 12 and cHras2IF (5⬘-CTGCGGTTCCTCCGG-3⬘) for
codon 61 with a TaKaRa Taq Cycle Sequencing Kit.
For the SSCP analysis, PCR reactions were performed using Ampli Taq
Gold and primers were labeled with [γ-32P]ATP and T4 polynucleotide kinase
(TaKaRa). Primers for exon 1 were rHras1F (5⬘-GCGATGACAGAATACAAGCT-3⬘) and rHras1R (5⬘-GAGCTCACCTCTATAGTGGG-3⬘), and for
exon 2 were cHras2IF and cHras2IR (5⬘-CACCTGTACTGGTGGATGTC-3⬘).
The rat c-Ha-ras gene was amplified at annealing temperatures of 55°C for
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exon 1 and 51°C for exon 2, and labeled products were analyzed in 5% nondenaturing acrylamide gels (49:1) with 5% glycerol at 4 or 20°C. DNAs
demonstrating mutations were isolated from acrylamide gels for SSCP. Base
substitutions were analyzed with a TaKaRa Taq Cycle Sequencing Kit under
the same conditions as described above. Amplified and labeled DNAs were
analyzed in 6% denaturing acrylamide gels.
Statistical analysis
Comparisons between transgenic and wild-type rats were made with the
Mann–Whitney U-test using Stat View 4.5J for the Macintosh, P ⬍ 0.05
being considered significant.
Results
Intake of basal diet and drinking water (~15 g/day) during
carcinogenic treatment and body weights did not show significant difference between Hras128 and wild-type rats. Clinical
signs for the late-stage bladder carcinomas such as gross
hematuria and emaciation were not noted in either case.
Although incidences of bladder tumors including transitional
cell papillomas and TCCs did not differ, the number and
volume of grossly evident total tumors per rat were significantly
larger in Hras128 rats (number, 13.2 ⫾ 6.9/rat; volume, 836
⫾ 937 mm3/rat) as compared with the wild-type cases (number,
7.1 ⫾ 5.1/rat; volume, 130 ⫾ 180 mm3/rat; P ⬍ 0.01) (Figure
1). The number of TCCs per animal also showed a significant
increase in Hras128 rats (4.7 ⫾ 4.2/rat as compared with
1.3 ⫾ 1.1/rat; P ⬍ 0.05) (Table I). Furthermore, in two
transgenic animals, lesions demonstrated clear invasion of the
muscular layer (T2⬍), pathological grade 3 (Figure 2). Most
of the carcinomas were classified into the superficial type (Ta),
pathological grade 2. Twenty-one tumors (nine papillomas and
12 TCCs) from transgenic rats and 25 tumors (12 papillomas
and 13 TCCs) from wild-type littermates were examined.
RFLP analysis revealed that only two TCCs out of 21 tumors
(9.5%) in transgenic rats had mutations in codon 12 (Figure
3A). GGC→GAC base substitutions in the transgenes were
detected by direct sequencing (Figure 3B). Only one mutation
(4.0%) in a transitional cell papilloma at exon 1 of the
endogenous c-Ha-ras gene was found in lesions from wildtype rats by SSCP (Figure 4). This was a base substitution
from GGA to GAA in codon 12. The two TCC cases with
muscle invasion did not show any mutations in either the
transduced or the endogenous c-Ha-ras genes.
Immunoblot analysis of normal bladder mucosa revealed
untreated transgenic rats to express 2.1-fold the level of total
c-Ha-ras protein found in wild-type (Figure 5).
Discussion
The present study revealed that the Hras128 rat has enhanced
susceptibility to BBN bladder carcinogenesis. Thus, the number
and volume of macroscopic tumors including transitional cell
papillomas and TCCs per rat were significantly greater than
in their wild-type counterparts, and the numbers of carcinomas
per rat were also significantly greater in Hras128 rats. Furthermore, two carcinoma lesions exhibited obvious invasion of
the muscular layer in Hras128 rats. Since we did not perform
periodic killing, data regarding latent period are not available.
However, since tumor multiplicity was ~2-fold greater in
Hras128 as compared with wild-type rats at week 20, it might
have been slightly shorter in the transgenic rats, as observed
previously for mammary carcinogenesis cases (22).
Mutation analysis revealed that only two TCCs out of 21
tumors (9.5%) showed a mutation in codon 12 of the transgene,
and one papilloma out of 25 tumors (4.0%) in wild-type rats
Bladder carcinogenesis in c-Ha-ras transgenic rat
Fig. 1. Macroscopic findings for representative bladders. The tumor number and volume are clearly larger in a Hras128 rat (A) as compared with in a wildtype counterpart (B).
Table I. Incidences and multiplicity of bladder tumors in Hras128 and non-transgenic rats treated with BBN
Line
Hras128
non-Tge
No. rats
20
15
Papillomas
Tumors (papillomas ⫹ carcinomas)a
Carcinomas
No. rats (%)
No./ratb
No. rats (%)
No./ratb
No. rats (%)
No./ratb
Vol./rat (mm3)
19 (95.0)
15 (100)
6.9 ⫾ 4.2
4.5 ⫾ 3.1
14 (70.0)
11 (73.3)
4.7 ⫾ 4.2c
1.3 ⫾ 1.1
19 (95.0)
15 (100)
13.2 ⫾ 6.9d
7.1 ⫾ 5.2
836 ⫾ 937d
130 ⫾ 180
aMacroscopic
findings.
⫾ SD.
cStatistically significant by Mann–Whitney U-test (P ⬍ 0.05).
dStatistically significant by Mann–Whitney U-test (P ⬍ 0.01).
eNon-transgenic rats.
bMeans
had a mutation in codon 12 of the endogenous c-Ha-ras gene.
We have reported that this Hras128 rat is highly susceptible to
mammary carcinogenesis induced by N-methyl-N-nitrosourea
(MNU). All the Hras128 rats were found to rapidly develop
multiple and large mammary carcinomas within an 8 week
period after MNU injection, whereas wild-type rats had no
or only small carcinomas. Although the large majority of
carcinomas (86.4%) contained cells with mutations involving
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T.Ota et al.
Fig. 4. Representative results of SSCP analysis for exon 1 (codon 12) of the
endogenous c-Ha-ras gene. One tumor in a wild-type rat demonstrates a
mobility-shifted band (arrow).
Fig. 2. Invasive bladder carcinoma induced by BBN in a Hras128 rat.
Carcinoma cells show obvious invasion of the muscle layer. Magnification
100⫻.
Fig. 5. Comparison of levels of the transgene and rat endogenous c-Ha-ras
protein expression in the bladder epithelium from Hras128 and wild-type
rats (arrow). The total c-Ha-ras protein (Ras) value for transgenic rats is
2.1-fold that for the wild-type.
Fig. 3. Representative results of RFLP analysis for codon 12 of the
transgene. (A) Lanes 1–11, bladder tumors induced in Hras128 rats; lane 12,
normal liver of a Hras128 rat; lane 13, T24 having a codon 12 GGC→GTC
mutation. Lanes 6 and 7 demonstrate an MspI-resistant band indicating the
presence of a mutation in codon 12. (B) Results of direct sequencing
analysis of an MspI-resistant mutant band indicating a codon 12
GGC→GAC mutation (arrow).
codon 12 in exon 1, the non-mutated cell population in each
of the carcinomas was in the majority. In contrast, wild-type
rats had mutations in codon 12 but not in codon 61 of the
endogenous c-Ha-ras gene in 28.6% of mammary carcinomas
(22). These and the current results for bladder carcinogenesis
indicate that the presence of the human c-Ha-ras gene clearly
enhances mammary and bladder carcinogenesis and that
mutations of the transduced human c-Ha-ras proto-oncogene
do not play a major role.
In human cases, 5–18% of bladder cancers have been
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reported to have mutations in the c-Ha-ras gene (18–20), but
enhanced expression of ras protein p21 is frequently observed
(24) especially in advanced-grade cancers, correlating with
tumor progression (25–27). In BBN-induced rat bladder carcinogenesis, c-Ha-ras gene mutations have been observed only
in 10–48% of tumors, while enhanced expression of ras p21
protein was also found in ⬎80% of tumors (28,29). Similar
mutation frequencies of the endogenous c-Ha-ras gene have
been reported without obvious correlation to any stage in
carcinoma development in bladder tumors induced by N-[4(5-nitro-2-furyl)-2-thiazolyl]formamide (30). Our Hras128 rats
demonstrated enhanced tumor growth with only a limited
population of cells with mutations of the transgene. These
observations support the conclusion that integration of the cHa-ras transgene may be of greater importance than a mutation
itself for bladder carcinogenesis.
About 70% of human urinary bladder cancer cases are of
superficial type. They can be removed by transurethral resection, but recurrence occurs in the lesion of 60% of patients
within 5 years, and ~10% exhibit muscular invasion or remote
metastasis, in spite of preventive therapy such as intravesical
instillation of chemotherapeutic agents or Bacillus Calmette–
Guerine (15). Therefore, it is important to elucidate the
underlying mechanisms of recurrence, progression and metastatic nature of superficial bladder cancer. With regard to rat
urinary bladder models, although abundant information is
available about the biological sequence from preneoplasia
to tumors, little is known regarding conversion to greater
malignancy due to a lack of appropriate experimental models
(31–38). In our Hras128 rats, it is of interest that the two cases
demonstrating obvious invasion of muscular tissue lacked
mutations in either the transgene or the endogenous c-Ha-ras
gene. On the other hand, overexpression of either wild or
mutated Ha-ras genes in RT-4 cells, achieved with a gene
transfection technique, was found to cause alteration from a
superficial to an invasive phenotype (21). This is in line with
the fact that mouse bladder carcinomas, which frequently
Bladder carcinogenesis in c-Ha-ras transgenic rat
exhibit a high incidence of invasion, generally lack mutations
of the c-Ha-ras gene (39).
Although, we have not analyzed c-Ha-ras protein levels in
tumor tissue, results with bladder epithelium from untreated
transgenic and wild-type rats indicated ⬎2-fold increase in
total protein derived from both endogenous and transduced
genes in the former. This may support the hypothesis that
overexpression of c-Ha-ras protein is important for the
enhanced susceptibility.
Thus, the Hras128 rat treated with the bladder carcinogen
BBN might serve as a good model for analysis of the correlation
between c-Ha-ras gene overexpression and malignant progression.
Acknowledgements
We thank Mikiko Oba (Morinaga Milk Industry Co., Japan) for assistance
regarding production of transgenic rats, and Akira Ando (postgraduate student
of Nihon University) and Hiroki Suzuki (undergraduate student of Nihon
University) for their assistance. We also thank Dr Malcolm A.Moore for his
kind advice during preparation of the manuscript. This study was supported
in part by a Grant-in-Aid for the Second Term Comprehensive 10-Year
Strategy for Cancer Control, a Grant-in-Aid for Cancer Research from the
Ministry of Health and Welfare of Japan, a Grant-in-Aid from the Ministry
of Education, Science, Sports and Culture of Japan, a research grant from the
Princess Takamatsu Cancer Research Fund, and a CREST (Core Research for
Evolutional Science and Technology) Grant-in-Aid from the Japan Science
and Technology Corporation (JST). T.O. was the recipient of a fellowship
from the Foundation for Promotion of Cancer Research, Tokyo, Japan when
this work was performed.
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Received December 16, 1999; revised March 27, 2000; accepted March
29, 2000