Carcinogenesis vol.20 no.4 pp.
715–718, 1999
LOH and mutational analysis of p53 alleles in mouse urinary
bladder carcinomas induced by N-butyl-N-(4-hydroxybutyl)
nitrosamine
Keiichirou Morimura2, Shinji Yamamoto,
Takashi Murai1, Satoru Mori1, Tian-Xin Chen,
Hideki Wanibuchi and Shoji Fukushima
First Department of Pathology, Osaka City University Medical School,
1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585 and 1Aburahi Laboratories,
Shionogi Research Laboratories, Shionogi Co. Ltd, Gonda, Koka-chou,
Koka-gun, Shiga 520-34, Japan
2To whom correspondence should be addressed
Email: m3886644@misc.med.osaka-cu.ac.jp
In human urinary bladder carcinogenesis, alterations in
the p53 tumor suppressor gene are common events. We
have previously reported that they are also frequent in
invasive urinary bladder carcinomas induced by N-butyl-
N-(4-hydroxybutyl)nitrosamine (BBN) in NON/Shi mice.
To further investigate the significance of the p53 gene status
for mouse urinary bladder carcinogenesis, we examined
both allele loss and mutational alterations in urinary
bladder cancers of (NON/ShiHC3H/He/Shi) F1 hybrid
mice exposed to the carcinogen for 12 weeks and then
maintained for a further 9 weeks without treatment. An
intragenic silent polymorphism within exon 7 of the p53
gene between NON/Shi and C3H/He/Shi mice allows assess-
ment of allele loss of the p53 gene and determination of
the parental origin of mutated and/or lost alleles. A tissue
microdissection method was employed to obtain carcinoma
samples without excessive contamination with normal tis-
sue. Allele losses were detected in one of 14 tumors (7.1%)
and nine mutations in eight of 14 (57%) tumors were found
in exons 5–8 by polymerase chain reaction–single strand
conformation polymorphism followed by DNA direct
sequencing analysis. All mutations involved one base substi-
tution with an amino acid change, although the types of
base substitution were random. In conclusion, the high
incidence of p53 alterations suggests a significant role in
the genesis of invasive urinary bladder tumors in BBN-
treated mice.
Introduction
Tumor development is generally accepted to involve the
inactivation of specific tumor suppressor genes (1). In human
urinary bladder cancers, deletions in several chromosome
regions (e.g. chromosomes 4p, 8p, 9p, 9q, 11p, 13q, 17p) have
been described in a number of reports (2–7). The locus of
chromosome 17p which bears the p53 tumor suppressor gene
is known to be one of the most frequently deleted loci, with
a frequency up to 70% in high grade and/or invasive cases
(8–11), suggesting a strong association between p53 gene loss
of heterozygosity (LOH) and grade or stage. The p53 gene is
Abbreviations: BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; LOH, loss of
heterozygosity; PCR, polymerase chain reaction; SCC, squamous cell
carcinoma; SSCP, single strand conformation polymorphism; TCC, transitional
cell carcinoma.
© Oxford University Press
also frequently mutated in urinary bladder carcinomas, especi-
ally in more malignant lesions (11–14).
Downloaded from http://carcin.oxfordjournals.org at University of DundeeGardyne Road Library on June 24, 2010
Experimental carcinogenic rodent models are useful tools
to ascertain how these genetic alterations act in mammalian
cells in vivo, and analysis of differences and similarities of
molecular events between humans and rodents is therefore
important. Regarding urinary bladder carcinogenesis, several
experimental models have been established using chemical
carcinogens (15–21). N-butyl-N-(4-hydroxybutyl)nitrosamine
(BBN) induces urinary bladder carcinomas in rats and mice,
although the histological types and invasive behavior of the
carcinomas differ considerably (17,22). Those in the rat are
generally papillary and histologically of transitional and super-
ficial type (18,19). In contrast, mouse urinary bladder carcin-
omas are non-papillary and tend to demonstrate invasion
(16,17). In a recent study, we observed that invasive urinary
bladder carcinomas with metastasis induced by BBN in NON/
Shi mice (23) had a high frequency of p53 mutations, similar
to the case for human high-grade invasive carcinomas (16,24).
In the present study, to further investigate the involvement
of p53 gene status in mouse urinary carcinogenesis, we
analyzed both LOH and gene mutations in BBN-induced
mouse urinary bladder carcinomas. We employed (NON/
Shi3C3H/He/Shi) F1 hybrid mice, with which we could detect
not only mutations but also allelic losses based on the intragenic
silent polymorphism of exon 7 among NON/Shi mice by
polymerase chain reaction (PCR)–single strand conformation
polymorphism (SSCP) followed by direct sequencing.
Materials and methods
Animals and carcinoma induction
Male F1 hybrid mice (NON/Shi3C3H/He/Shi) were bred in Aburahi Laborat-
ories (Shionogi, Shiga, Japan) and on reaching 5 or 6 weeks of age were
treated with BBN (Tokyo Kasei Kogyo, Tokyo, Japan) at a concentration of
0.05% in drinking water for 12 weeks. They were then maintained without
any treatment for a further 9 week period. Male hybrid mice, five animals in
total, served as controls without any chemical supplement throughout the
experiment. All surviving mice, 23 animals in total, were killed under ether
anesthesia at the end of experimental week 21. Their urinary bladders were
excised and then fixed in acetone at 4°C. Samples including carcinoma tissues
were embedded in paraffin, serially sectioned at a thickness of 5–8 mm and
used for both nucleic acid extraction and for histological examination after
staining with H&E.
PCR–SSCP analysis and direct sequencing
Of the 23 surviving animals, 14 provided adequately preserved material that
was informative for gene analysis. A microdissection method was employed
to obtain selected populations of invasive carcinoma cells from acetone-fixed,
paraffin-embedded tissue, with orientation by comparison with adjacent serial
sections stained with H&E.
PCR–SSCP analysis followed by direct sequencing was carried out to
screen for mutations of exons 5–8 of the p53 gene as previously described
(16). The sequences of the primers are indicated in Table I. Most primers
included both exon and intron portions to avoid amplification of pseudogenes.
Small areas of SSCP gels corresponding to the positions of mobility shifted
bands were cut out and sequenced as previously described (16). In cases for
which mutations were found in exons 5, 6, or 8, PCR products were designed
to include both mutation and polymorphic site and amplified from DNAs
using the primers listed above. PCR products were subcloned into the pCR-
715
K.Morimura et al.
Fig. 1. Intragenic polymorphism in p53 exon 7 of the NON/Shi strain and
the corresponding sequence of the C3H/He/Shi strain.
Table I. Oligonucleotide primers used for the p53 gene amplification
Exon 5 (214 bp) 59-TCTCTTCCAGTACTCTCCTC-39a (sense)
59-AGGCGGTGTTGAGGGCTTAC-39 (antisense)
Exon 6 (181 bp) 59-GGCTTCTGACTTATTCTTGC-39 (sense)
39-CAACTGTCTCTAAGACGCAC-39 (antisense)
Exon 7 (170 bp) 59-TCACCTGGATCCTGTGTCTT-39 (sense)
59-CAGGCTAACCTAACCTACCA-39 (antisense)
Exon 8 (279 bp) 59-ACTGCCTTGTGCTGGTCCTT-39 (sense)
59-GGAGAGGCGCTTGTGCAGGT-39 (antisense)
aParts of primers included in introns are in bold.
Script SK(1) vector (Stratagene, La Jolla, CA) and 10 or more recombinant
colonies were picked up and amplified in 3 ml of LB culture medium. Double
strand DNA was extracted using a plasmid mini kit (Qiagen, Chataworth,
CA) and sequenced with a DNA Sequencing System (Model 373A; Applied
Biosystems, Foster City, CA) using the designed, T3 or T7 promoter primers.
Identification of the parental origin of mutational or lost alleles
In the case of exon 7, the parental origin of mutated alleles could be determined
by performing direct sequencing, based on intragenic polymorphism in that
exon among maternal NON/Shi mice (Figure 1). In a similar way, we could
directly discriminate the lost allele, comparing the mobility shifted band
patterns of the remaining allele to the patterns detected for control parents
maintained without any chemical supplement.
Statistical analysis
Significance of differences between histological types and frequencies of p53
gene mutations were analyzed using Fisher’s exact probability test (Stat View
SE1 Graphics; Abacus Concepts, Berkeley, CA).
Results
Histological findings
The 14 urinary bladder carcinomas induced by BBN were all
of the invasive type. One of them (sample 6) invaded the
prostate and two (samples 11 and 12) macroscopically pro-
truded from the urinary bladder, although no metastatic sites
were found. Histopathological assessment revealed eight to be
squamous cell carcinomas (SCCs) and six transitional cell
carcinomas (TCCs) as shown in Table II. Carcinoma in
situ (CIS) and/or dysplasia were commonly present in areas
adjacent to the invasive carcinomas.
Mutational analysis following direct sequencing and identi-
fication of the parental origin of the mutated and/or lost allele
Within the 110 bases of exon 7, the third letter of codon 248
in the NON/Shi strain shows a strain-specific ATC (Ile) to
ATT (Ile) silent polymorphism (Figure 1). This base substitu-
tion results in a mobility shift in SSCP gels and, therefore,
allows discrimination of the NON/Shi allele from the C3H/
He/Shi allele. Using DNAs prepared from both paternal and
maternal strain controls maintained throughout without chem-
ical supplement, we confirmed that there was no other poly-
morphism in the other exons examined (exons 5, 6 and 8), by
716
comparing the results with previous sequencing data (25).
PCR–SSCP analysis was carried out to screen for mutations
in exons 5–8 of the p53 gene and to detect the lost allele
(Figure 2). As indicated in Table II, mutations were found in
eight of 14 (57%) urinary bladder carcinomas. There were two
(14%), two (14%) and five (35%) mutations in p53 exons 5,
6 and 7, respectively, with no mutations found in exon 8.
Direct sequencing of mobility shifts of exon 7 on SSCP gels
revealed all except one mutation in exon 7 to involve the
paternal allele. In sample 12, the long PCR fragment encom-
passing exons 5–7 contained both a mutation in exon 5 (codon
Downloaded from http://carcin.oxfordjournals.org at University of DundeeGardyne Road Library on June 24, 2010
149) and a silent polymorphism in exon 7 (codon 248). Also,
we could detect the loss of the maternal NON/Shi allele in a
single carcinoma (one out of 14; 7.1%), harboring no mutation
on the remaining paternal allele. Only one tumor (sample 8)
contained two p53 mutations in different exons. All mutations
in p53 exons 5, 6 and 7 involved single base-pair substitutions
with an amino acid change. There were no evident hot-spots.
Of the total of nine mutations, seven were transversions and
the remaining two were transitions. No statistically significant
relation to pathological type was noted.
Discussion
The present investigation of p53 gene alterations in BBN-
induced urinary bladder carcinomas in (NON/Shi3C3H/He/
Shi) F1 hybrid mice revealed rare allelic losses and a high
frequency of mutations.
Tumor suppressor genes are inactivated by various mechan-
isms. In general, for loss of function of suppressor genes, it is
necessary that both alleles are disrupted. However, some
mutated forms of the p53 protein can disturb the wild-type
protein action, and missense mutations are believed to exert
trans-dominant negative effects on the normal allele (26),
although recent studies have demonstrated that the suppression
may be incomplete (27). Our recent results for BBN-induced
urinary bladder carcinomas in NON/Shi mice indicated that
the carcinomas, in particular those with metastasis, tended to
show more frequent p53 gene mutations than those which
were less malignant (24), suggesting that a mutant protein
contributed to tumor development and/or progression. In the
present study a high frequency of mutation but infrequent
allelic loss of the p53 gene was detected. Therefore, complete
disruption of this suppressor gene seems not to be necessary
for invasive bladder carcinogenesis in mice but a trans-
dominant negative effect of the mutated p53 protein may play
an important role.
In human bladder carcinomas, clarification of the histological
type is important because it is closely related to the prognosis.
Human urinary bladder urothelial cell carcinomas can be
classified into two types: papillary superficial and non-papillary
invasive carcinomas. The former have a good prognosis despite
frequent recurrences, while the latter often metastasize and
have a very poor prognosis. Mouse urinary bladder carcinomas
induced by chemical carcinogens show flat growth and invas-
iveness similar to the more malignant human type (17,23). In
this study, all of the BBN-induced tumors were invasive. Thus,
the mouse urinary bladder carcinoma model would appear to
have distinct advantages for studies of the invasive type of
human bladder tumors.
From the viewpoint of analysis of molecular alterations in
carcinogenesis, the model is important. Spruck et al. (28) have
suggested the participation of two distinct molecular pathways
 Role of p53 suppressor gene in urinary bladder carcinogenesis

Downloaded from http://carcin.oxfordjournals.org at University of DundeeGardyne Road Library on June 24, 2010
Fig. 2. PCR–SSCP analysis of exons 5, 6 and 7 of the p53 gene. No mobility band shifts were found for exon 8. N and C indicate control DNAs from
animals without chemical supplement, the mobility shift noted for exon 7 being due to the intragenic polymorphism of exon 7 in the NON/Shi strain.
Numbers for samples harboring mobility shift bands are underlined. *Animals with allele loss.

Table II. Summary of data for allele losses and p53 mutations found in mouse urinary bladder carcinomas

Tumor no. Histology LOH Lost allele Mutation Mutated allele Exon Codon Base change Amino acid change

1 SCC – –
2 TCC – 1 C3H 7 248 ATC→AAC Ile→Asn
3 TCC – 1 ND 6 205 GAC→GCC Asp→Ala
4 TCC – 1 ND 5 164 CAG→AAG Gln→Lys
5 SCC – 1 C3H 7 251 ATC→AGC Ile→Ser
6 TCC – –
7 SCC – 1 C3H 7 248 ATC→AAC Ile→Asn
8 SCC – 1 ND 6 192 ATC→ACC Ile→Thr
NON 7 248 ATT→AGT Ile→Ser
9 SCC 1 NON –
10 TCC – –
11 SCC – 1 C3H 7 258 AGT→AGG Ser→Arg
12 SCC – 1 NON 5 149 CCA→CAA Pro→Gln
13 TCC – –
14 SCC – –

ND, not detectable; NON, NON/Shi; C3H, C3H/He/Shi.

in human urinary bladder carcinogenesis. Papillary non-invasive
tumors as well as the invasive disease exhibit loss of chromosome
9 which bears the p16 tumor suppressor gene, while carcinomas
in situ contain a p53 gene mutation without chromosome 9 allelic
loss, suggesting participation of p53 gene alterations in the
development of high grade lesions with greater malignant poten-
tial. Moreover, there have been reports that LOH involving the
p53 gene is strongly associated with the invasive phenotype of
urinary bladder carcinomas (8,10). Recently, Ogawa et al. (29)
examined p53 mutations and LOH of chromosome 4 in BBN-
induced invasive mouse urinary bladder tumors. The mouse p16
gene is located on chromosome 4. They reported frequent p53
mutations and occasional LOH of chromosome 4. In our F1
mouse model, similar data were obtained concerning p53 muta-
tion, providing further evidence for the molecular comparability
of this model to human invasive urinary bladder carcinomas. To
further understand urinary bladder carcinogenesis, it is necessary
to investigate LOH of the p53 gene in tumors of greater malignant
potential, such as those with metastases.
In conclusion, the present study using a (NON/Shi3C3H/He/
Shi) F1 hybrid mouse bladder carcinogenesis model revealed
frequent p53 gene mutations but rare allelic loss of the gene.
Taking into account our findings and previous ones in the literat-
ure, p53 gene alterations may play an important role in tumor
progression of urinary bladder carcinogenesis in both man and
rodents.
717
K.Morimura et al.
References
1. Levine,A.J. (1993) The tumor suppressor genes. Ann. Rev. Biochem., 62,
623–651.
2. Knowles,M.A., Elder,P.A., Williamson,M., Cairns,J.P., Shaw,M.E. and
Law,M.G. (1994) Allelotype of human bladder cancer. Cancer Res., 54,
531–538.
3. Cairns,P., Proctor,A.J. and Knowles,M.A. (1991) Loss of heterozygosity
at the RB locus is frequent and correlates with muscle invasion in bladder
carcinoma. Oncogene, 6, 2305–2309.
4. Cairns,P., Shaw,M.E. and Knowles,M.A. (1993) Initiation of bladder cancer
may involve deletion of a tumour-suppressor gene on chromosome 9.
Oncogene, 8, 1083–1085.
5. Cairns,P., Shaw,M.E. and Knowles,M.A. (1993) Preliminary mapping of
the deleted region of chromosome 9 in bladder cancer. Cancer Res., 53,
1230–1232.
6. Tsai,Y.C., Nichols,P.W., Hiti,A.L., Williams,Z., Skinner,D.G. and
Jones,P.A. (1990) Allelic losses of chromosomes 9, 11, and 17 in human
bladder cancer. Cancer Res., 50, 44–47.
7. Fearon,E.R., Feinberg,A.P., Hamilton,S.H. and Vogelstein,B. (1985) Loss
of genes on the short arm of chromosome 11 in bladder cancer. Nature,
318, 377–380.
8. Habuchi,T., Ogawa,O., Kakehi,Y., Ogura,K., Koshiba,M., Sugiyama,T. and
Yoshida,O. (1992) Allelic loss of chromosome 17p in urothelial cancer:
strong association with invasive phenotype. J. Urol., 148, 1595–1599.
9. Oka,,K., Ishikawa,J., Bruner,J.M., Takahashi,R. and Saya,H. (1991)
Detection of loss of heterozygosity in the p53 gene in renal cell carcinoma
and bladder cancer using the polymerase chain reaction. Mol. Carcinogen.,
4, 10–13.
10. Miyamoto,H., Shuin,T., Ikeda,I., Hosaka,M. and Kubota,Y. (1996) Loss of
heterozygosity at the p53, RB, DCC and APC tumor suppressor gene loci
in human bladder cancer. J. Urol., 155, 1444–1447.
11. Fujimoto,K., Yamada,Y., Okajima,E., Kakizoe,T., Sasaki,H., Sugimura,T.
and Terada,M. (1992) Frequent association of p53 gene mutation in
invasive bladder cancer. Cancer Res., 52, 1393–1398.
12. Hollstein,M., Sidransky,D., Vogelstein,B. and Harris,C.C. (1991) p53
mutations in human cancers. Science, 253, 49–53.
13. Olumi,A.F., Tsai,Y.C., Nichols,P.W., Skinner,D.G., Cain,D.R., Bender,L.I.
and Jones,P.A. (1990) Allelic loss of chromosome 17p distinguishes high
grade from low grade transitional cell carcinomas of the bladder. Cancer
Res., 50, 7081–7083.
14. Sidransky,D., Von Eschenbach,A., Tsai,Y.C. et al. (1991) Identification of
p53 gene mutations in bladder cancers and urine samples. Science, 252,
706–709.
15. Asamoto,M., Mann,A.M. and Cohen,S.M. (1994) p53 mutation is
infrequent and might not give a growth advantage in rat bladder
carcinogenesis in vivo. Carcinogenesis, 15, 455–458.
16. Yamamoto,S., Masui,T., Murai,T., Mori,S., Oohara,T., Makino,S.,
Fukushima,S. and Tatematsu,M. (1995) Frequent mutations of the p53
718
gene and infrequent H- and K-ras mutations in urinary bladder carcinomas
of NON/Shi mice treated with N-butyl-N-(4-hydroxybutyl)nitrosamine.
Carcinogenesis, 16, 2363–2368.
17. Tamano,S., Hagiwara,A., Suzuki,E., Okada,M., Shirai,T. and Fukushima,S.
(1991) Time- and dose-dependent induction of invasive urinary bladder
cancers by N-ethyl-N-(4-hydroxybutyl)nitrosamine in B6C3F1 mice. Jpn.
J. Cancer Res., 82, 650–656.
18. Ito,N., Hiasa,Y., Tamai,A., Okajima,E. and Kitamura,H. (1969) Histo-
genesis of urinary bladder tumors induced by N-butyl-N-(4-
hydroxybutyl)nitrosamine in rats. Gann, 60, 401–410.
19. Fukushima,S., Hirose,M., Tsuda,H., Shirai,T. and Hirao,K. (1976)
Histological classification of urinary bladder cancers in rats induced by
N-butyl-N-(4-hydroxybutyl)nitrosamine. Gann, 67, 81–90.
Downloaded from http://carcin.oxfordjournals.org at University of DundeeGardyne Road Library on June 24, 2010
20. Masui,T., Don,I., Takada,N., Ogawa,K., Shirai,T. and Fukushima,S. (1994)
p53 mutations in early neoplastic lesions of the urinary bladder in rats
treated with N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis, 15,
2379–2381.
21. Cohen,S.M., Arai,M., Jacobs,J.B. and Friedell,G.H. (1979) Promoting
effect of saccharin and DL-tryptophan in urinary bladder carcinogenesis.
Cancer Res., 39, 1207–1217.
22. Hirose,M., Fukushima,S., Hananouchi,M., Shirai,T. and Ogiso,T. (1976)
Different susceptibilities of the urinary bladder epithelium of animal
species to three nitroso compounds. Gann, 67, 175–89.
23. Murai,T., Mori,S., Hosono,M., Takeuchi,Y., Ohara,T., Makino,S.,
Takeda,R., Hayashi,Y. and Fukushima,S. (1991) Renal pelvic carcinoma
which shows metastatic potential to distant organs, induced by N-butyl-
N-(4-hydroxybutyl)nitrosamine in NON/Shi mice. Jpn. J. Cancer Res., 82,
1371–1377.
24. Yamamoto,S., Chen,T., Murai,T. et al. (1997) Genetic instability and p53
mutations in metastatic foci of mouse urinary bladder carcinomas induced
by N-butyl-N-(4-hydroxybutyl)nitrosamine. Carcinogenesis, 18, 1877–
1882.
25. Bienz,B., Zakut-Houri,R., Givol,D. and Oren,M. (1984) Analysis of the
gene coding for the murine cellular tumour antigen p53. EMBO J., 3,
2179–2183.
26. Vogelstein,B. and Kinzler,K.W. (1992) p53 function and dysfunction. Cell,
70, 523–526.
27. Frebourg,T., Sadelain,M., Ng,Y.S., Kassel,J. and Friend,S.H. (1994) Equal
transcription of wild-type and mutant p53 using bicistronic vectors results
in the wild-type phenotype. Cancer Res., 54, 878–881.
28. Spruck,C.R., Ohneseit,P.F., Gonzalez-Zulueta,M. et al. (1994) Two
molecular pathways to transitional cell carcinoma of the bladder. Cancer
Res., 54, 784–788.
29. Ogawa,K., Uzvolgyi,E., St John,M.K., de Oliveira,M.L., Arnold,L. and
Cohen,S.M. (1998) Frequent p53 mutations and occasional loss of
chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-
hydroxybutyl)nitrosamine in B6D2F1 mice. Mol. Carcinogen., 21, 70–79.
Received June 23, 1998; revised November 19, 1998;
accepted December 4, 1998