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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 Morimura 2 , Shinji Yamamoto, Takashi Murai 1 , Satoru Mori 1 , 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 1 Aburahi Laboratories, Shionogi Research Laboratories, Shionogi Co. Ltd, Gonda, Koka-chou, Koka-gun, Shiga 520-34, Japan

2 To 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/Shi C3H/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). 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/ Shi C3H/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/Shi C3H/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-

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Downloaded from K.Morimura et al. Fig. 1. Intragenic polymorphism in p53 exon 7 of the

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)

5 -TCTCTTCCAGTACTCTCCTC-3 a (sense)

Exon 6 (181 bp)

5 -AGGCGGTGTTGAGGGCTTAC-3 (antisense) 5 -GGCTTCTGACTTATTCTTGC-3 (sense)

Exon 7 (170 bp)

3 -CAACTGTCTCTAAGACGCAC-3 (antisense) 5 -TCACCTGGATCCTGTGTCTT-3 (sense)

Exon 8 (279 bp)

5 -CAGGCTAACCTAACCTACCA-3 (antisense) 5 -ACTGCCTTGTGCTGGTCCTT-3 (sense) 5 -GGAGAGGCGCTTGTGCAGGT-3 (antisense)

a Parts of primers included in introns are in bold.

Script SK( ) 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 SE 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

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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 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/Shi C3H/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

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Role of p53 suppressor gene in urinary bladder carcinogenesis

of p53 suppressor gene in urinary bladder carcinogenesis Fig. 2. PCR–SSCP analysis of exons 5, 6

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

C3H

7

248

ATC AAC GAC GCC CAG AAG ATC AGC

Ile Asn Asp Ala Gln Lys Ile Ser

3

TCC

ND

6

205

4

TCC

ND

5

164

5

SCC

C3H

7

251

6

TCC

7

SCC

C3H

7

248

ATC AAC ATC ACC ATT AGT

Ile Asn Ile Thr Ile Ser

8

SCC

ND

6

192

 

NON

7

248

9

SCC

NON

10

TCC

11

SCC

C3H

7

258

AGT AGG CCA CAA

Ser Arg Pro Gln

12

SCC

NON

5

149

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/Shi C3H/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.

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Received June 23, 1998; revised November 19, 1998; accepted December 4, 1998