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Carcinogenesis, Vol. 21, No. 10, 1891-1897, October 2000
© 2000 Oxford University Press

Carcinogenesis

p53 knockout mice (–/–) are more susceptible than (+/–) or (+/+) mice to N-methyl-N-nitrosourea stomach carcinogenesis

Masami Yamamoto, Tetsuya Tsukamoto5, Hiroki Sakai, Norimitsu Shirai, Hiroko Ohgaki1, Chie Furihata2, Lawrence A. Donehower3, Kenji Yoshida4 and Masae Tatematsu

Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa, Nagoya 464-8681, Japan,
1 Unit of Molecular Pathology, International Agency for Research on Cancer, F-69372 Lyon Cedex 08, France,
2 College of Science and Engineering, Aoyama Gakuin University, 1-1 Morinosato-Aoyama, Atsugi, Kanagawa 243-0123, Japan,
3 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA and
4 The First Department of Oral and Maxillofacial Surgery, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa, Nagoya 464-8650, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations of the p53 tumor suppressor gene constitute one of the most frequent molecular changes in a wide variety of human cancers. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we have investigated the susceptibility of p53 nullizygote (–/–), heterozygote (+/–) and wild-type (+/+) mice to N-methyl-N-nitrosourea (MNU) gastric carcinogenesis. p53 knockout mice were treated with 30 p.p.m. MNU in the drinking water 1 week on and 1 week off and killed after 5 weeks. The numbers of pepsinogen-altered pyloric glands (PAPG), putative preneoplastic lesions, were 1.8, 1.7 and 22.6 in p53 (+/+), (+/–) and (–/–) mice, respectively. In a 15 week experiment, adenomas were found in 0 of 19 (+/+) (0%), 2 of 21 (+/–) (9.5%) and 6 of 10 (–/–) (60.0%) animals. Also, one well-differentiated adenocarcinoma was observed in a p53 (–/–) mouse. After 40 weeks treatment with 120 or 30 p.p.m. MNU there was no significant difference in the incidence of gastric tumors between p53 (+/+) and (+/–) mice. However, mortality from carcinogen-induced lymphomas, leukemias and sarcomas was very much greater in the latter group. Homozygous knockout animals could not be maintained long term. PCR–single strand conformation polymorphism analysis of exons 5–8 of the p53 gene of DNA extracts from 68 gastric tumors consisting of 16 and 20 30 p.p.m. MNU-treated p53 (+/+) and (+/–) mice and 14 and 18 120 p.p.m. MNU-treated p53 (+/+) and (+/–) mice demonstrated no mutations. These results suggest that p53 may not be a direct target of MNU but rather play an important role as a gatekeeper in mouse stomach carcinogenesis induced by this direct acting agent.

Abbreviations: KO, knockout; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MNU, N-methyl-N-nitrosourea; Pg 1, pepsinogen 1; PAPG, pepsinogen 1-altered pyloric glands; SSCP, single strand conformation polymorphism.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Cancer is recognized as a disease arising from accumulation of genetic alterations, including activation of oncogenes and inactivation of tumor suppressor genes (1). Identifying carcinogens using rodent bioassays and limiting exposure is one approach to the problem of reducing risk. Recently, genetically altered mice have attracted attention for their potential utility for analysis of chemical toxicity in the short term. Transgenic mice, including p53-deficient mice, may provide advantages in shortening the time required for bioassays and improving the accuracy of carcinogen identification (2–5).

The p53 tumor suppressor gene is reported to be frequently mutated in a wide variety of human cancers (6,7). The functions of the p53 protein include a contribution to G1 cell cycle arrest to allow DNA repair as well as induction of apoptosis after DNA damage (8). For functional analysis, p53 knockout (KO) mice have been established by Donehower et al. (9) as a powerful tool. With nullizygous p53 (-/-) KO mice, a relative lack of DNA repair or apoptosis may facilitate causation of malignant tumors: by the age of 4.5 months, approximately half of the homozygotes developed tumors and by 10 months of age all the animals had died or developed tumors (10). These tumors were not epithelial in origin but usually lymphomas and sarcomas. Ozaki et al. (11) reported that p53 (+/-) mice were more sensitive to carcinogen-induced urinary bladder tumorigenesis than the p53 (+/+) genotype. Kemp et al. (12) showed that a reduction in p53 genes was not related to an increase in initiation or promotion but enhanced malignant progression of chemically induced skin tumors. Tennant et al. (2) revealed that p53 (+/-) KO mice were susceptible to genotoxic carcinogens but not to non-genotoxic carcinogens. For analysis of gastric carcinogenesis in a mouse model, we have established a system using N-methyl-N-nitrosourea (MNU) administered in the drinking water with successful development of adenomas and carcinomas (13).

In studies to determine the pathogenesis of gastric cancer, determination of the nature of preneoplastic changes is one of the most important fields for research. The enzyme pepsinogen 1 (Pg 1) (14,15) has received attention as a marker of preneoplasia. Pg 1 expression is preferentially decreased or disappears in pyloric mucosa during the early stages of rat N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cacinogenesis before distinct morphological preneoplastic changes (16–18). The alteration in Pg 1 expression (pepsinogen 1-altered pyloric glands, PAPG) can be readily detected immunohistochemically in normal-looking pyloric mucosa. Consistent with this observation, decreased Pg 1 expression is observed in adenomas and carcinomas (19,20). PAPG were found to be dose-dependently induced in MNNG-treated rats and MNU-treated mice, where a higher proliferative activity was found compared with their normal counterparts (20–22). Thus PAPG are now generally accepted as preneoplastic changes in the glandular stomach.

We have reported that MNU-induced mouse stomach tumors only rarely demonstrate p53 gene mutations (23) despite more frequent mutations of the p53 gene in human stomach cancers (24). The aims of the present study were to examine: (i) whether p53 KO mice, including the heterozygous (+/-) and nullizygous (-/-) genotypes, were more susceptible to MNU-induced tumorigenesis compared with wild-type (+/+) mice and clarify the role of p53 in stomach cacinogenesis; (ii) whether PAPG could be detected in these genotypes and how p53 gene dosage affected PAPG induction; (iii) whether p53 KO mice are useful for short term analysis of stomach carcinogenesis in comparison with other non-transgenic strains. Included was an analysis of p53 mutations.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animals
p53 KO mice produced by Donehower et al. (9) were maintained at the Animal Facility of the Aichi Cancer Center Research Institute. They were housed in plastic cages with hardwood chips in an air-conditioned room with a 12 h light–12 h dark cycle and were given basal diet (Oriental NMF; Oriental Yeast Co., Tokyo, Japan) and water ad libitum. For genotyping of each mouse, DNA samples were extracted from the tail using a QIAamp tissue kit (Qiagen, Tokyo, Japan). The 25 µl PCR reaction mixture consisted of 1.25 U Taq DNA polymerase (Takara Shuzo Co., Shiga, Japan), 1x buffer provided, 200 µM dNTP, 200 nM each 5'- and 3'-primers (10681, 10480, 10588 and 10930 as listed in Table IGo) and 2.5 µl of genomic DNA. PCR was performed using a Takara PCR Thermal Cycler MP (Takara) as follows: 94°C for 1 min; 35 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 1 min; 72°C for 10 min.


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  		Table I. PCR primers for genotyping and for SSCP analysis of mouse p53 analysis
 
Experimental design
The experimental design is shown in Figure 1Go. MNU (Sigma, St Louis, MO) was dissolved in distilled water and freshly prepared three times per week. p53 wild-type (+/+), heterozygous (+/-) and nullizygous (-/-) male mice were given drinking water ad libitum containing 30 p.p.m. MNU in light-shielded bottles on alternate weeks (total exposure was 2 weeks in experiment I and 5 weeks in experiment II) and then normal tap water until weeks 5 and 15 in experiments I and II, respectively. Survival of p53 (-/-) mice was poor but a long term observation was also performed using male and female p53 (+/+) and (+/-) mice which were given drinking water containing 30 or 120 p.p.m. MNU on alternate weeks for a total exposure of 5 weeks and killed at week 40. Matching numbers of each mouse genotype were given distilled water for the same period as controls. Necropsies were performed on all animals which died or were killed when they became moribund. At the end of the experiment surviving mice were killed and autopsied. The excised stomachs were fixed in sublimated formaldehyde for Pg 1 immunohistochemistry (22) or 4% paraformaldehyde in phosphate-buffered saline for histology. Samples were cut into eight strips, routinely processed and embedded in paraffin. Other tissues were carefully checked with the naked eye and tumors and related lesions were fixed in buffered formalin and embedded in paraffin.



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  		Fig. 1. Experimental design. Five-week-old wild-type (+/+), heterozygous (+/–) and nullizygous (–/–) p53 KO mice were used in experiments I and II. Animals were (+/+) and (+/–) in experiment III. MNU at 120 (black) or 30 p.p.m. (hatched) and water (white) as a control were administered. Exp, experiment; S, death.

 
Histopathological analysis
Tissue sections were stained with hematoxylin and eosin for histological diagnosis. Serial sections were stained with anti-Pg 1 antibody as described earlier (22). Pyloric glands were evaluated for PAPG, showing weak or negative staining for Pg 1, as putative precancerous lesions. The numbers of PAPG and pyloric glands with reduced Pg 1 expression per 100 pyloric glands were calculated for each animal in experiments I and II.

PCR–single strand conformation polymorphism analysis (SSCP)
PCR–SSCP was conducted basically as described (25). Briefly, genomic DNA was extracted from tumor areas in paraffin sections with DEXPAT (Takara) or from frozen tissues as detailed elsewhere (26). Four pairs of PCR primers for mouse p53 exons 5–8 were designed based on the published sequences (27) as listed in Table IGo. PCR was performed with a Takara PCR Thermal Cycler MP and products electrophoresed in 0.625x MDE polyacrylamide gels (FMC, Rockland, ME) with 5% glycerol. These were run at room temperature for 18 h at 8 W, dried and applied to imaging plates, which were then analyzed with a BAS 2500 (Fuji Film, Kanagawa, Japan).

Direct sequencing
Sequencing was performed using an AmpliCycle Sequencing Kit (Perkin Elmer) as described previously (28).

LA PCR
For samples with positive PCR–SSCP analysis in the (+/-) mice, LA PCR was performed to specifically amplify the wild-type and mutant alleles with PCR primers 10681 and C/10930, respectively, in combination with the antisense primer for exon 8 using a TaKaRa LA PCR kit (Table IGo). The PCR conditions were as follows: 94°C for 1 min; 35 cycles of 94°C for 1 min, 65°C for 1 min, 72°C for 4 min; 72°C for 10 min. The products were subjected to direct sequencing.

Statistical analysis
For labeling indices and numbers of PAPG, the two-tailed t-test was applied to establish significant differences (29). The incidences of early stomach lesions and tumors were analyzed using Fisher's exact probability test (29) and ridit analysis (30). Survival curves were drawn by the Kaplan–Meier method and analyzed using the log rank test (29).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Short term experiment I
In experiment I, histological sections partly showed slightly irregular glands and weak hyperplasia or atrophy, especially in the p53 (-/-) mice. Pg 1 staining revealed PAPG putative precancerous lesions even in normal looking mucosa in MNU-treated p53 (-/-) mice (Figure 2BGo), in contrast to the control mice in which Pg 1 was consistently present in pyloric gland cells, mucous neck cells and chief cells in the fundic mucosa of the glandular stomach (Figure 2AGo). The frequency of PAPG (Figure 3Go) was 22.6 ± 14.9% (mean ± SD) in MNU-treated p53 (-/-) mice and significantly higher (P < 0.001) compared with the values for MNU-treated p53 (+/+) and p53 (+/-) mice (1.8 ± 1.0 and 1.7 ± 0.8%, respectively). In the control group, the frequencies were 1.0 ± 0.4, 1.0 ± 0.3 and 1.2 ± 0.6% in p53 (+/+), (+/-) and (-/-) mice, respectively.



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  		Fig. 2. Pg 1 staining showing PAPG. (A) Normal-looking pyloric glands retaining positive Pg 1 immunoreactivity. (B) Pyloric glands in MNU-treated p53 (-/-) mice displaying partly negative or reduced Pg 1 expression (PAPG).

 


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  		Fig. 3. Frequency of PAPG in experiment I. In MNU-treated (+/+), (+/-) and (-/-) groups the PAPG frequencies were 1.8 ± 1.0, 1.7 ± 0.8 and 22.6 ± 14.9, respectively. In the control groups the values were 1.0 ± 0.4, 1.0 ± 0.3 and 1.2 ± 0.6. That of MNU-treated (-/-) mice was significantly higher (aP < 0.001) than MNU-treated (+/+) and (+/-) mice and control (-/-) mice.

 
Middle term experiment II
p53 (-/-) mice were compared with (+/-) and (+/+) mice for their survival and tumor incidence. Survival curves till 20 weeks of age are shown in Figure 4Go. In the control groups, most (-/-) mice developed lymphomas or sarcomas as described earlier (9,10,31). MNU strongly affected p53 (-/-) mice, causing mortality (P < 0.001), whereas effects on p53 (+/+) and (+/-) mice were less severe (statistically not significant). Data for PAPG at experimental week 15 are shown in Figure 5Go. The frequency was 37.5 ± 16.5% in MNU-treated p53 (-/-) mice and significantly higher (P < 0.001) than in MNU-treated p53 (+/+) or (+/-) mice (11.3 ± 4.4 and 12.4 ± 5.4%, respectively). In the control groups, the frequencies were 4.1 ± 2.1, 5.0 ± 0.4 and 6.2 ± 3.2 in p53 (+/+), (+/-) and (-/-) mice, respectively. PAPG frequencies in MNU-treated groups were significantly higher (P < 0.001) than that of control groups in each genotype. Stomach lesions were found only in MNU-treated groups and were histologically diagnosed as described (13,22; Figure 6Go). Representative histologies for hyperplasia (Figure 6BGo), adenoma (Figure 6CGo) and adenocarcinoma (Figure 6, D–FGo) are shown, in contrast to normal pyloric glands (Figure 6AGo). Incidences for each lesion type in the glandular stomach are summarized in Table IIGo. Adenomas were found only in p53 (+/-) and (-/-) mice. A well-differentiated adenocarcinoma was observed in a sole p53 (-/-) mouse. The trend of these lesions was revealed to be significantly shifted towards malignancy in p53 (-/-) mice compared with p53 (+/+) and (+/-) mice using ridit analysis (P < 0.001 each) (30,32).



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  		Fig. 4. Survival curves of mice treated with MNU in experiment II. p53 (+/+), (+/-) and (-/-) mice were treated with 30 p.p.m. MNU or water as a control. p53 (-/-) mice survived less with (aP<0.001) or without (bP<0.05) MNU treatment but the latter showed a higher death rate (cP<0.05).

 


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  		Fig. 5. Frequency of PAPG in experiment II. In the MNU-treated (+/+), (+/-) and (-/-) groups the PAPG frequencies were 11.3 ± 4.4, 12.4 ± 5.4 and 37.5 ± 16.5, respectively. In the control groups the values were 4.1 ± 2.1, 5.0 ± 0.4 and 6.2 ± 3.2. That of MNU-treated (-/-) mice was significantly higher (aP<0.0001) than MNU-treated (+/+) and (+/-) mice. MNU treatment increased PAPG in all genotypes compared with the control [(-/-), aP < 0.0001; (+/-), bP < 0.01; (+/+), cP < 0.01].

 


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  		Fig. 6. (A) Normal pyloric glands in a (+/-) male control animal. (B) Hyperplasia of pyloric mucosa in a 30 p.p.m. MNU-treated (+/-) male at week 15. (C) Adenoma developing in a 30 p.p.m. MNU-treated (+/-)female at week 40. (D) Invasive well-differentiated adenocarcinoma in a 120 p.p.m. MNU-treated (+/-) male at week 40. (E) Higher magnification of (D). (F) Signet ring cell carcinoma in a 30 p.p.m. MNU-treated (+/-) male at week 40.

 

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  		Table II. Incidence of gastric lesions at experimental week 15
 
Long term experiment III
Survival curves until week 40 are shown in Figure 7Go, mortality after carcinogen treatment being observed in a dose-dependent manner. Death occurred earlier in p53 (+/-) animals, due to carcinogen-induced lymphomas, leukemias or sarcomas, as compared with their (+/+) counterparts. The incidences of glandular stomach lesions at week 40 are given in Table IIIGo. There were no significant differences between p53 (+/+) and (+/-) mice. Fifty-one animals were observed to have adenocarcinomas, which included 44 well-differentiated, four poorly differentiated and three signet ring cell carcinomas. The lesions developed mainly in the pyloric mucosa and occasionally at the fundopyloric border. Intestinal metaplasias were not observed in any of the groups.



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  		Fig. 7. Survival curves of mice treated with MNU in experiment III. p53 (+/+) and (+/-) mice were treated with 120 or 30 p.p.m. MNU or water as a control. p53 (+/-) mice survived less than (+/+) (aP < 0.01). A dose-dependent carcinogen influence was observed for both sexes (b,cP < 0.0001). M, male; F, female.

 

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  		Table III. Incidence of gastric tumors at experimental week 40
 
PCR–SSCP analysis of the p53 gene in tumors
The 68 gastric tumors which developed in experiments II and III were subjected to PCR–SSCP analysis of p53 exons 5–8. SSCP conditions were set so as to show mobility shifts of PCR products amplified with a plasmid harboring a mutation produced by spontaneous Taq polymerase error as described earlier (25). These products from all the gastric tumors appeared to migrate at the same speed as the normal controls, while one lymphoma developed in a p53 (+/-) mouse, for example, was revealed to possess a mutant p53 allele in exon 7 as representatively shown in Figure 8Go. Allele-specific amplification of the p53 gene followed by direct sequencing revealed a TGC (Cys)->TAC (Tyr) mutation at codon 239 in the wild type allele in this lymphoma, indicating loss of function of the p53 protein. Two of eight lymphomas (five MNU-treated and three controls) and one of eight sarcomas (four MNU-treated and four controls) demonstrated mutations in p53(+/-) mice. These mutations were found only in the MNU-treated group.



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  		Fig. 8. Representative results of PCR–SSCP analysis of p53 exon 7. No band shifts were observed in gastric tumors compared with normal controls, in contrast to a lymphoma showing different mobility, with a TGC (Cys)->TAC (Tyr) mutation at codon 239. M, mutant control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study has demonstrated that nullizygous p53 KO mice are more susceptible to stomach carcinogenesis induced by MNU than their heterozygous and wild-type counterparts by analyses of PAPG and tumor incidences. PAPG are considered to be precancerous lesions in both the mouse (22) and rat (20,21,33–36) stomach carcinogenesis models. Alteration of Pg 1 expression has been reported to be detectable biochemically (16–18) and immunohistochemically (19,20,22) before morphologically distinct preneoplastic changes become evident. Furthermore, these changes were consistent in gastric tumors. In both short and middle term experiments conducted in the present study (experiments I and II), the PAPG incidences in MNU-treated p53 (-/-) mice were markedly and significantly higher than in control p53 (-/-) or MNU-treated p53 (+/+) and (+/-) mice. The results coincided well with the fact that tumors in p53 (-/-) mice developed within 15 weeks. Thus while survival was impaired to the extent that homozygous KO mice could not be included in the long term study, a clear conclusion of enhanced susceptibility was possible.

While there was no significant difference between (+/+) and (+/-) gastric tumor incidences in both middle term and long term experiments (experiments II and III) for males and females, the reduction in p53 gene dosage was associated with earlier mortality due to induced lymphomas and sarcomas. Since stomach cancer developed in p53 (+/+) mice in this experiment and a previous study (22), p53 loss is not necessary for stomach carcinogenesis. The present PCR–SSCP analysis supports the idea of no essential role by revealing no mutations in exons 5–8 of the p53 gene in 68 stomach tumors, as analyzed in BALB/c mice and reported by Furihata et al. (23). However, complete loss of wild-type p53 expression greatly enhanced stomach carcinogenesis. Thus the p53 gene might not be a direct target of MNU but the p53 protein may act as a gatekeeper (37,38) for initiation of neoplasia, as shown by the PAPG data and tumor promotion and progression as reflected in more advanced stomach lesion development in p53 (-/-) mice. In contrast, spontaneous sarcomas (39) and dimethylbenzanthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin squamous cell carcinoma (12) needed only a partial reduction in p53 gene dosage in (+/-) mice compared with (+/+) mice for cancer promotion and progression. In the latter case, progression rate was also greater in (+/-) than in (+/+) mice and was associated with loss of the remaining wild-type allele. Urinary bladder transitional cell carcinomas were also found to be enhanced in N-butyl-N-(4-hydroxybutyl)nitrosamine-treated p53 (+/–) mice as compared with the C57BL/6 parental strain (11). The data thus suggest that squamous cell and transitional cell epithelia may be more influenced by a decrease in p53 protein amount or that the p53 gene could be a direct target in these tissues. Dose dependency has been noted in the p53 (+/+) and (+/-) genotypes in other strains (13).

In experiment III, MNU up to 120 p.p.m. was applied to p53 (+/+) and (+/-) mice. The heterozygotes were significantly more susceptible to the carcinogen effects. Causes of death were not stomach lesions but rather sarcomas and lymphomas, in some of which p53 was completely inactivated by point mutations in the wild-type allele, as revealed by PCR–SSCP and allele-specific sequencing. Tissue targets of MNU were as described earlier (40–42), sarcomas and lymphomas.

In conclusion, the present study has shown the importance of p53 using p53 (-/-) mice in both 5 week PAPG precancerous lesion and 15 week tumorigenesis assays. p53 KO mice are one of several genetically altered mice whose use may increase the sensitivity and decrease the time and cost of rodent carcinogenicity bioassays. Consideration must also be given to the genetic background of the mouse strain and the impact of strain variability on disease and toxicity models. Despite these potential limitations, p53 KO mice provide a powerful tool for short term carcinogenicity studies.


   Notes
 
5 To whom correspondence should be addressed Email: ttsukamt{at}aichi-cc.pref.aichi.jp Back


   Acknowledgments
 
This work was supported in part by Grants-in-Aid from CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation, by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan and by a grant from the Society for Promotion of Pathology of Aichi, Japan. We also thank Dr Yoichi Ishida and Ms Michiyo Tominaga for expert technical assistance and Ms Rie Takabayashi for administrative tasks.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 10, 2000; revised June 28, 2000; accepted June 30, 2000.


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