Elevated susceptibility of the p53 knockout mouse esophagus to methyl-N-amylnitrosamine carcinogenesis

Norimitsu Shirai1,2,3, Tetsuya Tsukamoto1,5, Masami Yamamoto1, Takeshi Iidaka1,2,3, Hiroki Sakai1,2, Tokuma Yanai2, Toshiaki Masegi2, Lawrence A. Donehower4 and Masae Tatematsu1

1 Division of Oncological Pathology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa, Nagoya 464-8681,
2 Department of Veterinary Pathology, Gifu University, Yanagido 1-1, Gifu, 501-1193,
3 Nagoya Laboratories, Pfizer Global Research and Development, 5-2 Taketoyo, Aichi 470-2393, Japan and
4 Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA


    Abstract
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 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, including those in the esophagus. Mice deficient in p53 have recently attracted attention for their potential to identify chemical genotoxins. In this study we investigated the susceptibility of p53 nullizygous (–/-), heterozygous (+/-) and wild-type (+/+) mice to methyl-N-amylnitrosamine (MNAN), which specifically induces esophageal tumors in mice and rats. The p53 (+/-) and (+/+) mice were treated with 5 or 15 p.p.m. MNAN in their drinking water for 8 weeks then maintained without further treatment for an additional 7 or 17 weeks, being killed at experimental weeks 15 or 25. An additional group of p53 (–/-) mice were given 5 p.p.m. MNAN for 8 weeks and killed at week 15. At 15 weeks in the 5 p.p.m. groups, squamous cell carcinomas (SCCs) were observed in 10/12 (83.3%) p53 (–/-) and 1/15 (6.7%) p53 (+/-) mice, but in none of the p53 (+/+) mice. In the animals receiving 15 p.p.m., 2/14 (14.3%) p53 (+/-) and 1/11 (9.1%) p53 (+/+) mice developed SCCs. At 25 weeks, the incidence of SCCs was 7/16 (43.8%) and 8/14 (57.1%) in p53 (+/-) mice and 1/13 (7.7%) and 2/10 (20.0%) in p53 (+/+) mice at 5 and 15 p.p.m., respectively. Of the SCCs examined by PCR–single strand conformation polymorphism analysis, 61% (14/23) from p53 (+/-) and 50% (6/12) from p53 (+/+) mice demonstrated mutations in the p53 gene (exons 5–8). These results indicate the order of susceptibility to MNAN-induced esophageal tumorigenesis to be as follows: nullizygotes (–/-) > heterozygotes (+/-) > wild-type (+/+), and provide strong evidence of involvement of p53 mutations in the development of esophageal SCCs.

Abbreviations: KO, knockout; (+/–), heterozygous; (–/–), nullizygous; (+/+), wild-type; MNAN, methyl-N-amylnitrosamine; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism; SCC, squamous cell carcinoma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The p53 gene, encoding a transcriptional regulator that prevents the propagation of genetically damaged cells (1), is frequently mutated in a wide range of human cancers (2,3). In addition to somatic mutations of p53 in sporadic cancers, germline mutations in the p53 gene are associated with the Li–Fraumeni syndrome, a familial autosomal dominant disease characterized by a predisposition to development of a variety of tumors (4). The role of p53 includes a contribution to G1 cell cycle arrest and induction of DNA repair genes in response to DNA damage, as well as activation of genes promoting apoptosis (1,5).

Since homozygous or heterozygous p53-deficient mice were first described, demonstrating early onset of spontaneous tumors (6), they have attracted interest as a model for assessing the significance of loss of p53 for tumor development and as experimental animals for assays of carcinogenic potential (7–9). By the age of 4.5 months, approximately half of nullizygote p53-deficient (–/-) mice develop tumors and by 10 months of age the incidence is 100%. Most of the tumors are lymphomas or sarcomas, rapidly causing mortality. In contrast, the heterozygotes (+/-) show only a low background incidence of spontaneous tumors until almost 12 months of age and have much longer life spans (6,10). This low background tumor incidence, combined with elevated susceptibility to chemical induction of tumors, make the p53 (+/-) mouse useful for short-term bioassays (11,12), as well as providing a model for the human Li–Fraumeni syndrome.

In most countries of the world, SCCs constitute the most common esophageal cancers in humans, with mutations in the p53 gene frequently detected (13,14). Thus p53 alteration may be a crucial event in esophageal carcinogenesis. The aims of the present study were to examine whether p53 knockout (KO) mice, including heterozygotes (+/-) and nullizygotes (–/-), might be more susceptible to methyl-N-amylnitrosamine (MNAN)-induced esophageal tumorigenesis compared with wild-type (+/+) mice, and to determine whether deficiency in p53 plays a role in esophageal tumor development.


    Materials and methods
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 Materials and methods
 Results
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Animals
p53 knockout mice on a C57BL/6 genetic background, produced by Donehower et al. (15), were maintained at the Animal Facility of Aichi Cancer Center Research Institute. Six-week-old males were used in the experiment. They were housed in plastic cages with hardwood chips in an air-conditioned room with a 12 h light:12 h dark cycle and given basal diet (Oriental NMF, Oriental Yeast Co., Tokyo, Japan) and drinking water ad libitum. For genotyping of each mouse, the following procedure was performed as described earlier (16): DNA samples were extracted from the tail using QIAamp tissue kit (QIAGEN, KK, Tokyo, Japan). The 25 µl PCR reaction mixture consisted of 1.25 units of Taq DNA polymerase (Takara Shuzo Co. Ltd, Shiga, Japan), 1x provided buffer, 200 µM dNTP, 200 nM each of 5'- and 3'-primers (10681, 10480, 10588 and 10930 as listed in Table IGo), and 2.5 µl of genomic DNA. PCR was performed as follows: 94°C 1 minx1 cycle, 94°C 1 min–65°C 1 min–72°C 1 minx35 cycles, 72°C 10 min using a Takara PCR Thermal Cycler MP (Takara).


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Table I. PCR primers for genotyping and for SSCP analysis of mouse p53 analysis
 
Carcinogen treatment
MNAN was purchased from Sakai Rikagaku Institute (Fukui, Japan) and dissolved in drinking water weekly to achieve the desired concentrations. Black bottles were filled with drinking water containing 5 or 15 p.p.m. of MNAN and provided ad libitum to p53 (+/+) and (+/-) mice for 8 weeks. Mice were then maintained without further treatment for an additional 7 or 17 weeks, and killed at week 15 or 25. An additional group of p53 (–/-) mice were also treated with 5 p.p.m. MNAN for 8 weeks and killed at week 15. Three groups of control animals with p53 (+/+), p53 (+/-) or p53 (–/-) received unsupplemented drinking water (Figure 1Go).



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Fig. 1. Experimental design. Six-week-old wild-type (+/+), heterozygous (+/-) and nullizygous (-/-) p53 KO mice were used. MNAN at 15 (black) or 5 (hatched) p.p.m. and water (white) as a control were given. MNAN, methyl-N-amylnitrosamine; S, killed.

 
Histopathological analysis
At necropsy, the esophagus of each animal was resected from the larynx to the stomach, and slit longitudinally along the midline of the dorsal wall. After fixation in 4% paraformaldehyde in phosphate buffered saline they were embedded in paraffin, each was sectioned and stained with hematoxylin and eosin for microscopic examination. The total numbers of tumors were counted and the total areas and distances of each from the larynx were measured by computerized image analysis, using a microscope equipped with an image processor for analytical pathology (IPAP; Sumika Technos, Takarazuka, Japan).

PCR–single strand conformation polymorphism analysis (SSCP)
Nineteen tumors from p53 (+/+) mice and 35 from p53 (+/-) mice were subjected to PCR–SSCP. Each tumor was identified by means of 1–19 and 101–135 sequential numbers, respectively. PCR–SSCP was conducted basically as described previously (17). Briefly, genomic DNA was extracted from tumor areas in paraffin sections with DEXPAT (Takara) (18) or from frozen tissues (19) as detailed elsewhere. Four pairs of PCR primers for mouse p53 exons 5–8 were designed based on the published sequence (16) as listed in Table IGo. PCR was performed with a Takara PCR Thermal Cycler MP (Takara) and products were electrophoresed in 0.625xMDE 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 BAS 2500 (Fuji Film, Kanagawa, Japan).

Direct sequencing
Sequencing was performed using an AmpliCycle Sequencing Kit (Perkin Elmer) as described previously (20).

LA PCR
For freshly collected and frozen samples with positive PCR–SSCP results from p53 (+/-) mice, LA PCR was performed specifically to amplify the wild-type and mutant alleles with PCR primers 10681 or C/10930 in combination with the antisense primer for exon 8 using a Takara LA PCR kit (16) (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
Data for incidence of histopathological lesions were analyzed by the Fisher’s exact test method. The numbers and areas of tumors were analyzed with the Mann–Whitney rank sum test. Survival curves were drawn by the Kaplan–Meier method and analyzed using the log rank test (21).


    Results
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 Materials and methods
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 References
 
Mortality of each genotype
Administration of 5 and 15 p.p.m. MNAN in drinking water was well tolerated by both p53 (+/-) and p53 (+/+) mice. Survival was not significantly different between p53 (+/-) and p53 (+/+) mice. However, p53 (–/-) mice demonstrated a high mortality rate (Figure 2Go).



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Fig. 2. Survival curves of p53 (+/+), (+/-) and (-/-) mice treated with 5 p.p.m. MNAN (A) and those of p53 (+/+) and (+/-) mice at 15 p.p.m. (B). At week 15, all remaining p53 (-/-) mice were killed. p53 (-/-) mice survival was less than in the (+/+) (aP < 0.01) or (+/-) cases (bP < 0.05). The total numbers of mice are given in parentheses. The number of animals at the beginning of each experimental group (‘No. animals’) and at the scheduled killing (‘No. examined’) are as listed in Table IIGo.

 
Histopathological analysis
Administration of MNAN induced a 100% incidence of esophageal diffuse hyperplasia characterized by thickening of the squamous epithelium in p53 (–/-), p53 (+/-) and p53 (+/+) mice. This was often accompanied by subepithelial inflammatory infiltration (Figure 3BGo) in clear contrast to normal mucosa (Figure 3AGo).






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Fig. 3. Photomicrographs of normal mucosa at 200x (A), diffuse hyperplasia at 200x (B), a papilloma at 100x (C) and a squamous cell carcinoma at 100x (D) in the esophagus of p53 (+/-) mice treated with MNAN.

 
Papillomas characterized by nodular mucosal elevation of proliferating epithelial cells appeared to develop within diffuse hyperplasias (Figure 3CGo). Squamous cell carcinomas (SCCs) showed invasive growth of atypical squamous epithelial cells (Figure 3DGo). Neither adenocarcinomas nor Barrett’s esophagus, in which squamous epithelium is replaced by a columnar epithelium, were detected in any of the mice. There was no regional preference for tumor development with the esophageal mucosa.

In addition to esophageal lesions, occasionally nodular thickening of the mucosa was observed in the forestomach in mice of all three genotypes. However, no lesions were detected in the glandular stomach.

Incidence, number and size of esophageal tumors
In the mice treated with 5 p.p.m. MNAN, the incidence (Table IIGo) of SCCs was significantly higher (P < 0.001) in p53 (–/-) (83.3%) than p53 (+/-) (6.7%) and p53 (+/+) (0%) mice at 15 weeks after starting treatment. At 25 weeks, the SCC incidence in p53 (+/-) (43.8%) was significantly increased (P < 0.05) when compared with p53 (+/+) (7.7%) mice. Only one SCC developed in a p53 (+/+) mouse at 25 weeks. The total number of tumors per mouse (left box of Figure 4AGo) (mean ± SD) was also higher in the p53 (–/-) (4.8 ± 1.4) than the p53 (+/-) (1.5 ± 1.2) and p53 (+/+) ( 0.8 ± 0.9) groups at 15 weeks (P < 0.001) and in p53 (+/-) (4.4 ± 2.6) than in p53 (+/+) (2.7 ± 1.5) mice at 25 weeks (P < 0.05). The size of the tumors (left box of Figure 4BGo) (mean ± SD) was larger in p53 (–/-) (0.3 ± 0.05 mm2) than p53 (+/-) (0.1 ± 0.1 mm2) and p53 (+/+) (0.07 ± 0.03 mm2) mice at 15 weeks (P < 0.005). There were no significant differences in tumor size between p53 (+/-) and p53 (+/+) mice at 15 or 25 weeks.


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Table II. Incidences of esophageal lesions
 


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Fig. 4. (A) Average number of esophageal tumors in p53 (+/+), (+/-) and (-/-) mice treated with 5 (left) or 15 (right) p.p.m. MNAN for 8 weeks then maintained without further treatment for an additional 7 or 15 weeks. aP < 0.001 versus (+/+) and (+/-), bP < 0.05 versus (+/+). (B) Sizes of esophageal tumors in p53 (+/+), (+/-) and (-/-) mice of 5 (left) or 15 (right) p.p.m. MNAN treatment groups. aP < 0.005 versus (+/+) and (+/-), bP < 0.05 versus (+/+). Vertical bars indicate SE of the mean. Open columns, 15 weeks after starting treatment; closed columns, 25 weeks after starting treatment.

 
In the mice receiving 15 p.p.m. MNAN, there was a trend for greater SCC incidences in p53 (+/-) as compared with p53 (+/+) mice at 15 and 25 weeks; 14.3 and 9.1%, respectively, at 15 weeks, and 57.1 and 20.0%, at 25 weeks (Table IIGo). Average tumor size (right box of Figure 4BGo) (mean ± SD) in the p53 (+/-) case (0.8 ± 2.1 mm2) was significantly larger (P < 0.05) than in p53 (+/+) (0.2 ± 0.2 mm2) mice at 25 weeks, although there was little difference in the number of the tumors (right box of Figure 4AGo).

PCR–SSCP analysis of the p53 gene in tumors
PCR–SSCP and sequencing analyses for exons 5–8 of the p53 gene were performed, representative results being illustrated in Figure 5Go. p53 mutations were identified in six out of 12 SCCs (50%) and 14 out of 23 SCCs (61%) in p53 (+/+) (Table IIIGo) and p53 (+/-) (Table IVGo) mice, respectively. There were three SCCs (tumor IDs: 109, 129 and 133) with more than one mutation in p53 (+/-) mice. Only one of 19 papillomas examined (tumor ID: 10) had a mutation. DNA sequencing demonstrated eight mutations in exon 5 (33%), six in exon 6 (25%), eight in exon 7 (33%), and two in exon 8 (8%) of the total of 24 mutations identified in 20 SCCs. Of these mutations two (tumor IDs: 11 and 133) were silent and one (tumor ID: 4) was of nonsense type. All the others were missense mutations. There were 19 transitions (79%) and five transversions (21%). G:C->A:T transitions at non-CpG sites accounted for approximately half of all mutations. A total of 20 SCCs exhibiting p53 mutation varied widely in size from 0.13 to 9.34 mm2; 1.7 ± 2.7 mm2 (mean ± SD) and p53 mutation rate was comparable in smaller and larger carcinomas.



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Fig. 5. Representative results of PCR–SSCP analysis and DNA sequencing of p53 exons 5–8. Arrows indicate shifted bands on SSCP analysis and mutations of the p53 gene at exons 5, 6, 7, and 8 on DNA sequencing.

 

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Table III. p53 gene mutations identified in esophageal tumors in p53 (+/+) mice
 

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Table IV. p53 gene mutations identified in esophageal tumors in p53 (+/–) mice
 
Frozen tissues were available from two tumors (tumor IDs: 101 and 126) from p53 (+/-) mice in which p53 mutations were observed. LA PCR-amplified DNAs from these samples exhibited missense mutations not in the mutant but rather in the wild-type allele, indicating loss of functional p53 protein. The other tumors developing in p53 (+/-) mice could not be subjected to LA PCR analysis due to poor preservation of genomic DNA in paraffin blocks.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study demonstrated that nullizygous and heterozygous p53 KO mice are more susceptible to esophageal tumorigenesis induced by MNAN, a genotoxic carcinogen (22), than their wild-type counterparts, as indicated by an increased incidence and tumor size of SCCs. Furthermore PCR–SSCP analysis revealed a high frequency of missense mutations in p53 with evidence of loss of functional p53 protein in esophageal malignancies. These results are consistent with the observation that p53 (+/-) mice are generally susceptible to genotoxic carcinogens (12). In addition, accelerated tumor development with chemical exposure in p53 (+/-) mice has been reported with regard to lymphomas (23), mesotheliomas (24), skin tumors (12), vascular tumors (25), urinary bladder tumors (12,26) and lung tumors (27). Taken together, the results support the hypothesis that mutational inactivation of the retained wild-type allele or loss of p53 heterozygosity, with consequent loss of p53 function, eventually results in development of neoplasias as occurs with the human Li–Fraumeni syndrome (28). The lack of any increased susceptibility of p53 (+/-) mice to hepatocarcinogenesis (29,30), gastric carcinogenesis (18) and mammary carcinogenesis (31) may reflect organ/tissue-specific dependence in the requirement for the p53 gene product in tumorigenesis. While the mechanism may involve an additional ‘hit’ to inactivate the second normal allele, Venkatachalam et al. (32) have proposed that reduction of the p53 gene products may be sufficient to promote tumorigenesis.

There is growing evidence that esophageal adenocarcinoma and Barrett’s esophagus are related to the reflux of duodenal content in humans (33–35). Both of these conditions can be induced in the lower esophagus by reflux of duodenal content in rats (36). Fein et al. reported that total gastrectomy with esophagojejunostomy caused esophageal adenocarcinomas as well as dysplasia of the squamous epithelium in the operated p53 (–/-) mice (37). However, in addition to no regional preference in squamous tumor development, no adenocarcinomas were detected in the current study, although a high percentage of p53 (–/-) mice developed squamous cell carcinomas. These results are consistent with no evidence of reflux.

Mutations in the p53 gene commonly occur at hot spots in human cancers (3), but the mutation database for laboratory animals is limited. Our results point to a high frequency of p53 mutations in esophageal malignancies, the most common being missense, as reported for human cancer (38). In contrast to the frequent p53 mutations in SCCs even in small carcinomas, only one mutation was detected in 19 papillomas analyzed. This might simply be a reflection of p53 mutations occurring preferentially in malignant lesions. Similar findings have been reported in murine skin tumors induced with benzo[a]pyrene in which the majority of p53 mutations were identified in squamous cell carcinomas; they were rare in papillomas (39). Moreover, abnormal p53 protein is infrequently identified in human esophageal squamous cell papillomas (40) while even dysplastic lesions exhibit p53 mutations (41,42). It is worth noting that there might also have been normal cell contamination of the papilloma samples, although the proportion of normal cells derived from the interstitium and margin of a papilloma, for example, can be estimated at 50% of a sample at most. When one out of two alleles is mutated in the remaining 50%, the proportion of the mutant allele would correspond to 25% of the original sample. Theoretically, any mutant allele would be detected as a mobility shift by PCR–SSCP (17).

Regarding the location of p53 mutations, they were randomly distributed through exons 5–8, with more than one mutation detected at codons 164 (2/25, 8%), 219 (4/25, 16%), 232 (2/25, 8%) and 250 (4/25, 16%). Approximately half of the mutations observed in our study were G:C->A:T transitions at non-CpG sites. Retrospective analyses of p53 gene mutations in human esophageal cancers have also shown a predominance of G:C->A:T transitions (43,44), indicating the advantage of our MNAN-induced esophageal carcinogenesis model to mimic the human disease.

In conclusion, the present study demonstrated an increased susceptibility to esophageal tumorigenesis by a genotoxic agent in p53 nullizygotes (–/-) and then p53 heterozygotes (+/-) as compared with wild-type (+/+) mice, providing strong evidence of involvement of p53 mutations in the development of esophageal SCCs. Although consideration must be given to carcinogen and/or tissue specificity, p53 KO mice provide a powerful tool for identification and understanding of human carcinogens.


    Notes
 
5 To whom correspondence should be addressed Email: ttsukamt{at}aichi-cc.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, Labor and Welfare and by Grants-in-Aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 4, 2002; revised May 22, 2002; accepted May 23, 2002.





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