Xeroderma pigmentosum group A gene action as a protection factor against 4-nitroquinoline 1-oxide-induced tongue carcinogenesis

Fumio Ide1,2, Hideaki Oda1, Yoko Nakatsuru1, Kaoru Kusama2, Hideaki Sakashita3, Kiyoji Tanaka4 and Takatoshi Ishikawa1,5,6

1 Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033,
2 Department of Oral Pathology and
3 Second Department of Oral and Maxillofacial Surgery, Meikai University School of Dentistry, Saitama 350-0283,
4 Division of Cellular Genetics, Institute for Molecular and Cellular Biology, Osaka University, Osaka 565-0871 and
5 National Institution for Academic Degrees, Faculty of University Evaluation and Research, 2-1-1 Hitotsubashi, Chiyoda-ku, Tokyo 101-0003, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
To test the hypothesis that nucleotide excision repair (NER) plays a protective role in chemical carcinogenesis in internal organs, xeroderma pigmentosum group A gene-deficient (XPA–/–) mice, heterozygous (XPA+/–) and wild-type (XPA+/+) mice were orally administered 0.001% 4-nitroquinoline 1-oxide (4NQO) in their drinking water and compared. After 50 weeks of 4NQO exposure, tongue squamous cell carcinomas (SCCs) occurred in XPA–/– mice only, no tumors being observed in XPA+/– and XPA+/+ animals. Of the XPA–/– mice 86% had tumors and 100% demonstrated multiple foci of dysplastic epithelium in the tongue. Accumulation of p53 protein was immunohistochemically detected in 56% of the SCCs. Mutational analysis of the p53 gene (exons 4–10) in carcinoma DNA revealed missense mutations in exons 5 and 9 in four of 20 samples. Our results clearly demonstrate that the NER gene XPA acts as a defensive factor against 4NQO-induced tongue carcinogenesis in vivo.

Abbreviations: NER, nucleotide excision repair; 4NQO, 4-nitroquinoline 1-oxide; SCC, squamous cell carcinoma; XP, xeroderma pigmentosum; XPA, XP group A; XPC, XP group C.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Nucelotide excision repair (NER) is a versatile DNA repair process responsible for the elimination of a broad spectrum of chemically and structurally distinct DNA lesions (1,2). Deficiency in a single NER gene has been shown to be associated with the human hereditary cancer-prone disease xeroderma pigmentosum (XP) (3,4). Xeroderma pigmentosum patients are hypersensitive to UV and appear to have a 1000–2000-fold increased risk of skin cancer compared with normal individuals (36). In addition, information gathered from a literature survey suggests that those who are under 20 years of age have a 10–20-fold higher incidence of several types of internal tumor (5). The relative rarity of internal tumor development might be due to the fact that XP patients rarely survive beyond the third decade of life, a consequence of the dramatic development of skin cancer (5). However, an increase in frequency of internal tumors is not readily explained as being solely due to UV-induced DNA damage.

The importance of NER in the prevention of UV- or chemical carcinogen-induced skin carcinogenesis has been well established by experimental studies in mice generated via gene targeting to mimic the phenotype of human XP (710). These XP-deficient mice clearly provide a useful model for investigating the role of NER in carcinogenesis in organs not exposed to UV. However, in contrast to skin carcinogenesis, little is known about the magnitude of the effect of the NER deficiency on internal tumors in either XP patients or XP-deficient mice.

The NER gene XPA encodes a 273 amino acid protein with sequence hallmarks of a DNA-binding zinc-finger domain which is implicated in DNA damage recognition (7). To evaluate the protective role of XPA against chemical carcinogenesis, XPA-deficient mice were exposed to a very low dose of 4NQO by oral administration. We also analyzed the tumors obtained by both immunohistochemistry and DNA sequence analysis to explore the involvement of the p53 gene in murine tongue carcinogenesis. As a result of the chronic exposure regimen, tongue SCCs only developed with high frequency in XPA–/– mice. The tongue cancer proneness is highly comparable with that observed in XP patients (5,6,11), although almost all of the tongue tumors in XP patients appear to be sunlight induced. This striking result supports the hypothesis that the XPA gene indeed protects against chemical carcinogenesis in organs other than the skin.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice
XPA–/–, XPA+/– and XPA+/+ mice were generated by mating heterozygous mice as described previously (8). Regarding genetic background, these mice originated from crosses between CBA, C57BL/6 and CD-1. Genotypes were determined by PCR using tail-tip DNA samples. The animals were maintained in a facility with a 12 h light/dark cycle, a temperature of 23°C and a relative humidity of 48.9%. The room lamps do not emit any measurable UV radiation and no daylight entered the room. The mice received NMF diet (Oriental Yeast, Tokyo, Japan) and sterilized tap water ad libitum.

Experimental protocol
Sixty XPA–/–, 30 XPA+/– and 30 XPA+/+ mice were available at the start of the experiment. Each group consisted of approximately equal numbers of male and female 6-week-old mice. 4-Nitroquinoline 1-oxide (Iwai Chemical, Tokyo, Japan) was dissolved and diluted with sterilized tap water to obtain a concentration of 0.001% (12). Water containing 4NQO was administered orally as drinking water ad libitum from light-shielded, polyvinyl bottles for 50 weeks. The bottles were refilled with fresh 4NQO solution once a week and the consumption of 4NQO solution was recorded to estimate intake of 4NQO. Animals were carefully checked weekly. At week 50 of 4NQO exposure, the mice were killed and a complete autopsy was performed. Tissues from all major organs were fixed in 10% buffered formalin and paraffin-embedded sections were stained with hematoxylin and eosin (H&E) for light microscopic examination.

Immunohistochemical staining of p53 protein
Accumulation of p53 protein was investigated using a polyclonal antibody, CM5 (Novocastra Laboratory, distributed by Dia-Iatron, Tokyo, Japan) at a dilution of 1:1000. The bound primary antibody was visualized by streptavidin–biotin–peroxidase detection using a Histofine SAB-PO (R) kit (Nichirei, Tokyo, Japan) according the manufacturer's instructions. 3',3-Diaminobenzidine tetrahydrochloride was employed as the color-developing agent (Wako Pure Chemical, Osaka, Japan) and slides were counterstained with hematoxylin. A tumor was classified as positive when at least 30% of the tumor nuclei were immunoreactive.

RNA extraction, RT–PCR and PCR amplification
At autopsy, portions of tumor and non-tumor tissues were separately excised, frozen in liquid nitrogen and stored at –80°C until use. RNA was extracted by the acid–guanidine thiocyanate phenol–chloroform method (13). RT–PCR for cDNA synthesis was performed essentially as described elsewhere (14,15). The primers used in this study covered the conserved regions of exons 4–10 of the mouse p53 gene (15). All amplifications included negative controls with de-ionized water in place of template cDNA.

Subcloning and sequencing analysis of the p53 gene
The amplified PCR products were purified by electrophoresis using low melting point agarose. The purified products were then subcloned into the EcoRV site of a T-tailed pBluescript II SK(–) (Stratagene, La Jolla, CA) as described previously (16). The sequences of DNA from mixed recombinant colonies, at least 50 subclones, were determined for both strands by the dideoxyribonucleotide chain termination method (17), using a T7 Sequencing kit (Pharmacia, Uppsala, Sweden). When a mutation was identified, PCR, cloning and sequencing analysis were repeated using the original cDNA to confirm the results.


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Tumor induction
There were no significant differences in mean body weights and total intake of 4NQO per mouse among the three genotypes (data not shown). Thus, all mice were exposed to the same amount of 4NQO. Of 60 XPA–/– mice given 4NQO solution, three died during the administration period due to toxic renal failure. The incidences and time course of development of tongue tumors are summarized in Table IGo and Figure 1Go. They were found in XPA–/– mice only with an average percentage of tumor-bearing mice of 86% (80% in males and 91% in females). The locations of tumors were the dorsal, ventral and lateral surfaces of the middle or posterior tongue (Figure 2Go). There was no evidence of tongue tumor development in any of the XPA+/– and XPA+/+ mice. As shown in Figure 1Go, the first tongue tumor was observed in experimental week 32. Tumors were also observed in the palate and/or gingiva in a few XPA–/– mice (five of 49 tongue tumor bearing mice). No tumors were observed in other organs. Only a few small lung tumors were detected in all three genotype groups (<1%).


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Table I. Frequencies of tongue tumors in XPA-deficient mice with 4NQO administration
 


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Fig. 1. Time course of tongue tumor development in XPA–/– (male, {bigcirc}; female, •) and XPA+/+ ({blacktriangleup}) mice.

 


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Fig. 2. Tongue tumors in 4NQO-treated XPA–/– mice.

 
Histopathological and immunohistochemical analysis of the tongue tumors
All 49 resected tongue tumors were diagnosed as SCCs (Figure 3A and BGo). There was no induction of papillomas or sarcomas. In all XPA–/– tongues, multiple foci of severe epithelial dysplasia or carcinoma in situ were observed (Figure 3CGo) and similar dysplastic changes were also detected in pharyngeal and esophageal epithelium (three of 49 tongue tumor bearing mice). Epithelial changes other than atrophy were not observed in XPA+/– and XPA+/+ tongues (Figure 3DGo). The smallest identifiable lesion in XPA–/– mouse tongues was the moderate epithelial dysplasia. There was no metastasis, as reported previously (12). Besides small pulmonary adenoma or alveolar cell hyperplasia, no neoplastic lesions in other organs were evident in any mice.



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Fig. 3. (A) Histological section through a tongue SCC. Magnification, x20. (B) Histology of a well-differentiated SCC. Magnification, x200. (C) Histology of tongue dysplastic epithelium. Magnification, x100. (D) Normal tongue epithelium. Magnification, x100.

 
Twenty-four of 49 tongue carcinomas (56%) contained cancer cells that were positively stained with p53 antibody (Figure 4AGo), but the staining pattern varied considerably. We also observed clusters of p53-positive cells in dysplastic epithelium surrounding carcinomas (Figure 4BGo). Histologically normal epithelium did not contain cells with positively stained nuclei (Figure 4CGo).



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Fig. 4. (A) Immunohistochemical staining of p53 protein accumulation in a tongue SCC. Magnification, x100. (B) Positive staining for p53 protein in dysplastic epithelium. Magnification, x200. (C) Negative staining for p53 protein in normal epithelium. Magnification, x200.

 
p53 mutations of the tongue carcinomas
As shown in Table IIGo, analysis of exons 4–10 of the p53 gene demonstrated point mutations leading to amino acid substitutions in four of 20 (20%) tongue carcinomas. Codons 139, 176 (exon 5) and 275, 279 (exon 9) were involved. Among nucleotide substitutions resulting in amino acid changes, three were transversions (two C-to-A and one C-to-G) and one was a transition (C-to-T). All mutations were heterozygous and these samples showed immunohistochemial positivity for p53 protein. No mutations were detected in non-tumorous tongue tissues of any mice of the three genotypes.


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Table II. Mutations of the p53 gene in tongue carcinomas in XPA-deficient mice
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
The protective role of an efficient NER in UV-induced skin carcinogenesis is best illustrated by the study of XP patients (3,4,18). Measuring the importance of NER for internal tumor development has been hampered by the limited data available and the fact that the XP patients examined belonged to different complementation groups with variable NER capacity (5,6,11). Although recently developed mouse models for XP can serve to answer this question (19,20), no firm conclusions could hitherto be drawn as to whether XP-deficient mice are indeed prone to cancer development in internal organs. There have been only three indications of chemical carcinogenesis in internal organs using XP-deficient mice (2022). de Vries et al. (21) demonstrated that XPA–/– mice are predisposed to induction of lymphomas by exposure to benzo[a]pyrene. The same group briefly mentioned that XPA–/– mice treated with 2-acetylaminofluorene developed tumors in 100% of female livers and in 90% of male bladders (20). They also observed 2-amino-1-methyl-6-phenylimidazo (4,5-b)-pyridine (PhIP)-induced adenomas of the small intestine in XPA–/– mice, although at lower frequency (20). Another experimental study showed that XPC-deficient mice, which are also defective in NER, are highly sensitive to liver and lung carcinogenesis after treatment with 2-acetylaminofluorene (22). In the present study, we found that chronic 4NQO exposure lead to tongue carcinomas, with high frequency, only in XPA–/– mice. Although the location of tongue SCC in XPA–/– mice is somewhat different from those of XP patients where the tumors occur on the tip of the tongue, it is noteworthy that the frequency of tongue cancer was found to be increased 1x104–2x104-fold in XP patients (5,11) and also affected individuals under the age of 20 are reported to have a 400-fold increased risk of extra-glossal oral cancers (5).

Recently, we observed that XPA–/– mice treated with intratracheal instillation of benzo[a]pyrene had a statistically significant increase of lung tumors (23). Interestingly, human XP cells are hypersensitive to killing and hypermutation by benzo[a]pyrene in vitro (24). Moreover, a study in our laboratory using aflatoxin B1 showed that the frequency of liver tumors was significantly higher in XPA–/– mice (unpublished data). These pooled data from XP-deficient mice, in line with our present observations, suggest that a deficiency in NER makes the animals sensitive to chemical induction of tumors in internal organs. It is generally accepted that the increase in internal tumors in XP patients appears to be primarily central nervous system tumors (3,46,11). The lack of spontaneous brain tumors in our XPA–/– mice might be due, in part, to the fact that under controlled conditions mice are not exposed to any known carcinogens. Conversely, the high incidence of internal tumors in XP patients may be closely associated with exogenous or endogenous NER-linked DNA damaging agents present in the environment (5,6,11,25). Further investigation as to whether the exposure to agents causing oxidative damages only accelerates internal tumor incidence in XPA mice are essential.

The potent carcinogenicity of 4NQO depends on the formation of DNA adducts (N2-guanine, C8-guanine and N6-adenine adducts), in addition to the exertion of oxidative stress in target cells (26,27). Nucleotide excision repair can efficiently eliminate bulky adducts (1,2) and also repair DNA damaged by oxygen radicals (25). 4NQO has been shown to produce oral SCC in rodents, mostly in palatal or tongue models (12,28,29). It can be applied either by topical painting (28), or by drinking (12,29), and both methods result in almost 100% of SCCs in the oral mucosa. There is a sequence of epithelial changes from simple hyperplasia through various epithelial dysplastic changes to invasive SCC in this model (12,28). Interestingly, the present study found no induction of benign epithelial hyperplasia or papillomas. Thus, XPA–/– mouse tongue SCCs arise de novo without malignant conversion from pre-existing papillomas.

Susceptibility to 4NQO-induced oral carcinogenesis is highly variable among animal species. In general, rats are the most susceptible (12,29), and hamsters the most resistant (30). Repeated painting of 4NQO to murine oral mucosa induces SCCs at high incidence (28), but comparable studies employing mice with 4NQO administered in the drinking water do not appear to have been performed. However, the possibility of producing oral epithelial dysplasia in mice has been documented (31). We consider that mice are resistant, like hamsters, to low concentrations of 4NQO (0.001%) in the drinking water, since none of our control mice (XPA+/– and XPA+/+) developed oral neoplastic lesions of any kind after up to 2 years (data not shown). Although d.M.von Pressentin et al. (32) observed a high mutagenic frequency in the Lac Z transgenic mouse tongue, studies into 4NQO-induced oral carcinogenesis using gene-targeted mice have not yet been published.

It is interesting to note that 4NQO is a UV-mimetic agent (33,34) and UV-sensitive cells from XP patients are also highly sensitive to 4NQO in vitro (3537). In contrast to XPA-deficient mice (deficient in both global genome and transcription-coupled repair pathways), XPC-deficient mice have no sensitivity to 4NQO (9,10). Although the reason for this difference is as yet unknown, transcription-coupled repair was found to be normal in XPC cells (2,10,38). The precise functions of the XPC gene remain largely speculative (2,9,10).

Mutations in the tumor suppressor p53 gene have been implicated in development of ~50% of all human tumors (39). Previous studies showed p53 mutation frequencies of 21–91% in human head and neck SCCs (40). In 7,12-dimethylbenz[a]anthracene-induced hamster oral carcinogenesis models, 25–40% of SCCs have exhibited p53 mutations (41,42). Mutational inactivation of the p53 gene was also demonstrated in 48% of UV-induced XPA-deficient mouse skin tumors (43). On the other hand, de Vries et al. (44) detected p53 mutations in only 8% of UV-induced skin tumors in XPA-deficient mice. Differences in frequencies of p53 mutations seem to be influenced by UVB dose and the mouse genetic background. In our study, only 20% of tongue carcinomas had detectable mutations in the p53 gene. This relatively low frequency is in keeping with the lack of such mutations in 4NQO-induced tongue tumors of normal rats (45). Another interesting observation was the presence of clusters of p53-positive cells in dysplastic lesions, suggesting that p53 alteration may be an early event in oral cancer. Using the same XPA-deficient mice, Takeuchi et al. (43) reported that no p53 positively staining cells were detected in either hyperplastic or dysplastic lesions of UV-exposed skin. The reason for this disparity is not clear. One conceivable explanation is that differences in the experimental protocols, with 4NQO versus UV and oral mucosa versus skin, are responsible.

In summary, our evidence suggests that the development of 4NQO-induced tongue cancer is critically dependent on the presence or absence of the NER gene XPA, since the amounts of 4-hydroxyaminoquinoline 1-oxide–DNA adducts demonstrated in histological sections by an immunohistochemical method (46) showed no significant differences among the three XPA genotypes (data not shown). Further experiments with p53-deficient mice and p53- and XPA-deficient mice are now in progress to determine the relative importance of the p53 gene for oral carcinogenesis.


    Notes
 
6 To whom correspondence should be addressed Email: pathonak{at}m.u-tokyo.ac.jp Back


    Acknowledgments
 
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture, the Ministry of Health and Welfare of Japan and the Smoking Research Foundation.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received August 30, 2000; revised December 22, 2000; accepted January 4, 2001.