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Molecular Cancer Research 4:401-410 (2006)
© 2006 American Association for Cancer Research

Model Organisms

p53 Transgenic Mice Are Highly Susceptible to 4-Nitroquinoline-1-Oxide-Induced Oral Cancer

Zhongqiu Zhang1, Yian Wang1, Ruisheng Yao1, Ronald A. Lubet2 and Ming You1

1 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri and 2 Chemoprevention Agent Development Research Group, National Cancer Institute, Bethesda, Maryland

Requests for reprints: Ming You, Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: 314-462-9294; Fax: 314-362-9366. E-mail: youm{at}wustl.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
In this study, we did a bioassay employing mice with a dominant-negative p53 mutation (p53Val135/WT) to assess whether a germ-line p53 mutation predisposed mice toward the development of squamous cell carcinomas (SCC) in the oral cavity. Treatment of the mouse oral cavity with 4-nitroquinoline-1-oxide produced a 66%, 91%, and 20% tumor incidence in the oral cavity, esophagus, and forestomach/stomach, respectively, in p53Val135/WT mice. In contrast, only a 25%, 58%, and 4% tumor incidence was observed in oral cavity, esophagus, and forestomach/stomach, respectively, in wild-type littermates (p53WT/WT). The most striking difference between p53Val135/WT and p53WT/WT mice following the carcinogen treatment was the higher prevalence and more rapid development of SSC in p53Val135/WT mice than in wild-type mice. To identify the precise genes or pathways involved in these differences during tumor development, we examined gene expression profiles of 4-nitroquinoline-1-oxide-treated normal tongues as well as tongue SCC in p53Val135/WT and p53WT/WT mice. Microarray and GenMAPP analysis revealed that dominant-negative p53 (135Valp53) affects several cellular processes involved in SCC development. Affected processes included apoptosis and cell cycle arrest pathways, which were modulated in both tumor and normal epithelium. These results showed that reduction of p53-dependent apoptosis and increases in cell proliferation might contribute to the observed increase in oral cavity and gastroesophageal malignancies in p53Val135/WT mice as well as to the more rapid growth and progression of tumors. (Mol Cancer Res 2006;4(6):401–10)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Head and neck cancer accounts for 3% to 5% of all malignancies in Western countries, with cancer of the oral cavity accounting for 30% of all head and neck cancers. Squamous cell carcinoma (SCC) of the oral cavity is the sixth most frequent cancer in the world and ~30,000 new cases are diagnosed annually in the United States (1). The development of oral SCC shows a positive correlation with exposure to tobacco and alcohol. Multiple genetic changes are thought to contribute to the development of SCCs of oral cavity (2-5).

The molecular pathways involved in the development of oral SCC are poorly understood. Mutations in the p53 gene are the most prevalent mutations in human cancer and can be sporadic or germ-line in nature (6, 7). The p53 protein is present in normal cells and induces cell cycle arrest or apoptosis in the presence of DNA damage. Mutant p53 protein lacks this property. Germ-line mutations in p53 are associated with the Li-Fraumeni family cancer syndrome, which predisposes individuals to multiple, unpredictable, aggressive, and often lethal tumors (8). We have shown previously that p53 transgenic mice with a dominant-negative p53 mutation are highly susceptible to carcinogen-induced lung adenoma/adenocarcinoma, uterine sarcoma, and colon adenocarcinoma (9-12). Mutation in the p53 gene is also commonly seen in SCCs of oral mucosa (13-16). Abnormal staining of p53 protein is a frequent finding (17-19).

The objective of this study was to determine the role of p53 tumor suppressor in mouse oral SCC development. A bioassay using dominant-negative p53 transgenic (p53Val135/WT) mice was done to assess if there is a correlation between germ-line p53 mutation and susceptibility toward SCC in the oral cavity. Treatment of mouse oral epithelia with 4-nitroquinoline-1-oxide (4NQO) is a well-established model for human head and neck SCCs (20, 21). The developing tumors are moderate to well-differentiated SCCs that morphologically resemble human tumors (19). We found that p53 transgenic mice are highly susceptible to 4NQO-induced oral cancer and that the resulting tumors are more likely to progress. In addition, we examined that the molecular pathways involved in the tumor development include apoptosis and cell cycle arrest.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Effect of p53 Germ-line Mutation (Ala135Val) on 4NQO-Induced Tumorigenesis
To determine whether the p53Val135/WT mice would be more susceptible to chemical induction of upper gastrointestinal tumors than wild-type littermates, a 4NQO-induced tumor bioassay was conducted. Six-week-old p53Val135/WT and p53WT/WT mice were treated with 4NQO. After 16 weeks of treatment and 32 weeks postinitiation, p53Val135/WT mice had developed significantly more and larger upper gastrointestinal tumors, including oral cavity, esophagus, and forestomach/stomach tumors, than their p53WT/WT counterparts.

As shown in Table 1 and Fig. 1A , in 4NQO-treated groups, the survival rate of wild-type mice was significantly higher than the p53 transgenic mice. Most p53Val135/WT mice were either dead or moribund before the end of the experiment due to advanced oral/upper gastrointestinal tumors. All tumor phenotypes, including those from the mice either dead or moribund, were included in Table 1. As shown in Table 1, treatment of p53Val135/WT mice with 4NQO produced more than 66%, 91%, and 20% incidences of oral mucosa, esophagus, and forestomach/stomach tumors, respectively, whereas only 25%, 58%, and 4% tumor incidences were observed in the oral mucosa, esophagus, and forestomach/stomach, respectively, in wild-type mice treated with the same carcinogen (Table 1). 4NQO produced an average of ~0.3 tongue SCC and ~1.0 esophageal SCC in p53WT/WT mice, whereas treatment of p53Val135/WT mice produced an average of ~1.0 tongue SCC and ~3.2 esophageal SCC (Table 1).


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  		Table 1. 4NQO-Induced Tumorigenesis in p53 Transgenic (p53Val135/WT) Mice

 

Figure 1
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  		FIGURE 1. 4NQO-induced mouse tongue tumorigenesis in p53Val135/WT and p53WT/WT mice. A. Survivorship of p53Val135/WT and p53WT/WT mice treated with 4NQO. Six-week-old UL53-3 mice were randomized into four groups according to the p53 genotypes (p53WT/WT or p53Val135/WT). All mice were treated with 4NQO or vehicle control thrice weekly for 16 weeks. The experiment was terminated 32 weeks after the last treatment with 4NQO. B. Histopathology of 4NQO-induced tumors in p53Val135/WT mice. a and b, light photomicrographs of tongue SCC at x40 and x400 magnification from p53Val135/WT mice, respectively; c and d, light photomicrographs of esophageal SCC at x40 and x400 magnification from p53Val135/WT mice, respectively; e and f, light photomicrographs of forestomach SCC at x40 and x400 magnification from p53Val135/WT mice, respectively.

 
Morphologically, oral cavity lesions included lip, tongue, palate, and larynx tumors. Some tumors were grossly enlarged with invasion to surrounding tissues. In some advanced cases in the p53Val135/WT group, the entire oral cavity was diffusely swollen and the bottom of the oral cavity was grossly enlarged, with head and neck lymph node involvement. Sometimes, enlarged oral SCC could be easily palpated through the neck or protruded through the mouth. Histopathologically, these tumors were oral cavity, esophageal, and forestomach SCCs (Fig. 1B). Most SCCs showed proliferation into the submucosa and exhibited features of squamous cell differentiation, such as keratin pearls. The majority of p53Val135/WT mice developed tumors more rapidly than the wild-type mice and the tumors displayed more obvious features of malignancy, including large size, lymph node involvement, more nuclear atypia, and invasiveness. Furthermore, histopathology data showed that SCCs had relatively poorly differentiated cells in p53Val135/WT tumors when compared with p53WT/WT tumors. Some poorly differentiated SCCs are composed of spindle-shaped cells and show no keratinization. No tumors were found in either p53WT/WT or p53Val135/WT vehicle control mice. Because spontaneous upper gastrointestinal tumors are rare in mice, p53Val135/WT mice have an increased susceptibility to 4NQO-induced oral and upper gastrointestinal SCCs. No significant differences between the body weights of any of the groups treated with 4NQO compared with controls occurred in either experiment. However, the majority of p53Val135/WT mice were terminated before the end of the experiment due to overt SCCs. The survivorship data are shown in Fig. 1A.

Microarray Analysis of Normal and Tumor Tissues from Tongues Treated with 4NQO
To identify the precise genes or pathways responsible for these differences in tumor susceptibility and growth, four to five normal and tumor samples from both p53WT/WT and p53Val135/WT groups were examined. In normal tongue tissues, microarray analysis revealed 271 genes differentially expressed between p53WT/WT and p53Val135/WT mice treated with 4NQO. Among them, 67 genes were underexpressed, whereas 204 genes were overexpressed in p53Val135/WT normal tissues when using 2-fold change and P < 0.05 (t test) as cutoff (Fig. 2A ). Moreover, when we compared the expression of tongue SCCs, 913 genes were found, which showed differential expression between p53WT/WT and p53Val135/WT tumors. Among them, 377 genes were underexpressed, whereas 536 genes were overexpressed in p53Val135/WT tumors (Table 2 ; Fig. 2B). We also ranked the genes in the normal-to-normal and tumor-to-tumor comparisons based on fold expression difference in Tables 3 and 4 . We then verified the finding from microarray analysis that the expression of Rassf1A was decreased in tumors from p53Val135/WT mice when compared with those from p53WT/WT mice. We conducted methylation-specific PCR (MSP) analysis to obtain a methylation profile of Rassf1A in SCC. As shown in Fig. 3 , 25% of tumors from p53WT/WT mice and 75% of the tumors from p53Val135/WT mice showed Rassf1A promoter methylation. No Rassf1A methylation was detected in paired normal tissues. These results indicate a correlation between array data and hypermethylation status of the Rassf1A gene in SCC (Table 2).


Figure 2
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  		FIGURE 2. Microarray analysis 4NQO-induced mouse tongue tumors. A. Differentially expressed genes found in normal tissues of p53Val135/WT mice. More than 271 differentially expressed genes were detected in normal tongue tissues induced by 4NQO; 67 genes were underexpressed and 204 genes were overexpressed in p53Val135/WT mice. Green, expression below the mean for the gene; black, near the mean; red, above the mean. B. Differentially expressed genes found in tumors of p53Val135/WT mice. Over 913 differentially expressed genes were detected in tongue SCCs induced by 4NQO; 377 genes were underexpressed and 536 genes were overexpressed in p53Val135/WT mice.

 

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  		Table 2. Microarray Analysis for Differentially Expressed Genes between p53WT/WT and p53Val135/WT in Early and Later Stages from Mouse Oral Tumorigenesis

 

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  		Table 3. Microarray Analysis for Top 50 Differentially Expressed Genes between p53WT/WT and p53Val135/WT Mouse Normal Oral Tissues

 

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  		Table 4. Microarray Analysis for Top 50 Differentially Expressed Genes between p53WT/WT and p53Val135/WT Mouse Oral Tumors

 

Figure 3
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  		FIGURE 3. MSP analysis of Rassf1A in 4NQO-induced tumors. M, presence of methylated Rassf1A alleles in tumors; U, presence of unmethylated Rassf1A alleles in tumors or normal tissues. In vitro methylated DNA (lane 1) and normal tissue (lane 2) were used as positive and negative controls for Rassf1A promoter methylation, respectively. Lanes 3 to 5, representative MSP analysis on tumors from p53WT/WT mice; lanes 6 to 9, representative MSP analysis on tumors from p53Val135/WT mice. H2O was also included in each reaction as negative control (lane 10).

 
Finally, we used GenMAPP to illustrate all the available pathways containing differentially expressed genes associated with p53 status in tongue tumorigenesis. Several cellular pathways, such as apoptosis, cell cycle, transforming growth factor-ß, and Ras-mitogen-activated protein kinase pathways, showed alterations in both 4NQO-treated normal tissue and tumors from p53WT/WT versus p53Val135/WT mice. Among them, the cell cycle and apoptotic signaling pathways played an important role in 4NQO-induced tongue tumorigenesis. As shown in the cell cycle regulation pathway (Fig. 4A ), the expression of p53, Atm, Bub1, Tgfb1, Cdc25b, Orc21, and Cdc7 was differentially expressed in normal tissues of p53WT/WT versus p53Val135/WT mice. In addition, the expression Mdm2, Ccna1, Abl1, and Tfdp1 was differentially expressed in tumors of p53WT/WT versus p53Val135/WT mice. In the apoptotic pathways, expression of Bid and p53 was altered in normal tissues of the p53Val135/WT mice, whereas several death receptors or activators of cell death, such as Tnf, Tnfsf10, Prf1, Tradd, Ripk1, Apaf1, Mdm2, Bcl2, and Casp7, were found altered in p53Val135/WT tumors (Fig. 4B). These results indicate that deregulation of cell cycle and reduction of apoptosis activity may, in part, contribute to the increased oral malignancy in p53Val135/WT mice on carcinogen exposure.


Figure 4
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  		FIGURE 4. A and B. GenMAPP cell cycle regulation and apoptosis pathways integrate the expression data (cutoff of P < 0.05; fold change >1.5). Yellow, changes found in normal tissues from the p53Val135/WT mice; blue, changes found in 4NQO-induced p53Val135/WT tumors when compared with those from p53WT/WT mice; gray, the selection criteria were not met, but the gene is represented on the array. White boxes, the gene was not present on the chip.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
In the present study, we induced upper gastrointestinal malignancy with the carcinogen 4NQO, which was documented to induce oral cavity tumors in various species and multiple strains of mice (20-22). Using p53 transgenic mice, we found that introduction of a dominant-negative p53 mutant (Ala135Val) into the germ-line significantly enhanced tumorigenesis in the oral mucosa and upper gastrointestinal system on 4NQO treatment. The most striking distinction between p53Val135/WT and p53WT/WT mice on carcinogen treatment was that the majority of p53Val135/WT mice developed tumors more rapidly than the wild-type mice and the tumors displayed more obvious histologic features of malignancy. Thus, at 9 months following the initial 4NQO treatment, ~60% of the p53Val135/WT mice had died, whereas <15% of the p53WT/WT mice had died. In contrast, survival was >95% for control mice of either genotype. This implies that the mutant p53 allele greatly increased SCC susceptibility possibly through a promotion function of the missense protein. The wild-type p53 protein facilitates the regulation of genomic stability by preventing cell cycle entry and initiates apoptotic cell death in response to DNA damage (23, 24). Thus, loss of normal function of p53 leads to deregulation of the cell cycle and continued cell cycle progression after DNA damage resulting in accumulation of genetic damage. Our results strongly suggest that p53 plays a role in proliferation and apoptosis of squamous cells exposed to chemical carcinogens. Altered p53 protein expression has been associated with increased proliferative rates in other tumors, such as human breast cancer (25). The observation in this study is the profound effect of the mutant p53 transgene on development of upper gastrointestinal SCC in p53Val135/WT mice, strongly indicating that upper gastrointestinal SCC formation is highly p53 dependent.

Our findings are consistent with the results from Gallo et al. (26) in which they detected a germ-line p53 mutation in head and neck cancer patients with multiple malignancies and in their first-degree relatives. In addition, Rowley et al. (27) and Qin et al. (28) reported that the p53 gene is an early target for mutation in oral tumor development, with mutations being detected in 40% to 50% of premalignant lesions and SCCs. More recently, Ide et al. (29) found that p53 haploinsufficiency profoundly accelerates the onset of tongue tumors in mice lacking the xeroderma pigmentosum group A gene. All together, these results suggest that p53 plays a major and early role in head and neck tumor development. Our finding would seem to make this in situ mouse model particularly relevant for studying the role of p53 in head and neck tumorigenesis as well as for screening potential preventive or therapeutic agents for head and neck cancer.

To profile genes or pathways responsible for enhanced susceptibility to SCC in the oral cavity on 4NQO induction as well as the more rapid tumor growth and progression, altered gene expression in tongue tumors and normal tissues was investigated. Microarray studies with the GenMAPP analysis revealed that germ-line p53 mutation affects several cellular processes involved in the tumorigenesis possibly through the interplay among apoptosis, cell cycle arrest, transforming growth factor-ß signaling pathway, and Ras-mitogen-activated protein kinase pathway. For example, our data showed that wild-type p53 expression levels in both normal and tumor tissues were significantly decreased in p53Val135/WT mice, which regulate multiple cell type–dependent intracellular and cellular events. Thus, cross-talk of these cellular processes may be involved in the tumorigenic process itself. Furthermore, microarray results showed that cell proliferation significantly increased, whereas apoptosis activity decreased in p53Val135/WT samples compared with that from p53WT/WT mice. On exposure p53Val135/WT mice to 4NQO, the wild-type p53 could not function properly because of the dominant-negative effect of the mutant transgene, resulting in higher cell proliferation and decreased apoptosis relative to p53WT/WT mice. In p53WT/WT mice, high levels of wild-type p53 protein were induced, resulting in cell cycle arrest that should subsequently allow cells to recover from damage or undergo apoptosis. These results suggested that reduction of p53-dependent cell cycle inhibition and apoptosis might contribute to the observed increase in upper gastrointestinal tumor incidence in p53Val135/WT mice.

In summary, the present study and our previous in vivo studies have shown that in situ tumors with a germ-line p53 mutation can be induced in a variety of organ sites, including lung, uterus, colon, and upper gastrointestinal system (9-12). Microarray analyses began to offer clues for the mechanisms of tumor development associated with inactivation of p53. Our findings show that the combined use of mice with a dominant-negative p53 mutation combined with the carcinogen 4NQO yields a model of SCC of the upper gastrointestinal tract system that seems relevant to human oral SCC.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Agent
The chemical carcinogen 4NQO (Sigma Chemical Co., St. Louis, MO) was dissolved in propylene glycol to a final concentration of 5 mg/mL. The solution was stored at 4°C and a fresh aliquot was used each application session.

Animals
p53Val135/WT mice carrying a 135Valp53 mutation in exon 5 were obtained from the National Institute of Environmental Health Sciences (Research Triangle Park, NC). Mice were housed four per cage in plastic cages with hardwood bedding and dust covers in a HEPA-filtered, environmentally controlled room (24 ± 1°C with a 12-hour light/12-hour dark cycle).

p53 Genotype
p53Val135/WT mice were developed by microinjection of FVB/J mouse oocytes with a BALB/c mouse genomic clone of the p53 gene containing a point mutation in codon 135 (Ala-to-Val) at exon 5. The mutation, a C-to-T transition, created a RFLP with a new HphI restriction enzyme cleavage site (recognition site: GGTGA). This mutation was used to genotype p53Val135/WT mice using PCR-RFLP method as described previously (9).

Tumor Development
Six-week-old p53Val135/WT mice were randomized into four groups according to the p53 genotypes (Table 1). Mice in groups 3 and 4 were lightly anesthetized by inhalation of methoxyflurane vapor. The palate was stroked once from the soft palate to the incisive papilla with a no. 3 camel hairbrush, which had been dipped once in 4NQO solution. Mice in groups 1 and 2 were given vehicle control (propylene glycol). All mice were treated by direct application thrice weekly for 16 weeks. Thirty-two weeks after the last treatment with 4NQO, animals from all four groups were euthanized by CO2 asphyxiation. A portion of the upper gastrointestinal tumors and normal tissues were isolated, placed in individual tubes, and immediately frozen in liquid nitrogen. The remaining tumors and normal tissues were fixed in 10% neutral-buffered formalin overnight followed by 70% ethanol and paraffin embedding. Tissue sections (5 µm) were stained with H&E for histopathologic evaluation. A gross necropsy was done.

RNA Isolation and Amplification
Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified using the RNeasy Mini kit and RNase-free DNase Set (Qiagen, Valencia, CA) according to the manufacturer's protocols. In vitro transcription-based RNA amplification was then done on each sample. cDNA for each sample was synthesized using a SuperScript cDNA Synthesis kit (Invitrogen, Carlsbad, CA) and a T7-(dT)24 primer: 5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3'. The cDNA was cleaned using phase-lock gel (Fisher, Hampton, NH) phenol/chloroform extraction. Then, the biotin-labeled cRNA was transcribed in vitro from cDNA using a BioArray High-Yield RNA Transcript Labeling kit (ENZO Biochem, New York, NY) and purified again using the RNeasy Mini kit.

Affymetrix GeneChip Probe Array and Cluster
The labeled cRNA was applied to the Affymetrix Mu74Av2 GeneChip (Affymetrix, Santa Clara, CA), which contains >12,000 genes and expressed sequence tags on one array according to the manufacturer's recommendations. Every gene or expressed sequence tag is represented by a probe set consisting of ~16 probe pairs (oligonucleotides) of 25-mer oligonucleotides. One sequence of a probe pair represents the complementary strand of the target sequence, whereas the other has a 1-bp mismatch at the central base pair position. This mismatch sequence serves as an internal control for specificity of hybridization.

Four or five independent samples were collected for each group. Array normalization and gene expression estimates were obtained using Affymetrix Microarray Suite version 5.0 software. The array mean intensities were scaled to 1,500. These estimates formed the basis for statistical testing. Differential expression was determined on the combined basis of statistical testing using Student's t test and based on a ratio with a cutoff of P < 0.05 and fold change ≥2 called positive for differential expression. For the selected genes, expression indices were transformed across samples to a N(0,1) distribution using a standard statistical Z-transform. These values were put into the GeneCluster program of Eisen et al. (30) and genes were clustered using average linkage and correlation dissimilarity.

Rassf1A Methylation Analysis
The methylation status of the Rassf1A promoter region was determined by chemical modification of genomic DNA with sodium bisulfite and MSP. Bisulfite treatment converts cytosine bases to uracil bases but has no effect on methylcytosine bases. Bisulfite-treated DNA was used as a template for the MSP reaction. Primers for unmethylated reaction were forward 5'-GGTGTTGAAGTTGTGGTTTG-3' and reverse 5'-TATTATACCCAAAACAATACAC-3'. Primers for methylated reaction were forward 5'-TTTTGCGGTTTCGTTCGTTC-3' and reverse 5'-CCCGAAACGTACTACTATAAC-3'. The reaction was incubated at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute for 35 cycles. The methylation fragment was 213 bp, and the nonmethylation fragment was 204 bp. DNA from normal skin was used as a control for unmethylated Rassf1A, and normal skin DNA treated with SssI methyltransferases was used as a control for methylated Rassf1A. H2O was used as negative control. Each PCR (25 µL) was loaded onto a 6% nondenaturing polyacrylamide gel, stained with ethidium bromide, and pictured under UV light.

GenMAPP
Signal transduction pathways, metabolic pathways, and other functional groupings of genes were evaluated for differential regulation using the visualization tool GenMAPP (University of California at San Francisco; http://www.genmapp.org). GenMAPP is a recently reported tool for visualizing expression data in the context of biological pathways (31). We imported the statistical results of our data set into the program and used GenMAPP to illustrate pathways containing differentially expressed genes. Differential gene expression was based on p53 genotype status (P < 0.05; fold change >1.5).

Statistical Analysis
Fisher's exact test was used to determine the difference in incidence between p53Val135/WT and p53WT/WT mice. Nonparametric Wilcoxon's rank-sum test was used to determine the difference in tumor multiplicity between p53Val135/WT and p53WT/WT mice.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Dr. Roger W. Wiseman for providing the p53 transgenic mice and Drs. Daolong Wang and William J. Lemon for statistical assistance. Raw data are available upon request.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Grant support: NIH grants R01CA58554 and N01CN25104.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Z. Zhang and Y. Wang contributed equally to this work.

Received 2/ 1/06; revised 3/28/06; accepted 4/13/06.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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S. Miyamoto, Y. Yasui, M. Kim, S. Sugie, A. Murakami, R. Ishigamori-Suzuki, and T. Tanaka
A novel rasH2 mouse carcinogenesis model that is highly susceptible to 4-NQO-induced tongue and esophageal carcinogenesis is useful for preclinical chemoprevention studies
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