o-Nitrotoluene-induced large intestinal tumors in B6C3F1 mice model human colon cancer in their molecular pathogenesis

R.C. Sills1,4, H.L. Hong1, G. Flake1, C. Moomaw1, N. Clayton1, G.A. Boorman1, J. Dunnick2 and T.R. Devereux3

1 Laboratory of Experimental Pathology, 2 Laboratory of General Toxicology and 3 Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences. PO Box 12233, Research Triangle Park, NC 27709, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the previous 500 2-year chemical bioassays within the National Toxicology Program, large intestinal tumors (cecal carcinomas) related to chemical exposure have not been observed in B6C3F1 mice. The recently completed o-nitrotoluene study provided the first cecal tumor response and an opportunity to evaluate the morphology and molecular profile of oncogenes and tumor suppressor genes that are relevant to humans. Morphologically, the carcinomas were gland-forming tumors lined by tall columnar epithelial cells that were positive for cytokeratin 20 and negative for cytokeratin 7. Using immunohistochemistry ß-catenin (encoded by Catnb) protein accumulation was detected in 80% (8/10) of the cecal carcinomas, while increased cyclin D1 and p53 protein expression was detected in 73% (8/11), respectively. There was no difference in adenomatous polyposis protein expression between normal colon and cecal carcinomas. All tumors examined exhibited mutations in exon 2 (corresponds to exon 3 in humans) in the Catnb gene. Mutations in p53 were identified in nine of 11 carcinomas, and all were in exon 7. Analysis of the K-ras gene revealed mutations in 82% (9/11) of carcinomas; all had specific G -> T transversions (Gly -> Val) at codons 10 or 12. The alterations in cancer genes and proteins found in the mouse large intestinal tumors included mutations that activate signal transduction pathways (K-ras and Catnb) and changes that disrupt the cell-cycle and bypass G1 arrest (p53, cyclin D1). These alterations, which are hallmarks of human colon cancer, probably contributed to the pathogenesis of the large intestinal carcinomas in mice following o-nitrotoluene exposure.

Abbreviations: APC, adenomatous polyposis; CK, cytokeratin; LOH, loss of heterozygosity; SSCP, single stranded conformation polymorphism


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon cancer, the third leading cause of cancer deaths in the US is a multi-step process involving activation of oncogenes, inactivation of tumor suppressor genes and defects in repair enzymes (13). Although the adenomatous polyposis (APC) tumor suppressor gene is often mutated in familial adenomatous polyposis (FAP) colorectal cancer, a subset of colon cancers that lack APC mutations have CTNNB1 (ß-catenin) mutations (4,5). Activating mutations of the CTNNB1 gene or inactivating mutations in the APC gene are early steps in the development of colon cancer (2,6,7). These mutations disrupt the phosphorylation dependent breakdown of ß-catenin by ubiquitination, thus allowing ß-catenin to accumulate and Wnt signaling to accelerate. The progression of FAP colorectal cancer has been well documented by histological changes that are linked to a series of molecular events including genetic alterations in the APC gene, KRAS oncogene, p53 tumor suppressor gene and TGFß growth regulatory genes (1,8,9).

In addition to genetic factors that have been well documented in human colon cancer, environmental factors may contribute to the estimated 130 000 new cases of colon cancer per year in the US (10). In the history of the National Toxicology Program (NTP), which has conducted over 500 cancer studies in rodents, the o-nitrotoluene study is the first 2-year bioassay in which large intestinal tumors were detected in B6C3F1 mice (11). o-Nitrotoluenes are high production chemicals with over 30 million pounds produced in the US each year (US EPA, 2000). o-Nitrotoluene is used to synthesize agricultural and rubber products, azo and sulfur dyes, and dyes for cotton, wool, silk, leather and paper (12). Environmental surveys have detected low levels of o-nitrotoluene in rivers and drinking water (US EPA, 1976).

Over the past decade, our research has focused on identifying alterations in cancer genes such ras protooncogenes (1315), the p53 tumor suppressor gene (16,17) and the Catnb gene that occur predominantly as single alterations in mouse tumors (18,19). The aim of this study was to examine these major cancer genes that have been associated with colon cancer in humans for mutations and/or expression changes. Mutations in KRAS occur in 50% of human colon tumors while mutations in the p53 and APC or CTNNB1 with subsequent over-expression of Cyclin D1 occur at rates of 75 and 50%, respectively (1,2,20).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Neoplasms
Male and female B6C3F1 mice were exposed to 1250, 2500 or 5000 p.p.m. o-nitrotoluene in feed for 2 years (12). At necropsy, cecal carcinomas (large intestinal tumors) were observed mainly in the 1250 p.p.m. groups (males, 12/60; females, 1/60) and 2500 p.p.m. groups (males, 9/60; females, 4/60) and rarely in 5000 p.p.m. groups (males, 0/60; females, 3/60). The carcinomas were fixed in 10% neutral-buffered formalin, routinely processed, embedded in paraffin, sectioned to a thickness of 5 µm, and stained with hematoxylin and eosin. Subsequently, eight unstained serial sections, 10 µm thick, were prepared from each paraffin block containing cecal carcinomas for isolation of DNA for PCR-based assays. Eleven o-nitrotoluene-induced cecal carcinomas and two normal regions of the cecum provided adequate amounts of DNA and tissue for assessing the genes of interest and for conducting immunohistochemical studies. Generally, the cecal carcinomas that were not used in these molecular studies, including the three carcinomas in 5000 p.p.m. females, were too small because of early deaths due to a high incidence of hemangiosarcomas (12). The early deaths and small size of some tumors limited the analysis to the 11 tumors that had adequate tissue. There were no cecal carcinomas in control mice from this study, or within our historical database of over 500 studies.

Immunohistochemistry: cytokeratin 20 and cytokeratin 7
Cecal carcinomas were screened for cytokeratins (CK) as human colon adenocarcinomas are commonly CK 20 positive and CK 7 negative (21). Briefly, after two rinses in phosphate-buffered saline (PBS)/detergent (0.05% Brij), endogenous peroxidase activity was quenched by incubating each section in a 3% H2O2/methanol solution for 5 min. After a second series of two PBS/detergent rinses, non-specific binding of reagents was blocked by incubation with 2x concentration of the protein solution recommended and provided in the M.O.M kit (Vector Laboratories, Burlingame, CA) for 60 min. After two 2-min washes in PBS/detergent, slides were incubated for 5 min in M.O.M diluent. Excess diluent was decanted and the CK 20 or CK 7 primary mouse monoclonal antibodies (Research Diagnostics, Flanders, NJ) were diluted in M.O.M diluent at dilutions of 1:10 and applied for 30 min. Species-matched, isotype-matched, concentration-matched negative control antibodies were also applied to replicate positive control tissue sections for 30 min. Subsequently, after two 2-min PBS/0.05% Brij washes, all slides were incubated for an additional 10 min in a 1:10 dilution of the recommended working solution of the M.O.M. biotinylated IgG reagent. Following incubation with the ‘ABC’ Elite reagent for 30 min, 3,3-diaminobenzidine was applied for 2 min as substrate or the peroxidase reaction. Slides were counterstained with 50% hematoxylin for 30 s, Richard Allen bluing for 1 min, dehydrated and cover slipped for light microscopic evaluation.

Immunohistochemistry: ß-catenin, APC, p53 and Cyclin D1 protein
o-Nitrotoluene-induced cecal carcinomas and normal cecal regions were screened for ß-catenin, APC, p53 and cyclin D1 protein expression. Localization of ß-catenin expression was investigated using a polyclonal goat ß-catenin antibody at a dilution of 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA) (18). Non-immune rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as the negative control at equivalent conditions in the place of the primary antibody. Positive control for the ß-catenin protein was a mouse hepatoblastoma where a ß-catenin point mutation was confirmed by sequencing. APC immunohistochemistry was conducted using a rabbit polyclonal anti-APC antibody that was raised against a peptide corresponding to the last 20 amino acids of the C-terminus of the human APC protein, at a dilution of 1:200 (Santa Cruz Biotechnology) as described previously (22). The immunohistochemical staining for expression of mutant p53 protein was performed as described previously (16,23). Affinity purified rabbit polyclonal cyclin D1 antibody was obtained from Santa Cruz Biotechnology and immunohistochemical staining was performed as described previously (24).

DNA isolation and amplification
DNA was isolated from paraffin-embedded sections containing cecal carcinomas and normal cecal regions, using a DNeasy tissue kit (Qiagen, Valencia, CA). DNA was amplified by the polymerase chain reaction (PCR), and the use of nested primers for K-ras has been described (25). Touchdown PCR was performed for exons 5–8 of the p53 gene, and PCR/sequencing primers and annealing temperature profile of the cycles have been reported previously (17). The use of nested primers for Catnb have been described previously (18). Normal controls for K-ras, p53 or Catnb genes and no DNA controls were run with all sets of reactions.

Cycle sequencing
To identify mutations, samples were sequenced utilizing a cycle sequencing kit (US Biochemical, Cleveland, OH) which incorporates [{alpha}-33P]dideoxynucleotide (ddNTP) terminators (A, C, G, T) into the sequencing products (25,26). Prior to sequencing, PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen). The amplification primers also served as sequencing primers. Mutation identification was confirmed with two amplification reactions from original DNA.

Loss of heterozygosity analysis
To determine loss of heterozygosity (LOH) in the o-nitrotoluene-induced cecal carcinomas at marker D6MCO12 (27) near the K-ras gene on chromosome 6, single stranded conformation polymorphism (SSCP) analysis was utilized. SSCP distinguished a single nucleotide polymorphism in the amplified PCR product between the C3H(H) and C57BL/6(B) alleles. Allelic loss was identified by comparing the difference in density of the C57BL/6 band to the C3H band of the marker in the tumors to those from controls of normal B6C3F1, C57BL/6 or C3H mouse tissues. A semi-nested PCR technique was used to amplify the D6MC012 marker with outer primers 4F (5'-CCGAGGAGGAAGATGTCAAAC-3') and 3R (5'-CCGGGTTAGTTCTTCATGATT-3') for 25 cycles using 2 µl of the DNA extracted from 11 cecal carcinomas in a 20 µl reaction, and primers1F (5'-GATGTCAAACGTGAGAGTGTC-3') and 3R for an inner reaction of 28 cycles. One microliter of the outer PCR product was added to a 10 µl reaction that contained 0.2 µl [33P]dATP (10 µCi/µl, ICN Biomedicals, Costa Mesa, CA). Unlabeled dATP was diluted 1 in 40 or 1 in 100 in the inner PCR reaction mixture. Upon completion of the amplification, two sets of the PCR products were diluted 1:10 in sequencing stop solution with 5% 1 M methylmercury hydroxide, the samples denatured at 85°C for 5 min and 5 µl loaded onto a MDE gel (Cambrex BioScience Rockland, Rockland, ME). The gel was electrophoresed at 3 W at room temperature for 14.5 h to separate the alleles. The gels were dried and exposed to X-ray film overnight.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Eleven cecal carcinomas from B6C3F1 mice exposed to o-nitrotoluene were examined microscopically and analyzed for genetic alterations and altered expression of several genes associated with colon cancer in humans. Morphologically, the cecal tumors consisted of moderately to poorly differentiated adenocarcinomas that invaded into and through the muscular wall (Figure 1a and b). The glandular epithelium was often of the tall columnar type (Figure 1c), and all 11 of the tumors were CK 20 positive and CK 7 negative (Figure 1d). These cellular characteristics are consistent with that reported for human colon adenocarcinomas (http://www.immunoquery.com) (21,28).



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Fig. 1. Histopathology of cecal carcinomas in B6C3F1 mice exposed to o-nitrotoluene in the diet for 2 years. (a) Neoplastic glands infiltrate the muscularis mucosa and submucosa and extend into the muscular wall (arrows), hematoxylin and eosin (H&E), 4x. (b) High magnification of (a) (arrows) showing malignant glands within the muscular wall, 20x. (c) Note the tall columnar epithelial cells (arrows) lining the malignant glands, 40x. (d) Cecal carcinoma with positive cytoplasmic staining for cytokeratin 20 (CK 20+) and negative staining for cytokeratin 7 (CK7-).

 
The carcinomas were first examined for mutations in exon 2 of the Catnb (ß-catenin) gene, the region that contains potential phosphorylation sites for glycogen serine threonine kinase. Catnb mutations were detected in all 11 cecal carcinomas examined (Table I, Figure 2). Ten of the cecal carcinomas had deletions in the Catnb gene at the beginning of exon 2 and/or codon 5 (Table I). Most of the deletions involved the splice site and were in frame deletions of two or more codons (Figure 2a). Point mutations in Catnb were also detected at codons 15, 25, 37 or 41 (Table I, Figure 2b). As Catnb mutations were identified in all of the o-nitrotoluene-induced cecal carcinomas examined, and there was no difference in APC protein expression between normal colon and carcinomas (data not shown), the APC gene was not evaluated further for mutations.


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Table I. Summary of K-ras, cyclin D1, p53 and Catnb mutations in cecal carcinomas following o-nitrotoluene exposure

 


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Fig. 2. Example of Catnb deletion mutations in o-nitrotoluene-induced cecal carcinomas from B6C3F1 mice by cycle sequencing analysis. Sequencing panel (a) is normal ß-catenin sequence, and panel (b) is sequence with deletions at codons 5–6 and exon 2 splice site from sample 9 cecal carcinoma. (b) Identification of Catnb point mutation in o-nitrotoluene-induced cecal carcinomas from B6C3F1 mice by cycle sequencing analysis. A, C, G and T represent the 4 nt in DNA, adenine, cytosine, guanine and thymine, respectively. Sequencing panel (a) is normal ß-catenin sequence, and panel (b) is mutated sequence at indicated codon 41 from sample 4 cecal carcinoma.

 
Eight of ten o-nitrotoluene-induced cecal carcinomas examined exhibited increased expression of ß-catenin protein by immunohistochemistry (Figure 3a and b, Table I). Positive immunostaining was localized primarily to the cell membranes, except for sample number 6, which also had positive nuclear staining (Figure 3b). ß-Catenin membrane staining was most prominent in the tumor cells that were more anaplastic and less differentiated. Cyclin D1 nuclear protein expression was present in eight of 11 (73%) cecal carcinomas (Figure 3c and d).



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Fig. 3. Immunohistochemical analysis of ß-catenin, cyclin D1 and p53 expression in cecal carcinomas of B6C3F1 mice exposed to o-nitrotoluene in the diet for 2 years. (a) (Sample 1, Table I): note the prominent ß-catenin protein expression (brown chromagen) in the cecal carcinoma; neoplastic cells invading the cecal wall are also positive for the ß-catenin protein (arrows), 10x. (b) (Sample 6): high magnification showing both membrane and nuclear staining for the ß-catenin protein within the cecal carcinoma, 80x. (c) (Sample 6): cyclin D1 expression within nuclei of malignant cecal cells, 132x. (d) Note the lack of cyclin D1 staining in the negative control, 132x. (e) (Sample 11): note the marked accumulation of the p53 protein within the cecal carcinoma and invading cells (arrows), 10x. (f) (Sample 4): high magnification showing the localization of the p53 protein within the nucleus of neoplastic cells, 100x.

 
Nine of eleven (82%) o-nitrotoluene-induced cecal carcinomas had K-ras mutations, all nine had G -> T transversions (Gly -> Val) at codons 10 or 12 (Table I, Figure 4a). Sample number two had double K-ras mutations at codons 12 and 13. Mutations at K-ras codon 61 were not detected. Allelic loss on chromosome 6 near K-ras gene was observed in 36% (4/11) of o-nitrotoluene-induced cecal carcinomas (Table I), and examples of LOH detection by SSCP were shown in Figure 4b. Tumor samples 2 and 11 (Table I) lost the C57(B) allele, while samples 8 and 10 lost the C3H(H) allele. Loss of the wild-type allele of K-ras in samples 2 and 10 by sequencing (Figure 4a) correlated with chromosome 6 LOH as assessed by SSCP (Table I, Figure 4b). K-ras mutations were not identified in samples 1 or 6, and also chromosome 6 LOH was not detected in these samples.



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Fig. 4. (a) Identification of K-ras codons 10–13 mutations in o-nitrotoluene-induced cecal carcinomas from B6C3F1 mice by cycle sequencing of amplified exon 1. Sequencing panels are from left to right: (a) normal K-ras codon 10 sequence GGA, codon 12 sequence GGT, and codon 13 sequence GGC. (bd) Mutated sequences from cecal carcinomas samples 10, 5 and 2, respectively. Arrows point to mutant bands in each sequence. The wild-type allele is visible in panel (c). (b) SSCP analysis at marker D6MCO12 to assess LOH on chromosome 6 near K-ras gene in cecal carcinomas induced by o-nitrotoluene. Lanes 3 and 4 represent normal (N) C3H (H) and C57BL/6 (B) inbred mouse strains and lanes 2 and 5 are normal (N) tissues from B6C3F1 (F1) mice. Lane 1 (tumor sample 2) showed loss of C57 (B) allele; lane 6 (tumor sample 10) showed loss of C3H (H) allele. Lanes 6 (tumor sample 10) and 1 (tumor sample 2) with LOH also exhibit K-ras mutation and wild-type allele loss in panels (b) and (d) of (a), respectively.

 
Eight of eleven (73%) (Table I) cecal carcinomas exhibited positive immunohistochemical staining of p53 (Figure 3e and f). Missense mutations in p53 exons 5–8 were detected in nine of 11 (82%). Mutations in p53 were identified at codons 224, 231, 236, 238, 239, 240, 243 and 244 (Table I).

No mutation of Catnb, K-ras or p53 was detected in non-tumor regions of the cecum from two o-nitrotoluene exposed mice or untreated controls. At least one mutation in the Catnb, K-ras or p53 genes was identified in all o-nitrotoluene-induced cecal carcinomas examined.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The o-nitrotoluene-induced mouse cecal carcinomas represent the first mouse model to our knowledge in which multiple mutations of the Ras, p53 and Wnt signaling genes have been identified in the same tumors. The finding that all of the cecal carcinomas examined in the study had Catnb mutations indicates that deregulation of ß-catenin protein expression plays an important role in the pathogenesis of these tumors (2,6,7,29) as it does in human colon cancer. Deregulation of ß-catenin and increased Wnt signaling most often occurs in human colon cancer following mutation and loss of APC, an important component of the complex that controls ß-catenin expression. However, mutation of the CTNNB1 gene without loss of APC also results in accumulation of ß-catenin protein and increased Wnt signaling in a subset of colon cancers (1,30,31). The o-nitrotoluene-induced large intestinal carcinomas appear to follow this pattern.

We observed numerous short deletion mutations of Catnb in the cecal carcinomas that probably resulted in a growth advantage based on the accumulated ß-catenin protein and increased cyclin D1 staining in nuclei. This region of frequent mutations in the Catnb gene contains critical sites for phosphorylation and ubiquination of the ß-catenin protein. While most of the deletion mutations did not include these regulatory sites, the truncated protein created by the mutations may have altered structures that were protected from degradation. Deletion mutations of CTNNB1/Catnb resulting in high expression of truncated ß-catenin proteins have been detected previously in tumors from other studies, including mouse hepatocellular neoplasms and hepatoblastomas (18,19) and human hepatocellular carcinomas and hepatoblastomas (3235). Strong nuclear staining of ß-catenin and cyclin D1 was observed in the mouse hepatoblastomas that had deletion mutations, suggesting that the truncated products, also observed on western blots, were active in Wnt signaling (24). Thus, we consider the presence of short in-frame deletion mutations and missense mutations in the Catnb gene to be an important contribution to the early stages of mouse cecal carcinogenesis (4,32).

The predominant ß-catenin membrane staining identified in cecal carcinomas and nuclear accumulation of the ß-catenin protein in one cecal carcinoma is indicative of the dual roles (cell adhesion and transcription) ß-catenin plays in the carcinogenesis process. Although the ß-catenin protein was identified in the nucleus of only one carcinoma, expression of cyclin D1, known to be transcriptionally activated by Wnt signaling, was observed in the nuclei of 73% of the cecal carcinomas. One explanation for the rare identification of nuclear ß-catenin staining is that only a small increase in ß-catenin/Tcf transcription complex, less than detectable by immunohistochemistry, is necessary for a large effect on Wnt signaling characterized by the nuclear activation of cyclin D1. Cyclin D enhances cell proliferation by advancing the rate of the cell cycle through the G1/S checkpoint (33,36). In addition, the accumulation of ß-catenin at the cell membrane of neoplastic cells may alter the cell adhesion properties of the cells and also contribute to the malignant phenotype. Both enhanced cell proliferation and alterations in cell adhesion probably contributed to the invasive properties of the cecal carcinomas.

The high frequency of K-ras codon 12 G -> T transversions is consistent with a genotoxic event, and this mutation is most probably associated with o-nitrotoluene-induced adduct formation (37). In addition, transversion mutations occur infrequently in spontaneous tumors, further highlighting the importance of this mutation identified in the chemically induced cecal carcinomas. In other animal experiments, G -> T transversions have been linked to adduct formation by genotoxic chemicals such as benzo[a]pyrene and by oxidative DNA damage (3840). The findings of G -> T transversions in K-ras in all cecal carcinomas examined suggests that this mutation was the result of a relatively early event, and following clonal expansion, was maintained as a signature mutation within the tumors. Interestingly, we found in the sequencing results that the wild-type allele of K-ras was lost in two carcinomas. Further LOH analysis demonstrated allelic loss on chromosome 6 near the K-ras gene in four cecal carcinomas. In the cecum of the B6C3F1 mouse, which contains two resistant K-ras alleles, the wild-type K-ras allele may be acting as a tumor suppressor gene as was recently demonstrated for the mouse lung (41).

The predominance of exon 7 p53 mutations in o-nitrotoluene-induced cecal carcinomas provides neoplastic cells with a selective advantage for unregulated growth and avoidance of apoptosis, given the well accepted fact that p53 regulates cell cycle progression and apoptotic cell death (4244). It would be interesting to determine if mismatch repair defects, often associated with microsatellite instability, also contributed to the high percentage of mutations in the cecal tumors in mice (45,46). Another possibility for the large number of critical gene mutations is that o-nitrotoluene caused both genotoxic and random oxidative damage (40). The finding of both a signature mutation pattern of K-ras, but a random pattern of base changes in Catnb and p53 supports this idea.

Aside from the finding of APC mutations in human colon cancer and ß-catenin mutations in the mouse cecal carcinomas, these chemically induced tumors model human cancer remarkably well both in their molecular alterations and cellular characteristics, such as CK markers. The molecular alterations and expression changes identified in major cancer genes in the large intestinal tumors of the B6C3F1 mouse affect the same pathways as those discovered in human colon cancer, suggesting that the response in the mouse is of relevance to humans (2,3,47). Specifically, a high frequency of colorectal cancers have mutations in K-RAS and p53, and up-regulation of ß-catenin and Cyclin D1. In both humans and mice alterations in the ß-catenin/Wnt signaling pathway, Ras/map kinase pathway and cell-cycle checkpoint genes (cyclin D1, p53) are all important in the progressive conversion of normal cells into cancer cells (2,6,48,49). The formation of o-nitrotoluene large intestinal tumors are probably the result of the combined interaction of multiple genetically altered pathways, where the K-ras oncogene provides self sufficiency in growth signals, the p53 gene is associated with unregulated growth/avoidance of apoptosis, and the combined interaction of the Catnb and ras activation result in increased cyclin D1 expression (50,51). The data also support the hypothesis that as in humans, mouse large intestinal cells must acquire multiple genetic alterations, and it is the continued interaction between these pathways that results in the full malignant phenotype of the cecal carcinomas.


    Notes
 
4 To whom correspondence should be addressed Email: sills{at}niehs.nih.gov Back


    Acknowledgments
 
The authors thank Dr Ronald Melnick and Dr Robbert Slebos for their critical review of the manuscript. We also thank the professional and technical staff from the laboratories that conducted the study and the professional staff at NIEHS. Dr Carolyn Moyer is thanked for her expertise in conducting and interpreting the CK studies. In addition Ms Maureen Puccini of Experimental Pathology Laboratories is thanked for her excellent technical assistance in the preparation of photomicrographs.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received April 22, 2003; revised October 8, 2003; accepted November 26, 2003.





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