Preliminary analysis of azoxymethane-induced colon tumorigenesis in mouse aggregation chimeras
Qian-Shu Wang,
AnneMarie Walsh1,
Jennifer S. Goldsby,
Alexandros Papanikolaou,
Andrew B. Bolt and
Daniel W. Rosenberg2
Toxicology Program, Department of Pharmaceutical Sciences, University of Connecticut, 372 Fairfield Road, Storrs, CT 06269-2092 and
1 Department of Biochemical Genetics, Rockefeller University, New York, NY 10021, USA
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Abstract
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Inbred mice exhibit differential susceptibility to colon carcinogens. The following study addresses the possibility that differences are intrinsic to colonic mucosa (cell autonomous) or are mediated by extracolonic systemic factors (e.g. liver activation of carcinogens). Our approach was to construct mouse aggregation chimeras, mice whose tissues are a mosaic of cells derived from two parental genotypes, from a susceptible (SWR) and a resistant (DBA/2) strain. Forty-five embryo aggregations yielded 11 viable pups, four of which were chimeric by coat color. Six-week-old SWR
DBA/2 chimeras were injected i.p. with azoxymethane (AOM) once a week for 8 weeks (5 and 7.5 mg/kg body wt for 2 weeks followed by 10 mg/kg for 6 weeks) and tumor incidence in distal colon was evaluated 15 weeks after the last injection. Additional groups of parental mice received the same treatment. In the parental SWR treatment group, 1.7 ± 0.82 tumors/colon were found. No tumors were observed in AOM-treated DBA/2 mice. In SWR
DBA/2 chimeras exposed to AOM, 2.8 ± 2.1 tumors/colon were found. Tumor lineage was examined in paraffin sections stained with Dolichos biflorus agglutinin-peroxidase, a cell surface specific marker that stains intestinal endothelial cells of SWR and epithelial cells of DBA/2. Cellular lineage of tumors was further evaluated by microsatellite analysis of DNA isolated by microdissection. There was no significant difference in tumor incidence between SWR parental and chimera treatment groups. Histochemical analysis of tumor tissue in chimeras suggested that most tumors were derived from SWR. However, subsequent genetic analysis of tumors indicated mixed parental composition. These preliminary studies suggest that DBA/2 resistance mechanisms are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of AOM.
Abbreviations: AOM, azoxymethane; DMH, 1,2-dimethylhydrazine; H&E, hematoxylin and eosin; PBS, phosphate-buffered saline.
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Introduction
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Repetitive treatment with methylating carcinogens such as 1,2-dimethylhydrazine (DMH) and azoxymethane (AOM) produces tumors in rodents that are similar to those associated with `sporadic' (non-familial) forms of colorectal cancer (13). As in human populations, the genetic background of laboratory animals plays a significant role in carcinogenesis. Inbred mouse lines have been identified that vary in their susceptibility to the organotropic properties of colon carcinogens such as DMH (46). Genetic susceptibility is characterized by tumor incidence in susceptible strains, such as SWR, that approaches 100%. In contrast, strain DBA/2 is largely resistant to the carcinogenic properties of DMH and <5% develop tumors after repetitive exposures (46). We have recently extended this pattern of genetic susceptibility in inbred mice to a direct metabolite of DMH, AOM (7).
Differential susceptibility to these colon carcinogens is most likely controlled through multiple genetic loci (8,9). However, it is not known whether these heritable differences in sensitivity are intrinsic to the colonic mucosa (i.e. cell autonomous) or are indirectly mediated by extracolonic humoral or systemic factors, e.g. liver activation or detoxification of carcinogens. These possibilities can be directly tested in mouse aggregation chimeras (mice whose tissues are a mosaic of cells derived from two parental genotypes) constructed between susceptible and resistant strains. Chimeric mice are produced by in vitro aggregation of 8- to 16-cell stage embryos (10). Aggregated blastocysts are then transferred to the uterus of a pseudopregnant foster mother where they develop normally. Each of the tissues in the offspring, however, are comprised of cells derived in varying proportions from the two component lineages. By selecting embryos that differ maximally in tumor susceptibility, the intestinal epithelium will then be comprised of an interspersed patchwork of susceptible and resistant cells. Based on our understanding of the clonal composition of the adult intestine, large cellular domains comprised of a single genotype will be found, except at mosaic patch boundaries, where component lineages will directly interact (11). In chimeras treated with carcinogen, the mosaic colonic epithelium will thus be exposed in vivo to an identical profile of extracolonic and systemic factors (e.g. liver-activated mutagens).
In the following studies, we have tested the hypothesis that colon cells derived from tumor-resistant DBA/2 mice may protect adjacent SWR epithelium by producing diffusible factors that act beyond the DBA/2 component epithelium. We have also tested the possibility that protection may occur at the level of the whole organism, whereby DBA/2 produces humorally derived factors that elicit a protective effect on the SWR/J-derived epithelium. Lineage-specific histochemical and genetic markers have been used to establish parental composition of component cells within the colonic epithelium. If intestinal crypts derived from DBA/2 produce diffusible protective factors or DBA/2 tissue produces systemic protective factors, a reduction in the number of tumors within the SWR-derived epithelium is predicted. The absence of protection would indicate that the differences in colon tumor susceptibility in SWR and DBA/2 mice must be autonomous to the individual colonic epithelial cells.
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Materials and methods
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Materials
AOM, human chorionic gonadotropin, Gestyl, Dolichos biflorus agglutinin, 3,3'-diaminobenzidine and all other analytical reagents were purchased from Sigma (St Louis, MO).
Production of mouse aggregation chimeras
Initial breeding pairs of SWR and DBA/2 mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed in the Laboratory Animal Research Center (LARC) at Rockefeller University (New York, NY). Mice were housed 5 animals/cage in a temperature controlled (23 ± 1°C), light cycled (12 h light/dark) room and fed Purina laboratory chow 5001 ad libitum. Allophenic chimeras were created by in vitro aggregation between SWR and DBA/2 embryos, using a modification of an earlier technique (10). After induction of estrus with 5 U Gestyl, 34-week-old female mice were superovulated with 5 mg of human chorionic gonadotropin. Forty-eight hours after mating, 8-cell stage embryos were harvested by oviduct flushing. After flushing, embryos were transferred to wells containing M16 medium and covered with light mineral oil. Following removal of the zona in acid Tyrode's solution, embryos were aggregated and incubated overnight at 37°C or until the embryo had compacted. The resultant blastocysts were then surgically transferred to the uterus of a pseuodopregnant CD-1 mouse and brought to term in microisolator cages. Chimeric pups were born within 17 days after uterine transfer. After weaning at LARC, mice were shipped to the University of Connecticut (Storrs, CT) for subsequent treatment and analysis.
Treatment of animals and evaluation of tumors
Mice were housed at the University of Connecticut in a ventilated, temperature controlled (23 ± 1°C) and light cycled (12 h light/dark) room and allowed free access to Purina laboratory rodent chow (5001) and water up to the time of death. At 6 weeks of age, chimeric mice and the parental strains, SWR and DBA/2, were injected i.p. once a week for a total of 8 weeks with AOM (5 and 7.5 mg/kg, respectively, for the first 2 weeks and then 10 mg/kg body wt for the remaining 6 weeks). Fifteen weeks after the last injection, mice were killed. The AOM-treated and vehicle control mice in both the chimera and parental groups gained body weight to a similar extent throughout the entire experimental period (data not shown). Upon killing, colons were removed and flushed with ice-cold phosphate-buffered saline (PBS). Tissues were then opened longitudinally, pinned flat and examined under a dissecting microscope before being fixed in 10% buffered formalin. Tumors were counted without prior knowledge of genotype to eliminate bias. Liver samples from each mouse were snap frozen in liquid nitrogen and stored at 80°C for subsequent genotype analysis.
Genotyping of chimeras
Using liver DNA, PCR was used to identify chimeric mice. A microsatellite polymorphism of the mouse map marker D11Mit4 was used to generate PCR products of the following sizes: DBA/2, 300 bp; SWR, 244 bp. The following primer pairs were used: sense, 5'-CAGTGGGTCATCAGTACAGCA-3'; antisense, 5'-AAGCCAGCCCAGTCTTCATA-3'. The PCR reaction was carried out in 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dATP, dGTP, dCTP and dTTP, 0.25 µM each sense and antisense primers and 0.625 U Taq DNA polymerase (Gibco BRL, Gaithersburg, MD) in a final volume of 25 µl. The reaction mixture was first heated at 95°C for 5 min and amplification was performed over 35 cycles, at 94°C for 45 s, 55°C for 45 s and 72°C for 1 min, followed by an incubation for 10 min at 72°C. The PCR products were electrophoresed through a 1.8% agrose gel in 0.5x TBE buffer and visualized with ethidium bromide staining.
Dolichos biflorus agglutinin staining of colon tissue
We used lectin histochemistry to distinguish parental composition of tissues within chimeras. Dolichos biflorus agglutinin provides an especially informative marker for these studies. This cell surface marker stains colonic endothelial cells of SWR and epithelial cells of DBA/2. In DBA/2 colons, lectin binding sites are expressed in columnar epithelial cells, goblet cells and Paneth cells (11). Colonic tissue was opened longitudinally, fixed flat in 10% buffered formalin for 24 h and embedded in paraffin. Tissue sections were stained with D.biflorus agglutinin. Briefly, after deparaffinization and rehydration, tissue sections were placed in 1% hydrogen peroxide for 30 min to quench endogenous peroxidase activity. Non-specific binding was blocked by incubating the sections with 1% bovine serum albumin for 30 min at room temperature. Tissue sections were then incubated with 5 µg/ml of peroxidase-conjugated D.biflorus agglutinin at 4°C overnight. Peroxidase activity was detected with 3,3'-diaminobenzidine as substrate. Sections were counterstained with Harris hemotoxylin. To confirm the specificity of D.biflorus agglutinin staining, parental SWR and DBA/2 colon tissues were stained in parallel as positive controls. As a negative control, PBS was substituted for D.biflorus agglutinin, which showed no staining (data not shown).
Estimation of percentage of chimerism in the distal colon
The distal part of the colons (~2.5 cm in length measured from the anus) from chimeric mice were cut into two sections and embedded in parallel in paraffin as described above. Serial 4 µm sections (1020) were taken at each of three levels spaced ~400 µm apart throughout the tissue block and mounted to provide a set of slides for each animal. A representative section from each of the three levels was stained with D.biflorus agglutinin. The percentage of chimerism of the distal colon was estimated by measuring the lectin staining pattern within each section under a microscope. No correction was made for the effect of the plane of the section, i.e. whether crypts were cut transversely or longitudinally in different areas within the specimen.
Genotyping of tumors from paraffin sections
DNA extraction from paraffin sections was performed as described (12). Briefly, serial 4 µm sections were microdissected, using the D.biflorus agglutinin stained sections as a template. Tumor regions with varying lectin staining patterns, which contained both epithelial and non-epithelial cells, were carefully scraped into microcentrifuge tubes containing xylene and deparaffinized by vortexing gently for 20 min. After centrifuging for 5 min at 8000 g, the pellet was suspended in 150 µl of 100% ethanol and vortexed gently for 20 min, followed by centrifugation for 5 min. Dried pellets were resuspended in 100 µl of digestion buffer (50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20) containing 200 µg/ml of proteinase K and the suspension was incubated at 55°C for 12 h. The suspension was then incubated at 95°C for 10 min to inactivate proteinase K and centrifuged at 8000 g for 1 min. The supernatant was used as a DNA template for PCR analyses using the primer sets described above. Lineage analysis of a dysplastic lesion was further evaluated by PCR analysis using DNA from laser capture microdissection, a recently developed method that overcomes many of the drawbacks associated with microdissection of tissue sections (13). Briefly, a thin transparent film was placed over the hematoxylin and eosin (H&E) stained tissue section. A selected area of dysplastic lesion was captured and attached to the film using a short duration pulse from an infrared laser. The film with adherent tissue was then placed into an Eppendorf tube containing 50 µl of digestion buffer (described above). Samples were then incubated at 37°C for 12 h, followed by centrifugation at 6000 g for 5 min. After heating at 95°C for 10 min to inactivate proteinase K, the solution was vortexed briefly and used for PCR analysis using the D11Mit4 microsatellite marker.
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Results
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Forty-five aggregations were set up between SWR and DBA/2 mice, yielding a total of 60 blastocysts. Each blastocyst was then surgically transferred into pseudopregnant surrogates, producing 11 live offspring. The percentage contribution of each genotype to the coat color of chimeras was estimated visually from the proportion of white (SWR) and gray (DBA/2) fur. The relative coat color composition for each of the mice produced by embryo aggregation is shown in Table I
. Four chimeras, identified on the basis of coat color (DS.4, DS.7, DS.8 and DS.10) were produced. However, it is not possible to extrapolate lineage composition of individual organs on the basis of coat color alone. Therefore, the mice were divided into two treatment groups. One group, including three coat color chimeras, as well as five additional mice derived from embryo aggregation, received injections with AOM. The remaining coat color chimera and two additional mice produced by embryo aggregation were used as controls and received saline alone. In addition, groups of mice from SWR and DBA/2 parental strains received either AOM or vehicle alone and were treated concurrently for comparison.
Fifteen weeks after the last injection with AOM, distal colons were analyzed macroscopically for tumors. As shown in Table I
, no tumors were found in any of the untreated control mice derived from embryo aggregation. Tumor incidence in the uniform coat color mice ranged from 1 to 11 (4.80 ± 4.18, mean ± SD), whereas in the mixed coat color chimeras, the incidence of tumors ranged from 0 to 4 (2.33 ± 2.08, mean ± SD). There was no significant difference in tumor multiplicity between these two treatment groups. Further analysis of tumor multiplicity and size in chimeras treated with AOM is shown in Table II
. Additional groups of parental mice treated concurrently with AOM or saline are shown for comparison. Colon tumors were absent in each of the saline treatment groups, as well as in the DBA/2 mice treated with AOM. SWR mice treated with carcinogen had an average total number of tumors of 1.7 ± 0.82 per colon, somewhat fewer than reported earlier in our laboratory for this strain (7). This may reflect the relatively reduced time interval during which the animals were maintained after the last injection with carcinogen (15 weeks). The shorter post-exposure interval is also reflected by the small number (0.2 ± 0.41) of tumors exceeding a diameter of 3 mm in size. Overall, there are no significant differences between SWR parental and chimera treatment groups in either tumor incidence, multiplicity or size.
To provide additional information regarding chimerism for each of the 11 mice derived from embryo aggregation, genetic analysis of liver DNA using the D11Mit4 microsatellite polymorphism was performed. A representative gel is shown in Figure 1
. Three mice demonstrated mixed hepatocellular lineage (DS.7, DS.8 and DS.10). To assess the extent of chimerism within the distal colon, tissues were analyzed histochemically by their D.biflorus staining patterns. Lectin binding to the colonic epithelium is shown in representative tissue sections of distal colon in Figure 2
. Also shown are lectin binding patterns in the distal colons of an SWR and a DBA/2 parental control (Figure 2A and B
, respectively). In Figure 2C
, normal colon mucosa from an SWR
DBA/2 chimera shows positive epithelial staining of several isolated crypts derived from DBA/2. An area of chimeric mucosa that demonstrates the juxtaposition of both SWR and DBA/2 lineages is shown in Figure 2D
. Several tumors were also examined for their lectin staining patterns. As pictured in Figure 2E and F
, two adenomas from an SWR
DBA/2 chimera showed a lack of epithelial staining, indicating cellular composition derived from SWR cells. These tumors formed despite the presence of nearby DBA/2-derived lineage. An adenoma derived from the DBA/2 lineage (animal DS.5) is also shown in Figure 2G
. This tumor retained some lectin binding within the epithelium. Finally, a carcinoma from animal DS.4 showed intermittent endothelial staining of blood vessels, even within the tumor mass (Figure 2H
).

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Fig. 1. Genotyping of liver samples. DNA was isolated from liver samples as described in Materials and methods. A microsatellite marker D11Mit4 was used to generate PCR products of the following sizes: SWR, 244 bp; DBA/2, 300 bp. Mice DS.7, DS.8 and DS.10 showed both the SWR and DBA/2 genotypes, while DS.5 demonstrated only the DBA/2 trait. The remaining samples had only the SWR genotype. PCR products were electrophoresed through a 3% agarose gel and stained with ethidium bromide.
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To confirm the lineage of cells comprising tumor tissue, DNA was microdissected from colon sections containing tumors and subjected to microsatellite analysis using the D11Mit4 microsatellite marker. A representative gel is shown in Figure 3
. Mixed lineage is indicated by the presence of two bands at 244 (DBA/2) and 300 bp (SWR). In lane 1, DNA was isolated from the tumor from DS.5 that had only retained its partial epithelial staining for lectin (Figure 2G
). Microsatellite analysis found this tumor to be indeed comprised entirely of DBA/2 genotype. The DNA from three additional tumors produced in the distal colons of SWR
DBA/2 chimeras were also genotyped. Despite the retention of endothelial staining, indicative of SWR, genetic analysis revealed the presence of two bands (Figure 3
), indicating mixed cellular lineage. Finally, a colon section containing a dysplastic lesion was also examined for cellular lineage using laser capture microdissection. As shown in Figure 4
, DNA was isolated from a single colon crypt compartment and genotyped using the D11Mit4 microsatellite marker. The presence of only SWR lineage in this lesion was detected using this methodology. This analysis was repeated on pooled tissues of the same dysplastic crypt isolated from five consecutive sections to further confirm the cellular lineage of this lesion, and the same single SWR genotype was revealed. Table III
summarizes the analysis performed in the chimera group regarding lectin staining of the colon tissue and genotyping of liver and tumor samples. As shown in Table III
, liver genotype was correlated with coat color in each of the mice examined with the exception of animal number DS.4, in which no DBA/2 trait was detected in the liver specimen. In addition, two of the liver chimeras (DS.7 and DS.8) displayed mosaicism by D.biflorus staining within the distal colon. However, animal number DS.10 did not demonstrate lectin binding, although microsatellite analysis of the liver as well as the coat color showed the presence of both SWR and DBA/2 traits. Five of the remaining AOM-treated mice demonstrated no evidence of DBA/2-positive cells in either the liver or colon, whereas one mouse was found to be derived entirely from the DBA/2 lineage.
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Table III. Summary of D.biflorus agglutinin staining in mouse colon tissues and genotyping of liver and tumor samples
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Discussion
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These studies describe AOM-induced colon tumorigenesis in a mouse aggregation chimera model. Our approach was to examine the response of colonic epithelium derived from mixed parental genotypes to repetitive exposures of the organotropic colon carcinogen, AOM. This agent produces colon tumors in SWR mice at a frequency that approaches 100% (7). In contrast, DBA/2 mice are largely resistant to the carcinogenic properties of AOM and its parent compound, DMH, and <5% develop tumors and preneoplastic lesions in response to carcinogen treatment (47,14). Thus, while genetic background is an important determinant of cancer risk, it is not known whether heritable differences in susceptibility are intrinsic to the colonic mucosa (i.e. cell autonomous) or are indirectly mediated via extracolonic humoral/systemic factors, e.g. liver activation or detoxification of carcinogens. Factors that may contribute to differential cancer susceptibility include relative rates of carcinogen activation, the extent of DNA alkylation or efficiency of repair and the balance between proliferation and apoptosis of initiated colonic crypt cells (1518). We have considered the possibility that the rate of activation of indirect-acting procarcinogens underlies genetic susceptibility. In a series of studies using inbred mice, however, we found comparable levels of key metabolic enzymes, including cytochrome P450 isoforms, glutathione S-transferases and alcohol dehydrogenase within the colonic mucosa (1921). More importantly, we recently reported a similar pattern of DNA methyl adduct formation in the colons of susceptible and resistant mice shortly after treatment with AOM (22). These observations suggest that carcinogen metabolism and acute DNA alkylation may not be primary determinants of differential susceptibility in this murine model.
In this study, embryos were aggregated from mice that differ maximally in colon tumor susceptibility, producing an adult intestinal epithelium comprised of distinct interspersed colonic crypt compartments. By examining the response of colonic mucosa derived from these mixed parental genotypes to a chemical carcinogen, a goal of these studies was to define the relative importance of systemic/humoral factors as modifiers of cancer risk. Several lines of evidence suggest that colon-specific carcinogen-induced tumors are autonomous to the large bowel. In an earlier study, Gennaro et al. (23) surgically transposed sections of rat colon and small intestine and then exposed the animal to AOM. None of the surgically transposed small bowel loops developed tumors, whereas all of the transposed colons had tumors, regardless of their geographical location within the gut. Further evidence for the selective action of carcinogens on colonic mucosa was provided by an acute analysis of the effects of methylazoxymethanol on rat intestine (24). In this study, acute carcinogen effects were limited to the regions of the distal large bowel that would subsequently develop tumors.
Critical to our lineage analysis of epithelial cells comprising tumor tissue is the ability to identify parental origin directly within the intact tissue architecture. Of the various strategies available for histochemical analysis of chimeric tissue, the cell surface carbohydrate polymorphism recognized by the lectin D.biflorus agglutinin was selected to mark cellular lineage. This polymorphism is specified by co-dominant alleles of the Dlb-1 gene and has been used successfully to mark descendent clones of cells in aggregation chimeras (2527). In fact, we (28) and others (29) have shown that SWR are vascular endothelium positive and intestinal epithelium negative, whereas DBA/2 exhibits a reverse pattern. In our earlier study, we also reported that DBA/2 mice retained homogeneous staining patterns throughout the entire colon, a particular advantage to using this resistant mouse line for producing aggregation chimeras with SWR (28). A potential limitation to the use of D.biflorus agglutinin, however, has been suggested by the results of a carcinogenesis study in CBA mice treated with DMH (29). In this study, some loss of lectin binding was observed in several dysplastic crypts (29). We have also found partial loss of epithelial lectin binding in the single DBA/2-derived tumor (Figure 2
). To circumvent this problem, we further examined the tumor lineage using a microsatellite polymorphism-based PCR analysis on selected tumor sections. Except for one tumor which showed only DBA/2 lineage, the other tumors examined showed both DBA/2 and SWR genotypes. It is possible that the microdissected tumor tissues were contaminated with surrounding tissues which were derived from mixed lineages. It may also be ascribed to the fact that the tumor tissues contained stromal cells which were derived from a different lineage. In these cases, the laser capture microdissection technique should provide a solution. On the other hand, it is possible that some of the tumors may indeed contain mixed genotypes resulting from coalescence of adjacent tumors, as reported in an earlier study using a mouse chimera model (30).
An analysis of cellular origin in adenomas and carcinomas induced by DMH has also been performed in C3H-HeN
BALB/c chimeras by Tatematsu et al. (30). In this study, cellular origin of colon tumors and preneoplastic lesions (focal atypias) were examined immunohistochemically using a specific antibody to the C3H strain. Unlike our experimental design, this experiment set up aggregations between inbred mouse lines of equivalent tumor susceptibility. Similar to our findings in SWR
DBA/2 chimeras, some of the adenomas and adenocarcinomas were found to be comprised of two cell types. In the cases where two phenotypes were mixed, there was a non-random distribution of lineages within discrete regions (30). Analysis of focal areas of atypia, however, revealed only a single lineage in every case (30). Although we found a similar result in a dysplastic lesion analyzed in our study, our sample size needs to be greatly expanded in order to draw a similar conclusion regarding cellular lineage of these preneoplastic lesions.
In summary, we have examined AOM-induced colon tumorigenesis in a mouse chimera model. We believe that this model provides a useful experimental system for studying the pathogenesis of colon cancer and the potential interaction of resistant and susceptible cells that share overlapping intestinal structures. Although our sample size is small, our preliminary data suggest that resistance mechanisms that protect the DBA/2 mice from AOM-induced colon tumors are not sufficient to protect adjacent SWR-derived epithelium from the tumorigenic effects of this agent. Further studies are needed with additional aggregation chimeras to enable a quantitative assessment of lineage interactions in this model.
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Acknowledgments
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The authors would like to gratefully acknowledge Dr David Thaler at The Rockefeller University for his many helpful suggestions regarding our experimental design. These studies were supported in part by a New Investigator grant from the Donaghue Medical Research Foundation, NIH grant DK49805-01 and NIEHS Training grant 1T32ES07163.
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Notes
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2 To whom correspondence should be addressed Email: rosenber{at}uconnvm.uconn.edu 
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Received August 14, 1998;
revised October 27, 1998;
accepted December 1, 1998.