Short Title: p53 and colitis-associated neoplasia

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Loss of p53 enhances the induction of colitis-associated neoplasia by dextran sulfate sodium

Short Title: p53 and colitis-associated neoplasia

Wen-Chi L. Chang1 *, Renata A. Coudry1, Margie L. Clapper1, Xiaoyan Zhang1,

Kara-Lynn Williams1, Cynthia S. Spittle1, Tianyu Li1, and Harry S. Cooper2

1Division of Population Science and 2Division of Medical Science,

Fox Chase Cancer Center, Philadelphia, PA 19111

* To whom requests for reprints should be addressed: Wen-Chi L. Chang, Ph.D., Division of

Population Science, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111

Phone: 215-728-5368

FAX: 215-214-1622

E-mail: [email protected]

Abbreviations: APC, adenomatous polyposis coli; AOM, azoxymethane; DSS, dextran sulfate

sodium; CRC, colorectal cancer; IBD, inflammatory bowel disease; LCM, laser capture

microdissection; LOH, loss of heterozygosity; UC, ulcerative colitis

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] Carcinogenesis Advance Access published June 8, 2007 by guest on February 3, 2014

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Abstract

Loss of p53 function is an early event in colitis-associated neoplasia in humans. We assessed the

role of p53 in a mouse model of colitis-associated neoplasia. Colitis was induced in p53-/-, p53+/-,

and p53+/+ mice using 3 or 4 cycles of dextran sulfate sodium followed by 120 days of water.

Mice were examined for incidence, multiplicity, and types of neoplastic lesions. Lesions were

examined for mutations in ? -catenin (exon 3), K-ras (codons 12/13), and p53 (exons 5-8) by

sequencing and for cellular localization of ? -catenin by immunohistochemistry. The incidence

of neoplastic lesions was 57%, 20%, and 20% in p53-/-, p53+/-, and p53+/+ mice, respectively (P =

0.013). p53-/- mice had a greater number of total lesions (P < 0.0001), cancers (P = 0.001), and

dysplasias (P = 0.009) per mouse than either p53+/- or p53+/+ mice. Flat lesions were associated

with the p53-/- genotype, while polypoid lesions were associated with the p53+/- and p53+/+

genotypes (P < 0.0001). ? -catenin mutations were present in 75% of lesions of p53+/+ mice and

absent in lesions from p53? /? mice (P = 0.055). Nuclear expression of ? -catenin was seen only in

polypoid lesions (91%). No K-ras or p53 mutations were detected. These data indicate that loss

of p53 enhances the induction of colitis-associated neoplasia, particularly flat lesions, and

dysregulation of ? -catenin signaling plays an important role in the formation of polypoid lesions

in this mouse model. As observed in humans, p53 plays a protective role in colitis-associated

neoplasia in the dextran sulfate sodium model.

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Introduction

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease,

affects approximately one million individuals in the U.S. each year. Patients with UC have a

relative risk that is 8 times higher than the normal population for developing colorectal cancer

(CRC) (1). The risk of CRC and dysplasia in patients with UC increases with the extent of

colonic involvement, age of onset, duration of disease (2) and histological severity of

inflammation (3). Recent data suggest that the cumulative risk for CRC in UC patients is below

1% in the first 8-10 years, and rises in annual increments of 0.5-1.0% thereafter, reaching 5-10%

after 20 years and 15-20% after 30 years (4). After 40 years of colitis, 25-40% of all patients

who have not had a prophylactic colectomy will develop CRC (5).

Recent studies suggest that loss of p53 function (mutation and loss of heterozygosity

(LOH)) is an early event in the development of colitis-associated neoplasia (6-10). The

frequency of G:C to A:T transition mutations in p53 is increased within nonneoplastic inflamed

colon tissue from colitis patients as compared to that of normal colon tissue from healthy

controls (8). The frequency of p53 mutations in different grades of dysplasias has also been

determined (11). Examination of 278 colon samples from colectomy specimens of 7 patients

with long-standing ulcerative pancolitis (duration of colitis: 12 ? 6.5 years) revealed that 19%

(40/212) of the specimens without dysplasia, 41% (17/29) of the specimens indefinite for

dysplasia, 38% (9/24) of the specimens with low-grade dysplasia and 63% (5/8) of the specimens

with high-grade dysplasia were positive for mutant p53. In addition, LOH of p53 was observed

in approximately 25% of samples that were nondysplastic, actively inflamed and indefinite for

dysplasia, 45% of informative dysplastic lesions and 70% of cancers (9,10). These emerging

data suggest that colitis-associated dysplasia and CRC develop along a defined pathway that is

distinct from that of sporadic CRC and that loss of p53 function is an early critical event in the

pathway from dysplasia to cancer (12).


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The model of dextran sulfate sodium (DSS)-induced colitis (13) has been widely used to

study colitis-associated neoplasia in the mouse (14-16). However, the role of p53 in the DSS

model remains unclear. Some studies report little or no expression of nuclear p53 protein in the

dysplasias and cancers obtained from this model (14-16), while other studies report

overexpression of nuclear p53 protein in carcinomas and dysplasias (17) as well as in

nonneoplastic crypts (18). p53 mutations were found in colitis-associated adenocarcinomas

from ? 2micro-/- x IL-2-/- mice (19) but not in tumors from IL-10-deficient mice (20). Based on

these inconsistent data, it is difficult to conclude the importance of p53 in mouse models of

colitis-associated neoplasia.

Recently, Fujii et al. (14) reported that DSS-treated p53-/- mice have a significantly greater

incidence and multiplicity of cancers and dysplasias than DSS-treated p53+/- and p53+/+ mice,

with nuclear localization of ? -catenin present in 82.6% of colonic neoplasias. However,

mutation of ? -catenin and p53 was not examined. In the current study, we not only report

similar results with the highest tumor incidence and multiplicity observed in DSS-treated p53-/-

mice, but also find that flat lesions are significantly associated with the p53-/- genotype, while

polypoid lesions are significantly associated with p53+/- and p53+/+ genotypes. In addition,

? -catenin mutations are found more frequently in p53+/+ mice, and polypoid neoplasia is

significantly associated with nuclear localization of ? -catenin.

Materials and methods

Animals

Ten-week-old wild-type (p53+/+) and p53-deficient (p53-/- and p53+/-) C57BL/6J mice were

obtained from the Laboratory Animal Facility at Fox Chase Cancer Center (FCCC). p53-

deficient mice were originally developed by Donehower and Bradley (21), and a colony was

established in-house by inbreeding three male p53-/- mice (N4) with six female p53+/- mice (N5)

(Taconic Farms, Inc., Hudson, NY). This colony has been maintained at FCCC for more than 10


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years. Animals were housed in a temperature- and humidity-controlled room and received PMI

rodent chow (PMI Nutrition International, Cat#5013) and drinking water ad libitum. During the

experimental period, no murine viruses, Mycoplasma pulmonis, parasites, pathogens (including

H. hepaticus and H. bilis) or Pneumocystis carinii were detected. All animal treatments were

approved by the Institutional Animal Care and Use Committee at FCCC.

Induction of colitis

Colitis was induced by providing animals with drinking water containing DSS (molecular weight

30-40,000; ICN, Costa Mesa, CA) as described previously (13,16). DSS was administered in a

cyclic fashion, with each cycle consisting of 4 days of DSS and 17 days of untreated water

(Figure 1). p53-deficient (p53+/- and p53-/-) and wild-type (p53+/+) mice received 3 and 4 cycles

of DSS, respectively, followed by 120 days of untreated water. Results from previous

experiments (22) indicate that 4 cycles of DSS are required to induce neoplasia in p53+/+ mice on

a C57BL/6 background. p53-/- and p53+/- mice without DSS treatment served as controls and

were maintained in parallel until 183 days of age. A subset of mice from each treatment group

was sacrificed after 3 cycles of DSS treatment to assess the level of colorectal inflammation

present. Animals were monitored for gross rectal bleeding and weight loss throughout the study.

Histopathology

At the time of sacrifice, the colon and rectum were excised, opened longitudinally, rinsed with

phosphate-buffered saline, and examined for gross morphologic changes. All lesions were

counted and recorded. The colon/rectum was divided into equal thirds (proximal, middle and

distal), and fixed in 70% ethanol or 10% formalin for 12-72 h. The entire colon was cross-

sectioned at 2 mm intervals, embedded in paraffin and submitted for histopathologic processing.

All slides were stained with hematoxylin and eosin (H&E) and reviewed in a blinded manner.

Inflammation scores were determined as described previously (16). Briefly, the severity of

inflammation was graded semiquantitatively on a scale from 0 to 3 and multiplied by the extent


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of involvement (0-100%) to yield a score ranging from 0-300. Following the assignment of a

score to each piece of tissue on the slide, the total score was computed by summing the scores of

all pieces and dividing by the number of pieces of tissue evaluated.

The histopathologic evaluation of dysplasia was based on the classification scheme

developed by the Inflammatory Bowel Disease-Dysplasia Morphology Study Group (23).

Lesions were scored as negative, indefinite, or positive for dysplasia. Carcinomas and dysplasias

were classified as polypoid or flat. Polypoid lesions had an elevated growth pattern seen either

grossly or microscopically. Flat lesions had no elevated growth component and were less than

twice the height of the adjacent nonneoplastic mucosa. Lesions were diagnosed as

adenocarcinomas when neoplastic cells had invaded into the submucosa or beyond, and were

classified according to the World Health Organization publication of the classification of tumors

of the digestive system (24). “Pseudoinvasion” was defined as displacement of nonneoplastic

glands into the deeper layers of the intestinal wall as reported by Boivin et al. (25).

Laser capture microdissection (LCM) and DNA isolation

Paraffin-embedded colon tissues were sectioned (5 µm thickness), rehydrated, incubated with

Staining Solution® (Arcturus Bioscience, Inc., Mountain View, CA), and dehydrated with a

series of gradient alcohols and xylene. Slides were air-dried for 5 min and LCM was performed

immediately. Two thousand cells from each lesion (dysplasia or cancer) were microdissected

using the Pixcell® II Automated System (Arcturus). DNA was isolated from laser-

microdissected samples using the PicoPure™ DNA kit (Arcturus).

Mutational Analyses

Because greater than 95% of k-ras, p53 and ? -catenin mutations in human cancers are found at

codons 12/13 (26), exons 5 to 8 (27) and exon 3 (28), respectively, mutational analyses focused

on these regions.


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K-ras. A 167 bp region of exon 1 of the mouse K-ras gene, containing codons 12 and

13, was amplified by PCR. Primers were designed based on genomic sequence obtained from

Genbank (accession number S39586) – krasF - 5´TTA TTA TTT TTA TTG TA, krasR - 5´GCA

CGC AGA CTG TAG AG. The reverse primer was 5´-biotinylated to facilitate single-strand

DNA template isolation for the pyrosequencing reaction. Each PCR reaction contained 20 ng of

genomic DNA, 10 pmoles of each primer, and 25 ? l of Jumpstart Readymix RedTaq Polymerase

(Sigma-Aldrich, St Louis, MO) in a total volume of 50 ? l. Thermal cycling was carried out as

follows: 95ºC for 5 min, 35 cycles of 95ºC for 30 s, 45ºC for 30 s, 72ºC for 45 s, and a final

extension for 5 min at 72ºC. Successful and specific amplification of the region of interest was

verified by visualizing 10 ? l of PCR product on a 2% agarose gel containing ethidium bromide.

The single-stranded DNA template for pyrosequencing was prepared using streptavidin-

coated Sepharose beads (Amersham Biosciences, Piscataway, NJ) and processed using the

PSQ 96 Sample Preparation Kit (Biotage, Westborough, MA) according to the manufacturer’s

instructions. The template was incubated with 16 pmol sequencing primer (5?-

CTTGTGGTGGTTGGAGC) at 80ºC for 2 min. The sequencing-by-synthesis reaction of the

complementary strand was performed on a PSQ 96MA instrument (Biotage) at room temperature

using PyroGold reagents (Biotage).

The PCR amplicon generated for pyrosequencing was also sequenced on the ABI Prism®

3100 system for confirmation using Big Dye® Terminator chemistry (Applied Biosystems, Inc.,

Foster City, CA). Sample sequences were aligned with wild-type sequence using Accelrys

SeqWeb version 2 (San Diego, CA).

? -catenin and p53. Exon 3 of the mouse ? -catenin gene and exons 5-8 of the mouse

p53 gene were amplified by PCR in a 50 ? l reaction with 20 ng of genomic DNA and 10 pmoles

of each primer using Jumpstart Readymix RedTaq Polymerase (Sigma-Aldrich). Primers for

? -catenin and p53 were designed based on the gene sequence found in the Celera database

(#mCG22702) and Genbank (#AF367373), respectively. Primers for ? -catenin, ? -cateninF -

5´GCC TTT CTA ACA GTA TT and ? -cateninR - 5´GCC CTT CCT TCA ACC CA, generated


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a 408 bp PCR product. Primers for p53 were: exon5F - 5´TAC TCT CCT CCC CTC AAT,

exon5R - 5ĆGG GAG ATG GGA GGC TGC; exon6F - 5´GGG GTT AGG ACT GGC AGC,

exon6R - 5ĆTA AGA CGC ACA AAC CAA; exon7F- 5ĆTG TAG TGA GGT AGG GAG,

exon7R - 5´GGA ACA GAA ACA GGC AGA; exon8F - 5ĆTT ACT GCC TTG TGC TGG,

exon8R- 5´GAG GTG ACT TTG GGG TGA. Thermal cycling conditions were as follows: 95ºC

for 5 min, 35 cycles of 30 s at 95ºC, 45 s at the amplicon-specific annealing temperature and

1 min at 72ºC, followed by a final extension for 5 min at 72ºC. Annealing temperatures for each

amplicon were: ? -catenin=50ºC, p53exon5=56ºC, p53exon6=54ºC, p53exon7=53ºC, and

p53exon8=56ºC. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen,

Valencia, CA). Sequencing was performed with forward and reverse PCR primers on an ABI

Prism® 3100 using Big Dye® Terminator chemistry (Applied Biosystems). Sample sequences

were aligned with wild-type sequence using Accelrys SeqWeb version 2. Base changes were

classified as mutations if found in both the forward and reverse sequencing reactions. All

mutations were confirmed in replicate tumor samples.

Immunohistochemistry of ? -catenin

Expression of ? -catenin was evaluated by immunohistochemistry in paraffin-embedded tissues.

A rabbit polyclonal antibody raised against ? -catenin peptide amino acids 768-781 (C-2206) was

purchased from Sigma-Aldrich and used at a dilution of 1:2000. Immunohistochemistry was

performed on a Ventana XT automated immunohistochemistry stainer. Sections were incubated

with the primary antibody for 32 min at 37°C. The standard Ventana XT protocol was used

except that biotinylated sheep anti-rabbit secondary antibody (Vector Laboratories, Inc.,

Burlingame, CA) was substituted for the standard Ventana XT secondary antibody. Normal

colon/rectum and neoplastic colorectal lesions from mice possessing a germ line mutation in the

adenomatous polyposis coli (Apc) gene served as a positive control for ? -catenin expression.

The negative reagent control consisted of nonimmune rabbit IgG at the same protein


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concentration. Cells were scored as positive or negative for membranous and nuclear staining of

? -catenin.

Statistical analysis

The Wilcoxon Signed Rank test was used to compare differences between groups with respect to

number of lesions and inflammation score. Fisher’s exact test was used to compare tumor

incidence, type of lesions, mutation status of genes, and staining pattern of ? -catenin between

groups. All tests were performed using SAS software (SAS Institute, Cary, NC).

Results

Incidence, multiplicity, and types of lesions

The percentage of p53+/+ and p53+/- mice completing the DSS treatment regimen (4 or 3 cycles of

DSS, respectively, plus 120 days of untreated water) was 77% (10/13) and 71% (89/125),

respectively. However, only 34% (14/41) of p53-/- survived the DSS treatment (mean of 26 days

after cycle 3 of DSS; range from 3 to 120 days), with the remainder euthanized prematurely due

to sickness. Necropsy results indicated that 50% of the p53-/- mice had developed lymphoma,

while an additional 14% had developed sarcoma. The incidence of cancer in DSS-treated p53-/-

(42.9%, 6/14) mice was significantly higher than that of p53+/- (5.6%, 5/89) and p53+/+ (0%, 0/10)

mice (P = 0.0006). DSS-treated p53-/- mice had a significantly higher incidence of colorectal

dysplasia (50.0%, 7/14) as compared to DSS-treated p53+/- and p53+/+ mice (15.7% (14/89), and

20% (2/10), respectively) (P = 0.02). The incidence of colorectal neoplasia (dysplasia and

cancer) was 57% (8/14), 20% (18/89), and 20% (2/10) in p53-/-, p53+/-, and p53+/+ mice,

respectively (P = 0.013). No colorectal lesions were found in control mice not receiving DSS.

Mouse genotype affected not only tumor incidence, but also the multiplicity of colorectal

lesions and types of lesions (flat vs. polypoid). DSS-treated p53-/- mice had significantly greater


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numbers of total lesions (P = 0.0001), dysplasias (P = 0.009) and carcinomas (P = 0.001) per

mouse compared to DSS-treated p53+/- and p53+/+ mice (Table I). In the DSS-treated p53-/- mice,

84.6% of the colorectal lesions were flat, while 15.4% of the lesions were polypoid. In contrast,

83.3% of the colorectal lesions in DSS-treated p53+/- mice were polypoid and only 16.7% of the

lesions were flat and 100% of the colorectal lesions in DSS-treated p53+/+ mice were polypoid

(P < 0.0001) (Figure 2). Irrespective of mouse genotype, 8 of 11 cancers (72.7%) were flat and 3

of 11 cancers (27.3%) were polypoid.

Pathology of colitis-associated neoplasia

Polypoid lesions tended to be larger in size than flat lesions. All polypoid cancers arose from a

background of polypoid dysplasia (Figure 3), while flat cancers tended to invade from the

bottom of the crypts, often without dysplastic changes in the full thickness of the overlying

mucosa (Figure 3). This histopathology and pattern of invasion from the crypt base is identical

to that of low-grade tubuloglandular adenocarcinomas seen in human IBD (29). Flat cancers

exhibited small numbers of invasive malignant glands (Figures 3 and 4), which occasionally

disappeared upon deeper sectioning (see below). There were 11 invasive adenocarcinomas, 6 of

which were associated with contiguous acellular mucin pools. Nine cancers invaded into the

submucosa and 2 into the muscularis propria.

A few DSS-treated mice (1 p53-/-, 3 p53+/-, but no p53+/+) had pools of acellular mucin

within the submucosa and muscularis propria. These findings are similar to colitis cystica

profunda as seen in patients with UC (30). Serial and deeper histological sections (H&E) were

obtained from all cases with acellular mucin pools; in one of the cases, deeper sections revealed

a carcinoma. In another case of adenocarcinoma, the carcinoma was absent in deeper H&E

sections, leaving only acellular mucin pools.


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Pathology of colitis and inflammation score

At the end of 3 cycles of DSS, all 3 groups of mice (p53+/+, p53+/-, and p53-/-) showed similar

classic histopathologic changes of chronic active colitis with chronic inflammation, crypt

distortion, ulceration, and regenerative epithelium. p53+/- mice had higher inflammation scores

than p53+/+ and p53-/- mice for both the entire colon/rectum (P = 0.02 and 0.06 for p53+/+ and

p53-/- mice, respectively) and for the distal colon/rectum (P = 0.01 and 0.02 for p53+/+ and p53-/-

mice, respectively). Inflammation scores of p53+/+ and p53-/- mice did not differ significantly. At

the end of 3 cycles of DSS (4 cycles for p53+/+ mice) plus 120 days of water, the p53+/+ and p53+/-

mice showed almost total healing and normalization of the mucosa with focal areas of minimal

crypt changes. However, these changes could not be compared to those of p53-/- mice since p53-/-

mice had to be sacrificed early (mean - 26 days following completion of cycle 3). The only p53-/-

mouse that completed 120 days of water showed histopathologic changes identical to those of

p53+/+ and p53+/- mice sacrificed after 120 days of water.

Mutation of K-ras, p53 and ? -catenin

Tumor cells were laser capture microdissected from 21 colorectal lesions (4 p53+/+, 12 p53+/- and

5 p53-/- mice) for mutation analyses. The mutational status of K-ras and ? -catenin was evaluated

in all 21 samples, while similar analyses for p53 were performed only on lesions from p53+/- and

p53+/+ mice. Mutation of K-ras (codons 12/13) or p53 (exons 5-8) was not detected in any of the

samples. ? -catenin mutations (exon 3) were detected in p53-/-, p53+/- and p53+/+ mice at an

incidence of 0% (0/5), 33.3% (4/12) and 75% (3/4), respectively (P = 0.055). Hot spots included

codons 32 (n = 1), 33 (n = 1), 34 (n = 3) and 37 (n = 2) of exon 3 of ? -catenin (Table II).

Irrespective of the p53 genotype, 7 of 16 (43.7%) polypoid lesions had ? -catenin mutations

compared to 0 of 5 (0%) flat lesions. However, this difference was not statistically significant

(P = 0.12).


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Immunohistochemistry of ? -catenin

Nuclear expression of ? -catenin was observed in 18 of 19 (91.3%) polypoid lesions vs. 0 of 7 flat

lesions (0%) (P < 0.0001) (Figure 4). The number of neoplastic cells positive for nuclear

? -catenin varied from 5 to 50%. Both cancers and dysplasias exhibited a similar nuclear

localization of ? -catenin. No significant relationship was observed between mouse genotype and

the nuclear expression of ? -catenin in polypoid lesions (P = 0.84). Of the 7 flat lesions with

membranous ? -catenin, 5 had sufficient material for ? -catenin mutational analysis, and no

? -catenin mutations were detected. Of the 18 polypoid lesions expressing nuclear ? -catenin, 15

had sufficient material for mutational analysis. Six cases had ? -catenin mutations and 9 cases

lacked ? -catenin mutations. All lesions with ? -catenin mutations expressed nuclear ? -catenin.

Discussion

In this study we have shown that loss of function of p53 is a critical event in the DSS mouse

model of colitis-associated neoplasia, similar to that reported in human colitis-associated

neoplasia (8,10). An increased frequency of G:C to A:T transition mutations in p53 have been

observed within the nonneoplastic inflamed colorectal tissues of colitis patients as compared to

normal colorectal tissue from healthy controls (8). Takaku et al. (7) found a high percentage

(50%) of p53 mutations in nonneoplastic mucosa near UC-associated cancers. Yoshida et al. (6)

microdissected individual crypts from patients with long-standing UC and found mutations in

66.7% and 33.3% of crypts that were regenerative or indefinite for dysplasia, respectively.

Holzmann et al. (11) reported that p53 mutations were present in 19% (40/212) of colon

specimens without dysplasia, 41% (17/29) of specimens indefinite for dysplasia, 38% (9/24) of

specimens of low-grade dysplasia and 63% (5/8) of specimens of high-grade dysplasia. In

addition, LOH of p53 was observed in approximately 25% of samples that were nondysplastic,


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actively inflamed and indefinite for dysplasia, 45% of informative dysplastic lesions and 70% of

cancers (9,10).

In this study, the incidence of colitis-associated neoplasia (dysplasias and cancers) was

significantly higher in p53-/- mice (57%) as compared to both p53+/- and p53+/+ mice (20%) (P =

0.013). This significance held true when dysplasias (P = 0.02) and carcinomas (P = 0.0006)

were examined independently. p53-/- mice had significantly greater numbers of total lesions (P =

0.0001), dysplasias (P = 0.009) and carcinomas (P = 0.001) per mouse compared to p53+/- and

p53+/+ mice. This difference may be underestimated because p53-/- mice were euthanized

prematurely due to illness. Compared to the results for DSS-treated p53-/- mice reported recently

by Fujii et al. (14), we report a higher incidence of invasive carcinoma (42.9% vs 3.0%), while

they report a higher incidence of neoplasia (100% vs. 57%) and numbers of lesions per mouse

(5.0 vs. 0.93). In addition, we observed that flat lesions were associated with the p53-/- genotype,

while polypoid lesions were associated with the p53+/- and p53+/+ genotypes (P < 0.0001). In

contrast to our finding, Fujii et al. reported that flat lesions were associated with the p53+/-

genotype. There are several possible explanations for these differences. First, the mice used in

the present study were inbred on a C57BL/6 background, while in Fujii’s study the p53-/- and

p53+/+ mice were on a C57BL/6 x CBA background and p53+/- mice were obtained by

backcrossing p53-/- mice with wild-type C57BL/6 mice. It is well known that mouse strains

differ in their susceptibility to DSS (31), which could lead to different levels of inflammation as

well as incidence and numbers of neoplastic lesions (16). Second, although animals in both

studies were maintained under specific pathogen-free conditions, the intestinal microflora is most

likely different. Bacterial flora has been shown to affect the type and magnitude of the

inflammatory response to DSS (32,33). Third, different DSS treatment regimens were used in

these two studies. In the present study, mice were treated with DSS for 3 cycles (4 days of 4%

DSS plus 17 days of untreated water per cycle), followed by 120 days of untreated water; while

Fujii et al. (14) used 2 cycles of DSS, (each cycle consisting of 7 days of 4% DSS followed by

14 days of water) followed by 84 days of water.


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Nuclear ? -catenin has been observed in DSS-induced tumors (16) and is usually attributed

to the mutation of members of the Wnt pathway. Because of this, lesions from the present study

were examined for mutation and subcellular localization of ? -catenin by direct sequencing and

immunohistochemistry, respectively. Mutations in exon 3 of ? -catenin were detected in 0% of

p53-/-, 36.4% of p53+/- and 75% of p53+/+ mice (P = 0.055). This direct relationship between

incidence of ? -catenin mutations and increasing copy number of the wild-type p53 gene suggests

that ? -catenin mutations are probably important for colorectal tumorigenesis in mice with wild-

type p53 but not in those mice with loss of p53 function. In the present study, ? -catenin

mutations were detected in codon 32 (1 lesion), codon 33 (1 lesion), codon 34 (3 lesions) and

codon 37 (2 lesions) at or near the GSK3? phosphorylation sites. Similarly, ? -catenin mutations

have been detected in tumors induced by azoxymethane (AOM) (at codons 33, 37, and 41) (34),

DSS/AOM (codons 32, 33, 34) (15), and DSS/PhIP (codons 32 and 34) (35). To date, only one

study has reported on mutation of ? -catenin in human colitis-associated neoplasia (36), and no

mutations (exon 3) were found in 30 UC-associated carcinomas. It should be noted that this

study also reported a lower frequency of the APC mutations in sporadic CRC (26%) than is

usually reported in the literature. Four to 15% of all sporadic colorectal adenomas and cancers

harbor ? -catenin mutations primarily in exon 3 (37). Johnson et al. (38) recently reported that

? -catenin mutations (exon 3) were present in 18.2% of cancers from hereditary nonpolyposis

colon cancer patients but absent in microsatellite stable (N = 78) and unstable (N = 34) sporadic

colorectal cancers. Immunohistochemical analysis of ? -catenin expression revealed that 91.3%

of polypoid lesions expressed nuclear ? -catenin compared to 0% of flat lesions (P < 0.0001).

These data are identical to those of our previous study in DSS-treated Swiss Webster mice (16).

In contrast, Fujii et al. (14) observed similar expression of nuclear ? -catenin in polypoid and flat

lesions of DSS-treated p53? /?, p53+/+, and p53+/+ mice. One possible explanation for the lack of

nuclear ? -catenin in flat lesions in the present study is that the small size of these lesions

hindered identification of foci positive for nuclear localization of ? -catenin. All 7 mice with

? -catenin mutations expressed nuclear ? -catenin, while 9 mice expressed nuclear ? -catenin but


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lacked ? -catenin mutations. From the latter group 3 were p53-/- mice, 5 were p53+/- mice and 1

was a p53+/+ mouse. Several studies (39,40) have shown that cellular expression of ? -catenin can

be regulated by p53. Cagatay et al. (41) convincingly demonstrated that nuclear accumulation of

? -catenin in hepatocellular carcinomas is due to mutation of p53. Wild-type p53 has been shown

to induce transcription of the Siah-1 gene, which encodes a protein involved in the ubiquitin-

mediated degradation of ? -catenin (39). The loss of regulation of ? -catenin by Siah-1 could

explain the observed presence of nuclear ? -catenin in colorectal lesions from p53-/- mice lacking

? -catenin mutations. Castellone et al. (42) recently reported that PGE2, through its EP2 binding

site, can prevent degradation of ? -catenin in cells with activated Wnt signaling, resulting in

nuclear accumulation of ? -catenin. Since PGE2 is upregulated in UC (43), this could be another

explanation for the nuclear localization of ? -catenin in the absence of ? -catenin mutations. In

human studies, a significantly higher incidence of nuclear ? -catenin staining has been observed

in sporadic CRC as compared to UC-associated cancers (44,45). Wong (46) reported nuclear

localization of ? -catenin in 45% of UC-associated CRC as compared to 85% of sporadic CRC.

However, in none of these studies were comparisons made between polypoid and flat lesions.

The results of this study suggest that flat and polypoid lesions arise from different

molecular pathways. First, the presence of flat lesions was significantly associated with loss of

p53 function. Second, nuclear localization of ? -catenin was present in 91.3% of the polypoid

lesions while none of the flat lesions showed this pattern. This result is in agreement with our

previous study, which revealed a significant association between nuclear ? -catenin and polypoid

colitis-associated neoplastic lesions in DSS-treated Swiss Webster mice (16). Third, the

differential response of flat and polypoid lesions to chemopreventive agent exposure has been

noted. In the AOM/DSS model of colitis-associated neoplasia, treatment with celecoxib

decreased the multiplicity of polypoid lesions but not flat lesions (47). Lastly, recent

examination of the gene expression profile of flat and polypoid colitis-associated adenomas by

cDNA array revealed the differential regulation of 27 genes (48). Sixteen genes (e.g. p21,

Smad4, IGF-2) were upregulated and 11 genes (e.g. Cdc42, p120, Ras-GAP) were


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downregulated significantly in the flat as compared to the polypoid lesions. These data, when

combined with the results from the present study, provide strong support for the emergence of

flat and polypoid lesions via different molecular pathways.

No mutations were found in codons 12/13 of K-ras in any of the neoplastic lesions from

DSS-treated p53-/-, p53+/-, and p53+/- mice. Similarly, K-ras mutations are uncommon in UC-

associated neoplasia in the human (49) and have not been reported in DSS colitis-associated

neoplasia in the mouse (14,17).

In an attempt to explain the mechanism of colitis-associated neoplasia in those mice

without ? -catenin mutations or loss of p53 function (p53-/- mice), lesions from DSS-treated p53+/-

and p53+/+ mice were analyzed for p53 mutations (exons 5-8) by direct sequencing. However, no

mutations were found. In other mouse models of colitis-associated neoplasia, no p53 mutations

were found in the IL-10-/- mice, while C:G ? T:A transitions at codon 229 have been observed

in IL-2-/- x ? 2micro-/- mice (19,20). Although no mutations of p53 were found in DSS-treated

p53+/- and p53+/+ mice, we still cannot rule out the possibility of loss of function of p53 by

methylation or mutation/methylation of other members of the p53 pathway (i.e., MDM2

overexpression (50)).

Levels of inflammation have been shown to play an important role in both the type (flat vs

polypoid) and incidence of colitis-associated neoplasia in mouse models (16) and in humans (3).

In order to investigate if the increased incidence and multiplicity of neoplasia in the p53-/- mice

could be due to differences in inflammation, we studied inflammation scores in all 3 genotypes.

After 3 cycles of DSS, p53+/- mice were found to have significantly higher inflammation scores

than p53-/- and p53+/+ mice. No significant difference in the inflammation scores of p53-/- and

p53+/+ mice was observed. At the end of 120 days of water, little if any inflammation was

present in p53+/- and p53+/+ mice and the colorectal mucosa was healed. p53-/- mice of the same

age could not be compared at this point in time, as 13 of 14 mice were sacrificed (due to illness)

at a mean of 26 days after the third cycle of DSS. These findings indicate that the higher


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incidence and multiplicity of colitis-associated neoplasia in p53-/- mice may not have any

association with levels of inflammation.

In this study we have characterized colitis-associated neoplasia in p53-/-, p53+/-, and p53+/+

mice using the DSS model of mouse colitis. Our results show that loss of p53 enhances the

induction of colitis-associated colorectal neoplasia, in particular flat lesions, and dysregulation of

? -catenin signaling plays an important role in the formation of polypoid lesions in this mouse

model of colitis-associated dysplasia. Because p53 function appears to play a protective role in

colitis-associated neoplasia in the human and in this DSS mouse model, we believe this model

provides an excellent system in which to study both the pathogenesis and chemoprevention of

colitis-associated neoplasia. The contribution of ? -catenin mutations and different molecular

pathways to the formation of polypoid and flat lesions is worthy of further investigation in the

human.


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Acknowledgments

The authors would like to thank Drs. Maureen Murphy and Andres Klein-Szanto for reviewing

this manuscript; Denise Green, Lisa Landi and Sherene Cohen for histology services; Wendy

Davis, Cheryl Koch and Rose Adams-McDonnell for performing the immunohistochemistry;

Rita Michielli for assistance in mutation analyses; and Gerry Terlecky and Maureen Climaldi for

assistance in preparing manuscript.

This work was supported by USPHS grants CA099122 and CA-06927 from the National

Cancer Institute, an ACS Institutional Research Grant, and by an appropriation from the

Commonwealth of Pennsylvania. Its contents are solely the responsibility of the authors and do

not necessarily represent the official views of the National Cancer Institute or the American

Cancer Society.

This project utilized the following Core facilities at FCCC: Laser Capture Microdissection

Unit, Laboratory Animal Facility, Cancer Biomarker and Genotyping Facility, Biostatistics

Facility, DNA Sequencing Facility, and Biochemistry and Biotechnology Facility.


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Figure Legends

Figure 1. Experimental Protocol. DSS-treated p53-/- and p53+/- mice were administered 3 cycles

of DSS, with each cycle consisting 4 days of 4% DSS in the drinking water plus 17 days of

untreated water. p53+/+ mice were treated with 4 cycles of DSS. After DSS treatment, animals

were maintained for a maximum of 120 days. Untreated control p53-/- and p53+/- mice were aged

in parallel with DSS-treated p53-/- and p53+/- mice.

Figure 2. Colorectal Lesions in DSS-treated Mice Categorized by Subtype of Lesion. In DSS-

treated p53-/- mice, 38.5% of cancers and 46.1% of dysplasias were flat, 15.4% of cancers were

polypoid, and 0% of dysplasia was polypoid. In DSS-treated p53+/- mice, 16.7% of cancers were

flat, 0% of dysplasias were flat, 5.6% of cancers were polypoid, and 77.8% of dysplasias were

polypoid. In DSS-treated p53+/+ mice, 100% of the lesions were polypoid dysplasias.

Figure 3. Colorectal Lesions Observed in DSS-treated p53-/-. A) Low power (4X) view of

polypoid adenocarcinoma. Square areas show invasive carcinomas. B) High power (40X) view

of malignant glands in submucosa (black square in panel A). C) High power (40X) view of

adenocarcinoma with mucin production (green square in panel A). D) Low power view (10X) of

flat carcinoma. E) High power view (40X) of malignant invasive glands (black square in panel

D). F) High power view (40X) of acellular mucin pool (green square area in panel D).

Figure 4. Expression of ? -catenin in Colorectal Lesions. A) High power view (40X) of

? -catenin staining in normal colonic mucosa. ? -catenin is limited to the cell membrane; B) Low

power view (4X) of a polypoid lesion (H&E). C) High power view (40X) of panel B showing

nuclear localization of ? -catenin (approximately 50% of cells express nuclear ? -catenin).

D) Low power view (10X) of flat invasive cancer (H&E). Arrows indicate the invasive


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adenocarcinoma. E and F) High power view (40X) of membranous ? -catenin staining in

invasive adenocarcinoma from panel D.


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Table I. Colorectal Tumor Multiplicity in DSS-treated Mice

(Mean ± SEM)

p53 genotype N Cancer Dysplasia Total

+/+ 10 0 ± 0 0.40 ± 0.32 0.40 ± 0.32

+/− 89 0.06 ± 0.03 0.17 ± 0.04 0.22 ± 0.05

−/− 14 0.50 ± 0.18* 0.57 ± 0.18** 1.07 ± 0.28§

* Significantly different from p53+/- and p53+/+ mice, P = 0.001.

** Significantly different from p53+/- mice, P = 0.009 § Significantly different from p53+/- and p53+/+ mice, P < 0.0001.


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Table II. β-catenin Mutation (Exon 3) in Colorectal Lesions Obtained from DSS-treated Mice

Sample Type of Lesion ββββ-cateninmutation

GeneAlteration

Amino Acid Substitution

p53−/− 1 Flat cancer No mutation

2 Flat cancer No mutation

3 Flat dysplasia No mutation

4 Polypoid cancer No mutation


p53+/− 1 Polypoid dysplasia 32 codon GAT → GTT Asp → Val

2 Polypoid dysplasia 34 codon GGA → GAA Gly → Glu


4 Polypoid dysplasia 37 codon TCT → TTT Ser → Phe

5 Polypoid dysplasia No mutation




9 Polypoid cancer No mutation




p53+/+ 1 Polypoid dysplasia 34 codon GGA → GAA Gly → Glu


3 Polypoid dysplasia 34 codon GGA → GAA Gly → Glu



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Fig. 1 60x20mm (600 x 600 DPI)


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Fig. 2 62x43mm (600 x 600 DPI)


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Fig. 3 182x89mm (300 x 300 DPI)


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Fig. 4 182x97mm (300 x 300 DPI)


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Short Title: p53 and colitis-associated neoplasia

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