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[CANCER RESEARCH 62, 6367– 6370, November 15, 2002]
Advances in Brief
Microadenomatous Lesions Involving Loss of Apc Heterozygosity in the Colon of
Adult ApcMin/⫹ Mice1
Yasuhiro Yamada,2 Kazuya Hata, Yoshinobu Hirose, Akira Hara, Shigeyuki Sugie, Toshiya Kuno, Naoki Yoshimi,
Takuji Tanaka, and Hideki Mori
Department of Pathology, Gifu University School of Medicine, Gifu 500-8705 [Y. Y., K. H., Y. H., A. H., S. S., T. K., H. M.]; and Department of Pathology, Faculty of Medicine,
University of the Ryukyus, Okinawa 903-0213 [N. Y.]; and Department of Pathology, Kanazawa Medical University, Ishikawa 920-0293 [T. T.], Japan
Abstract
Mutations in the human adenomatous polyposis coli (APC) gene are
causative for familial adenomatous polyposis (FAP), a rare condition in
which numerous colonic polyps arise during puberty and, if left untreated,
lead to colon cancer. Mouse model for human FAP, ApcMin/⫹ mouse,
contains a truncating mutation in the Apc gene and spontaneously develops intestinal adenomas. However, the distribution of tumors along the
intestine found in ApcMin/⫹ mice is different from that in human FAP. A
majority of intestinal polyps in the ApcMin/⫹ mice is known to be located
in the small intestine but rarely detected in the colon. We report here that
adult ApcMin/⫹ mice develop dozens of microadenomatous lesions in the
colon (>20 lesions/colon). Surprisingly, the vast majority of such adenomatous lesions consisting of colonic crypts were <300 ␮m in their greatest
dimension, whereas lesions in the small intestine showed various sizes. The
allelic loss analysis revealed that these adenomatous crypts in the colon
have already lost the remaining allele of Apc. Such findings suggest that,
in contrast to tumorigenesis in the small intestine, loss of heterozygosity
of the Apc gene is not sufficient for tumor development in the colon of
ApcMin/⫹ mice. Our results may give an account for the low incidence
of colonic tumors in ApcMin/⫹ mice.
Introduction
The colorectal carcinogenesis is known to have multistep processes
(1). Because of its gradual evolution toward malignancy, colorectal
cancer might serve as an excellent paradigm to examine the genes
involved in tumorigenesis. In humans, APC,3 ␤-catenin (CTNNB1),
Ki-ras (KRAS1) oncogene, and p53 (TP53) genes play important roles
at different stages of colorectal carcinogenesis (2, 3). Of these, mutations in APC gene found in the earliest stages of the adenomacarcinoma pathway are considered to play a gate-keeping role in
tumor formation and progression (4). Moreover, germ-line mutations
in the APC gene are recognized to be responsible for FAP, a dominantly inherited autosomal condition characterized by formation of
multiple colonic adenomatous polyps with a high likelihood to develop colon carcinomas (5, 6). Somatic APC mutations are also
implicated in most of the colonic tumors (4, 7) and other neoplasms
in the digestive tracts (8).
Mutant mouse lineage being predisposed to Min is regarded as one
of the models for colorectal tumorigenesis (9). Originally, this lineage
was established from an ethylnitrosourea-treated C57BL/6J (B6) male
mouse, and its phenotype is a fully penetrate autosomal dominant
trait. The dominant mutation is known to be located in Apc, the mouse
homologue of the human APC gene, resulting in truncation of the gene
product at amino acid 850 (10). It is suggested that, although homozygous ApcMin/Min mice die as embryos, ApcMin/⫹ mice develop multiple
intestinal neoplasias in the intestinal tracts within a few weeks after
birth. APC is now recognized as a recessive tumor suppressor gene,
and its inactivation of both alleles is considered to be necessary for
tumor formation (11). In mice heterozygous for a mutant allele of Apc,
the loss of Apc function occurs almost exclusively by LOH (12, 13).
Although it appears to be true that inactivation of both APC alleles is
necessary for tumorigenesis, it is still not clear whether such mutations
are sufficient. We have assumed that many precursors of colonic tumor
could be recognized in the colonic mucosa of aged ApcMin/⫹ mice if the
inactivation of both Apc alleles were insufficient for the tumorigenesis.
To test this possibility, we screened intramucosal colonic lesions of aged
ApcMin/⫹ mice by analysis using en face and serial histological sections.
In such analysis, we have reported the presence of newly identified early
appearing lesions of colon cancer, which are hard to be detected in whole
mount preparations (14, 15). Because it is known that a majority of
ApcMin/⫹ mice live no longer than 150 days, ApcMin/⫹ mice ⬎20 weeks
of age were examined in this study. LMM and LPC were used to analyze
allelic loss of the Apc gene.
Materials and Methods
Experimental Procedure. ApcMin/⫹ mice were obtained from The Jackson
Laboratory (Bar Harbor, ME). They were bred and maintained on a basal diet,
CE-2 (CLEA Japan Inc., Tokyo, Japan), until termination of the study. Sixteen
ApcMin/⫹ mice (9 males and 7 females) and 7 Apc⫹/⫹ mice (4 males and 3
females), which were ⬎20 weeks of age (20 –33 weeks of age), were used in
the present study. The colons were removed, cut open along their longitudinal
axis, and fixed flat in 10% buffered formalin for 24 h at room temperature.
Colon tumors identified macroscopically were also fixed in 10% buffered
formalin and processed for histopathological evaluation by routine procedures
(16). To identify small tumors, mucosal surface with methylene blue staining
was observed under a microscope. In brief, fixed colons were placed in 0.5%
solution of methylene blue in distilled water. They were then placed mucosal
side up on a microscope slide and observed through a light microscope at a
magnification of ⫻40.
Tissue Preparation. To identify intramucosal lesions, colonic mucosa was
examined in histological sections. Colons (the length of 6 cm from the anal
ring) were divided into three segments and were embedded in paraffin. The
Received 8/5/02; accepted 10/2/02.
middle part of the small intestine was also cut into small segments and
The costs of publication of this article were defrayed in part by the payment of page
embedded in paraffin. A total of 69 segments from the colon and 23 segments
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
from the small intestine were examined by using an en face preparation and
1
Supported in part by Grants-in-Aid for Cancer Research from the Ministry of Health,
3–5-␮m thick serial sections (14, 15, 17). For each case, 10 –20 serial sections
Labor and Welfare and the Grants-in-Aid from the Ministry of Education, Culture, Sports,
were used to investigate the intramucosal lesions.
Science and Technology, Japan.
2
LMM and LPC. In the current study, DNA for the analysis of the Apc
To whom requests for reprints should be addressed, at Department of Pathology, Gifu
University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan. Phone: 81-58allelic loss was extracted from cells isolated with LMM and LPC (zref18). For
267-2235; Fax: 81-58 -265-9005; E-mail: [email protected].
laser capturing, the slides were put into xylene for 30 min to dissolve the
3
The abbreviations used are: APC, adenomatous polyposis coli; FAP, familial adeparaffin that otherwise interferes with LMM. Next, the slides were washed for
nomatous polyposis; Min, multiple intestinal neoplasm; LMM, laser microbeam micro10 min in 100% ethanol. After staining with H&E, sections were dehydrated
dissection; LPC, laser pressure catapulting; LOH, loss of heterozygosity.
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MICROADENOMATOUS LESIONS IN ADULT APCMIN/⫹ MICE COLONS
Fig. 1. Microadenomatous lesions found in the colon of aged ApcMin/⫹ mice. A, at lower magnification of en face sections, intramucosal adenomatous crypts are frequently detectable
in the colonic mucosa of aged ApcMin/⫹ mice (arrowheads). Note that all lesions are ⬍300 ␮m in their greatest dimension. B, in the small intestine, various sizes of adenomatous lesions
are seen in cross-sections. C–E, higher magnifications of microadenomatous crypts in the colon. The crypts bear basophilic cytoplasm and hyperchromatic nuclei. Mucin production
is almost absent in those crypts. F, a cross-section of a single adenomatous crypt in the small intestine, which have been described as the earliest lesion in the small intestine (13, 24).
Bars, 300 ␮m (A and B), 100 ␮m (C, D, and F), 50 ␮m (E)
in 100% ethanol, incubated 2 min in xylene, and dried at room temperature.
Sections were captured in one session using the PALM Robot-MicroBeam
system (P.A.L.M. Mikrolaser Technology AG, Bernried, Germany). The LPCcollected cells were solved in 20 ␮l of lysis buffer.
Apc Allelic Loss Analysis. A total of 62 microdissected tissues (28 normalappearing crypts, 14 microadenomatous lesions in the colon, 5 tumors in the
small intestine, 10 colonic tumors in ApcMin/⫹ mice, and 5 normal crypts in
Apc⫹/⫹ mice) were selected at random. They were digested overnight at 50° in
20 ␮l of lysis buffer containing 500 ␮g/ml proteinase K, 10 mmol/liter
Tris-HCl (pH 8.0), 50 mmol/liter KCl, 0.45% NP40, and 0.45% Tween 20. The
proteinase K was heat inactivated (10 min at 95°). The tubes were centrifuged
for 5 min, and the supernatant was transferred to new tubes. LOH of the Apc
gene was checked using PCR with mismatched primers, as described previously (12). Briefly, the amplification of the ApcMin allele resulted in a 155-bp
PCR product with one HindIII site, whereas the 155-bp product from the Apc⫹
allele contained two HindIII sites. HindIII digestion of PCR-amplified DNA
from ApcMin/⫹ heterozygous tissue resulted in a 123-bp product from the Apc⫹
allele and a 144-bp product from the ApcMin allele. Therefore, PCR products
from tissue with LOH displayed only one band (144-bp) from the ApcMin
allele. Samples were assayed at least twice, independently.
mice developed at least 20 lesions/colon. The histological features of
such dysplastic crypts resembled those of adenomas; the crypts bore
basophilic cytoplasm and hyperchromatic nuclei, and they were associated with an increase of the nuclear:cytoplasmic ratio (Fig. 1,
C–E). Mucin production was almost absent in the microadenomatous
crypts (Fig. 1E). Interestingly, the vast majority (⬎95%) of the
adenomatous lesions in the colon were ⬍300 ␮m in their greatest
dimension (Fig. 1A; Fig. 2). Most of such lesions consisted of one to
Results
Microadenomatous Lesions in the Colon of ApcMin/⫹ Mice.
Unlike the frequency of tumors in the small intestine (30 – 60 tumors/
small intestine), only small numbers of tumors (0 –5 tumors/colon)
were evident in the colon of adult ApcMin/⫹ mice. Histological sections of these colonic tumors were examined for tumor types, and they
were classified as adenomas or adenocarcinomas. In the sections
stained with H&E, dysplastic crypts were detected frequently in the
colonic mucosa of all of the ApcMin/⫹ mice (Fig. 1A), whereas the
lesions were absent in the colons of Apc⫹/⫹ mice. Adult ApcMin/⫹
Fig. 2. Size distribution of adenomatous lesions in the colon and small intestine.
Representative example of each greatest dimension of adenomatous lesions including
tumors is shown.
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MICROADENOMATOUS LESIONS IN ADULT APCMIN/⫹ MICE COLONS
Table 1 Adenomatous lesions in the colon and small intestine
Lesion/area (/cm )
Mean diameter (␮m)
17.85 ⫾ 9.86a,b
8.07 ⫾ 3.06
176.04 ⫾ 410.84c
1286.96 ⫾ 1069.78
2
Colon
Small intestine
Mean ⫾ SD.
The incidence of colonic lesions was significantly higher than that in the middle
portion of the small intestine (P ⬍ 0.001).
c
The mean diameter of colonic lesions was significantly smaller than that in the small
intestine (P ⬍ 0.001).
a
b
six adenomatous crypts. The greatest dimensions of the adenomatous
lesions, including microadenomatous lesions, adenomas, and adenocarcinomas, in both colon and small intestine are summarized in
Fig. 2. In contrast to the colon, adult ApcMin/⫹ mice developed a
number of adenomatous lesions with a variety of sizes in the small
intestine (Fig. 1, B and F; Fig. 2). The numbers of the lesion/area in
the colon and small intestine were 17.85 ⫾ 9.86/cm2 and 8.07 ⫾ 3.06/
cm2, respectively (Table 1). The incidence of colonic lesions was
significantly higher than that of the small intestine (P ⬍ 0.001). The
mean dimensions of colonic lesions and small intestinal lesions
were 176.04 ⫾ 410.84 ␮m and 1286.96 ⫾ 1069.78 ␮m, respectively
(Table 1). The size of colonic lesions was smaller than that of the
small intestine (P ⬍ 0.001).
Apc Allelic Loss in Colonic Lesions. Twenty-eight normalappearing crypts, 14 microadenomatous lesions in the colon, and 15
intestinal tumors were randomly selected, and picked individually
from the histological sections of adult ApcMin/⫹ mice. Fig. 3 represents the results of nondenatured acrylamide gel electrophoresis. After
HindIII digestion of PCR-amplified DNA, two DNA bands (144-bp
and 123-bp) appeared on gels from phenotypically normal crypts of
ApcMin/⫹ mice, whereas only one band at 123-bp was evident in
normal crypts of the Apc⫹/⫹ mice (Fig. 3). All of the PCR products
from tumors revealed a single band (144-bp) deriving from the ApcMin
allele (Fig. 3; Table 2). Remarkably, the majority of microadenomatous lesions (12 of 14 lesions) showed a single band at 144 bp,
suggesting that such small lesions have lost Apc⫹ allele already
(Fig. 3; Table 2).
Discussion
Min mice are heterozygous for a nonsense mutation in the Apc
gene. They develop spontaneously Mins similarly to the FAP syndrome in humans. However, it is well demonstrated that the distributing pattern of intestinal tumors in ApcMin/⫹ mice is different from
that in human FAP. Most adenomatous polyps in FAP patients arise
in the colon and, if left untreated, lead to colonic cancers. In contrast,
the highest frequency of tumors in ApcMin/⫹ mice is seen in the small
intestine, whereas lower numbers are located in the colon (19). In the
current study, we found that there are many microadenomatous lesions that are hardly identified in the whole mount preparations in the
colonic mucosa of the ApcMin/⫹ mice. It is noteworthy that adult
ApcMin/⫹ mice developed a number of lesions not only in the small
intestine but also in the colon.
It has been demonstrated that loss of Apc function plays a pivotal
role in colorectal carcinogenesis. It is also known that all of the
intestinal tumors in mice heterozygous for a mutant allele of Apc have
lost the Apc function by LOH (12, 13). In this study, microadenomatous crypts in the colon were found to have lost the remaining allele
of Apc, indicating that loss of Apc function has already occurred in
such crypts. Accordingly, it seems to be reasonable to apply Knudson’s “two-hit” theory (20) to the formation of microadenomatous
lesions in the colon of ApcMin/⫹ mice. Importantly, in this study, the
number of colonic adenomatous lesions per area was much higher
than that of lesions in the small intestine, suggesting that LOH of the
Apc occurs frequently in the colonic crypts as well as epithelium in the
small intestine.
It is also interesting that almost all of the intramucosal adenomatous
lesions in the colon were ⬍300 ␮m in their greatest dimension.
Because ApcMin/⫹ mice used in this experiment were ⬎20 weeks of
age, such microadenomatous crypts are suggested to be self-limiting
lesions and not grow into colonic tumors. Conditional knockout mice
of the Apc are reported to develop only 6.7 colon tumors on average,
although numerous colonic cells in the mice ought to be lacking in
both alleles of Apc (21). The results in the present study, together with
previous findings, suggest that inactivation of the Apc is not sufficient
for development of colonic tumors. It is noteworthy that mutation of
K-ras seems to be correlated with the development of large, dysplastic
adenomatous polyps in humans (1, 22). K-ras mutation may be
associated with the tumor development in the colon of ApcMin/⫹ mice.
However, no ras mutations have been found in intestinal tumors of
ApcMin/⫹ mice (23), indicating that the mutational activation of K- or
H-ras is not a common event in the formation of intestinal adenomas
in ApcMin/⫹ mice. Therefore, it is possible that another genetic and/or
epigenetic event except ras mutations occurs in those colonic tumors.
In contrast to colonic lesions, it is quite interesting to note that the
adenomatous lesions in the small intestine had various sizes, and the
mean size of the lesions was significantly larger than in the colon. We
detected different adenomatous lesions, which have been reported in
the stages of polyp development (13, 24), including a single adenomatous crypt. It has been demonstrated that loss of the Apc gene is also
involved in the earliest lesions in the small intestine of mice heterozygous for a mutant allele of Apc (13). It is possible that, in the small
Table 2 Apc allelic loss in the ApcMin/⫹ mouse
Small
intestine
Colon
Normal-appearing crypts
0/28 (0%)
Microadenomatous crypts
Tumors
Tumors
12/14 (85.7%)
10/10 (100%)
5/5 (100%)
Fig. 3. Apc allelic loss assay. Two DNA bands
(144-bp and 123-bp) appear on gels from phenotypically normal crypts of ApcMin/⫹ mice, whereas
only one band at 123-bp is evident in normal crypts
of the Apc⫹/⫹ mice. PCR products from tumors
revealed a single band (144-bp), which were derived from the ApcMin allele. Remarkably, microadenomatous lesions also showed a single band at
144 bp, suggesting that such crypts already have
lost Apc⫹ allele.
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MICROADENOMATOUS LESIONS IN ADULT APCMIN/⫹ MICE COLONS
intestine, a dominant-negative mechanism by the loss of function of
Apc is directly responsible for the formation of microadenomas that
could develop into intestinal tumors. Thus, our results may explain
why the ApcMin/⫹ mouse develops intestinal tumors preferably in the
small intestine and suggest that mechanisms of tumorigenesis involved in the small intestine may differ from those in the colon.
In conclusion, it was shown that there are a number of microadenomatous lesions together with a few tumors in the colon of aged
ApcMin/⫹ mice. Such microadenomatous lesions have already lost the
remaining allele of Apc, indicating that LOH of the Apc gene has
occurred in the crypts. These findings suggest that loss of Apc function is responsible for the formation of microadenomatous lesions in
the colon but is not sufficient for the development of colonic tumors
in ApcMin/⫹ mice.
Acknowledgments
We thank Misato Yasuda for steady and highly capable technical assistance
in the allelic loss assay. We also thank Kyoko Takahashi, Tomoko Kajita,
Hisae Shibazaki, and Yoshitaka Kinjyo for assistance.
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Microadenomatous Lesions Involving Loss of Apc
Heterozygosity in the Colon of Adult ApcMin/+ Mice
Yasuhiro Yamada, Kazuya Hata, Yoshinobu Hirose, et al.
Cancer Res 2002;62:6367-6370.
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