[CANCER RESEARCH 61, 3472–3479, April 15, 2001]
Mouse Intestinal Goblet Cells Expressing SV40 T Antigen Directed by the MUC2
Mucin Gene Promoter Undergo Apoptosis upon Migration to the Villi1
James. R. Gum, Jr.,2 James. W. Hicks, Anne Marie Gillespie, Jose L. Rius, Patrick A. Treseler, Scott C. Kogan,
Elaine J. Carlson, Charles J. Epstein, and Young S. Kim
Department of Veterans Affairs Medical Center, San Francisco, California 94121 [J. R. G., J. W. H., J. L. R., Y. S. K.], and Departments of Anatomy [J. R. G.], Pathology
[P. A. T., Y. S. K.], Pediatrics [A. M. G., E. J. C., C. J. E.], Medicine [Y. S. K.], and Laboratory Medicine [S. C. K.], University of California at San Francisco School of Medicine,
San Francisco, California 94143
ABSTRACT
Mucinous colorectal cancers exhibit a characteristic set of molecular
genetic alterations and may be derived from progenitor cells committed to
the goblet cell lineage. Previously, we demonstrated that the MUC2 mucin
gene promoter drives transgene reporter expression with high specificity
in small intestinal goblet cells of transgenic mice. On the basis of these
experiments, we reasoned that the MUC2 promoter could be used to drive
SV40 T antigen (Tag) expression in the same cell type, decoupling them
from their normal antiproliferative controls. A line of mice was established (MUCTag6) that expressed Tag in intestinal goblet cells as determined by RNA blot and immunohistochemical analysis. These goblet cells
were markedly involuted however, most notably in the villi. Endogenous
intestinal MUC2 message levels were reduced to about one third the
normal level in these mice. However, absorptive cell lineage markers were
comparable with nontransgenics. Bromodeoxyuridine-positive S-phase
cells are limited to crypts in nontransgenic intestine but are present in
both crypts and villi in MUCTag6. In contrast, mitotic cells were not
present in the villi, indicating that MUCTag6 villi goblet cells do not
progress into M phase. Apoptotic cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling were increased
more than fourfold in MUCTag6 villi (P < 0.0001), and apoptotic goblet
cells were evident. Electron microscopic examination of MUCTag6 intestinal villi revealed the presence of degraded cell remnants containing
mucin goblets together with other cell debris, further indicating apoptosis
of the goblet cell lineage. Thus, the expression of Tag in intestinal goblet
cells releases them from normal antiproliferative controls, causing their
inappropriate entry into S phase even after they transverse the crypt/
villus junction. They do not, however, progress to M phase. Instead, they
undergo apoptosis with a high degree of efficiency in S or G2 phase. These
experiments demonstrate that apoptosis effectively blocks inappropriate
goblet cell proliferation in the intestine, supporting its proposed role as an
antineoplastic mechanism.
INTRODUCTION
Colorectal cancers arise from stem or progenitor cells via several
pathways involving both genetic alterations as well as epigenetic
events (1, 2). These cancers manifest several histological subtypes
that exhibit different molecular and clinical features. Mucinous
CRCs3 are characterized by their high content (⬎50% of tumor
volume) of extracellular mucus (3, 4), almost always present as a
consequence of MUC2 gene expression (5). Compared with nonmucinous CRC, mucinous CRC is more likely to exhibit microsatellite
Received 11/6/00; accepted 2/13/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
Supported by the Department of Veterans Affairs Medical Research Service, the
Theodora Betz Foundation, the California Cancer Research Program, the National Cystic
Fibrosis Foundation, and NIH Grant DK-47766.
2
To whom requests for reprints should be addressed, at GI Research Laboratory (151
M2), Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121. Phone: (415) 750-2095; Fax: (415) 750-6972; E-mail: jgum@
maelstrom.ucsf.edu.
3
The abbreviations used are: CRC, colorectal cancer; Tag, SV40 T antigen; MUC2,
mucin gene MUC2; BrdUrd, bromodeoxyuridine; hGH, human growth hormone; Rb,
retinoblastoma gene product; TUNEL, terminal deoxynucleotidyl transferase-mediated
dUTP-biotin nick end-labeling.
instability and K-ras gene mutations and is less likely to contain p53
gene mutations and to express detectable levels of P53 protein (6 – 8).
Thus, mucinous and nonmucinous CRCs appear to follow different
pathways to malignancy.
Several mouse models are available for the genetic analysis of
tumorigenesis in the colon (9). The first to be described were Min
mice, which have a truncating mutation in codon 850 of the Apc
gene (10, 11). These mice rarely if ever develop invasive intestinal
cancer. Instead tumor progression is limited to adenoma formation.
Moreover, tumorigenesis in Min mice occurs predominately in the
small intestine as opposed to the colon. This seemingly detracts
from the attractiveness of these mice as a model for CRC, although
it has the advantage that the crypt/villus junction in the small
intestine is an unmistakable landmark for studies involving histological analysis. Because of its importance in human CRC cancer,
targeted mutagenesis of Apc has been performed as well (9).
Notably, mice bearing a more distal truncating mutation at codon
1638 produce early stage carcinomas of the intestine at reasonable
frequency (12). Other mouse models of CRC involve disruption of
DNA mismatch repair genes (9); interestingly, these mice produce
nonintestinal tumors as well as early stage carcinomas and adenomas throughout the gastrointestinal tract.
In previous studies we and others have cloned and characterized
the MUC2 goblet cell mucin gene and its promoter both in vitro
and in vivo (13–16). Experiments examining the expression of a
⫺2864 to ⫹17 bp MUC2 promoter/hGH reporter construct in
transgenic mice have demonstrated highly specific expression in
goblet cells of the distal small intestine (16). On the basis of these
experiments, we reasoned that the MUC2 promoter could be used
to drive SV40 Tag expression in these same cells in transgenic
mice. Because of the ability of Tag to decouple the cell cycle from
its normal antiproliferative controls (17, 18), we hypothesized that
intestinal goblet cells in such mice would experience uncontrolled
growth. Depending on other antineoplastic mechanisms in force in
this tissue and cell type, mice bearing a MUC2 promoter/Tag
hybrid oncogene could manifest several possible phenotypes up to
and including the development of malignant, mucinous tumors of
the small intestine.
This article describes the effects of expressing Tag under the
control of the MUC2 promoter in mice. In particular, a line of mice
was established that continuously express Tag in intestinal goblet
cells. Unlike intestinal epithelial cells in normal mice, these Tagexpressing goblet cells continue in the cell cycle and proceed into S
phase even after they migrate past the crypt/villus junction. They do
not, however, undergo mitosis, and intestinal neoplasms do not develop. Rather, these cells are deleted from the intestinal epithelium by
a very efficient apoptotic process. Thus, this study provides direct
experimental evidence for the antiproliferative effects of apoptosis in
the intestinal epithelium. Moreover, the mice provide a model for
future studies of apoptosis in the intestine in a highly efficient,
genetically modifiable in vivo system.
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APOPTOSIS IN INTESTINAL GOBLET CELLS
MATERIALS AND METHODS
Preparation of MUC2 Promoter/Tag Hybrid Oncogene and Transgenic
Mice. SV40 DNA was obtained from Life Technologies, Inc., digested with
BglI, treated with S1 nuclease, and digested with BamHI, and the SV40 early
region fragment encoding the large and small Tags was isolated for cloning
into BamHI/EcoRV-digested pBluescript (SK⫺). A fragment containing bases
⫺2864 to ⫹19 of the MUC2 promoter was retrieved from the previously
described pGL2-basic construct (15) by digesting with HindIII, Klenow treating, and digesting with XhoI. This fragment was then cloned into the Tag/
pBluescript (SK⫺) construct that had been digested with ClaI, Klenow treated,
and digested with XhoI, allowing the MUC2 promoter/Tag hybrid oncogene to
be retrieved by digesting with XbaI/XhoI. This procedure resulted in linkage
between the MUC2 promoter and the SV40 Tag gene with the following
sequence: 5⬘-GCCCCTGCAAGCTCGATAAGCTTGATCGGCCTCTGAG,
where the MUC2 5⬘-untranslated region is underscored, the linker is plain text,
and the Tag 5⬘-untranslated region is boldface. The linkage region and the
regions near the ends of the construct were confirmed by sequence analysis.
Transgenic mice were prepared using C57BL/6J ⫻ DBA/2J F2 hybrid
zygotes as previously described (16). Transgene was detected in BamHIdigested mouse tail DNA using bases ⫺2864 to ⫹19 of the MUC2 promoter
as a probe for blot analysis. Transgene copy number was estimated from the
intensity of the 331-bp BamHI fragment internal to the MUC2 promoter using
NIH Image software (16).
RNA Blot Analysis. RNA was extracted from mouse tissues using Tri
reagent (Molecular Research Center, Cincinnati, OH), and 10-␮g aliquots were
subjected to electrophoresis and transferred to nylon membranes as previously
described (16). A probe for Tag was obtained by digesting the initial TagpBluescript (SK⫺) plasmid described above with BamHI and SalI. A probe for
intestinal fatty acid binding protein spanning bases 343–543 (19) was prepared
by PCR of mouse intestinal cDNA. Probes for mouse Muc2 (16) and glyceraldehyde-3-phosphate dehydrogenase and dipeptidylpeptidase IV (20) were
described elsewhere.
BrdUrd Labeling of S-phase Cells. Mice were injected i.p. with 120 ␮g
BrdUrd (Sigma, St. Louis, MO) in PBS per gram body weight (21). After 1 h,
the mice were killed and segments of their intestine were opened longitudinally, rinsed in PBS, fixed in buffered formalin, and embedded in paraffin.
After rehydration and endogenous peroxidase inactivation with 3% H2O2 for
10 min, sections were treated in 2 N HCl at 40°C for 1 h, treated with 25 ␮g/ml
proteinase K for 10 min at 37°C, and blocked using serum-blocking solution
(Histostain-Plus kit; Zymed, South San Francisco, CA). Monoclonal antiBrdUrd (Clone BU 33; Sigma) was applied at a 1:50 dilution for 2 h at 37°C,
and its binding was detected using the Histostain-Plus protocol and reagents.
Hematoxylin was used as a light counterstain.
Goblet Cell Involution and Apoptosis. Goblet cells were visualized using
periodic acid-Schiff/Alcian blue/hematoxylin staining. Depth of mucin goblets
were measured using a Zeiss Universal Research Microscope fitted with a
Hitachi video camera using Scion Image software. Apoptosis was visualized
after H&E staining using standard morphological criteria (22) and using the
TUNEL assay (23). For quantitative analysis of TUNEL-positive cells per villi,
a cell was scored TUNEL positive only if it was in the epithelial layer and had
apoptotic morphology. Villi were scored only if they occupied at least 50% of
a ⫻400 microscopic field, ensuring reasonable longitudinal orientation. Immunohistochemical staining with monoclonal antibody against single-stranded
DNA was also used to assess apoptosis in tissue sections (24, 25). Monoclonal
antibody F7–26 was obtained from Chemicon International Inc., and singlestranded DNA was stained using the protocol suggested by the manufacturer.
Immunohistochemical Staining. For SV40 Tag detection, antigen retrieval was performed by microwaving rehydrated sections in 10 mM citric acid
(pH 6.0) four times for 5 min each with 1 min between heatings. Monoclonal
antibody against SV40 (Pab 108; Santa Cruz Biotechnology, Santa Cruz, CA)
was applied at a 1:40 dilution at 4°C overnight. Histochem-Plus kits were used
to detect bound primary antibody. Lysozyme immunohistochemistry was performed by the Moffitt Hospital pathology laboratory using polyclonal antibody
obtained from Cell Marque.
Electron Microscopy. Samples were fixed in 2.7% glutaraldehyde, 0.8%
paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at room
temperature. They were rinsed in distilled water and postfixed in 2% osmium
tetroxide for 1 h, dehydrated through graded alcohols to 100% ethanol, and
embedded in Eponate resin (Polysciences, Inc., Warrington, PA). Seventynanometer sections were cut and stained with uranyl acetate (10% in 50%
ethanol) and Reynold’s lead citrate (microwave method). A Philips Bioscan
Techni 10 was used at 80 kV for photography.
RESULTS
Production and Characteristics of Initial Hybrid Oncogene
Recipient Mice. A total of five mice were obtained that had one to
approximately eight copies of the MUC2 promoter/Tag hybrid oncogene incorporated into their genome. These mice all died within 26
weeks (Table 1). A mucinous occlusion occurred in the lumen of the
distal colon in mouse 28. This mass contained mucin-producing
(periodic acid-Schiff-positive) cells but did not appear to be firmly
attached to the bowel wall. Mouse 1 developed intraepithelial neoplasia in its submandibular gland and a sarcoma in its thorax. Mouse
17 developed high grade carcinoma in situ in its cervix as well as
intraepithelial neoplasia in its submandibular gland that had a small
foci of adenocarcinoma. Mouse 6 developed splenic and cecal tumors,
which will be described in the next section. Mouse 4 developed a
splenic tumor as well but died unexpectedly, compromising detailed
histological examination. In several cases RNA was extracted from
these tumors, and all tested samples expressed high levels of Tag (data
not shown). An attempt was made to propagate these mice by breeding to the C57BL/6 strain but was only successful in the case of mouse
6. Offspring of this mouse were designated line MUCTag6 and were
characterized in detail as described below. The mice used in this study
were from generations F1 to F3.
Description of the MUCTag6 Line. MUCTag6 mice develop to
adulthood normally and resemble their nontransgenic littermates in
appearance and behavior. Both males and females are fertile and
propagate the hybrid oncogene in an autosomal dominant fashion. As
shown in Fig. 1A, these mice express high levels of Tag in the middle
and distal portions of their small intestine and very low to undetectable levels in other tissues, including their colons. Thus, the tissuespecific pattern of MUCTag6 transgene (Tag) expression mimics the
pattern observed with multiple pedigrees of MUC2 promoter/hGH
Table 1 Tumor formation in hybrid oncogene recipient mice
All mice except mouse 4 were killed, and necroscopy was performed when they became moribund, according to institutional guidelines.
Mouse
Transgene
copy no.
Sex
Tumor site
Tumor type
1
8
F
4
6
1
2
F
F
17
1
F
28
2
M
Salivary gland
Thorax
Spleen
Spleen
Cecum
Cervix
Salivary gland
Colon
Intraepithelial neoplasia
Sarcoma
Unknowna
Histiocytic sarcoma
Histiocytic sarcomab
Carcinoma in situ
Intraepithelial neoplasia
Colonic occlusion, unknown origin
Age at death
(wk)
a
Mouse 4 died unexpectedly without overt symptoms, which precluded detailed histological examination.
b
Histiocytic sarcoma located in unencapsulated lymphoid tissue in cecum.
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10
13
26
13
12
APOPTOSIS IN INTESTINAL GOBLET CELLS
Fig. 1. RNA blot analysis of Tag expression in MUCTag6 mice. A, RNA was extracted
from the normal tissues of a F1 male 7 weeks of age and subjected to blot analysis. PSI,
MSI, DSI, proximal, middle, and distal small intestine, respectively. Stom, Liv, Kid,
stomach, liver, and kidney. Probe used is given left of the autorad. Probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to ensure that all samples contained
RNA. B, RNA was extracted from the tissues of various MUCTag6 mice (TG) or their
nontransgenic littermates (Non-TG). The sizes of the spleens and lymph nodes are given.
A separate blot was also probed for endogenous Muc2 gene expression. All samples taken
from an individual mouse are indicated by a line drawn under the GAPDH autorad.
reporter transgenic mice (16). The spleens and lymph nodes of young
MUCTag6 mice appear normal. However, they become enlarged with
aging in most animals. Six of seven of the spleens examined from 18to 27-week-old MUCTag6 mice expressed detectable levels of Tag
message, but the spleen of a 6-week-old mouse did not (Fig. 1B). In
general, the level of Tag expressed increased with the size of the
spleens. The enlarged lymph nodes found in MUCTag6 mice also
express Tag, whereas the nontransgenic spleens used as controls were
negative for expression. The salivary gland of a 27-week-old MUCTag6 mouse also failed to express detectable Tag. In contrast to
expressing Tag, the enlarged spleens and lymph nodes of MUCTag6
did not express endogenous Muc2 (Fig. 1B). On histological examination, the enlarged spleens and lymph nodes contained areas of
tumor cells that exhibited the typical morphology of histiocytic sarcoma. Two cases of histiocytic sarcoma in liver were also noted.
Although histiocytic sarcoma is a rare tumor in humans, it commonly
occurs in some strains of mice (26). The tumor cells were immunoreactive for lysozyme, confirming their histiocytic lineage. Moreover,
immunostaining also revealed Tag expression in these tumor cells (not
shown). As is commonly the case, the histiocytic tumor cells were
accompanied by extramedullary hematopoiesis. MUCTag6 mice become moribund with enlarged spleens as they reach ⬃5–7 months
of age.
S-Phase Cells and Goblet Cell Involution in MUCTag6 Small
Intestinal Villi. Intestinal epithelial cells in the normal mouse proliferate strictly in the crypts of Lieberkühn; i.e., cell cycle activity
ceases before the cells migrate past the crypt/villus junction (27, 28).
This withdrawal from the cell cycle appears to be caused by marked
decreases in cyclin D1 levels, resulting in the accumulation of unphosphorylated Rb, which binds and inactivates the E2F family of
transcription factors required for entry into S phase (17, 29). Tag,
however, inactivates Rb (17, 18). Thus, the expression of Tag in
intestinal goblet cells could cause abnormal proliferation of this cell
lineage. To test this possibility, we used BrdUrd labeling to identify
S-phase cells in the intestinal epithelium of MUCTag6 mice and their
nontransgenic littermates. As expected, S-phase cells were confined to
the crypts of nontransgenic mouse small intestine (Fig. 2A). In MUCTag6, however, S-phase cells were present in the crypts as well as in
the lower part of the villi (Fig. 2B). Some of the S-phase villi cells
could be clearly identified as goblet cells. Moreover, MUCTag6
goblet cells express Tag, which is detectable by immunohistochemistry (Fig. 2, C and D). These villi goblet cells are frequently misshapen, however, appearing involuted with small nuclei displaced
apically and with markedly diminished mucin vacuole content. Some
Tag-expressing nuclei appear not to be goblet cell associated in Fig.
2D. This could be caused by both goblet involution and by nuclei and
goblets lying in different planes of the section. Goblet cell involution
is clearly visualized with periodic acid-Schiff/Alcian blue/hematoxylin staining (Fig. 2, E and F). Although this aberrant goblet cell entry
into S phase and morphology was readily apparent, the MUCTag6
intestinal epithelium did not develop tumors. No gross alterations
were detected in MUCTag6 epithelial structure, and dysplastic features such as branched crypts or villi were not detected (30).
MUCTag6 Intestinal Villi Goblet Cells Are Deleted by Apoptosis. The experimentation described above indicates that
MUCTag6 intestinal villi goblet cells continue in the cell cycle, freely
entering S phase. Mitotic cells, however, although common in intestinal crypts, could not be detected in MUCTag6 villi. This and the fact
that what appeared to be involuted goblet cells were common in
MUCTag6 villi suggested the hypothesis that these cells were removed from the epithelium by apoptosis. Microscopic examination of
H&E-stained MUCTag6 intestine supported this hypothesis. The
MUCTag6 villi epithelium was laden with what appeared to be the
remnants of cells in various stages of apoptosis. Nuclei that were
apically displaced, condensed, distorted, blebbing, and/or fragmented
were common (Fig. 3, A–E). In some cases, remnants of mucus
goblets can be seen to be associated with these nuclei (Fig. 3, A–C).
We also used the TUNEL assay (23), which detects the fragmented
DNA characteristic of apoptosis, to characterize the MUCTag6 intestinal epithelium (Fig. 3, G–K). TUNEL-positive nuclei were common,
exhibiting a spectrum of apoptotic morphology similar to that observed using H&E staining. Antibody to single-stranded DNA, which
binds as a manifestation of nuclear protein degradation (24, 25),
confirmed that these structures were undergoing apoptosis (Fig. 3,
L–P). In contrast to the high rate of apoptosis observed in the MUCTag6 villi epithelium, apoptotic cells were rare in the intestinal crypts
of both MUCTag6 and nontransgenic mice.
Because of the alterations observed in MUCTag6 villi goblet cells,
we sought to determine whether absorptive cells, the major cell type
found in villi (27, 28), were also affected. Microscopic examination
did not reveal any alterations in absorptive cell morphology, and, by
all appearances they remained the major epithelial cell type of MUCTag6 villi (Figs. 2 and 3). To examine this in more detail, we
performed RNA blot analysis of cell lineage markers in MUCTag6
and nontransgenic mouse intestine (Fig. 4). Here, the steady state
mRNA levels of two absorptive cell markers, dipeptidyl peptidase IV
(20) and intestinal fatty acid binding protein (19), were compared. The
blots detected no differences in the level of these markers in MUCTag6 intestine versus nontransgenic. Hence, there was no discernable
affect of the hybrid oncogene on the absorptive cell lineage. Also in
Fig. 4, the expression of endogenous Muc2 (mouse) message was
examined. Levels of this goblet cell marker in MUCTag6 small
intestine were about one third that found in the nontransgenic control.
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Fig. 2. Goblet cell alterations in the MUCTag6 small intestinal epithelium. All samples were removed from the distal third of the small intestine of MUCTag6 mice (B, D, and F)
or a nontransgenic littermate (A, C, and E). Bars, 100 (A, B, E, and F) or 25 ␮m (C and D). S-phase cells identified by BrdUrd labeling in nontransgenic mice are confined to the crypts
(A); in MUCTag6 mice, however, S-phase cells persist into the villi (B). Inset in B, S-phase cells with clearly identifiable goblet cell morphology. Tag expression in the nuclear region
of MUCTag6 goblet cells is shown in D, whereas nontransgenic goblet cells fail to stain (C). Mucin goblets are stained blue in C and D with Alcian blue. E and F, periodic
acid-Schiff/Alcian blue/hematoxylin-stained sections from nontransgenic and MUCTag6 intestine, respectively; insets, enlarged views of goblet cells. D and F, involution of the goblet
cells in MUCTag6, compared with nontransgenic (C and E).
Although this is difficult to quantitate precisely because of the polydisperse nature of Muc2 mucin mRNA isolated using conventional
methods (31), it is consistent with the goblet cell involution observed
in histological sections. It is also possible that down-regulation of
Muc2 expression by Tag contributes to this effect. The absence of Tag
expression in nontransgenic mouse intestine is also shown in Fig. 4.
Quantitative analysis of goblet cell involution and apoptosis in
MUCTag6 villi was also performed. The total number of periodic
acid-Schiff-positive goblet cells per MUCTag6 villi was approximately the same as in nontransgenic villi (12.2 ⫾ 4.2 versus
10.4 ⫾ 3.3, n ⫽ 25 villi; data not shown). The slight increase in goblet
cell number observed in MUCTag6 could reflect an expected increase
in goblet cell proliferation in the crypts attributable to Tag expression.
Many of the MUCTag6 mucus goblets counted were very small,
however, as described above. Therefore, we measured both the mean
size of the mucus goblets and the average number of “normal-sized”
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Fig. 3. Apoptotic bodies in MUCTag6 distal small intestinal villi. Arrowheads, apoptotic bodies associated with remnants of mucus goblets; arrows, apoptotic bodies devoid of
identifiable mucus goblets. A–E, H&E-stained sections. G–K,
TUNEL-stained sections; counterstain is methyl green. L–P,
immunohistochemistry using anti-single-stranded DNA;
counterstain is hematoxylin. Size markers are given at bottom
of figure.
mucus goblets per villi in MUCTag6 and nontransgenic intestine.
Here, normal-sized mucus goblets were defined as goblets equal to or
larger than the mean minus SD of goblets found in nontransgenic
intestine (Fig. 5). When counted using this criteria, the number of
normal-sized goblet cells per villi in MUCTag6 was less than one fifth
Fig. 4. RNA blot analysis of cell lineage markers in MUCTag6 and nontransgenic
mouse intestine. RNA was isolated from intestinal segments, and blots were prepared and
hybridized to Tag, dipeptidyl peptidase IV (DPPIV), intestinal fatty acid binding protein
(FABPI), and mouse Muc2 probes. The final pair of autorads were probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
that of nontransgenic (Fig. 5). Moreover, almost all of the normalsized goblet cells counted in MUCTag6 were located near the crypt/
villus junction. Also shown in Fig. 5, the mean size of MUCTag6
goblets was approximately one half that of nontransgenic (measured
in length from the apical part to the basal part of the goblet). Because
living goblet cells are three-dimensional objects, opposed to the
two-dimensional measurements necessitated by histological sectioning, the actual difference is greater. We also counted the number of
TUNEL-positive apoptotic cells per villi and found approximately
fivefold more in the MUCTag6 villi versus nontransgenic (Fig. 5).
Thus, almost all MUCTag6 intestinal villi goblet cells are involuted,
and the epithelium contains dramatically elevated levels of cells
undergoing apoptosis.
Finally, electron microscopy was used to gain further insight into
cells undergoing apoptosis in MUCTag6 villi. Goblet cells in nontransgenic mouse epithelium are packed with apically located, tightly
packed mucus vacuoles (Fig. 6A). These cells also have well-developed, rough endoplasmic reticulum and Golgi, necessary for the
production and packaging of large quantities of secretory mucus.
Normal-appearing goblet cells were seldom encountered in MUC-
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both containing vacuoles associated with other condensed cell debris.
Also very common were the remnants of mucin goblets together with
other cell debris, including rough endoplasmic reticulum located near
the cusp of the epithelium (Fig. 6D). Thus, electron microscopic
examination of MUCTag6 intestinal villi revealed the presence of
degraded cell remnants including mucin goblets together with other
cell debris, further indicating apoptosis of the goblet cell lineage.
DISCUSSION
Fig. 5. Quantitative analysis of goblet cell involution and apoptosis in MUCTag6 distal
small intestinal villi. ⴱ, statistical significance at P ⬍ 0.0001 using the two-tailed t test. To
calculate normal-sized goblet cells per villi, mean mucus goblet depth was calculated
along with SD in 50 goblet cells from nontransgenic mouse villi. The resulting mean
minus SD (5.6 ␮m) was used as the lower limit of normal mucus goblet size, and cells
with mucus goblets greater than or equal to this size were counted from each of 25 distal
small intestinal villi from nontransgenic or MUCTag6 mice. Means were plotted; error
bars, SD. The center graph depicts the mean mucin goblet size from both nontransgenic
and MUCTag6 villi. The final graph shows the number of TUNEL-positive apoptotic cells
observed per villi in nontransgenic and MUCTag6 mice (100 villi of each were counted
using scoring criteria described in “Materials and Methods”).
Tag6 villi. Instead, apoptotic fragments and the remnants of involuted
goblet cells were common (Fig. 6, B–D). Figure 6B shows examples
of an involuted mucus goblet and a condensed vacuole containing cell
debris. Figure 6C shows two examples of degraded cell remnants,
The intestinal epithelium represents a continuum of cell division,
differentiation, migration, and removal that is maintained for the
lifetime of an animal. All intestinal epithelial cells are derived from a
small number of pluripotent stem cells located near the base of the
crypts of Lieberkühn (28, 32, 33). These cells are believed to give rise
to both long- and short-lived progenitor cells that retain the capacity
to divide but that can be committed to the generation of one of four
cell lineages (absorptive cells, goblet cells, enteroendocrine cells, and
Paneth cells; Refs. 28, 32, and 33). Paneth cells migrate downward to
occupy positions in the base of the crypts, whereas the other cell
lineages migrate upward. In the small intestine, all cell cycle activity
ceases before the cells reach the crypt/villus junction, with the cells
apparently arrested in G1 phase, as judged by their content of cyclin
E (29). Goblet cells differentiate while still deep in the crypts, forming
Fig. 6. Electron microscopic examination of MUCTag6 intestinal villi epithelium. Bars, 5 ␮m in A–C and 1 ␮m in D. A,
a normal goblet cell in a nontransgenic mouse villi. The apical
region of the cell is laden with mucus-containing vacuoles and
the peri-nuclear region contains abundant rough endoplasmic
reticulum and Golgi. These cells are rare in MUCTag6 villi;
instead apoptotic fragments and goblet cell debris is found. B,
involuted goblet with intact mucus vacuoles (arrow) and a
separate vacuole containing condensed cell debris (arrowhead)
in a MUCTag6 villi. C, examples of membrane-encapsulated
cell remnants containing vacuoles as well as condensed cell
debris (arrows). Goblet cell debris including mucus vacuoles
and rough endoplasmic reticulum located near the cusp of the
epithelium is shown in (D).
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APOPTOSIS IN INTESTINAL GOBLET CELLS
characteristic apically located mucus-containing goblets (34, 35).
They then migrate for 2–3 days until they reach the tip of the villi
where they are deleted by a sloughing process that may also involve
apoptotic mechanisms and the partial reclamation of cellular material
(22). The colonic epithelium experiences a pattern of cell renewal
similar to what occurs in the small intestine, although the process is
less well studied (36). In colon cancer, genetic and epigenetic events
occur that cause the dysregulation of genes involved in controlling
various aspects of this cell renewal process (1, 2, 37).
In this study we sought to perturb the regulation of intestinal goblet
cell proliferation via the expression of Tag under the control of the
MUC2 promoter in transgenic mice. We have previously shown that
this MUC2 promoter directs reporter gene expression to goblet cells of
the distal portion of the mouse small intestine (16). Reporter expression initiates deep in the crypts, where goblet cells are still actively
dividing (34, 35). The principle effect of Tag on the cell cycle is to
bind and inactivate the Rb protein, thus releasing E2F transcription
factor and driving the passage of cells from G1 to S phase (17, 29).
Our hypothesis was that this would cause the uncontrolled proliferation of goblet cells, leading to the expansion of the goblet cell lineage
as well as priming for further mutations that would ultimately lead to
the development of mucinous CRC. A further hypothesis was that the
expression of Tag in still-dividing, deep crypt cells, as opposed to
quiescent villi cells, may be important in the initiation of tumorigenesis. The formation of these hypotheses was guided by previous
studies in which intestinal cell lineage-restricted promoters were used
to drive Tag expression in mice (21, 38, 39). Use of the Fabpi
promoter resulted in Tag expression in the villi of the small intestine
(21). This caused reentry of quiescent villi absorptive cells into the
cell cycle but had no measurable effect on absorptive cell numbers or
villi cell lineage distribution, nor did neoplasms arise. Interestingly,
however, an increase in apoptosis was noted in the villi of these mice
(29, 30). Mice with SV40 Tag driven by the cryptdin-2 promoter
experienced Paneth cell ablation in the small intestine instead of
proliferation and surprisingly developed neuroendocrine tumors of the
prostate, derived from a second cell type in which the cryptdin-2
promoter is also active (38). A similar attempt using a glucagon
promoter resulted in endocrine tumors in the colon and the pancreas,
where Tag was expressed in endocrine cells (39).
Forced expression of Tag did release intestinal goblet cells from at
least one antiproliferative control. As shown in Fig. 2B, MUCTag6
goblet cells fail to undergo G1 arrest and continue into S phase
inappropriately as they migrate up the crypts and into the villi. This
alone, however, does not lead to intestinal neoplasia. It appears rather,
that these S-phase cells are removed by a very efficient apoptotic
process in lieu of entering mitosis. The evidence for this is as follows:
(a) the absence of mitotic cells in the MUCTag6 villi despite the
presence of S phase goblet cells; (b) the presence of large numbers of
TUNEL and anti-single-stranded, DNA-positive, and histologically
evident apoptotic cell bodies in MUCTag6 villi, some of which retain
morphological traces of their goblet cell origins (Figs. 3 and 6); and
(c) the involution of MUCTag6 mucin goblets (visualized by periodic
acid-Schiff/Alcian blue staining), resulting in their near-complete
removal as they migrate away from the crypt/villus junction (Figs. 2,
E and F, and 6). At the final stages of this process, some material from
the degraded cells, especially the mucus, appears to be extruded into
the intestinal lumen, whereas some apoptotic cellular material appears
to be engulfed by neighboring cells (Figs. 2, E and F, and 6). Thus, the
cellular debris from intestinal apoptosis, at least in this setting, is both
endocytosed and sloughed. This suggests the possibility that both
processes may be used to delete intestinal epithelial cells from the
extrusion zone near the villi tips at the end of their natural life span
(22).
The antineoplastic effects of apoptosis in the intestine has been
suggested by other studies, including analysis of cells in culture and
the observation that the proapoptotic gene BAX is frequently mutated
in microsatellite unstable CRCs (40, 41). It is also been observed that
both CRCs and adenomas have increased apoptotic indices (42, 43).
Furthermore, the colonic epithelium of azoxymethane-treated rats
exhibits abundant apoptotic indices (44). This study provides direct
experimental evidence for the antiproliferative effects of apoptosis in
the intestine in a well-defined, in vivo system. Because goblet cells are
morphologically distinct from other intestinal cell lineages, it is clear
that complete or near-complete ablation of these cells has occurred by
the time of their migration to the upper part of the villi. Thus,
intestinal apoptosis can be extremely efficient, and the bulk of the in
vivo evidence suggests that it occurs as an adaptive response to the
uncontrolled proliferation of intestinal epithelial cells.
The development of histiocytic sarcomas in MUCTag hybrid oncogene-bearing mice was unexpected. MUC2 gene expression is very
tightly regulated, and to the best of our knowledge MUC2 has not
been reported to be expressed in macrophages, the presumed precursors of histiocytic sarcomas. It is likely that the hybrid oncogene
expresses Tag in a population of macrophages and that this leads to
expansion of this cell lineage. The correlation of Tag expression with
spleen size in MUCTag6 supports this hypothesis (Fig. 1B). Further
epigenetic and genetic changes may then be required for histiocytic
sarcoma formation. The observation that endogenous Muc2 message
is not expressed in the histiocytic sarcomas (Fig. 1B) indicates that the
hybrid oncogene can be active in a population of cells, albeit a
selected population, that does not express endogenous MUC2. The
development of histiocytic sarcomas and ensuing limitation of life
span in MUCTag6 may preclude the possibility of intestinal neoplasm
formation, which could require antiapoptotic genetic changes. The
notion that additional genetic changes may be required for neoplasia
in Tag-expressing intestinal epithelial cells is supported by the observation that cell lines developed from Tag-expressing intestinal epithelial cells do not form colonies in soft agar or produce tumors when
transplanted into nude mice (45). Furthermore, decreased apoptosis
failed to increase intestinal tumor formation in the genetically initiated
Apc1638 mouse model (46). It is also possible that in the MUCTag6
model, Tag is not expressed early enough in the intestinal goblet cell
differentiation pathway to effect neoplasia formation, although deep
crypt goblet cells retain the ability to divide (34).
In addition to Rb inactivation, Tag is known to inactivate P53 (17,
18). Because P53 has proapoptotic activity (47), a cell expressing Tag
may be expected to be apoptosis deficient. On the other hand, P53independent pathways for apoptosis are known to be operative in
many cell types (48). Most relevant to this study, intestinal absorptive
cells forced to reenter the cell cycle by Tag expression in the villi
undergo apoptosis with equal efficiency in P53 wild-type and P53 null
mice (29, 30). Thus, it is most likely that the goblet cell apoptosis
demonstrated in this study proceeds via a P53- independent pathway.
In summary, this study demonstrates the effectiveness of apoptosis
in counterbalancing uncontrolled proliferation in the intestine using a
well-defined, in vivo system. MUCTag6 intestinal goblet cells continue into S phase during their migration from the crypts to the villi.
Rather than progressing to mitosis, however, these cells undergo
apoptosis, resulting in the near complete ablation of the goblet cell
lineage in the upper reaches of the villi. Thus, these experiments
demonstrate that apoptosis counteracts uncontrolled proliferation in
the intestine and support the hypothesis that apoptosis functions as an
important antineoplastic mechanism in this tissue. In addition, the
MUCTag6 line provides a model that will be useful for the examination of several parameters important in regulating apoptosis in the
lower gastrointestinal tract, including the roles of the BAX, APC, and
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APOPTOSIS IN INTESTINAL GOBLET CELLS
E2F1 gene products and the determination of the signaling pathways
that initiate the apoptotic process. These studies will have direct
relevance to understanding and perhaps enhancing through chemopreventive intervention the anticancer effects of apoptosis in the
human colon.
ACKNOWLEDGMENTS
We thank Sandra Huling and Ivy Hsieh for their aid with electron microscopy.
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Mouse Intestinal Goblet Cells Expressing SV40 T Antigen
Directed by the MUC2 Mucin Gene Promoter Undergo
Apoptosis upon Migration to the Villi
James. R. Gum, Jr., James. W. Hicks, Anne Marie Gillespie, et al.
Cancer Res 2001;61:3472-3479.
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