1 Department of Medicine, University of Liverpool, Liverpool L69 3GA; and 2 Royal Preston Hospital, Preston PR2 4HT, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Apoptosis plays an important role in homeostasis of intestinal epithelia and is also a stress response to toxic stimuli. Transgenic and knockout mice have provided insights into the regulation of intestinal epithelial apoptosis that could not have been obtained by cell culture techniques. Two broad types of apoptosis have been characterized: spontaneous apoptosis, which occurs continuously at low levels in the normal, unstressed intestine, and stress-induced apoptosis, which occurs after genotoxic insult such as exposure to gamma radiation or DNA-damaging drugs. Spontaneous apoptosis occurs at the base of the crypt at or near the position of epithelial stem cells. Knockout studies have shown that spontaneous apoptosis is independent of p53 and Bax in both small and large intestine, whereas Bcl2 only regulates spontaneous apoptosis in the colon. Little is known about the regulation of the specialized form of cell death at the villus tip. In contrast, knockout studies have demonstrated that both p53 and Bcl2 are important regulators of stress-induced apoptosis but that there are significant differences between early and late time points. Bax plays only a minor role in the regulation of stress-induced apoptosis. The cumulative effect of stress-induced apoptosis on tissue architecture is not straightforward, and cell cycle arrest also plays a critical role. Nevertheless, p53 is an important determinant of the histopathological damage induced by 5-fluorouracil in murine intestinal epithelium. These studies have important implications for the development of more effective treatment for inflammatory bowel disease and cancer.
radiation-induced apoptosis; cell cycle arrest; wild-type p53; DNA damage; clonogenic survival; hierarchical status; growth arrest; G1 checkpoint; Bax; Bcl2; 5-fluorouracil
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IT HAS BEEN KNOWN for several decades that programmed cell death is essential for the growth and development of multicellular organisms. However, it was not until 1972 that Kerr, Wyllie, and Currie (10) coined the term "apoptosis" and described its defining morphological features. Since then, it has become clear that apoptosis is also important in the development of the immune system, is a major mechanism of immune-mediated cytotoxicity, and participates in deletion of cells with potentially carcinogenic mutations. In disease pathogenesis, apoptosis can be either inappropriately excessive or deficient and has been implicated in a wide variety of gastrointestinal conditions including Helicobacter-associated gastritis, Shigella flexneri dysentery, inflammatory bowel disease, and colorectal neoplasia. Furthermore, anticancer drugs and nonsteroidal anti-inflammatory drugs induce apoptosis (25).
Apoptosis plays an important role in determining the architecture of intestinal epithelia and is also a part of the stress response of intestinal epithelial cells to toxic stimuli. Rather than being a single process, a number of mechanistically distinct pathways to apoptosis can be discerned in intestinal epithelia, which depend on the physical position of the cell along the crypt/villus axis, its level of differentiation, and the type of stimulus involved.
The study of apoptosis in gastrointestinal epithelium has been greatly hampered by slow progress in the development of suitable in vitro models of normal intestinal epithelial cells. All established gastrointestinal epithelial cell lines are abnormal by definition as they are immortalized with a disturbed balance between rates of cell death and proliferation. Such cell lines probably have abnormal apoptosis mechanisms. Also, many of the more commonly used cell lines are also fully transformed. Furthermore, the gastrointestinal epithelium is a complex tissue in which epithelial cells undergo a differentiation program and receive signals from extracellular matrix, neighboring cells, and circulating hormones, all of which potentially influence apoptosis. For these reasons, study of the regulation of apoptosis under normal physiological conditions has been largely restricted to techniques based on analysis of histological sections of intact epithelium. In this themes article, we shall review the information gained on the regulation of apoptosis from transgenic and knockout mice.
![]() |
STRUCTURE AND CELL KINETICS OF INTESTINAL EPITHELIUM |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The small intestine provides a unique opportunity for studying the
influence of differentiation pathways on apoptosis as cells at
different stages of differentiation can be identified simply through
their position along the crypt/villus axis. The intestinal epithelium
is a self-renewing monolayer arising from stem cells located at or near
the base of crypts (22). One can identify their position by counting
cell positions from the base of the crypt. In the small intestine, stem
cells are believed to be located at cell positions 3-5, whereas in
the large intestine they are located at cell positions 1-2.
Unfortunately, there are no biochemical or molecular markers for
intestinal stem cells and their positions have been implied from
indirect experimental techniques and mathematical modeling (22).
Daughter cells undergo four to six rounds of cell division to form a
cohort of transit cells that populate the midportion of the crypt.
These cells differentiate into four cell lineages. Absorptive
enterocytes, comprising 80% of all epithelial cells, goblet cells, and
enteroendocrine cells all continue to migrate up the crypt. Paneth
cells are a fourth lineage, located at the base of the crypt below the
putative position of stem cells. As cells exit the crypt onto the
villus, they stop cycling and become trapped in the G1
phase of the cell cycle as a result of downregulation of cyclin D1 and
cyclin-dependent kinase 2 (2). As they migrate up the crypt, they
continue to differentiate and express a new repertoire of proteins such
as brush-border hydrolases. After 2-3 days, they reach this villus
tip where, in the mouse, they are shed at a rate of 1,400 cells · villus1 · 24 h
1 (22). Thus the intestinal epithelium
has one of the most rapid turnover rates among mammalian tissues.
![]() |
GENETIC REGULATION OF APOPTOSIS IN INTESTINAL EPITHELIUM |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mice that have been rendered homozygously null for genes that regulate apoptosis have proved extremely valuable in determining the mandatory role of a protein in controlling apoptosis in intestinal epithelium. Experiments involving these mice have often provided evidence that confirms previous immunohistochemical studies, yet these experiments have the additional advantages of allowing damage-induced apoptosis to be easily studied while avoiding some of the technical problems associated with the immunohistochemical process.
Investigations of the levels and cell positions of spontaneous apoptosis can provide evidence for the roles played by these genes during homeostasis within the epithelium. In addition, studies of the apoptosis induced in the epithelium by gamma radiation and cytotoxic drugs can demonstrate how these genes influence the response of the intestine to damage. Genotoxic stimuli can also be used to investigate whether the genes that regulate acute apoptosis also modulate the long-term histopathological outcome of the epithelium in response to cytotoxic damage. One note of caution should be mentioned when interpreting any experiments involving "knockout" mice, namely, that any observed effects may have arisen not only from absence of the gene being investigated but also from compensatory changes in other gene products, which may have occurred during the animal's development. For example, Bcl2 knockout mice have severe developmental abnormalities of the kidneys, causing renal failure (19). It is at least theoretically possible that abnormalities in apoptosis in these mice are due to the sequelae of renal failure rather than to a direct result of Bcl2 deficiency.
To date, knockout mice have been used to determine how p53 and two Bcl2
family members, Bcl2 and Bax, affect both spontaneous and
damage-induced apoptosis in intestinal epithelia (see Table 1).. Obviously, these genes
are likely to represent only the tip of the iceberg, and similar
experiments using a range of other knockout mice will be necessary to
establish a comprehensive picture of gene-regulating apoptosis in
intestinal epithelium.
|
![]() |
APOPTOSIS MECHANISMS IN THE UNSTRESSED INTESTINE |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the unstressed intestine two apoptotic pathways have been identified. The first takes place in the crypt at the level of the stem and early transit cells and is sometimes referred to as "spontaneous" apoptosis, although in fact the trigger has not been identified. This spontaneous apoptosis takes place at a low rate and is thought to regulate the number of cells entering the crypt/villus axis. It is readily identified as apoptotic bodies in histological sections fixed in Carnoy's medium stained with hematoxylin and eosin. Mice that are p53 deficient (7) show similar levels of spontaneous apoptosis in the intestinal epithelium compared with their wild-type counterparts (3, 16). This suggests that p53 plays little part in normal homeostasis in this tissue.
Homozygous Bcl2-null mice (19, 20) demonstrate levels of
spontaneous apoptosis in small intestinal crypts similar to their wild-type counterparts. In the colon, however,
Bcl2/
mice show
elevated levels of spontaneous apoptosis, and this is concentrated at
cell positions 1-2 at the base of the crypt (17). This is the
location of Bcl2 protein expression found by
immunohis-tochemical studies and is also the presumed
location of colonic stem cells. This observation suggests that Bcl2
expression plays a part in homeostasis of normal colonic epithelium,
although its precise role remains to be established, since the actual
morphology of the colonic epithelium is normal in
Bcl2
/
mice (19, 20).
In contrast, Bax expression appears to have little effect on
homeostasis in normal intestinal epithelium, as
Bax
/
mice (11) show
the same levels of spontaneous apoptosis as their wild-type
counterparts in both small intestine and midcolon (24). This correlates
well with the observed immunohistochemical distribution of Bax
expression in those small intestinal cell lineages that are not
crucial for maintenance of epithelial renewal, namely,
differentiated Paneth cells and villus enterocytes (13).
Apoptosis may also participate in the shedding process at the villus tip (for review, see Ref. 14). Careful studies with electron microscopy have demonstrated that neighboring epithelial cells extend processes underneath the cell to be extruded and form a tight junction. This tight junction then migrates toward the lumen like a "zipper," pushing the cell to be shed out of the monolayer. Apoptotic morphology is only seen rarely during this extrusion process; the shed cells only take on apoptotic morphology once free in the lumen (27). It is not known at which point along the crypt/villus axis the process of apoptosis begins. This is because most of the important signal transduction events take place while the cell is morphologically normal. Nevertheless, at least some early apoptotic events must take place while the cell is within the monolayer, since activation of caspase-3, a member of the principal effector enzyme family of apoptosis, takes place at the villus tip (21).
Few data are available on the regulation of cell loss at the villus
tip. It is clear that this shedding process is tightly regulated. It is
unknown whether apoptosis itself is the regulated event or whether
apoptosis is merely secondary to detachment from basement membrane.
Bcl2 does not appear to regulate cell shedding, as villus dimensions
are normal in mice with forced overexpression of Bcl2 in villus
epithelial cells (6). A particularly fascinating point is that the rate
of cell loss at the villus tip must be tightly coupled with cell
production at the base of the crypt. This implies that there must be a
feedback loop between the crypt and villus tip, but the anatomic basis
for such a feedback loop is unknown. One possibility is that /
T
cells migrating from the villus tip act as messengers to the crypt
cells, as knockout studies have demonstrated that they are capable of
reducing crypt cell production rate (12). Glucagon-like peptide-2
(GLP-2) is another candidate for regulating the shedding process on the
villus. Raised levels of GLP-2 are known to increase villus length,
possibly through inhibition of apoptosis at the villus tip and
increased proliferation in the crypt (28). Very recently, the human and mouse GLP-2 receptor has been cloned and characterized as a member of
the G protein-coupled receptor superfamily (18). It is unknown whether
this receptor participates in the regulation of apoptosis.
Apoptosis is never seen along the length of the villus of the unstressed intestine except under highly artificial conditions. This is not because the villus cells are postmitotic, since forcing villus cells back into cycle through the overexpression of SV40 large T antigen does not restore radiation-induced apoptosis (5). Mice that express a dominant negative N-cadherin in their villus cells have spontaneous apoptosis along the length of the villus in addition to developing an inflammatory bowel disease reminiscent of Crohn's disease and colonic adenomas (9).
![]() |
STRESS-INDUCED APOPTOSIS IN INTESTINAL EPITHELIUM |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
p53.
Studies of the apoptosis induced in
p53/
mice by
administration of genotoxic stimuli have revealed that p53 is a crucial
determinant of the apoptosis that occurs within a few hours of damage.
The first observations of damage-induced apoptosis in this setting demonstrated that
p53
/
mice showed
absence of intestinal apoptosis, which is normally observed in p53
wild-type animals during the 3-4 h period following gamma
radiation (3, 16). This observation correlated well with
immunohistochemical studies in wild-type mice following the same
stimulus of gamma radiation, since analysis of the cell positional distribution of immunohistochemical p53 protein expression was coincident with the position of apoptotic cells in the small intestine following 8-Gy gamma radiation(16).
Bcl2 family members.
The Bcl2 family consists of both pro- and anti-apoptotic proteins (1).
A cell's capacity to undergo apoptosis is probably determined by the
ratio of all Bcl2 family members expressed within it. Knockout studies
uniquely enable the contribution of an individual family member to be
studied within the context of a complex tissue in vivo. Three to four
hours following stimulation by gamma radiation, Bcl2/
mice show
similar levels of small intestinal apoptosis as their wild-type
counterparts. However, as with spontaneous apoptosis, elevated levels
of damage-induced apoptosis are observed in the colon. This colonic
apoptosis again occurs specifically at those cell positions at the base
of colonic crypts, which are believed to harbor the colonic stem cells
(17). A similar picture is also seen using the alternative cytotoxic
stimulus of 5-fluorouracil, in that
Bcl2
/
mice
demonstrate significantly greater levels of apoptosis particularly at
the base of colonic crypts 4.5 h after 40 mg/kg 5-fluorouracil (24).
However, 24 h after 40 mg/kg 5-fluorouracil administration, there was
no significant difference in apoptotic yield between Bcl2 wild-type and
null mice, suggesting that, in this setting, Bcl2 expression serves to
delay the onset of apoptosis rather than prevent it completely (24).
The long-term effects of the protection of stem cells at positions
1-2 at the early time points after 5-fluorouracil administration
require a long-term toxicological study of gut integrity, as has been
described above for
p53
/
animals.
![]() |
ACKNOWLEDGEMENTS |
---|
A. J. M. Watson is supported by grants from the Cancer Research Campaign, the Association of International Cancer Research, and North West Cancer Research Fund.
![]() |
FOOTNOTES |
---|
* Seventh in a series of invited articles on Lessons From Genetically Engineered Animal Models.
Address for reprint requests and other correspondence: A. J. M. Watson, Dept of Medicine, Univ. of Liverpool, Daulby St., Liverpool L69 3GA, UK (E-mail: alastair.watson{at}liv.ac.uk).
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, J. M.,
and
S. Cory.
The Bcl-2 protein family: arbiters of cell survival.
Science
281:
1322-1326,
1998
2.
Chandrasekaran, C.,
C. M. Coopersmith,
and
J. I. Gordon.
Use of normal and transgenic mice to examine the relationship between terminal differentiation of intestinal epithelial cells and accumulation of their cell cycle regulators.
J. Biol. Chem.
271:
28414-28421,
1996
3.
Clarke, A. R.,
S. Gledhill,
M. L. Hooper,
C. C. Bird,
and
A. H. Wyllie.
p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation.
Oncogene
9:
1767-1773,
1994[ISI][Medline].
4.
Clarke, A. R.,
L. A. Howard,
D. J. Harrison,
and
D. J. Winton.
p53, mutation frequency and apoptosis in the murine small intestine.
Oncogene
14:
2015-2018,
1997[ISI][Medline].
5.
Coopersmith, C. M.,
and
J. I. Gordon.
Gamma-ray-induced apoptosis in transgenic mice with proliferative abnormalities in their intestinal epithelium: re-entry of villus enterocytes into the cell cycle does not affect their radioresistance but enhances the radiosensitivity of the crypt by inducing p53.
Oncogene
15:
131-141,
1997[ISI][Medline].
6.
Coopersmith, C. M.,
D. O'Donnell,
and
J. I. Gordon.
Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice.
Am. J. Physiol. Gastrointest. Liver Physiol.
276:
G677-G686,
1999
7.
Donehower, L. A.,
M. Harvey,
B. L. Slagle,
M. J. McArthur,
C. A. Montgomery, Jr.,
J. S. Butel,
and
A. Bradley.
Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.
Nature
356:
215-221,
1992[ISI][Medline].
8.
Hendry, J. H.,
W. B. Cai,
S. A. Roberts,
and
C. S. Potten.
p53 deficiency sensitizes clonogenic cells to irradiation in the large but not the small intestine.
Radiat. Res.
148:
254-259,
1997[ISI][Medline].
9.
Hermiston, M. L.,
and
J. I. Gordon.
Inflammatory bowel disease and adenomas in mice expressing a dominant-negative N-cadherin.
Science
270:
1203-1207,
1995[Abstract].
10.
Kerr, J. F. R.,
A. H. Wyllie,
and
A. R. Currie.
Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics.
Br. J. Cancer
26:
239-257,
1972[ISI][Medline].
11.
Knudson, C. M.,
K. S. Tung,
W. G. Tourtellotte,
G. A. Brown,
and
S. J. Korsmeyer.
Bax-deficient mice with lymphoid hyperplasia and male germ cell death.
Science
270:
96-99,
1995[Abstract].
12.
Komano, I.,
Y. Fujiura,
M. Kawaguchi,
S. Matsumoto,
Y. Hashimoto,
S. Obana,
P. Mombaerts,
S. Tonegawa,
H. Yamamoto,
S. Itohara,
M. Nanno,
and
H. Ishikawa.
Homeostatic regulation of intestinal epithelia by intraepithelial gamma delta T cells.
Proc. Natl. Acad. Sci. USA
92:
6147-6151,
1995
13.
Krajewski, S.,
M. Krajewska,
A. Shabaik,
T. Miyashita,
H. G. Wang,
and
J. C. Reed.
Immunohistochemical determination of in vivo distribution of Bax, a dominant inhibitor of Bcl-2.
Am. J. Pathol.
145:
1323-1336,
1994[Abstract].
14.
Mayhew, T. M.,
R. Myklebust,
A. Whybrow,
and
R. Jenkins.
Epithelial integrity, cell death and cell loss in mammalian small intestine.
Histol. Histopathol.
14:
257-267,
1999[ISI][Medline].
15.
Merritt, A. J.,
T. D. Allen,
C. S. Potten,
and
J. A. Hickman.
Apoptosis in small intestinal epithelia from p53-null mice: evidence for a delayed, p53-independent G2/M-associated cell death after gamma-irradiation.
Oncogene
14:
2759-2766,
1997[ISI][Medline].
16.
Merritt, A. J.,
C. S. Potten,
C. J. Kemp,
J. A. Hickman,
A. Ballmain,
D. P. Lane,
and
P. A. Hall.
The role of p53 in spontaneous and radiation-induced apoptosis in the gastrointestinal tract of normal and p53-deficient mice.
Cancer Res.
54:
614-617,
1994[Abstract].
17.
Merritt, A. J.,
C. S. Potten,
A. J. M. Watson,
D. Y. Loh,
K. Nakayama,
K. Nakayama,
and
J. A. Hickman.
Differential expression of Bcl-2 in intestinal epithelia: correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia.
J. Cell Sci.
108:
2261-2271,
1995
18.
Munroe, D. G.,
A. K. Gupta,
F. Kooshesh,
T. B. Vyas,
G. Rizkalla,
H. Wang,
L. Demchyshyn,
Z. J. Yang,
R. K. Kamboj,
H. Chen,
K. McCallum,
M. Sumner-Smith,
D. J. Drucker,
and
A. Crivici.
Prototypic G protein-coupled receptor for the intestinotrophic factor glucagon-like peptide 2.
Proc. Natl. Acad. Sci. USA
96:
1569-1573,
1999
19.
Nakayama, K.,
I. Negishi,
K. Kuida,
H. Sawa,
and
D. Y. Loh.
Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia.
Proc. Natl. Acad. Sci. USA
91:
3700-3704,
1994[Abstract].
20.
Nakayama, K.,
I. Negishi,
K. Kuida,
Y. Shinkai,
M. C. Louie,
L. E. Fields,
P. J. Lucas,
V. Stewart,
F. W. Alt,
and
D. Y. Loh.
Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice.
Science
261:
1584-1588,
1993[ISI][Medline].
21.
Piguet, P. F.,
C. Vesin,
Y. Donati,
and
C. Barazzone.
TNF-induced enterocyte apoptosis and detachment in mice: induction of caspases and prevention by a caspase inhibitor, ZVAD-fmk.
Lab. Invest.
79:
495-500,
1999[ISI][Medline].
22.
Potten, C. S.,
C. Booth,
and
D. M. Pritchard.
The intestinal epithelial stem cell: the mucosal governor.
Int. J. Exp. Pathol.
78:
219-243,
1997[ISI][Medline].
23.
Pritchard, D. M.,
C. S. Potten,
and
J. A. Hickman.
The relationships between p53-dependent apoptosis, inhibition of proliferation, and 5-fluorouracil-induced histopathology in murine intestinal epithelia.
Cancer Res.
58:
5453-5465,
1998[Abstract].
24.
Pritchard, D. M., C. S. Potten, S. J. Korsmeyer, S. A. Roberts, and J. A. Hickman.
Damage-induced apotosis in intestinal epithelia from bcl-2-null
and bax-null mice: investigations of the mechanistic determinants of
epithelial apoptosis in vivo. Oncogene. In press.
25.
Pritchard, D. M.,
and
A. J. M. Watson.
Apoptosis and gastrointestinal pharmacology.
Pharmacol. Ther.
72:
149-169,
1996[ISI][Medline].
26.
Pritchard, D. M.,
A. J. M. Watson,
C. S. Potten,
A. L. Jackman,
and
J. A. Hickman.
Inhibition by uridine but not thymidine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: evidence for the involvement of RNA perturbation.
Proc. Natl. Acad. Sci. USA
94:
1795-1799,
1997
27.
Shibahara, T.,
N. Sata,
S. Waguri,
T. Iwanaga,
A. Nakahara,
H. Fukotomi,
and
Y. Uchiyama.
The fate of effete epithelial cells at the villus tips of the human small intestine.
Arch. Histol. Cytol.
58:
205-219,
1995[ISI][Medline].
28.
Tsai, C. H.,
M. Hill,
S. L. Asa,
P. L. Brubaker,
and
D. J. Drucker.
Intestinal growth-promoting properties of glucagon-like peptide-2 in mice.
Am. J. Physiol. Endocrinol. Metab.
273:
E77-E84,
1997
29.
Wilson, J. W.,
D. M. Pritchard,
J. A. Hickman,
and
C. S. Potten.
Radiation-induced p53 and p21(WAF-1/CIP1) expression in the murine intestinal epithelium: apoptosis and cell cycle arrest.
Am. J. Pathol.
153:
899-909,
1998