HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Potten, C.S. Articles by Booth, C. Search for Related Content PubMed PubMed Citation Articles by Potten, C.S. Articles by Booth, C.

Regulation and Significance of Apoptosis in the Stem Cells of the Gastrointestinal Epithelium

CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Withington, Manchester, United Kingdom

Key Words. Apoptosis • Stem cell • Intestine • Epithelium

Correspondence: Dr. C.S. Potten, CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester M20 9BX, United Kingdom.

	Abstract

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
In rapidly proliferating tissues the stringent control of cell proliferation and cell death by apoptosis is central to the maintenance of tissue homeostasis. In the gastrointestinal tract most work studying the control of tissue cell number has traditionally focused on the growth factor control of proliferation, and the changes that occur during carcinogenesis. However, in recent years it has become increasingly apparent that the control of apoptosis is also crucial. Apoptosis is an important mechanism for eliminating both excess normal cells and those cells which have sustained damage; therefore maintaining a tissue, i.e., stem cells with preserved DNA integrity.

In this review the incidence of apoptosis in the stem cells of both the small and large intestine will be discussed in relation to the expression of a number of apoptosis regulating genes (e.g. p53, Bcl-2, bax) within these cells. The importance of apoptosis as a means of controlling stem cell number (and therefore cellular output) will be addressed, as will the mechanisms by which any alterations to this process may contribute to malignancy.

	Introduction

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
Apoptosis
Apoptosis is a form of programmed cell death (i.e., genetically controlled self-destruction or cell suicide) that is central to tissue morphogenesis in the developing animal and tissue homeostasis in the adult. It can be used by nature to remove unwanted but otherwise healthy cells (e.g., tissue restructuring during development), or to remove damaged single cell(s) in order to protect the embryo, an adult animal or tissue. It has also become apparent over recent years that cellular inability to undergo apoptosis may have important consequences during neoplastic transformation. Thus damaged, and possibly mutated cells, normally eliminated by apoptosis, are prevented from entering this pathway, and instead survive and may go on to divide and perpetuate or expand the mutated cells thereby increasing carcinogenic risk.

A classical apoptotic cell has a very distinctive morphology throughout the death process which is well described in the literature. Initially the cell cytoplasm shrinks and the cell becomes detached from its neighbors. The nucleolus disappears and the chromatin becomes condensed around the nuclear membrane (marginated) and consequently frequently appears as a crescent shape in histological cross section. The cell membrane takes on a "blebbed" appearance. The cell and its nucleus fragment into smaller membrane-bound vesicles which become phagocytosed by neighboring cells. This type of deliberate fragmentation and "bite size" packaging of the cell therefore eliminates the possibility of any cellular "leakage," subsequent inflammatory response and major tissue disturbance (unlike necrosis, in which physical injury destroys both organelle and cytoplasmic membrane integrity). Apoptosis is, therefore, a deliberate stepwise event that appears to be genetically controlled. It is an active sequence of events requiring the activation of specific "cell death" proteins, such as the ICE/CED3-related proteases [1]. One of the most distinctive features of apoptosis is the internucleosomal cleavage of DNA into specific-sized fragments by calcium/magnesium dependent endonucleases. These fragments of approximately 200 base pair multiples can be observed as a DNA ladder when separated by gel electrophoresis [2]. Some characteristically apoptotic cells, however, do not demonstrate internucleosomal cleavage but do show specific cleavage of DNA into larger fragments of 50 and/or 300 kbp in size [3]. The presence of such DNA fragments can be utilized in certain techniques to identify apoptotic cells. The widely used ISEL/TUNEL labeling techniques attach tagged molecules to the exposed ends of the DNA fragments in situ, and can therefore be used to support microscopic morphological identification [4]. Other cellular targets which are cleaved during apoptosis include the DNA repair enzyme poly(ADP-ribose) polymerase; cleavage of the enzyme can be demonstrated using Western blotting [5].

	The Intestinal Epithelium

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
Tissue homeostasis depends on both cell proliferation and cell death. Hence, in a rapidly renewing tissue, such as the small intestinal epithelium, cells are lost from the villus into the gut lumen and are generally replaced at an equal rate by the proliferation of cells in the crypts of Lieberkühn. There is therefore a linear migratory pathway from crypt base to villus tip. Most studies on this renewal process have focused on the control of cell proliferation and the growth factor/signal transduction changes that may result in hyperproliferation and ultimately transformation. However, it is now becoming increasingly apparent that the control of cell death is an equally, if not more, important regulator of cell number and susceptibility to neoplastic transformation. Thus, both decreased proliferation and/or increased cell death may reduce cell number, whereas increased proliferation and/or decreased death may increase cell number.

The intestinal epithelium is an ideal model for investigating the relationship between these two processes and their effects or controls within a tissue environment. For example, the small intestine has very high levels of proliferation and cell loss, which occur in a well-defined and polarized topographical organization in which the hierarchy or cellular "age" can be assessed by the position of that cell in the tissue [6]. Due to the unique structure of the crypt and villus, it is easy to record the ability, or inability, of any cell in the hierarchy to either proliferate or die (Fig. 1). For example, data from numerous cell kinetic and cell migration experiments have indicated that the epithelial stem cells in the small intestine are located four to five cell positions up from the crypt base, immediately above the differentiated Paneth cells, whereas in the regions of the large intestine they are located at the very base of the crypt. Thus, by assessing the incidence of cell death or DNA synthesis at these cell positions we can, by inference, determine the ability of the stem cells to undertake these options in a number of different scenarios. Similarly, frequency distributions can be generated in which the number of "events" at each position up the crypt (as seen in histological cross section, with position 1 referring to the cell at the very base of the crypt) can be measured (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Figure 1. Frequency of S-phase and apoptotic cells along the length of an intestinal crypt. Top: Small Intestine; Bottom: Large Intestine. ( ______ ): Frequency of cells labeled with tritiated thymidine at each cell position (unirradiated adult mouse); (__ __ __ ): frequency of spontaneously occurring apoptosis (unirradiated adult mouse); (- - - - -): frequency of radiation-induced apoptosis (4 1/2 hours after 1 Gy irradiation). Apoptosis can be seen to be most frequent in the small intestine at positions 4-6—thought to be the location of the stem cells (the stem cell position is indicated by the arrowhead).

	Spontaneous Apoptosis in the Normal Intestine

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

View larger version (130K): [in this window] [in a new window]   Figure 2. Spontaneous apoptosis in the small intestine. A: Human, formalin fixed. B: Mouse, Carnoy's fixed. The arrows indicate apoptotic cells, whereas the arrowheads indicate mitotic cells. The difficulties in distinguishing these two types of cell are clearly apparent. Pink secretory granules within the Paneth cells at the crypt base are also clearly visible in the human tissue.

Cell loss from the villus can also be termed anoikis ("homelessness") in which cell loss occurs via lost adhesion, which may in turn induce apoptosis. Altered adhesion signals have been shown to initiate apoptosis in epithelial tissues, either by loss of integrin function [19-21], or altered extracellular matrix composition [22-24]. It may be relevant that there is increased tenascin expression up the villus axis, an extracellular matrix component with anti-adhesive properties [16, 17]. Furthermore, the epithelial adhesion molecule E-cadherin, which is present in the enterocyte basement membrane, has been shown to moderate their death [25] (see discussion below).

If apoptosis is confirmed as the general method of cell removal, it may also explain how downwardly migrating epithelial cells (such as the small intestinal Paneth cells or the cells at the base of the stomach glands) are removed after fulfilling their differentiated function.

In the normal intestine, therefore, it appears that the ability of a healthy cell to undergo apoptosis is determined by the cell's positional (i.e., hierarchical) status and the possible changes that the cells encounter in the extracellular environment as they migrate to the villus tip. Positional influence on apoptosis can also be observed in the response to certain cytotoxics, but other studies with a range of cytotoxics have shown that all the epithelial cells are capable of undergoing apoptosis (see below).

Unlike the small intestine, spontaneous apoptosis in the proliferative zone of the large intestine is a rare event (about 10 times lower incidence). Furthermore, rather than being most prevalent in the stem cell zone, the apoptotic cells are observed scattered throughout the crypt. Thus, in the large bowel spontaneous apoptosis is unlikely to be an effective means of regulating stem cell numbers. However, like the small intestine it may be an effective means of removing senescent cells from the top of the crypts (plateau zones) at the lumenal face of the mucosa [11].

Measurements of proliferation kinetics have revealed few major differences in organization between the small and large intestine, and may indicate that proliferation per se is less relevant to cancer incidence than apoptosis (although the cell cycle in the large bowel is about two to four times longer [6]). The differences observed in positional apoptotic incidence in the small and large intestine, however, have direct implications on cancer incidence. If the small intestine is capable of removing excess stem cells, whereas the large intestine is not, the increased number of stem cells may result in hyperplastic crypts susceptible to transformation (the excess stem cells being the carcinogenesis target cells). This may therefore be one contributing factor to the observation that human colonic cancer is much more common than small intestinal cancer.

	Damage-Induced Apoptosis

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
Cytotoxic agents can induce apoptosis in many tissues. For example, low and high linear energy transfer ionizing radiation causes a dose-dependent increase in apoptosis in the small intestinal crypt within three to six hours of exposure [7, 26]. Radiation-induced apoptosis occurs predominantly within the stem cell region—again indicating the specific sensitivity of cells at the stem cell position to apoptosis. Indeed, doses as low as 1-5 cGy (roughly equivalent to just one DNA-damaging event per cell) can induce stem cell apoptosis [7]. As the dose increases, however, more cells die, up to a dose of about 1 Gy in the small intestine where about six cells per crypt are killed. These highly sensitive cells do not appear capable of repair, as evidenced by the lack of a dose rate effect [26]. Higher doses do not induce more cells to enter apoptosis even though they are heavily damaged. Many of the damaged cells appear to prematurely differentiate and emigrate onto the villus [27]. A more extensive investigation of this phenomenon carried out by Ijiri and Potten [28, 29] involved 18 different cytotoxic agents (later supplemented by four mutagens [30, 31]) and examined the resultant apoptosis at each crypt position. It was found that each agent tended to target a specific crypt position or region but when examined collectively, the study showed that apoptosis could be induced throughout the crypt [28, 29]. Further data supporting the hypothesis that all the epithelial cells are capable of apoptosis come from crypts isolated and placed in vitro which undergo apoptosis throughout the structure [32], and the fact that villus cells can be induced to apoptose by bacterial toxins [33] or by serial elution [34]. Similar interesting data have been produced in lymphocytes lacking p53 where radiation will not induce apoptosis, whereas other cytotoxics are potent apoptosis inducers [35, 36]. These findings support the hypothesis that normally it is the in vivo ability to suppress apoptosis that is important, and distinguishes the survival ability of the different epithelial populations. This may be due to selective gene expression and also changes in the growth signals received by the cells.

In the large intestine differences are again observed when damage-induced apoptosis is considered. The apoptosis occurs at lower levels when specific low doses are studied; it is not specifically located at the stem cell position at the crypt base, and the "plateau" in apoptotic yield seen at 1 Gy in the small bowel occurs at 6-8 Gy in the mid-colon.

	Genetic Regulation of Apoptosis in the Intestinal Epithelium

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
The reason why there is a differential susceptibility to stem cell apoptosis along the gastrointestinal (GI) tract is central to our understanding (and hence future ability to clinically manipulate) cancer incidence. This has led, therefore, to an enormous amount of research into the genes that control apoptosis. The mouse has proved to be an invaluable model in these studies and has allowed us and others to examine the effect of specific gene deletions/mutations in the whole animal.

This review will concentrate on the following gene families/groups: A) p53, which is involved in the response of cells to DNA damage; B) Bcl-2 family genes whose products are capable of both suppressing and promoting apoptosis; C) genes coding for cellular adhesion molecules, e.g., cadherin and adenomatous polyposis coli (APC) genes which are involved in controlling cell migration and position-dependent differentiation and D) the cyclooxygenase-2 (cox-2) gene which is responsible for prostaglandin synthesis.

p53 and the Bcl-2 family proteins are intimately linked as p53 has been shown to stimulate transcription of Bax [37]. We have investigated the expression of p53 and Bcl-2 family proteins in the GI tract in the mouse. Our studies show important regional differences in the expression of p53 and Bcl-2 family proteins between the large and the small intestine, which directly relate to tumor incidence in these regions of the GI tract [38, 39].

	p53

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
p53 plays an important role in damage surveillance, and as such has been dubbed the "guardian of the genome" [40]. It is normally expressed at very low levels, except during DNA synthesis [41]. Upon DNA damage, expression is upregulated and p53 is translocated to the nucleus where it binds to DNA, acting to regulate the transcription of a number of genes including p21/waf1 [42], mdm2 [43] and Bax [37].

p53 expression has been demonstrated to be controlled at the translational level which permits a rapid response to DNA damage (due to the nondependence on transcription) [44]. It may act to cause G₁ arrest, a process mediated by p21/waf-1 inhibition of cyclin dependent kinases (CDK) and proliferating cell nuclear antigen-dependent DNA replication. This allows time for the cell to make a decision on its fate (repair/apoptosis) prior to committing itself to S-phase. Entry into S-phase with damaged DNA can lead to daughter cells having unstable chromosomal structure allowing possible recombination events to occur. Alternatively stable mutations with the potential to promote malignancy may result. p53 may also act directly to initiate apoptosis. These various options may differ depending on the cell type. The response has recently been investigated by Polyak [45], who showed that cell lines which underwent p53-mediated apoptosis expressed dominant trans-acting factors which were capable of overcoming p21-dependent growth arrest. Induction of p21 by p53 has recently been shown to be codependent upon the action of another transcription factor, interferon regulatory factor 1, in embryonic fibroblasts [46]. The authors also showed the interferon regulatory factor 1 dependence of p53-mediated apoptosis in Ha-ras transformed embryonic fibroblasts. These are clearly important results and add an additional layer of complexity to p53 function.

	p53 and Spontaneous Apoptosis in the Intestinal Epithelium

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
The p53 gene is frequently mutated in cancers, including cancers of the GI tract [47-49]. Hence, one of the initial experiments we performed was to compare the levels of spontaneous apoptosis in normal and p53 knockout (transgenic) mice. Interestingly, in both cases the levels of spontaneous apoptosis were similar, indicating that this gene has little involvement in controlling the removal of excess, but undamaged, cells [38]. This is supported by the observation that there is very little wild type p53 expressed in the normal intestinal epithelium. Also consistent with this hypothesis that spontaneous apoptosis is p53-independent is the observation that p53 null mouse embryos develop normally ([50, 51], see later discussion). Furthermore, the continued terminal differentiation and loss of cells on the villus is also p53-independent, indicating that this too is a form of spontaneous apoptosis.

	The Bcl-2 Gene Family

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
One gene family that has been intensively studied over the last few years is the Bcl-2 family. Bcl-2 was first identified in B cell lymphoma patients, where a t14:18 chromosome translocation occurs, juxtaposing the Bcl-2 gene with the heavy chain immunoglobulin gene enhancer sequences [52, 53]. The resulting disregulated Bcl-2 expression led to enhanced B cell survival and development of lymphoma/leukemia [54, 55]. Since this initial discovery the number of Bcl-2-related proteins has increased dramatically. The family members are characterized by specific conserved domains; Bcl-2 homology regions are essential for the protein interactions with other members of this family [56]. The family can be split into two groups: those members which are involved in suppressing apoptosis, i.e., Bcl-2, Bclx_L [57], mcl1 [58], bag-1 [59], bfl-1 [60], brag-1 [61] and Bcl-w [62]; and those which promote apoptosis, i.e., bax [63], bad [64], Bclx_S [57], bak [65-67] and bik [68]. Two models have been proposed. In the first, homodimers of the pro-apoptotic protein BAX provide a dominant signal for apoptosis which can be suppressed by the competitive interaction of apoptosis-suppressing members to form heterodimers with BAX [69]. Thus, in the normal "healthy" cell the majority of the pro-apoptotic BAX would be in heterodimeric complexes with suppressors such as Bcl-2 or Bclx_L. In the second model, the apoptosis-suppressing proteins provide a dominant survival signal, independent of their binding to the apoptosis-promoting members of the family [70].

	Bcl-2 and Spontaneous Apoptosis

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
Bcl-2 is commonly overexpressed in colonic adenocarcinomas. Interestingly, although there is minimal expression in the small intestine (in both mouse and humans), there is expression at the base of the colonic crypts in both species, indicating this may be involved in overriding the apoptotic homeostatic mechanism in these cells [39] (Fig. 3). This hypothesis was supported by the finding that in Bcl-2 knockout mice the incidence of spontaneous apoptosis is dramatically increased in the stem cell region of the colon but unchanged in the stem cell region of the small intestine [39].

View larger version (170K): [in this window] [in a new window]   Figure 3. Bcl-2 immunoreactivity at the base of a human colonic crypt (magnification x 400).

View larger version (62K): [in this window] [in a new window]   Figure 4. Pattern of BAX expression in murine small intestine (A), which shows greatest immunoreactivity at the base of the crypts, and colon (B), which shows greatest immunoreactivity at the lumenal surface (magnification x 100).

	Genetic Regulation of Radiation-Induced Apoptosis

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

Experiments conducted in this laboratory and by others [38, 72], have shown that mice lacking functional p53 are insensitive to ionizing radiation-induced apoptosis in the GI tract (measured at 4 h postirradiation). This appears to confirm that radiation-induced apoptosis is p53-dependent. However, there are apparently conflicting reports showing that radiation can induce apoptosis in colorectal tumor cell lines containing mutated p53 [73]. This paper shows that cells do not apoptose in G₁, but appear to accumulate at G₂/M. We have now obtained data that agree with this observation showing that delayed damage-induced apoptosis can occur in the intestinal epithelium of null mice. Following irradiation, p53 null intestinal epithelial cells seem to undergo arrest at G₂/M and ultimately undergo apoptosis approximately 24 h postirradiation (unpublished data). These data are consistent with those of Hooper [74] who showed that the epithelial cells in the crypts of p53 null mice, although resistant to radiation-induced apoptosis, accumulate in G₂ and exhibit reduced DNA synthesis in the first few hours after exposure. Similar events have been described in vitro in cells that lack p21/WAF1. p21/WAF1-deficient cells failed to undergo G₁ arrest following DNA damage but arrested in G₂. They then carried out further rounds of DNA synthesis (S-phase) without undergoing any mitoses; the resulting polyploid cells eventually died by apoptosis [75].

The Bcl-2 Family and Radiation-Induced Apoptosis
The radiosensitivity of the small intestinal and colonic stem cells correlates well with the regional expression of different Bcl-2 protein family members (Figs. 3 and 4). As described above, Bcl-2 is expressed, albeit at low levels, in the colonic stem cell region, and mice lacking functional Bcl-2 have a higher incidence of apoptosis in the stem cell region. This is significantly increased following irradiation [39]. This increased apoptosis was concentrated at the base of the crypt, supporting the theory that Bcl-2 is normally responsible for preventing apoptosis in these cells. These experiments show that Bcl-2 expression is essential for the normal survival of colonic stem cells and can inhibit apoptosis in response to DNA damage. It has been reported that BAX expression is increased in the small intestinal epithelium following exposure to ionizing radiation [76], an observation which we have recently been able to confirm (unpublished observation). BAX protein levels were found to be elevated in the small bowel within 2 h of exposure to low dose ionizing radiation [76]. This is prior to the peak levels of apoptosis which occur at about 4 h postirradiation. Again, these results support the model of Oltavi and Korsmeyer [69]. We are currently examining the interaction of Bcl-2, p53 and now Bax in a series of doubly mutant animals.

	p53, Bcl-2 and Tumor Incidence

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
During our studies we noticed that the differential expression of Bcl-2 in the stem cell populations of the small and large bowel correlates well with the frequency of tumors observed in these regions [39]. This has now also been found to be true with regard to the expression of BAX, i.e., the sensitivity to apoptosis can govern the frequency of malignancy. We and others have also investigated the expression of p53 and the Bcl-2 family proteins in human colon tumor samples. Bcl-2 expression is associated with the onset of early stage disease. This is based on the evidence that Bcl-2 was found to be most frequently expressed in early stage, well-differentiated tumors, and increased levels of expression were observed in adjacent normal tissue. Late stage tumors that were moderately or poorly differentiated showed a greatly reduced frequency of Bcl-2 expression. Bcl-2 expression is probably reduced as tumor cells acquire other mutations which provide more dominant signals for cell survival or mutations which repress dominant signals that promote apoptosis. p53 was found to have a reciprocal pattern of expression as compared to Bcl-2, with the highest levels of expression being found in late stage disease [49]. p53 immunoreactivity observed in these studies is thought to probably be due to the expression of mutant rather than wild type protein.

Li-Fraumeni syndrome is an inherited disorder that is the result of germ line mutations in p53 [77, 78]. Sufferers show a greatly enhanced frequency of tumors (e.g., lymphomas, sarcomas and breast carcinoma) but levels of colonic tumors are not greatly increased [78]. However, it may be that Li-Fraumeni sufferers die of these other cancers before they have time to develop colonic cancers.

	p53 Function—Protection against Teratogenesis

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
The importance of p53 in eliminating damaged cells cannot be overemphasized. Its key role in both adult and embryonic tissue protection is becoming increasingly apparent. For example, it has recently been shown that although p53 null mice develop normally (again possibly indicating this gene does not have a role in spontaneous apoptosis), p53 does appear to have a role in controlling the viability of the embryo [51]. When pregnant, normal (wild type) and p53 null mice were irradiated in order to damage the embryos. The wild type mice aborted 60% of the embryos, compared to only 10% in the null mice. However, although more embryos survive in the null mice the majority have malformations. It therefore appears that p53 normally induces the abortion of damaged embryos, probably via a cellular "proofreading" mechanism operating using apoptosis. Thus as an extension of its role as "guardian of the genome" p53 also appears to be the "guardian of the embryo." In fact, it may have evolved initially to perform this function.

	Adhesion Molecules and Apoptosis

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
As mentioned previously, the genes which have a profound influence on the turnover of GI epithelial cells are those which code for cell surface adhesion molecules and the cytoplasmic proteins with which they interact to influence cell function. The cadherins are such proteins and are involved in cell-cell adhesion [79]. Their function has been demonstrated in an elegant series of experiments in chimeric mice expressing both wild type and mutant forms of cadherin in the GI epithelium [80, 25]. These studies showed that overexpression of E-cadherin greatly reduced cell proliferation and migration rates in small intestinal crypts and resulted in a small increase in the frequency of apoptosis in the transit cell population at the very top of the crypts. The architectural integrity of the tissue remained intact. In contrast, expression of a mutant N-cadherin, which lacks an extracellular domain, results in a complete breakdown of crypt/villus organization. This is the result of increased cell proliferation and apoptosis, failure of cells to differentiate, and increased and chaotic migration of cells from the crypts up the villi. These effects resulted in an inflammatory condition in the animals resembling Crohn's disease in humans and ultimately resulted in an increased frequency of tumors.

The E-cadherin cytoplasmic domain binds to the cytoplasmic protein ß-catenin as does the product of the APC gene. The APC gene is commonly mutated in human colorectal tumors [81, 82]. Mice bearing an equivalent mutation (multiple intestinal neoplasia [MIN]) also display an increase in GI tumors. The wild type APC gene product has been suggested to negatively regulate cell cycle progression by inhibiting CDK activity required for G₁ to S transition [83]. This inhibition of CDK activity could possibly be mediated by controlling expression of p21/WAF1. In normal intestine the expression of p21/WAF1 is restricted to the nonproliferating cell populations in both the small and large intestinal epithelium [84]. This regional distribution prompted the authors to suggest that p21/WAF1 expression could be modulated by signaling pathways linked to cell surface adhesion molecules [84]. It was found that in colonic adenomas there was greatly reduced and disrupted positional expression of p21/WAF1, and the later stage carcinomas expressed virtually no p21/WAF1. As discussed previously, loss of the G₁ checkpoint allows G₁ cells with DNA damage to progress into S-phase. Although most should die by apoptosis, there is always the possibility of chromosomal translocations occurring or single point mutations becoming stabilized, both of which could have the potential to result in the progression of malignancy.

In studies carried out by Clarke et al. [85] p53 gene mutation failed to increase the frequency of bowel tumors in MIN mice. This resulted in the authors questioning the role of p53 as an important protective factor against carcinogenesis in the GI tract. However, if the APC protein functions by regulating the activity of proteins that are also regulated by p53, such as p21/WAF1, then the failure to observe cooperation between p53 mutations and APC mutations may not be surprising.

APC and E-cadherin clearly have an important influence on signals required for normal tissue homeostasis with a role in controlling cell proliferation and migration. The studies on these molecules suggest that cell function is position dependent and that a cell's life span is probably programmed.

	Cyclooxygenase

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 
Another gene that has recently been identified as having a possible role in colonic neoplasia is the cox-2 gene. Cyclooxygenase exists as two isoforms, COX-1 and COX-2, and is responsible for the synthesis of prostaglandins and thromboxanes from arachidonic acid. Early studies have pointed to the possible clinical usefulness of employing drugs that inhibit prostaglandin synthesis in the prevention and treatment of colonic tumors [86-89]. More recent work has shown that both human colorectal adenocarcinomas and carcinogen-induced colonic tumors in rats exhibit elevated levels of COX-2 mRNA and protein [90-93]; COX-1 expression was not altered. Furthermore, animals fed diets rich in corn oil showed elevated levels of prostaglandin and thromboxane synthesis and enhanced tumor promotion following treatment with chemical carcinogens [88]. Artificial overexpression of COX-2 in rat intestinal epithelial cells has been shown to inhibit butyrate-induced apoptosis; this was associated with increased expression of both Bcl-2 and E-cadherin which could account for the anti-apoptotic effect observed [94]. Selective inhibition of COX-2 has now been shown to inhibit tumor promotion in rats [95]. These observations (Fig. 5) indicate that the prostaglandins are functioning as autocrine survival/growth factors for colonic tumor cells. The ability of prostaglandins to protect against cell death is not solely confined to the colon. Prostaglandins have also been shown to partially protect against paracetamol-induced cell death in hepatocytes [96].

View larger version (29K): [in this window] [in a new window]   Figure 5. Schematic diagram illustrating the effects of COX-2 and the relationship with apoptosis in the large intestine. The possible effects of inhibition of this enzyme by non-steroidal anti-inflammatory drugs are also shown.

	Conclusions and Prospects

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

Hence, in the small bowel the sensitivity of the stem cells to undergo apoptosis allows the effective removal of excess stem cells (produced by the occasional symmetrical division) and also the removal of damaged cells. Therefore, in the small intestine apoptosis has a role in both tissue homeostasis, by maintaining a stable stem cell population, and in protecting the tissue from cancer development (Fig. 6).

View larger version (38K): [in this window] [in a new window]   Figure 6. Summary of the observations and conclusions regarding apoptosis in the intestinal epithelium and their likely implications. SI: small intestine; LI: large intestine; AI: apoptotic index.

Continued dissection of the pathways controlling apoptosis will provide opportunities to understand the homeostatic control of the normal tissue and cancer incidence, and reveal novel clinical targets.

	Acknowledgments

 
All the authors are funded by the Cancer Research Campaign.

	References

Top Abstract Introduction The Intestinal Epithelium Spontaneous Apoptosis in the... Damage-Induced Apoptosis Genetic Regulation of Apoptosis... p53 p53 and Spontaneous Apoptosis... The Bcl-2 Gene Family Bcl-2 and Spontaneous Apoptosis Genetic Regulation of Radiation... p53, Bcl-2 and Tumor... p53 Function--Protection against... Adhesion Molecules and Apoptosis Cyclooxygenase Conclusions and Prospects References

 

1. Kumar S, Lavin MF. The ICE family of cysteine proteases as effectors of cell death. Cell Death Differ 1996;3:255-267.

2. Wyllie A. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1990;284:555-556.

3. Oberhammer F, Wilson JW, Dive C et al. Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to, or in the absence of, internucleosomal fragmentation. EMBO J 1993;12:3679-3684.[Medline]

4. Gravrieli Y, Sherman Y, Ben-Sasson A. Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation. J Cell Biol 1992;119:493-501.[Abstract/Free Full Text]

5. Kaufmann SH, Desnoyers S, Ottaviano Y et al. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 1993;53:3976-3985.[Abstract/Free Full Text]

6. Potten CS. Structure, function and proliferative organization of the mammalian gut. In: Potten CS, Hendry JH, eds. Radiation and Gut. Amsterdam: Elsevier, 1995:1-31.

7. Potten CS. Extreme sensitivity of some intestinal crypts to X and -irradiation. Nature 1977;269:518-521.[Medline]

8. Potten CS. The significance of spontaneous and induced apoptosis in the gastrointestinal tract of mice. Cancer Metastasis Rev 1992;11:179-195.[Medline]

9. Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls, and uncertainties: lessons for and from the crypt. Development 1990;110:1001-1020.[Abstract/Free Full Text]

10. Potten CS. A comprehensive study of the radiobiological response of the murine (BDF1) small intestine. Int J Radiat Biol 1990;58:925-973.[Medline]

11. Hall P, Coates PJ, Ansari B et al. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci 1994;107:3565-3577.

12. Shibahara T, Sato N, Waguri S et al. The fate of effete epithelial cells at the villus tip of the human small intestine. Arch Histol Cytol 1995;58:205-219.[Medline]

13. Potten CS, Allen TD. Ultrastructure of cell loss in intestinal mucosa. J Ultrastruct Res 1977;60:272-277.[Medline]

14. Polzar B, Zanotti S, Stephan H et al. Distribution of deoxyribonuclease I in rat tissues and its correlation to cellular turnover and apoptosis (programmed cell death). Eur J Cell Biol 1994;64:200-210.[Medline]

15. Barnard JA, Warwick GJ, Gold LI. Localisation of transforming growth factor beta isoforms in the normal murine small intestine and colon. Gastroenterology 1993;105:67-73.[Medline]

16. Probstmeier R, Martini R, Tacke R et al. Expression of the adhesion molecules L1, N-CAM and J1/tenascin during development of the murine small intestine. Differentiation 1990;44:42-55.[Medline]

17. Probstmeier R, Martini R, Schachner M. Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding. Development 1990;109:313-321.[Abstract]

18. Raff MC, Barnes BA, Burne JF et al. Programmed cell death and the control of cell survival: lessons from the nervous system. Science 1993;262:695-700.[Abstract/Free Full Text]

19. Boudreau N, Sympson CJ, Werb Z et al. Suppression of ICE and apoptosis in mammary epithelial cells by extracellular matrix. Science 1995;267:891-893.[Abstract/Free Full Text]

20. Montgomery AM, Reisfeld RA, Cheresh DA. Integrin alpha v beta 3 rescues melanoma cells from apoptosis in three dimensional dermal collagen. PNAS 1994;91:8856-8860.[Abstract/Free Full Text]

21. Bates RC, Lincz LF, Burns GF. Involvement of integrins in cell survival. Cancer Metastasis Rev 1995;14:191-203.[Medline]

22. Meredith JE, Fazeli B, Schwartz MA. The extracellular matrix as a survival factor. Mol Biol Cell 1993;4:953-961.[Abstract]

23. Frisch SM, Francis H. Disruption of epithelial cell-matrix interaction induces apoptosis. J Cell Biol 1994;124:619-626.[Abstract/Free Full Text]

24. Ruoslahti E, Reed JC. Anchorage dependence, integrins and apoptosis. Cell 1994;77:477-478.[Medline]

25. Hermiston ML, Gordon JI. Inflammatory bowel disease and adenomas in mice expressing a dominant negative N-cadherin. Science 1996;270:1203-1207.[Abstract/Free Full Text]

26. Hendry JH, Potten CS, Chadwick C. Cell death (apoptosis) in the mouse small intestine after low doses: effects of dose rate, 14.7 MeV neutrons and 600 MeV (maximum energy) neutrons. Int J Radiat Biol 1982;42:611-620.

27. Paulus U, Potten CS, Loeffler M. A model of the control of cellular regeneration in the intestinal crypt after perturbation based solely on local stem cell regulation. Cell Prolif 1992;25;559-578.[Medline]

28. Ijiri K, Potten CS. Response of intestinal cells of differing topographical and hierarchical status to ten cytotoxic drugs and five sources of radiation. Br J Cancer 1983;47:175-185.[Medline]

29. Ijiri K, Potten CS. Further studies on the response of intestinal crypt cells of different hierarchical status to eighteen different cytotoxic agents. Br J Cancer 1987;55:113-123.[Medline]

30. Li YQ, Fan CY, O'Connor PJ et al. Target cells for the cytotoxic effects of carcinogens in the murine small bowel. Carcinogenesis 1992;13:361-368.[Abstract/Free Full Text]

31. Potten CS, Li YQ, O'Connor PJ et al. A possible explanation for the differential cancer incidence in the intestine, based on distributions of the cytotoxic effects of carcinogens in the murine large bowel. Carcinogenesis 1992;13:2305-2312.[Abstract/Free Full Text]

32. Lifshitz S, Schwartz B, Polak-Charon S et al. Disruption of colonic cell-cell interaction induces apoptosis. Gastroenterology 1995;108:498a.

33. Keenan KD, Sharpnack DD, Collins H et al. Morphological evaluation of the effects of Shiga toxin and E. coli Shiga-like toxin on the rabbit intestine. Am J Pathol 1986;125:69-80.[Abstract]

34. Shaposhnikov Y, Maheshwari Y, Sykes DE et al. Intestinal cell apoptosis and Bcl-2 expression. Cell Death Differ 1996;3:125-130.

35. Lowe SW, Schmitt EM, Smith SW et al. P53 is required for radiation induced apoptosis in mouse thymocytes. Nature 1993;362:847-849.[Medline]

36. Clarke AR, Purdie CA, Harrison DJ et al. Thymocyte apoptosis induced by p53 dependent and independent pathways. Nature 1993;362:849-852.[Medline]

37. Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 1995;80:293-299.[Medline]

38. Merritt AJ, Potten CS, Kemp CJ et al. The role of p53 in spontaneous and radiation induced apoptosis in the gastrointestinal tract of normal and p53 deficient mice. Cancer Res 1994;54:614-617.[Abstract/Free Full Text]

39. Merritt AJ, Potten CS, Watson AJ et al. 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 1995;108:2261-2271.[Abstract]

40. Lane DP. p53 guardian of the genome. Nature 1992;358:15-16.[Medline]

41. Mosner J, Deppert W. p53 and mdm2 are expressed independently during cellular proliferation. Oncogene 1994;9:3321-3328.[Medline]

42. El-Deiry WS, Tokino T, Velculescu VE et al. WAF1, a potential mediator of p53 tumor suppression. Cell 1993;75:817-825.[Medline]

43. Wu X, Bayle JH, Olsen D et al. The p53-mdm-2 autoregulatory feedback loop. Genes Dev 1993;7:1126-1132.[Abstract/Free Full Text]

44. Mosner J, Mummenbrauer T, Bauer C et al. Negative feedback regulation of wild type p53 biosynthesis. EMBO J 1995;14:4442-4449.[Medline]

45. Polyak K, Waldman T, He T-C et al. Genetic determinants of p53-induced apoptosis and growth arrest. Genes Dev 1996;10:1945-1952.[Abstract/Free Full Text]

46. Tanaka N, Ishihara M, Lamphier MS et al. Cooperation of the tumor suppressors IRF-1 and p53 in response to DNA damage. Nature 1996;382:816-818.[Medline]

47. Hollstein M, Sidransky D, Vogelstein B et al. p53 mutations in human cancers. Science 1991;253:49-53.[Abstract/Free Full Text]

48. Levine AJ, Momand J, Finlay CA. The p53 tumor suppressor gene. Nature 1991;351:453-456.[Medline]

49. Watson AJM, Merritt AJ, Jones LS et al. Evidence for reciprocity of Bcl-2 and p53 expression in human colorectal adenomas and carcinomas. Br J Cancer 1996;73:889-895.[Medline]

50. Donehower LA, Harvey M, Slagle BL et al. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumors. Nature 1992;356:215-221.[Medline]

51. Norimura T, Katsuki M, Nomoto S et al. P53 dependent apoptosis suppresses radiation induced teratogenesis. Nature Medicine 1996;2:577-580.[Medline]

52. Tsujimoto Y, Finger LR, Yunis J et al. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 1984;226:1097-1099.[Abstract/Free Full Text]

53. Tsujimoto Y, Croce CM. Analysis of the structure, transcripts and protein products of Bcl-2, the gene involved in human follicular lymphoma. Proc Natl Acad Sci USA 1986;83:5214-5218.[Abstract/Free Full Text]

54. Chen-Levy Z, Nourse J, Cleary ML. The Bcl-2 candidate proto-oncogene product is a 24-kilodalton integral-membrane protein highly expressed in lymphoid cell lines and lymphomas carrying the t(14;18). Mol Cell Biol 1989;9:701-710.[Abstract/Free Full Text]

55. Vaux DL, Cory S, Adams JM. Bcl-2 gene promotes haemopoietic cell survival and co-operates with c-myc to immortalise pre-B cells. Nature 1988;335:440-442.[Medline]

56. Yin X-M, Oltavi ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerisation with bax. Nature 1994;369:321-323.[Medline]

57. Boise LH, Gonzalez-Garcia M, Postema CE et al. Bcl-x, a Bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 1993;74:1-20.[Medline]

58. Kosopas KM, Yang T, Buchan HL et al. Mcl-1, a gene expressed in programmed myeloid cell differentiation has sequence similarity to Bcl-2. Proc Natl Acad Sci USA 1993;90:3516-3520.[Abstract/Free Full Text]

59. Takayama S, Sato T, Krajewski S et al. Cloning and functional analysis of BAG-1: a novel Bcl-2 binding protein with anti-cell death activity. Cell 1995;80:279-284.[Medline]

60. Choi SS, Park I-C, Yun JW et al. A novel Bcl-2 related gene, Bfl-1, is overexpressed in stomach cancer and preferentially expressed in bone marrow. Oncogene 1996;11:1693-1698.

61. Das R, Reddy EP, Chatterjee D et al. Identification of a novel Bcl-2 related gene, BRAG-1, in human glioma. Oncogene 1996;12:947-951.[Medline]

62. Gibson L, Holmgreen SP, Huang DCS et al. Bcl-w, a novel member of the Bcl-2 family, promotes cell survival. Oncogene 1996;13:665-675.[Medline]

63. Oltavi ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerises in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609-619.[Medline]

64. Yang E, Zha J, Jockel J et al. Bad, a heterodimeric partner for Bcl-x_L and Bcl-2, displaces bax and promotes cell death. Cell 1995;80:285-291.[Medline]

65. Farrow SN, White JHM, Martinou I et al. Cloning of a Bcl-2 homologue by interaction with adenovirus E1B 19K. Nature 1995;374:731-733.[Medline]

66. Chittenden T, Harrington EA, O'Conner R et al. Induction of apoptosis by the Bcl-2 homologue Bak. Nature 1995;374:733-736.[Medline]

67. Keifer MC, Brauer MJ, Powers VC et al. Modulation of apoptosis by the widely distributed Bcl-2 homologue Bak. Nature 1995;374:736-739.[Medline]

68. Boyd JM, Gallo GJ, Elangovan B et al. Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene 1995;11:1921-1928.[Medline]

69. Oltavi ZN, Korsmeyer SJ. Checkpoints of dueling dimers foil death wishes. Cell 1994;79:189-192.[Medline]

70. Cheng EH-Y, Levine B, Boise LH et al. Bax-independent inhibition of apoptosis by Bclx_L. Nature 1996;379:554-556.[Medline]

71. Potten CS, Hendry JH. Clonal regeneration studies. In: Potten CS, Henry JH, eds. Radiation and Gut. Amsterdam: Elsevier, 1995:45-59.

72. Clarke AR, Gledhill S, Hooper ML et al. p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following -irradiation. Oncogene 1994;9:1767-1773.[Medline]

73. Bracey TS, Miller JC, Preece A et al. -irradiation induced apoptosis in human colorectal adenoma and carcinoma cell lines can occur in the absence of wild type p53. Oncogene 1995;10:2391-2396.[Medline]

74. Hooper ML. The role of the p53 and Rb-1 genes in cancer, development and apoptosis. J Cell Sci Suppl 1994;18:13-17.[Medline]

75. Waldmann T, Lengauer C, Kinzler KW et al. Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature 1996;381:713-716.[Medline]

76. Kitada S, Krajewski S, Miyashita T et al. -radiation induces upregulation of Bax protein and apoptosis in radiosensitive cells in vivo. Oncogene 1996;12:187-192.[Medline]

77. Malkin D, Li FP, Strong LC et al. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas and other neoplasms. Science 1990;250:1233-1238.[Abstract/Free Full Text]

78. Srivastava S, Zou Z, Pirollo K et al. Germ-line transmission of a mutated p53 gene in cancer-prone family with Li-Fraumeni syndrome. Nature 1990;348:747-749.[Medline]

79. Shapiro L, Fannon AM, Kwong PD et al. Structural basis of cell-cell adhesion by cadherins. Nature 1995;374:327-337.[Medline]

80. Hermiston ML, Wong MH, Gordon JI. Forced expression of E-cadherin in the mouse intestinal epithelium slows cell migration and provides evidence for nonautonomous regulation of cell fate in a self-renewing system. Genes Dev 1996;10:985-996.[Abstract/Free Full Text]

81. Moser AR, Pitot HC, Dove WF. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse. Science 1990;247:322-324.[Abstract/Free Full Text]

82. Powell SM, Zilz N, Beazer-Barclay Y et al. APC mutations occur early during colorectal tumourigenesis. Nature 1992;359:235-237.[Medline]

83. Baeg GH, Matsumine A, Kuroda T et al. The tumor suppressor gene product APC blocks cell cycle progression from G₀/G₁ to S phase. EMBO J 1995;14:5618-5625.[Medline]

84. El-Deiry WS, Tokino T, Waldman T et al. Topological control of p21^WAF1/CIP1 expression in normal and neoplastic tissues. Cancer Res 1995;55:2910-2919.[Abstract/Free Full Text]

85. Clarke AR, Cummings MC, Harrison DJ. Interaction between murine germline mutations in p53 and APC predisposes to pancreatic neoplasia but not to increased intestinal malignancy. Oncogene 1995;11:1913-1920.[Medline]

86. Narisawa T, Takahashi M, Niwa M et al. Involvement of prostaglandin E2 in bile acid-caused promotion of colon carcinogenesis and antipromotion by the cyclooxygenase inhibitor indomethacin. Jpn J Cancer Res 1987;78:791-798.[Medline]

87. Earnst DL, Hixson LJ, Alberts DS. Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J Cell Biochem Suppl 1992;161:156-166.

88. Rao CV, Simi B, Wynn TT et al. Modulating effect of amount and type of dietary fat on colonic mucosal phospholipase A2, phosphatidylinositol-specific phospholipase C activities, and cyclooxygenase metabolite formation during different stages of colon tumor promotion in male F344 rats. Cancer Res 1996;56:532-537.[Abstract/Free Full Text]

89. Alberts DS, Hixson L, Ahnen D et al. Do NSAIDs exert the colon cancer chemoprevention activities through the inhibition of mucosal prostaglandin synthetase? J Cell Biochem Suppl 1995;22:18-23.[Medline]

90. Gustafson-Svard C, Lilja I, Hallbook O et al. Cyclooxygenase-1 and cyclooxygenase-2 gene expression in human colorectal adenocarcinomas and in azoxymethane-induced colonic tumors in rats. Gut 1996;38:79-84.[Abstract/Free Full Text]

91. DuBois RN, Radhika A, Reddy BS et al. Increased cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors. Gastroenterology 1996;110:1259-1262.[Medline]

92. Rao CV, Rivenson A, Simi B et al. Chemoprevention of colon carcinogenesis by sulindac, a non-steroidal anti-inflammatory agent. Cancer Res 1995;55:1464-1472.[Abstract/Free Full Text]

93. Yoshimi N, Ino N, Suzui M et al. The mRNA overexpression of inflammatory enzymes, phospholipase A2 and cyclooxygenase, in the large bowel mucosa and neoplasms of F344 rats treated with naturally occurring carcinogen, 1-hydroxyanthraquinone. Cancer Lett 1995;97:75-82.[Medline]

94. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493-501.[Medline]

95. Takahashi M, Fukutake M, Yokota S et al. Suppression of azoxymethane-induced aberrant crypt foci in rat colon by nimesulide, a selective inhibitor of cyclooxygenase-2. J Cancer Res Clin Oncol 1996;122:219-222.[Medline]

96. Nasseri-Sina P, Fawthrop DJ, Wilson JW et al. Cytoprotection by iloprost against paracetamol-induced toxicity in isolated hamster hepatocytes. Br J Pharmacol 1992;105:417-423.[Medline]

This article has been cited by other articles:

				A. Bilger, R. Sullivan, A. J. Prunuske, L. Clipson, N. R. Drinkwater, and W. F. Dove Widespread hyperplasia induced by transgenic TGF{alpha} in ApcMin mice is associated with only regional effects on tumorigenesis Carcinogenesis, September 1, 2008; 29(9): 1825 - 1830. [Abstract] [Full Text] [PDF]

				S. Shangary and S. Wang Targeting the MDM2-p53 Interaction for Cancer Therapy Clin. Cancer Res., September 1, 2008; 14(17): 5318 - 5324. [Abstract] [Full Text] [PDF]

				J H SHIN, S K LEE, H-Y. SONG, J-S KIM, H CHOE, E-H KIM, I J LEE, T-H KIM, E-Y KIM, C-W WOO, et al. The effects of 188rhenium-filled balloon dilation following bare stent placement in a rabbit oesophageal model Br. J. Radiol., May 1, 2008; 81(965): 413 - 421. [Abstract] [Full Text] [PDF]

				S. Shangary, D. Qin, D. McEachern, M. Liu, R. S. Miller, S. Qiu, Z. Nikolovska-Coleska, K. Ding, G. Wang, J. Chen, et al. Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition PNAS, March 11, 2008; 105(10): 3933 - 3938. [Abstract] [Full Text] [PDF]

				A.-H. Zhang, J. N. Rao, T. Zou, L. Liu, B. S. Marasa, L. Xiao, J. Chen, D. J. Turner, and J.-Y. Wang p53-Dependent NDRG1 expression induces inhibition of intestinal epithelial cell proliferation but not apoptosis after polyamine depletion Am J Physiol Cell Physiol, July 1, 2007; 293(1): C379 - C389. [Abstract] [Full Text] [PDF]

				L. J. Cliffe, C. S. Potten, C. E. Booth, and R. K. Grencis An Increase in Epithelial Cell Apoptosis Is Associated with Chronic Intestinal Nematode Infection Infect. Immun., April 1, 2007; 75(4): 1556 - 1564. [Abstract] [Full Text] [PDF]

				Y. Cao, L. Chen, W. Zhang, Y. Liu, H. T. Papaconstantinou, C. R. Bush, C. M. Townsend Jr., E. A. Thompson, and T. C. Ko Identification of apoptotic genes mediating TGF-beta/Smad3-induced cell death in intestinal epithelial cells using a genomic approach Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G28 - G38. [Abstract] [Full Text] [PDF]

				J. C. Finley, B. J. Reid, R. D. Odze, C. A. Sanchez, P. Galipeau, X. Li, S. G. Self, K. A. Gollahon, P. L. Blount, and P. S. Rabinovitch Chromosomal Instability in Barrett's Esophagus Is Related to Telomere Shortening. Cancer Epidemiol. Biomarkers Prev., August 1, 2006; 15(8): 1451 - 1457. [Abstract] [Full Text] [PDF]

				I. Illa-Bochaca and L. M. Montuenga The regenerative nidi of the locust midgut as a model to study epithelial cell differentiation from stem cells J. Exp. Biol., June 1, 2006; 209(11): 2215 - 2223. [Abstract] [Full Text] [PDF]

				H. M. Zhang, K. M. Keledjian, J. N. Rao, T. Zou, L. Liu, B. S. Marasa, S. R. Wang, L. Ru, E. D. Strauch, and J.-Y. Wang Induced focal adhesion kinase expression suppresses apoptosis by activating NF-{kappa}B signaling in intestinal epithelial cells Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1310 - C1320. [Abstract] [Full Text] [PDF]

				W. Deng, M. J. Viar, and L. R. Johnson Polyamine depletion inhibits irradiation-induced apoptosis in intestinal epithelia Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G599 - G606. [Abstract] [Full Text] [PDF]

				S. N. Salm, P. E. Burger, S. Coetzee, K. Goto, D. Moscatelli, and E. L. Wilson TGF-{beta} maintains dormancy of prostatic stem cells in the proximal region of ducts J. Cell Biol., July 4, 2005; 170(1): 81 - 90. [Abstract] [Full Text] [PDF]

				P. E. Burger, X. Xiong, S. Coetzee, S. N. Salm, D. Moscatelli, K. Goto, and E. L. Wilson Sca-1 expression identifies stem cells in the proximal region of prostatic ducts with high capacity to reconstitute prostatic tissue PNAS, May 17, 2005; 102(20): 7180 - 7185. [Abstract] [Full Text] [PDF]

				J. A. Clark, R. H. Lane, N. K. MacLennan, H. Holubec, K. Dvorakova, M. D. Halpern, C. S. Williams, C. M. Payne, and B. Dvorak Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis Am J Physiol Gastrointest Liver Physiol, April 1, 2005; 288(4): G755 - G762. [Abstract] [Full Text] [PDF]

				H. Kitazawa, T. Nishihara, T. Nambu, H. Nishizawa, M. Iwaki, A. Fukuhara, T. Kitamura, M. Matsuda, and I. Shimomura Intectin, a Novel Small Intestine-specific Glycosylphosphatidylinositol-anchored Protein, Accelerates Apoptosis of Intestinal Epithelial Cells J. Biol. Chem., October 8, 2004; 279(41): 42867 - 42874. [Abstract] [Full Text] [PDF]

				T. Zou, J. N. Rao, X. Guo, L. Liu, H. M. Zhang, E. D. Strauch, B. L. Bass, and J.-Y. Wang NF-{kappa}B-mediated IAP expression induces resistance of intestinal epithelial cells to apoptosis after polyamine depletion Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1009 - C1018. [Abstract] [Full Text] [PDF]

				J. A. Rudolph, J. L. Poccia, and M. B. Cohen Cyclic AMP Activation of the Extracellular Signal-regulated Kinases 1 and 2: IMPLICATIONS FOR INTESTINAL CELL SURVIVAL THROUGH THE TRANSIENT INHIBITION OF APOPTOSIS J. Biol. Chem., April 9, 2004; 279(15): 14828 - 14834. [Abstract] [Full Text] [PDF]

				S. Bhattacharya, R. M. Ray, M. J. Viar, and L. R. Johnson Polyamines are required for activation of c-Jun NH2-terminal kinase and apoptosis in response to TNF-{alpha} in IEC-6 cells Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G980 - G991. [Abstract] [Full Text] [PDF]

				H. Yang, Y. Fan, and D. H. Teitelbaum Intraepithelial lymphocyte-derived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G629 - G637. [Abstract] [Full Text] [PDF]

				E. M. Dahly, M. B. Gillingham, Z. Guo, S. G. Murali, D. W. Nelson, J. J. Holst, and D. M. Ney Role of luminal nutrients and endogenous GLP-2 in intestinal adaptation to mid-small bowel resection Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G670 - G682. [Abstract] [Full Text] [PDF]

				S. N. O'Brien, N. M.A. Blijlevens, T. H. Mahfouz, and E. J. Anaissie Infections in Patients with Hematological Cancer: Recent Developments Hematology, January 1, 2003; 2003(1): 438 - 472. [Abstract] [Full Text] [PDF]

				E. M. Dahly, Z. Guo, and D. M. Ney Alterations in Enterocyte Proliferation and Apoptosis Accompany TPN-Induced Mucosal Hypoplasia and IGF-I-Induced Hyperplasia in Rats J. Nutr., July 1, 2002; 132(7): 2010 - 2014. [Abstract] [Full Text] [PDF]

				S. Ruiz-Santana, A. Lopez, S. Torres, A. Rey, A. Losada, L. Latasa, J. L. Manzano, and B. N. Diaz-Chico Prevention of Dexamethasone-Induced Lymphocytic Apoptosis in the Intestine and in Peyer Patches by Enteral Nutrition JPEN J Parenter Enteral Nutr, November 1, 2001; 25(6): 338 - 345. [Abstract] [PDF]

				L. A. Welniak, A. R. Khaled, M. R. Anver, K. L. Komschlies, R. H. Wiltrout, S. Durum, F. R. Ruscetti, B. R. Blazar, and W. J. Murphy Gastrointestinal Cells of IL-7 Receptor Null Mice Exhibit Increased Sensitivity to Irradiation J. Immunol., March 1, 2001; 166(5): 2923 - 2928. [Abstract] [Full Text] [PDF]

				J.M. Gee, H.P.J.M. Noteborn, A.C.J. Polley, and I.T. Johnson Increased induction of aberrant crypt foci by 1,2-dimethylhydrazine in rats fed diets containing purified genistein or genistein-rich soya protein Carcinogenesis, December 1, 2000; 21(12): 2255 - 2259. [Abstract] [Full Text] [PDF]

				D M K Keefe, J Brealey, G J Goland, and A G Cummins Chemotherapy for cancer causes apoptosis that precedes hypoplasia in crypts of the small intestine in humans Gut, November 1, 2000; 47(5): 632 - 637. [Abstract] [Full Text] [PDF]

				C. W. Houchen, W. F. Stenson, and S. M. Cohn Disruption of cyclooxygenase-1 gene results in an impaired response to radiation injury Am J Physiol Gastrointest Liver Physiol, November 1, 2000; 279(5): G858 - G865. [Abstract] [Full Text] [PDF]

				M. Y. Hong, J. R. Lupton, J. S. Morris, N. Wang, R. J. Carroll, L. A. Davidson, R. H. Elder, and R. S. Chapkin Dietary Fish Oil Reduces O6-Methylguanine DNA Adduct Levels in Rat Colon in Part by Increasing Apoptosis during Tumor Initiation Cancer Epidemiol. Biomarkers Prev., August 1, 2000; 9(8): 819 - 826. [Abstract] [Full Text]

				L. A. Davidson, R. E. Brown, W.-C. L. Chang, J. S. Morris, N. Wang, R. J. Carroll, N. D. Turner, J. R. Lupton, and R. S. Chapkin Morphodensitometric analysis of protein kinase C {beta}II expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis Carcinogenesis, August 1, 2000; 21(8): 1513 - 1519. [Abstract] [Full Text] [PDF]

				N. J. Toft, O. J. Sansom, R. A. Brookes, M. J. Arends, M. Wood, G. P. Margison, D. J. Winton, and A. R. Clarke In vivo administration of O6-benzylguanine does not influence apoptosis or mutation frequency following DNA damage in the murine intestine, but does inhibit P450-dependent activation of dacarbazine Carcinogenesis, April 1, 2000; 21(4): 593 - 598. [Abstract] [Full Text] [PDF]

				D. R. Cronk, D. C. Ferguson, and J. S. Thompson Malnutrition Impairs Postresection Intestinal Adaptation JPEN J Parenter Enteral Nutr, March 1, 2000; 24(2): 76 - 80. [Abstract] [PDF]

				C. Bai, B. Connolly, M. L. Metzker, C. A. Hilliard, X. Liu, V. Sandig, A. Soderman, S. M. Galloway, Q. Liu, C. P. Austin, et al. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster PNAS, February 1, 2000; 97(3): 1230 - 1235. [Abstract] [Full Text] [PDF]

				C. E. Shin, R. A. Falcone Jr., C. J. Kemp, C. R. Erwin, D. A. Litvak, B. M. Evers, and B. W. Warner Intestinal adaptation and enterocyte apoptosis following small bowel resection is p53 independent Am J Physiol Gastrointest Liver Physiol, September 1, 1999; 277(3): G717 - G724. [Abstract] [Full Text] [PDF]

				Y.-Y. Fan, J. Zhang, R. Barhoumi, R. C. Burghardt, N. D. Turner, J. R. Lupton, and R. S. Chapkin Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells Am J Physiol Cell Physiol, August 1, 1999; 277(2): C310 - C319. [Abstract] [Full Text] [PDF]

				N. R. Murray, L. A. Davidson, R. S. Chapkin, W. Clay Gustafson, D. G. Schattenberg, and A. P. Fields Overexpression of Protein Kinase C beta II Induces Colonic Hyperproliferation and Increased Sensitivity to Colon Carcinogenesis J. Cell Biol., May 17, 1999; 145(4): 699 - 711. [Abstract] [Full Text] [PDF]

				C. W. Houchen, R. J. George, M. A. Sturmoski, and S. M. Cohn FGF-2 enhances intestinal stem cell survival and its expression is induced after radiation injury Am J Physiol Gastrointest Liver Physiol, January 1, 1999; 276(1): G249 - G258. [Abstract] [Full Text] [PDF]

				T. Fujiwara, J. M. Stolker, T. Watanabe, A. Rashid, P. Longo, J. R. Eshleman, S. Booker, H. T. Lynch, J. R. Jass, J. S. Green, et al. Accumulated Clonal Genetic Alterations in Familial and Sporadic Colorectal Carcinomas with Widespread Instability in Microsatellite Sequences Am. J. Pathol., October 1, 1998; 153(4): 1063 - 1078. [Abstract] [Full Text] [PDF]

				H Ikeda, Y Suzuki, M Suzuki, M Koike, J Tamura, J Tong, M Nomura, and G Itoh Apoptosis is a major mode of cell death caused by ischaemia and ischaemia/reperfusion injury to the rat intestinal epithelium Gut, April 1, 1998; 42(4): 530 - 537. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints/Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Potten, C.S. Articles by Booth, C. Search for Related Content PubMed PubMed Citation Articles by Potten, C.S. Articles by Booth, C.