Carcinogenesis, Vol. 21, No. 3, 469-476,
March 2000
© 2000 Oxford University Press
Cancer Biology |
Stem cells: the intestinal stem cell as a paradigm
1 CRC Department of Epithelial Biology, Paterson Institute for Cancer Research and
2 Department of Surgery, Christie Hospital NHS Trust, Wilmslow Road, Manchester, UK
 |
Abstract |
|---|
 Top Abstract IntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Stem cell research provides a foundation for therapeutic advancement in oncology, clinical genetics and a diverse array of degenerative disorders. For example, the elucidation of pathways governing proliferative regulation and differentiation within cellular systems will result in medical strategies aimed at the root cause of cancer. At present the characterization of reliable stem cell markers is the immediate aim in this particular field. Over the past 30 years investigators have determined many of the physical and functional properties of stem cells through careful and imaginative experimentation. Intestinal stem cells reside at the crypt base and give rise to all cell types found within the crypt. They readily undergo altruistic apoptosis in response to toxic stimuli although their progeny are hardier and will regain stem cell function to repopulate the tissue compartment, giving rise to the concept of a proliferative hierarchy. Contention exists when deciding whether the full complement of cells within a crypt is derived from either a single or multiple stems. Evidence has also arisen to challenge the long held view that colorectal tumours arise from a single mutated stem cell, as early adenomas from a human XO/XY mosaic contained distinct clones. Mechanisms governing the stem cell cycle and subsequent proliferative activity largely remain obscure. The adenomatous polyposis coli gene product has, however, been shown to promote the degradation of ß-catenin, an enhancer of cell proliferation, thereby downregulating this activity in healthy individuals.
Abbreviations: APC, adenomatous polyposis coli; DBA, Dolichos biflorus agglutinin; 3HTdR, tritiated thymidine; TGF-ß, transforming growth factor ß.
|
Introduction |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Certain tissues, having adapted to fulfil a specialized role in the body, experience continuous cell loss either through high rates of mechanical attrition, as seen in the gut, or as a result of the terminal differentiation of cells with a short lifespan. These cells must be continually replaced at a rate that matches their rate of loss. Once fully differentiated, however, such cells often lose the ability to proliferate, as dramatically illustrated by the nuclear loss of erythrocytes and keratinocytes. It is clear that in many situations some cell divisions do occur before terminal differentiation, e.g. in erythroblasts. However, since erythroblasts eventually become erythrocytes, these blast cells must also be replaced. A subset of progenitor cells therefore exists, the function of which is to populate these cellular tissue compartments. We call these the stem cells.
The study of stem cells is of medical importance as: (i) homeostatic mechanisms of stem cell proliferation are the same processes that become disregulated in carcinogenesis. Discovery of these pathways therefore brings us a step closer to treating such uncontrolled proliferation, providing us with targets at which future cancer treatments including gene therapy can be aimed. (ii) The rapidly dividing tissues of bone marrow, gut and skin are the first to be affected by cancer treatment. Toxicity in these tissues is dose limiting for many chemotherapeutic agents or radiotherapeutic practises. Isolation of viable stem cells could be used as a therapeutic manoeuvre to repopulate such tissues following cancer therapy. Alternatively growth factor manipulation to alter their sensitivity to treatment or improve their regenerative potential could also have benefits. (iii) The culture of stem cells may eventually facilitate tissue engineering. Already, skin can be manufactured from its constitutive elements to provide cover following ulceration or burns. Hopefully this tissue and others may eventually be derived from clones of our own progenitor cells. (iv) Identification of stem cells by reliable markers may aid the development of stem cell gene therapy for conditions such as adenomatous polyposis coli (APC), hereditary non-polyposis colorectal cancer and inherited colorectal cancer.
The mammalian intestinal mucosa is a rapidly proliferating tissue and provides an excellent model for the study of proliferative hierarchies, regulation of cell division and differentiation. Our knowledge of intestinal stem cell function is largely based upon work carried out in the mouse. We will therefore describe how intestinal stem cells were characterized in this model with human correlates where they are known.
|
Defining stem cells |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The intestine is lined by a simple columnar epithelium, which is continually replaced as cells are shed into the gut lumen. Small intestinal villi and colonic intercrypt plates receive a constant supply of enterocytes from progenitor cells located within the lower poles of the crypts of Lieberkuhn. Each new cell will undergo four to six rounds of cell division as it rapidly migrates out of the crypt to the mucosal surface. The rate of cell replacement must mirror cell loss with dynamic control mechanisms able to operate under both steady state and stressed conditions.
Morphological criteria do not exist to identify stem cells in gut mucosa. They are instead defined by their characteristics. Potten and Loeffler (1) proposed that stemness was not a single property, but a number of properties or options that a cell has the capability to perform depending upon circumstances. It is now accepted that these properties are that a cell must be of a relatively undifferentiated type capable of proliferation and self-maintenance, producing a variety of cell lineages and capable of tissue regeneration following injury.
Self-maintenance is the fundamental stem cell requirement and describes a cell's ability to preserve its own population. When a stem cell undergoes mitosis it must be able to produce a single daughter stem cell. When both daughter cells are stem cells, via a `symmetrical division', the stem cell population increases, whereas the production of one stem cell with one daughter that differentiates is termed an `asymmetric division'. This is thought to be the average response under normal conditions and results in a stable stem cell population. If both cells go on to differentiate then the stem cell from which they arose will cease to exist. It is probable that stem cells have the ability to switch between these various options in response to environmental conditions, thereby regulating their own number and consequently that of the crypt as a whole.
Differentiation can be defined as qualitative changes in the cellular phenotype that are a consequence of the onset of synthesis of new gene products, i.e. the non-cyclic changes in gene expression that ultimately lead to functional competence.
|
Basic mechanics of crypt function |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Murine small intestinal crypts constitute an average of 250 cells in a test-tube like structure. When viewed in longitudinal cross section they are approximately 22 cells in height with 16 cells forming an average circumference at the widest point. The vertical dimension is overestimated in cross section, due to the three-dimensional configuration of the cells, and is actually nearer 16 once this has been taken into account (2). Approximately 30 fully differentiated Paneth cells occupy the very lowest crypt cell positions. The next 150 or so cells are actively proliferating as determined by incorporation of tritiated thymidine (3HTdR) or bromodeoxyuridine, with 75 of these in the S phase of the cell cycle at any one time. Analysis of the percentage of mitotic cells labelled with 3HTdR against time and 3HTdR grain dilution assays have demonstrated an average cell cycle time of 12–13 h for these rapidly proliferating cells. A small proportion of cells situated at the base of this band have a somewhat slower cell cycle time of ~24 h and it is proposed that these may be stem cells (3,4). The remaining cells occupying positions towards the lumenal pole of the crypt are relatively more differentiated and will usually undergo only one further cell division before emerging onto the villus surface.
The colonic crypts of BDF1 mice are larger than those found in the small intestine and do not contain Paneth cells. The rate of cell proliferation is again higher at the lower pole of the crypt with cell cycle times in the region of 33 h (5).
In humans the cell cycle times of stem cells are less well defined; however, they are generally thought to be between four and eight times longer (6).
|
Stem cell location |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Under steady state conditions the cellular migration pathways of small intestinal crypts arise from positions 4 to 6, i.e. above the Paneth cells, whereas in the colon they originate from the very base of the crypt (7,8). These data were obtained by measuring cell velocity as determined by changes in the position of 3HTdR-labelled cells with time. Large doses of irradiation or cytotoxic drugs (hydroxyurea, etoposide or arabinoside), used to induce significant cell death within intestinal crypts demonstrated that the crypt's proliferative regenerative response also arises from these positions (4,9). Similarly, exposure of these basally situated cells to a lethal dose of radiation derived from the filtered weak beams of ß particles from 147promethium, revealed that whole crypts were sterilized by doses of radiation that spared the middle and upper crypt regions (10). Regenerative clonogenic cells must therefore be located exclusively at the lower pole of the crypt.
|
Stem cell number |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The number of stem cells located within small intestinal and colonic crypts is not known precisely. However, estimates may be made based upon cell cycle times, tissue regeneration studies and the pattern of expression of cells of differing genotypes within a crypt. By altering the crypt microenvironment to perform such studies one may inherently affect the behaviour of stem cells, thereby bringing a degree of uncertainty to the results. This may be how individual investigators have produced widely different estimates of stem cell number (1).
|
Polyclonal versus monoclonal crypts |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Some investigators argue for the existence of a single stem cell from which all crypt cells derive. Chimeric mice strains with intestinal polymorphism in their lectin binding capacity of Dolichos biflorus agglutinin (DBA), which binds to the N-acetylgalactosamine residues present on blood group markers at the surface of epithelial cells, demonstrate either positive or negative crypt staining for this marker (11). One interpretation of these data is that adult crypts are derived during development from one stem cell. Examination of this marker distribution during the first 14 days of life, however, revealed that, initially, mixed DBA expression does occur, and crypts only become exclusively positive or negative at a later stage (12). DBA-expressing crypt stem cells may be mutated at this allele by treatment with ethylnitrosourea resulting in a progressive loss of staining. This staining loss eventually spreads throughout the entire crypt indicating that all cells have acquired the mutation, again arguing for the existence of a single stem cell (13,14). However, the interval between stem cell mutation and loss of DBA expression within the crypt as a whole is considerably longer than the cell turnover time of the crypt. An alternative explanation is that there is competition between multiple stem cells with the eventual dominance of a mutated stem cell in some situations following successive rounds of cell division (15). Adding weight to this hypothesis it was observed that 10 days after initial mutation the stripe of negatively stained cells migrating up villi was two cells wide. This accounts for 25% of the normal crypt cellular output and supports the existence of four stem cells per crypt, although a full complement of stem cells cannot be guaranteed following the administration of ethylnitrosourea, which is cytotoxic (16).
Examination of mutation patterns in separate marker genes such as glucose-6-phosphatase-dehydrogenase have produced comparable results (17,18).
Stem cell number based upon in vivo mouse studies
A combination of cell proliferation studies and mathematical modelling suggests that in the small intestine a crypt could be maintained under steady state conditions by between four and six ultimate stem cells with six generations of dividing transit cells (15,19). The situation is somewhat different in colonic epithelium as modelling can account for the observed patterns of 3HTdR labelling and mitoses based upon only one stem cell with eight generations of transit cells. However, it should be noted that a larger number of stem cells could also be supported by these data. Therefore, despite the greater size of colonic crypts it would appear that their stem cell quota might actually be the same or lower than that of small intestinal crypts.
|
Stem cell hierarchy |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The ability of stem cells to regenerate damaged tissue following injury has been used to study their functional characteristics. The microcolony clonogenic stem cell assay (20) measures the number of intestinal stem cells surviving exposure to radiation or cytotoxic therapy. The number of regenerating crypts is measured in cross-sections of mouse intestine following a range of enterotoxic treatment dosages. Crypt regeneration occurs where one or more functional stem cell survives the toxic insult. Repopulation of the crypt will begin over the course of 3 days enabling surviving crypts to be counted at day 4. By this time, crypts without surviving stem cells have largely disappeared or are reproductively sterile. Dose–response curves (survival curves) can then be generated. These data suggest that the number of clonogenic cells present within a crypt is dependent upon the level of damage induced within the crypt. As damage increases, so more cells appear to be recruited into the clonogenic compartment. At low doses of radiation there are approximately six clonogenic cells per crypt, a figure that corresponds closely to the ultimate stem cell number, under steady state conditions predicted by the mathematical model of Potten and Loeffler (1). At higher doses this number increases to 36 in both the small intestine and colon (21,22).
These studies have been complemented by the addition of data indicating the positional distribution of apoptosis in crypts following cytotoxic exposure (23).
A three-tiered hierarchical system of stem cell organization has been proposed based upon these studies. Cells at the base of small intestinal crypts are the first to undergo apoptosis following low-dose 
-irradiation (1 Gy). These are either cells at an early stage of the proliferative hierarchy if not the ultimate stem cells themselves. It is postulated that these stem cells prefer to apoptose rather than repair even quite minor damage to their DNA. This may serve to reduce the risk of propagating a mutated clone within the crypt. If all ultimate stem cells are destroyed then their more radioresistant daughter cells have the ability to assume stem cell functions and maintain the crypt.
Using split-dose techniques (24) and the administration of a second, higher dose of radiation (<9 Gy), the existence of this second stem cell tier, composed again of six cells, is revealed (25,26). These cells would normally be the asymmetric sisters of the ultimate stem cells, being dividing transit cells in the process of migrating towards the crypt's lumenal pole. At this early stage in their lineage development they appear to retain stem cell properties. Radiation doses >9 Gy reveal an additional third tier of about 24 stem cells with even greater repair capabilities and hence radioresistance. These ultimate and clonogenic stem cells will be concentrated around crypt cell positions 2–7 in the small intestine. The remaining 114 or so rapidly proliferating cells situated above this level appear to have no clonogenic or stem cell potential.
Similar experiments adapted to account for the inherent radioresistance and slower cell cycling times of the colon have demonstrated that there are a similar small number of ultimate stem cells, perhaps in the region of four to six cells with again up to 36 clonogenic cells in total (22).
|
Evidence for pluripotency |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Radiation experiments also indicate that a single surviving clonogenic cell can form a regenerative crypt containing all cell lineages (i.e. enterocytes, goblet cells, Paneth cells and endocrine cells). The surviving clonogenic cell was therefore probably pluripotent. Further evidence for pluripotency is that the subcutaneous injection of single cells from rat colonic adenocarcinoma into mice can give rise to tumours containing all cell lineages (27). The human HRA19 cell line has also been shown to produce a variety of cell types from a single cell in vitro (28). The pluripotency of intestinal stem cells has been reviewed by Wright (29).
Debate also exists over the issue of whether intestinal cell lineages develop directly from ultimate stem cells or via intermediate progenitors. In general the latter hypothesis was favoured although evidence for this was limited mainly to the fact that cells of intermediate phenotype are observed in mid-crypt positions. Recent work by Bjerknes and Cheng addresses this point (30). Chimeric mice heterozygous at the Dlb-1 locus which generates the intestinal binding site of DBA, were subject to somatic mutation in a proportion of the remaining wild-type alleles using the chemical mutagen ethylnitrosourea. Cells lacking a DBA binding site fail to stain with this lectin and so can be identified within intact crypt–villus units. Mutated progenitor cells give rise to a clone of similarly unstained progeny. The presence of multiple cell lineages within a mutated clone indicates pluripotency of the progenitor, whereas a clone composed of a single cell type is likely to be derived from a unipotent progenitor. By observing the incidence, position, size and cellular composition of these mutated clones with time, the existence of both short- (<10 days) and long-lived (>100 days) enterocytic and goblet cell lineage specific progenitor cells situated in the upper mid-crypt and lower crypt, respectively, was demonstrated. Two types of mixed clones also occurred. Most were short-lived and composed predominantly of two cell types, while others were long-lived, more frequently containing three or more cell types. In 90% of cases, these latter long-lived mixed clones contained mutated basally situated crypt columnar cells, the predicted site for ultimate stem cells. The authors discuss how this technique may be developed into an assay of growth factor effect upon individual crypt cell lineages.
|
Regulation of stem cell number |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The number of stem cells per crypt is governed by net production versus cell deletion. To maintain the stem cell population during mitosis, one stem cell gives rise to a further stem cell plus a daughter cell that will differentiate (asymmetrical division). The mechanisms underlying pathways of cellular differentiation as well as those responsible for continued stem cell function are not understood. Several hypotheses pitting cellular nature against nurture are, however, proposed. Stem cells may be selected by independently expressing a specific element of their genetic code following cell division or this decision may have taken place during mitosis resulting in a polarized cell division (31). These processes will result in autonomous stem cell function. Alternatively, progeny may be stimulated to behave as stem cells by environmental signals perhaps mediated through cell–extracellular matrix contacts. Movement of the cell outside a limited stem cell zone may bring about the loss of stem cell status and induce consequent cellular differentiation (32,33). It is, however, difficult to reconcile this theory in a crypt containing spatially separated stem cells with other cells in the niche. Recently, an initial breakthrough was made in determining the genetic regulation of stem cell function. The HMG (high-mobility-group) box transcription factor Tcf-4 was shown to be necessary for maintenance of the stem cell phenotype during the early stages of crypt development. Mice lacking this allele exhibited depletion of the small intestinal crypt stem cell compartment (34).
The genes mediating intestinal stem cell proliferation are considered in the section dealing with carcinogenesis, for it was through the investigation of disregulated cell growth that these genes were first identified.
One would speculate that on occasions an extra stem cell might result from the occasional symmetrical cell division. This could have dramatic consequences for the crypt, as each extra stem cell is capable of producing an entire lineage of up to 64–128 cells. To prevent such fluxes in the enterocyte population these supernumerary stem cells therefore require deletion. Examining untreated murine crypt sections we have indeed found a spontaneous apoptotic rate of ~5–10% in the stem cell region of the small intestine (35,36). The rate of spontaneous apoptosis remains unchanged in homozygously null p53 mice in comparison with wild-type controls indicating that the mechanism is p53 independent (37,38). In the colon one sees a much lower rate of spontaneous apoptosis that is not associated with the stem cell position. Again it appears that apoptosis is independent of p53 function. However, mice lacking the anti-apoptotic gene bcl-2 demonstrate increased rates of spontaneous apoptosis amongst the colonic stem cell population. Bcl-2 may therefore mediate the apparent resistance of these colonic stem cells to apoptosis (39). It is therefore possible that the spontaneous apoptosis seen in the small intestine represents part of the stem cell homeostatic process and that this process is compromised in the colon by bcl-2.
|
Maintaining the genomic integrity of stem cells |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
It is estimated that the small intestinal stem cells of mice undergo up to 1000 divisions in their lifetime. It has been noted that these cells divide more slowly with a cycle time of approximately twice that seen in their daughters within the transit cell compartment (3). It is possible that this occurs in order to minimize the risk of genetic mutation within the stem cell compartment and allows maximum time for detection and correction of replicative errors or the implementation of altruistic apoptosis. Heddle et al. (40) propose that the continued existence of stem cells throughout an organism's lifetime is not necessary for the purpose of populating tissue compartments. It is calculated that their progeny could create sufficient cells to adequately perform this function. Instead this group hypothesize that stem cells, by their comparatively low rate of division, serve to reduce the rate of spontaneous somatic mutation and, therefore, the risk of developing cancer. Actively proliferating cell-types are more prone to mutating events but their short lifespans prohibit the development of cancer.
|
The role of p53 and p21waf1/cip1 in maintaining stem cell integrity |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The response of murine intestinal stem cells to DNA damage has been determined following exposure to low doses of radiation and the cytotoxic agent 5-fluorouracil (5-FU). These studies revealed that very small doses of radiation (0.01–0.05 Gy) induced marked p53-dependent apoptosis amongst small intestinal stem cells within the first 24 h (38). The number of apoptotic cells increases as the dose is raised to 1 Gy. At this point approximately six cells per crypt are killed and above this dose few additional cells can be seen to die via apoptosis over the first few hours. Within colonic crypts a more diffuse pattern of apoptosis was seen, not specifically localized to the stem cell compartment. These data indicate that small intestinal stem cells have a lower threshold for initiating apoptosis in response to DNA damage and do not attempt to effect a repair. This may serve to protect the genome from mutation arising from environmental carcinogens and may explain in part the lower incidence of adenocarcinoma in small intestine.
In both the small intestine and colon, wild-type p53 protein is expressed 2–4 h after radiation exposure, and in the small bowel its expression, in terms of time and cell position, is coincident with that observed for apoptosis (38). However, it is not expressed in many of the apoptotic cells but can be found in other cells at the stem cell position. The p53-related gene, p21waf1/cip1 is also expressed at this time and broadly over the same cell positions as well as at additional, slightly higher positions within the crypt. This suggests a role for p21waf1/cip1 in the cellular repair mechanisms of stem cells, particularly the clonogenic stem cells. When the p53 gene is deleted (p53 knockout mice), radiation-induced apoptosis is completely absent indicating a role for this protein in the detection of DNA damage in clonogenic stem cells.
The enterotoxic anti-metabolite 5-FU in doses of 40 or 400 mg/kg, produces similar levels of acute, p53-dependent apoptosis in both the small intestine and colon (41). However, intestinal toxicity was only associated with the higher dose which, additionally, impaired DNA synthesis and consequently the mitotic index. This impairment correlated with the prolonged, p53-dependent expression of p21waf1/cip1. In p53 null mice much lower rates of apoptosis combined with a lack of cell cycle inhibition resulted in the maintenance of crypt integrity at the higher dose. These data suggest that both p53 and p21 are involved in the regulation of cell cycle checkpoints and repair in clonogenic cells.
|
The role of Bcl-2 in maintaining stem cell integrity |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The anti-apoptotic gene bcl-2 is expressed at the base of murine and human colonic crypts, whereas expression is not seen in the small intestine, supporting the view that bcl-2 increases the apoptotic threshold of colonic stem cells (39).
-Irradiation of Bcl-2 null mice significantly increased apoptotic cell death within the colon, compared with wild-type controls (39). In human adenomas, bcl-2 expression is increased while low levels are generally found in carcinomas (42). This may indicate that altered expression of the bcl-2 gene initially confers a survival advantage upon the cell that is later superseded by more potent factors. The converse tends to be seen with the survival gene, bcl-w, which is particularly evident in adenocarcinomas of the colon (43). |
Colorectal adenocarcinoma and stem cell function |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
This disease process is thought to originate as a series of genetic mutations within colonic stem cells accumulated over an extended period of time. Kinzler and Vogelstein (44,45) proposed that genetic mutations occurred in three vital areas: (i) gatekeeper functions; (ii) caretaker functions; and (iii) landscaper functions, which, respectively, refer to regulation of cell growth, DNA repair and cell–extracellular matrix interactions (44,45). In characterizing the genetic mutations responsible for disordered cell proliferation in the colon, investigators have identified some of the genes that mediate cellular proliferation within the crypt (gatekeepers).
|
The APC gene—a regulator of ordered cellular proliferation |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
APC gene mutations are the first to arise in colorectal cancer. The gene is mapped to chromosome 5q21 and is mutated in 80% of sporadic colorectal tumours while being inherited as a heterozygous germline mutation in all cases of familial adenomatous polyposis coli (FAP). Patients with this condition develop multiple adenomas of the colon in their second decade of life following a mutation in the remaining wild-type APC allele. This points to APC playing a pivotal role in the regulation of mucosal proliferation. APC is a classical tumour suppressor gene or gatekeeper in the Kinzler–Vogelstein model (44,45).
The APC gene codes for a 312 kDa protein comprising 2843 amino acids. APC inhibits members of the Wnt signalling pathway, which promote the expression of ß-catenin, an enhancer of cell division within crypts (46). APC specifically promotes the phosphorylation of ß-catenin and in this form the molecule is prone to degradation. The importance of this pathway is illustrated by the observation that a mutant of ß-catenin lacking this phosphorylation site produced a 4-fold increase in cell proliferation within murine crypts (47,48). An increase in E-cadherin, a component of intercellular adherens junctions and therefore a regulator of cell contacts, was also noted (49). This process may indicate how APC-mutated cells are able to manipulate their cellular contacts to remain within the crypt, thereby avoiding migration to the lumenal surface and consequent death/exfoliation.
ß-Catenin associates with members of the HMG box family of transcription factors, T-cell factor (Tcf) and lymphoid enhancer factor (LEF) (49,50). These complexes regulate the transcription of target genes in the nucleus. The presence of mutated APC has been shown to increase the transcriptional activity of targets containing a DNA-binding site recognized by Tcf family members (51,52). The targets of this ß-catenin/Tcf/LEF pathway remain to be elucidated in most instances, although the cyclin D1 gene promoter is transcriptionally activated through an LEF-1 binding site resulting in cell cycle entry (53). C-MYC, which is often upregulated in colorectal cancer, albeit without a defined role, is also activated by this pathway (54).
ß-Catenin therefore appears to be an important factor in determining cell adhesion and proliferative signalling. Increased, unregulated expression may prove to be a vital early factor in the development of colorectal cancer. Adding weight to this point, amongst the minority of sporadic colorectal tumours with wild-type APC gene function, 50% have a dominant mutation of the ß-catenin gene rendering it resistant to degradation (52,55,56).
|
Modifiers of APC gene function exerting an additional level of proliferative control |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Multiple intestinal neoplasia (Min) mice are frequently used to model the impact of APC mutation upon the intestinal tract. These mice have a germline truncation mutation of the APC gene and, as a consequence, develop numerous, mainly small intestinal, tumours (57). Interestingly, however, mice bred on a B6 genetic background have four times the tumour load of those bred on an AKR background, implying that there is a further level of regulation (58). One gene responsible has been mapped to chromosome 4 and designated Mom-1 (modifier of Min-1). The gene encoding secretory phospholipase A2 maps to the same region and has been shown to reduce tumour number and size (59). This enzyme is involved in the production of arachadonic acid, a substrate for prostaglandin and leukotriene synthesis. No mechanism has been discovered to account for the modifying effect of this gene and it yet remains to be definitively proven whether it is this gene, or another closely situated allele exhibiting linkage with the Pla2g2a site, that is responsible for modification of APC function.
|
Cyclooxygenase-2 (COX-2) |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
Loss of the COX-2 allele, responsible for conversion of arachadonic acid to prostaglandin derivatives, decreases the number of adenomas developing in the Min small intestine (60). COX-2 expression is found to be upregulated at an early stage of tumour evolution, indicating that this gene is also able to modulate APC function. The administration of a selective COX-2 inhibitor substantially reduces the development of chemically induced tumours in rats, supporting this view (61). This enzyme is also expressed at high levels in 85% of human adenocarcinomas and 45% of human adenomas (62). Non-steroidal anti-inflammatory drugs are known to reduce the relative risk of developing colorectal cancer by 40–50%, again adding weight to the proposed interaction of these gene products (63).
|
The transforming growth factor ß (TGF-ß) signalling pathway—also promoting ordered proliferation |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
The TGF-ß signalling pathway has been shown to inhibit intestinal epithelial proliferation, particularly in colonic mucosa (64). In vivo the role of TGF-ß may be to modulate cell cycle exit and the subsequent differentiation of enterocytes in the upper crypt or villus (65–67). Alternatively increased expression has also been reported in the proliferative zone of the crypt and it is hypothesized that this factor mediates the output of cells from this area (68). Loss of responsiveness to TGF-ß is commonly seen during the development of colorectal cancer and, indeed, under these circumstances TGF-ß may become a tumour promotor by stimulating angiogenesis, causing immunosuppression and encouraging the growth of extracellular matrix providing an environment conducive to tumour growth (69).
The signal transduction pathway of TGF-ß involves a family of proteins known as Smads. Following activation of the TGF receptor these Smads are phosphorylated before complexing with Smad4, an essential step prior to transcriptional activation of target genes (70). Mutation of the Smad4 gene on chromosome 18q inactivates the TGF-ß pathway (71). It is known that loss of, as yet undefined, genes on chromosome 18q is an important step in the development of colorectal cancer (72). The Smad4 locus, also known as deleted in pancreatic carcinoma, is one such candidate gene. Mutations specific to this gene have also been demonstrated in a few colorectal cancers (73,74). Mice bred to be heterozygous for a truncation mutation of APC
716 and a null Smad4 allele were shown to develop invasive neoplasms much more frequently than mice heterozygous for APC716 alone (75).
|
Glucagon-like peptide (GLP-2), a factor that can override crypt homeostatic mechanisms |
|---|
|
TopAbstractIntroductionDefining stem cellsBasic mechanics of crypt...Stem cell locationStem cell numberPolyclonal versus monoclonal...Stem cell hierarchyEvidence for pluripotencyRegulation of stem cell...Maintaining the genomic...The role of p53...The role of Bcl-2...Colorectal adenocarcinoma and...The APC gene—a regulator...Modifiers of APC gene...Cyclooxygenase-2 (COX-2)The transforming growth factor...Glucagon-like peptide (GLP-2), a...The clonality of colorectal...Future directions in stem...References |
|---|
GLP-2 is able to stimulate crypt proliferation, increasing crypt and villus height in the mouse (76). In this setting it was able to reduce the severity of experimentally induced colitis. In achieving this result the normal crypt homeostatic mechanisms must be overcome, as a compensatory increase in apoptosis did not occur. It is hoped that the protein may also be of use in treating intestinal mucosal hypoplasias resulting from either chemotherapy or radiotherapy. The receptor for GLP-2 has recently been cloned (77). This will allow its expression in tumour tissue to be determined, an important consid