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Carcinogenesis vol.21 no.3 pp.469–476, 2000
Stem cells: the intestinal stem cell as a paradigm
Simon P.Bach1,2, Andrew G.Renehan1,2 and
Christopher S.Potten1,3
1CRC
Department of Epithelial Biology, Paterson Institute for Cancer
Research and 2Department of Surgery, Christie Hospital NHS Trust,
Wilmslow Road, Manchester, UK
3To
whom correspondence should be addressed
Email: [email protected]
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 β-catenin, an enhancer
of cell proliferation, thereby downregulating this activity
in healthy individuals.
Introduction
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
Abbreviations: APC, adenomatous polyposis coli; DBA, Dolichos biflorus
agglutinin; 3HTdR, tritiated thymidine; TGF-β, transforming growth factor β.
© Oxford University Press
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
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
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S.P.Bach, A.G.Renehan and C.S.Potten
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.
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.
Basic mechanics of crypt function
Polyclonal versus monoclonal crypts
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).
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 Nacetylgalactosamine 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
Stem cell location
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 β
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Stem cell number
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).
Stem cells: the intestinal stem cell as a paradigm
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
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 γ-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
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 midcrypt 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 longlived, 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
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–
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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
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
The response of murine intestinal stem cells to DNA damage
has been determined following exposure to low doses of
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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
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). γIrradiation 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
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:
Stem cells: the intestinal stem cell as a paradigm
(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
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 β-catenin, an
enhancer of cell division within crypts (46). APC specifically
promotes the phosphorylation of β-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 βcatenin 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.
β-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 β-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).
β-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 β-catenin gene rendering it resistant
to degradation (52,55,56).
Modifiers of APC gene function exerting an additional
level of proliferative control
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)
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 β (TGF-β) signalling pathway—also promoting ordered proliferation
The TGF-β signalling pathway has been shown to inhibit
intestinal epithelial proliferation, particularly in colonic mucosa
(64). In vivo the role of TGF-β 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-β
is commonly seen during the development of colorectal cancer
and, indeed, under these circumstances TGF-β 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-β 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-β 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
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invasive neoplasms much more frequently than mice heterozygous for APC∆716 alone (75).
Glucagon-like peptide (GLP-2), a factor that can override
crypt homeostatic mechanisms
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 consideration if
it is to be administered to patients previously treated for
malignant disease.
The clonality of colorectal carcinoma
It is a well-held view that colorectal carcinomas are derived
from the clonal expansion of a single intestinal crypt cell (78).
This paper described monoclonal X chromosome expression
in both colorectal adenomas and carcinomas which contrasted
with the polyclonal expression found in intestinal epithelial
tissues as a whole (at an early stage in the development of the
female fetus, one copy of the X chromosome is randomly
inactivated within each cell). This observation favours the
view that tumours originate from a single mutated enterocyte.
However, it still remains possible that single tumours may
arise from several cells possessing a range of genetic mutations,
which confer differing growth advantages. Through a process
of clonal evolution the cell type that is optimally adapted to
proliferate under the prevailing conditions emerges to form
the tumour. This process has been observed to occur in a
chemically induced mouse fibrosarcoma (79).
An unusual patient found to possess both an XO/XY mosaic
and FAP was used to explore the relationship between adenoma
evolution and XO or XY expression, utilizing the technique
of in situ hybridization with a Y chromosome probe (80). The
intestinal crypts of normal tissue were indeed monoclonal, as
demonstrated in previous studies (11); however, 76% of
microadenomas, but not the larger adenomas were found to
be polyclonal. The clonality of adenomas has also been
assessed in a chimeric Min/ROSA mouse model (81). This
study found 79% of adenomas to be polyclonal. It is not
known precisely how these polyclonal tumours arise and what,
if any, interaction occurs between the genetically distinct cell
types during the early stages of tumour evolution.
Future directions in stem cell science
It is hoped that stem cell research will lead us into an era
when degenerative human tissues can be readily replaced by
newly synthesized genetically identical equivalents, negating
the need for organ donation, prosthetic implants and allowing
treatment of many previously untreatable disorders. To date,
human embryonic stem cells have been isolated and grown in
culture (82). These cells have demonstrated the ability to
differentiate and form gut, skin, muscle, bone and neural
epithelium when implanted under the skin of mice. It is not,
however, possible to direct the differentiation pathways from
these progenitor cells at present and the development of
474
malignant teratomas in recipients of these cells cannot be
ruled out.
Another strategy to bring us a step closer to the cloning of
viable human tissue is to attempt to isolate lineage-specific
stem cells. Although stem cells from several tissue types can
currently be grown in culture, contamination with other rapidly
proliferating cell types can occur; cells can also cease to
exhibit stem cell functions, a factor that is difficult to determine
quickly as there are no reliable stem cell markers. In addition
some stem cell properties, such as differentiation and crypt
regeneration, are not evident in vitro. Crypt cell suspensions
from adult mouse donors have been shown to undergo epithelial
differentiation and subsequent crypt formation when injected
subcutaneously into immunocompromised mice, indicating
that functional stem cells do survive isolation and culture
procedures (83). We therefore await the development of
markers to the aid their identification and purification. It is
still not known whether these seemingly committed progenitors
are capable of regenerating tissues of other lineages. Work
recently published by Peterson et al. (84) has shown that bone
marrow stem cells are able to regenerate injured liver in a rat
model. This raises the possibility that our own cells may be
reprogrammed to express dormant areas of the genetic code
and thereby regenerate physically distinct organs or tissues.
The second main area of potential therapeutic benefit could
arise from the determination of mechanisms underlying ordered
cellular growth and differentiation. As previously stated, colorectal cancer is currently thought to be a disease originating
within colonic stem cells. Once the activities of genes mutated
in colorectal cancer are identified then this should eventually
facilitate development of novel approaches to cancer therapy
aimed at limiting the proliferative capacity of mutant clones
either through gene therapy, the administration of antagonistic
growth factors or by immunotherapy directed towards unique/
overexpressed cellular markers. The great challenge facing
investigators will be to tailor treatment to minimize side effects
upon normal tissues and this can be best achieved by detailing
the specific differences that exist between ordered and disordered cellular growth.
Acknowledgements
We thank Dr Catherine Booth for her helpful advice during the preparation
of this manuscript. S.P.B. and A.G.R. are supported by the Christie Hospital
Endowment Fund and C.S.P. is supported by the CRC.
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Received August 6, 1999; accepted September 20, 1999