1 Epithelial Biology Department, Paterson Institute for Cancer Research,
Christie Hospital NHS Trust, Wilmslow Road, Manchester M9 4BX,UK
2 EpiStem Limited, Incubator Building, Grafton Street, Manchester M13 9XX,
UK
* Author for correspondence (e-mail: potten{at}epistem.co.uk )
Accepted 26 March 2002
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Summary |
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Key words: Stem cells, DNA strand segregation, Genome protection, Intestine
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Introduction |
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In the label-retaining experiments in skin, stem cell template strands
would become labelled if 3HTdR was administered over a time span that covers
the penultimate symmetric stem cell expansionary cell divisions associated
with skin development. At this time, new stem cells and new template strands
would be formed and are, therefore, capable of becoming permanently labelled.
In experiments performed in our laboratory in 1978, we demonstrated that,
following radiation injury that stimulates stem cell amplification, in the
small intestinal crypts, where new template strands would also be synthesised,
some LRCs could be observed at the specific position in the crypts for the
stem cells (Potten et al.,
1978).
In the same paper (Potten et al.,
1978), we produced evidence that in the dorsal epithelium of the
tongue of the mouse, a tissue where the stem cells are located at a specific
position beneath tongue proliferative units, the stem cells could be pulse
labelled with 3HTdR with a high frequency if several doses were
given around 3 am, because of the extremely strong circadian rhythm
(Hume and Potten, 1976
). These
stem cells were believed to have a cycle time of 24 hours, and after 48 hours
(two divisions) some cells at the stem cell location apparently became
completely unlabelled (non-radioactive). This observation is consistent with
the stem cells having their new strands of DNA labelled, and these newly
synthesised strands would then pass to the dividing transit daughter. Two cell
divisions would be required for the stem cells to `clean themselves' of
radioactivity (Fig. 1).
|
The crypts of the small intestine of the mouse have been well studied and
characterised in terms of cell proliferation and cellular organisation. Each
crypt in the steady state adult contains about 250 cells and is composed of
between four and six cell lineages, each with a lineage ancestral stem cell.
These cells are believed to divide once a day and produce lineages containing
six to seven dividing transit generations, and during the course of this
amplification up to about four distinct differentiated cell phenotypes can be
produced. It is believed that the ultimate commitment to differentiation only
occurs at the level of about the third generation in the lineage. Thus the
crypt contains a hierarchy of stem cells with four to six ultimate stem cells
that are responsible for all the day to day cell replacement and a population
of reserve potential stem cells in the first, second and third transit
generation that are capable of repopulating the crypt if the ultimate stem
cells are damaged or deleted (Potten et
al., 1997; Potten,
1998
). No marker exists for these early generation cells, but the
crypt represents an elegant cell biological model system since the stem cells
have a strict spatial distribution along the crypt-villus axis, with the
ultimate stem cells being located in an annulus of cells about four cell
positions from the base of the crypt, immediately above one of the
differentiated lineages, the Paneth cells, which are located at the crypt base
(Potten et al., 1997
;
Potten, 1998
)
(Fig. 2). The ultimate stem
cells appear to have an exquisite radiosensitivity such that a single hit
anywhere in their DNA molecule can trigger an altruistic apoptotic cell
deletion (Potten, 1997; Hendry et al.,
1982
; Potten and Grant,
1998
; Potten et al.,
1992
). These cells do not appear to have the option to repair DNA
damage but rather commit suicide, which can be easily compensated for by
division of either undamaged neighbouring ultimate stem cells or cells in the
much more resistant and repair-efficient potential stem cell compartment
(Fig. 2). Although
stem-cell-specific markers have not been described as yet, the possible
ability of these cells to retain 3HTdR label, if given under
appropriate conditions, does suggest one way in which stem cells could be
marked. Other possible approaches are also being developed (e.g. using an
antibody (C.S.P and H. Okano, unpublished) to the RNA-binding protein
Musashi-1 (Nakamura et al.,
1994
), which will greatly facilitate stem cell studies).
|
Attempts have been made previously
(Potten et al., 1978) to label
template strands in the ultimate stem cells of the small intestinal crypt
(i.e. create LRCs as in the skin). This can be achieved at the times when new
stem cells and new template strands are being produced, either during gut
development or following injury and destruction of the ultimate stem cell
population (e.g. by radiation) and the re-establishment of the crypt lineage
from the potential stem cell compartment. A dose of 8 Gy of Cs 137
radiation to an adult (10 to 12 week old) BDF1 mouse results in the killing or
reproductive sterilisation of all but about one of the potential stem cells
(Potten and Hendry, 1985
).
This single surviving stem cell repopulates the crypt via a clonal expansion
and hence is sometimes referred to as a clonogenic cell. During the course of
this clonal expansion, it re-establishes the entire stem cell lineage. This
process takes 2 to 3 days (Potten and
Hendry, 1985
). Our earlier work
(Potten et al., 1978
) showed
that 3HTdR administered repeatedly (every 6 hours) over the first
48 hours of this post-irradiation period resulted in regenerated crypts with
all the cells labelled by the 48 hour time point. If, however, time was
allowed to elapse, for example, for a further week beyond the 2 day labelling
time, a total of about four to eight LRCs per crypt were observed to be
distributed around the fourth position from the bottom of the crypt. During
this week after labelling, the stem cells would have divided at least eight
times, but probably more like 12 times, since their cycle time soon after
irradiation is shorter. Thus, the stem cells should have diluted their label
to sub-threshold levels, but they in fact retained abundant label
(Potten et al., 1978
). This
observation is consistent with, but does not prove, the Cairns' selective DNA
segregation hypothesis.
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Materials and Methods |
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In order to generate template strand labelling, the principles that have
been applied for label-retention studies in the skin have been adopted and
modified (Bickenbach, 1981;
Morris et al., 1985
;
Cotsarelis et al., 1990
). Two
distinct protocols were adopted in order to label DNA strands in stem cells at
a time when new stem cells are being made, either during development or during
post-irradiation regeneration of the tissue. The protocols are outlined in
Fig. 3.
|
Labelling during gut development (template labelling 1)
The first of the labellings (i.e. during development) involved labelling
juvenile animals with twice daily injections of 3HTdR (at 9 am and
9 pm) for three consecutive days. The doses of 3HTdR varied
depending on the age (and hence, size) of the animals. Each dose was 15 µC
for animals aged between 11 and 21 days post-natum, 25 µC per injection for
mice 21 to 37 days of age and 50 µC for mice older than 37 days. All
animals were sacrificed at 11 weeks of age, when the small intestinal tissue
was fixed (Carnoy's), sections prepared (haematoxylin and eosin staining) and
autoradiography undertaken (K5 emulsion). The exposure time varied depending
on the nature of the specific experiment but was generally 14 days. LRCs were
counted in transverse sections of the intestine and expressed as the number of
LRCs per intestinal circumference (Potten
and Hendry, 1985), having excluded from the counts any labelled
nuclei associated with the differentiated Paneth cell population. The
threshold for detecting LRCs was set at five or more grains per nucleus. The
experiment was repeated more than once, and four to six animals were used per
experimental group, and up to 10 intestinal circumferences were counted per
animal.
Labelling during post-irradiation crypt regeneration (template
labelling 2)
The second labelling protocol for LRCs (i.e. during regeneration) involved
irradiating 12 week old BDF1 mice with a dose of 8 Gy of Cs137
rays to the whole body (3.5 Gy/minute). Then during the crypt
regeneration phase, which takes 2-3 days, 3HTdR at a dose of 25
µC per injection was given every 6 hours for the first 48 hours
post-irradiation. The animals were then left for a period of 8 days, during
which the label present in the majority of the proliferating cells diluted to
sub-threshold levels, leaving only LRCs present in the crypt.
Double labelling of LRCs (new strand labelling)
A second series of experiments was then conducted that was designed to
label newly synthesised strands with BrdUrd in LRCs present at 11 weeks of age
using the juvenile labelling protocol and at 8 days post-irradiation using the
post-irradiation protocol. The BrdUrd labelling schedule involved injections
of 1 mg in 0.1 ml saline, with injections being delivered every 6 hours for 48
hours. Samples were then taken immediately after the end of the BrdUrd
labelling (40 minutes) and 2 to 10 days post-BrdUrd labelling
(Fig 3). In these experiments
the proportion of double-labelled LRCs was determined on a cell positional
basis (see below) over time to determine whether the 3HTdR and
BrdUrd labels behaved in the same fashion (diluted at the same rates).
Sections were dewaxed, rehydrated and the endogenous peroxidase was inactivated with 1% hydrogen peroxide in methanol for 30 minutes. DNA was denatured to single strands by immersing sections in 1M HCl at 60°C for 8 minutes and then neutralising in boric acid buffer for 6 minutes at room temperature. Sections were blocked using normal rabbit serum 1/20 for 30 minutes prior to the application of the anti-bromodeoxyuridine antibody (MAS 250b; Sera Labs, Crawley Down, Sussex, UK) diluted 1/5 for 1 hour. After washing in PBS, a peroxidase-conjugated rabbit anti-rat secondary antibody was applied at a dilution of 1/100 in 10% normal mouse seum for 1 hour. Sections were again washed in PBS and developed using 3,3 Diaminobenzidine (DAB). The sections were washed in deionised water overnight prior to autoradiography being performed. All the sections were counterstained with thionin prior to examination.
Scoring
All LRC scoring at the level of the crypts and all double-labelled LRC
scores were made on a cell positional basis, along the long axis of
longitudinally sectioned crypts. In this way a frequency plot of the labelling
pattern against cell position could be generated, and the labelling
characteristics at the stem cell position (cell position four) could be
assessed. Details of the gut bundling procedure that ensures good transverse
sections of the intestine and hence longitudinal sections of crypts have been
previously published (Potten and Hendry,
1985). Details of cell positional scoring have also been published
previously (Potten et al.,
1997
; Potten,
1998
; Potten and Grant,
1998
).
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Results and Discussion |
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Fig. 5 shows that the peak
of the labelling index, about 4%, occurs at cell position four for the LRCs.
The fourth cell position from the base represents the presumed positioned of
the stem cells (Fig. 2) [see
(Potten et al., 1997;
Potten, 1998
] for the cell
positional scoring technique]. The labelling index distribution is quite
broad, with a long tail to the right. The low percentage of LRCs at cell
position four, the stem cell position, is a consequence of (a) the low
frequency of ultimate stem cells per crypt (about four to six are distributed
between cell positions three and seven), (b) probable sub-optimal labelling
for these LRCs possibly because of highly variable cell cycle times and/or (c)
the possibility that in the 3-5 week interval that some stem cells still
retain a higher than 50% self-maintenance probability, that is, they are still
making new stem cells, randomly segregating their DNA and making new template
strands and, hence, diluting their label. The actual data are presented with
standard error limits rather than our usual presentation of a smoothed cell
positional frequency plot.
|
An alternative strategy for generating LRCs in the crypt is to use the
post-irradiation regeneration process described for experiments conducted in
1978 (Potten et al., 1978).
Such experiments have been repeated here using a dose of 8 Gy
-radiation delivered to 12 week old BDF-1 mice and a labelling protocol
of 25 µCi of 3HTdR delivered every 6 hours between 6 and 48
hours post-irradiation. Since cell cycle times in this situation are greatly
shortened compared with the steady state, this protocol labels virtually all
the cells in the crypt, including the regenerated ultimate stem cell
population. Eight days following the last 3HTdR injections, clearly
identified LRCs are apparent in the crypt. The autoradiographic background is
higher than usual for pulse labelling experiments because virtually all cells
contain some residual radioactivity resulting from the grain dilution process
following cell division. Nevertheless the LRCs are clearly discernible
(Fig. 6). The cell positional
distribution for these LRCs is shown in
Fig. 5, and the peak yield of
about 10% is also seen at cell position four with the same long tail to the
right. Label is retained in these cells in spite of up to 12 rounds of cell
division.
|
Thus, it is relatively easy to perform experiments to generate cells in the small intestinal crypt that retain the 3HTdR label in spite of numerous assumed rounds of division. To provide evidence that these cells progress through the cell cycle and enter mitosis is something of a `needle in a haystack' task. These LRCs are distributed in the crypt with their highest frequency at the positions along the crypt axis, which is presumed to be the location for the stem cells. If four out of the 16 cells in the crypt annulus of cells at cell position four were labelled stem cells, a cell position four labelling frequency (LI) of 25% might be expected. However, since cell position four is the average position for the stem cells and their actual location may be spread between cell positions three and seven, a lower frequency and a tail to the right of the LI distribution would be expected.
Stem cells can currently only be studied using functional tests; however
there are data that suggest that one property of the ultimate stem cells is an
exquisite sensitivity to DNA damage and, hence, sensitivity to radiation
(Potten, 1977;
Potten and Grant, 1998
). A low
dose of 1 Gy of
radiation induces a rapid (within 3-6 hours) apoptotic
response in four to six cells at cell position four, the stem cell position,
and these apoptotic cells have a cell-positional distribution similar to the
theoretical stem cell distribution and also to the distribution of LRCs. Thus
an additional `needle-in-the-haystack' experiment would be to determine
whether LRCs can be induced into apoptosis by a dose of 1 Gy. Owing to the
rarity of these events, quantitative data are difficult to obtain, but
Fig. 7 illustrates that LRCs
can clearly be labelled by an S phase marker such as bromodeoxyuridine
(BrdUrd) and that LRCs in mitosis can occasionally be identified. Indeed
images consistent with an asymmetric distribution of 3HTdR
autoradiographic grains at anaphase in LRCs can even be obtained. Although
difficult to photograph, 1 Gy of radiation delivered 16 days after LRCs,
generated by the 8 Gy regeneration protocol, also results in LRCs in apoptosis
at 4.5 hours (Fig. 7).
|
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The data presented in this paper are consistent with LRCs retaining
3HTdR label, because they contain tritium-labelled template strands
of DNA that are selectively segregated and retained in the ultimate stem cell
population of the crypt in spite of many rounds of cell division. If the newly
synthesised strands in such cells are subsequently labelled with BrdUrd, the
tritium and BrdUrd DNA markers in the LRCs behave with the passage of time in
totally different ways. The tritium assumed to be in the template strands is
retained, and the BrdUrd in the newly synthesised DNA strands is selectively
removed from the nucleus of the actual stem cells at, or around, the time of
their second division after labelling, consistent with the model proposed by
Cairns (Cairns, 1975) and the
preliminary data presented from our laboratory
(Potten et al., 1978
). The
mechanisms used by the cells to selectively retain the template strands remain
unclear.
The observations on strand segregation presented here, together with other
observations related to cells at the stem cell position, provide an
explanation for the rarity of cancers in the small bowel of mice and men. The
selective retention of template strands in the ultimate stem cells ensures
that replication-induced errors are effectively avoided. One possible
associated consequence of the evolution of this stem-cell-protective mechanism
is a prohibition of DNA excision repair processes, since these pose the risk
of sister chromatid exchanges. Exchange and recombination events would involve
a mixing of old and new strands and would thus compromise the protective
mechanism. The inability to undergo repair processes might be expected to
render these cells highly sensitive to genotoxic damage, since some enzymes
would be common to both processes. Indeed, it appears that cells at the stem
cell position do have an exquisite radiosensitivity. They are so sensitive
that doses as low as 1 to 5 cGy induce an apoptotic suicide in cells assumed
to be the ultimate stem cells, thus removing the potential genotoxic damage by
removing the cell (Potten,
1977; Hendry et al.,
1982
; Potten and Grant,
1998
; Potten et al.,
1992
). This represents a second highly effective protective
mechanism that would deal with random errors induced in the template strands
by background radiation or genotoxic chemicals.
The removal of an occasional stem cell as a consequence of incurring DNA damage is easily compensated for by either a symmetric division of a neighbouring ultimate stem cell or the recruitment of a potential stem cell from generation 1, 2 or 3 (depending on the level of cytotoxicity) transit cells (see circles in Fig. 2). In the former case, there is a risk associated with the regenerative division and the elevation of newly synthesised strands with potential errors to form new templates. However, the risk here is only that associated with a single round of DNA replication. In the latter case (the recruitment of a potential stem cell), the risk again is one associated with the elevation of newly synthesised strands to template status, but again only a minimum number of rounds of DNA synthesis (one to a maximum of three) would be involved. It is interesting that p53 plays a critical role in regulating the altruistic cell suicide following genotoxic damage and may also be playing an important role in regulating the asymmetric strand segregation (J. Cairns, personal communication). This hierarchy within the stem cell compartment (ultimate and potential stem cells) ensures that if many, or all, of the ultimate stem cells incur template errors and commit suicide, the crypt survives and is repopulated from the repair-efficient potential stem cells a fail-safe ultimate protective mechanism. It is, however, unlikely that in nature all ultimate stem cells die at one time, but this can be induced in the laboratory.
One final consideration related to the selective DNA strand segregation hypothesis in stem cells concerns the telomeretelomerase hypothesis for ageing. If the ultimate stem cells have a mechanism for selectively sorting the DNA template strands that they retain, it would be predicted that the telomeres on the template strands would not be subject to the same telomere strand-end replication problems that would be expected for the newly synthesized strand. This may help to account for the fact that there is little evidence for a decline in the proliferative capacity of the 4x106 small intestinal ultimate stem cells over the 1000 divisions estimated to occur during the lifetime of a laboratory mouse (possibly 5000-6000 divisions in each of approximately 2x108 ultimate stem cells in man).
The mechanisms involved in the selective sorting of all old and new DNA strands at mitosis remain to be elucidated. Possibilities here range from the existence of some linker molecule joining the ends of all old or new strands, such that moving one would pull them all (like a string of sausages), to an association of all old or new strands with the spindle proteins associated with old and new centrioles.
It is also unclear what mode of DNA segregation (random or selective) occurs in stem cells that are actively making extra stem cells (during development or tissue regeneration).
The question of what happens in terms of these protective mechanisms in
other tissues remains unanswered. However it does look as if selective
segregation must be occurring in the interfollicular and hair follicle bulge
stem cells to account for the label retention that is seen in those sites
(Bickenbach, 1981;
Morris et al., 1985
;
Cotsarelis et al., 1990
). There
is also some evidence in support of this concept for dorsal tongue epithelium
(Potten et al., 1978
). It is
possible that in other tissues different networks of genes operate to regulate
strand segregation (symmetric versus asymmetric divisions), DNA repair and
apoptosis. Certainly in the adjacent intestinal epithelium of the large bowel,
where data are not available at present for strand segregation and label
retention, the protective altruistic suicide in the ultimate stem cells is
compromised by the active expression of the bcl-2 protein
(Merritt et al., 1995
). Thus in
this region of the gut the evolutionary pressures have selected for survival
and repair of DNA damage in the crucial stem cells rather than apoptotic
suicide and replacement (regeneration) as a means of correcting the DNA
damage. This may be because many more genotoxic molecules are encountered in
the large bowel, and if the altruistic cell suicide mechanism were adopted,
repeated stem cell regeneration or replacement with the elevation of (error
prone) newly synthesised DNA strands to template status would be repeatedly
required. This may carry a greater risk than the possibility of errors
associated with the excision repair process. However, the abandonment of the
apoptosis protective mechanism, which in conjunction with the selective strand
segregation process provides such an effective protection against the genetic
defects leading to cancer in the small bowel, raises the risk of such events
occurring more frequently in the large bowel
(Potten et al., 1992
;
Merritt et al., 1995
).
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Acknowledgments |
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References |
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