Oncogene (1997) 14, 2759 ± 2766
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Apoptosis in small intestinal epithelia from p53-null mice: evidence for a
delayed, p53-indepdendent G2/M-associated cell death after g-irradiation
Anita J Merritt1,3,4, Terence D Allen2, Christopher S Potten1 and John A Hickman3
Cancer Research Campaign Departments of 1Epithelial Biology and 2Ultrastructure, The Paterson Institute for Cancer Research,
Christie Hospital Trust, Wilmslow Road, Manchester and 3Molecular and Cellular Pharmacology Group, School of Biological
Sciences, The University of Manchester, Manchester M13 9PT, UK
The death of small intestinal epithelial cells has been
characterized and quantitated after irradiation of mice
rendered homozygously null for the p53 gene. In wild-type
animals homozygous for p53 a rapid (4.5 h) elevation of
p53 protein was observed in the proliferative compartment
of the crypts after 8 Gy of irradiation. Cells underwent
cell death by apoptosis in this region. We had reported
previously a total repression of apoptosis in small
intestinal crypt epithelia 4.5 h after the g-irradiation
(8 Gy) of p53 homozygously null animals. Thus, while 400
apoptotic cells were observed in 200 half crypts taken
from wild-type animals at 4.5 h, this fell to background
levels (10 ± 30) in the p53 null animals (Merritt et al.,
1994) and did not increase by 12 h. However, we have
now found a delayed initiation of a p53-independent
apoptosis after 8 Gy of g-radiation: at 24 h, approximately 100 apoptotic cells were observed in 200 half
crypts. This late wave of apoptosis was not observed after
1 Gy of g-radiation. The morphological appearance of
this p53-independent apoptosis suggested that death may
have arisen as the result of aberrant mitosis. Analysis of
the regeneration of crypts 3 days after irradiation of mice
with between 11 and 17 Gy showed that there was no
signi®cant increase (P=0.135) in the potential of
clonogenic cells from the p53 null animals to repopulate
the crypts. The data support the idea that a p53independent apoptotic mechanism permits the engagement
of apoptosis, probably by a mitotic catastrophe, after
8 Gy of g-irradiation in vivo and that a loss of p53 does
not make these epithelial cells radioresistant in vivo to
doses of 8 Gy and above. In contrast, irradiation with
1 Gy failed to induce a p53-independent apoptosis in vivo,
suggesting that the p53 `sensor' of damage was more
sensitive than that engaging the p53-independent mechanism of cell death.
Keywords: p53; apoptosis; p53-independent apoptosis;
radiation; small intestine; G2/M checkpoint
Introduction
The initiation of death by apoptosis in cells which
have sustained DNA damage is a critical process
which can limit oncogenesis. The survival of cells with
Correspondence: JA Hickman
4
Current address: McArdle Laboratory for Cancer Research, 1400
University Avenue, Madison, Wisconsin 53706 ± 1599, USA
Received 16 August 1996; revised 27 February 1997; accepted
4 March 1997
DNA damage is permissive for subsequent malignant
transformation. The tumor suppressor p53 has been
suggested to play a key role in determining the fate
of cells which have received potentially carcinogenic
DNA damage (Lane, 1992). The amount of p53
protein is increased in DNA-damaged cells and it
may act either as a transcriptional activator or
repressor. According to cell type, the nature of
changes in gene expression and whether the cells are
proliferating or not, DNA damaged cells may either
undergo a G1 and/or, in some cases, a G2 cell cycle
`checkpoint' (Kastan et al., 1991; Kuerbitz et al.,
1992; Stewart et al., 1995; Agarwal et al., 1995;
Guillouf et al., 1995). This may allow repair of a
damaged DNA template or surveillance of damaged
spindles (Cross et al., 1995). Alternatively, p53 may
induce cell death by apoptosis (Yonish-Rouach et al.,
1993). In non-proliferating thymocytes from mice
which have been rendered genetically homozygously
null for p53 (7/7), radiation and other types of
DNA damage failed to initiate apoptosis in short
term assays of cell integrity (Clarke et al., 1993; Lowe
et al., 1993a). However, DNA damage-induced cell
death in mitogen stimulated, proliferating T lymphocytes from the same p53 7/7 mice was at the same
levels as in p53+/+ cells (Strasser et al., 1994) and
the clonogenic survival of irradiated primary fibroblasts from p537/7 mice also showed no di€erence
from the p53+/+ cells in their sensitivity to DNA
damage (Slichenmeyer et al., 1993). These latter
studies are important because they reported clonogenic assays of survival, removing the uncertainty that
a loss of p53 may have only a temporal e€ect on the
initiation of cell death, delaying it rather than
inhibiting it. They also provide a measure of the
maintenance of cellular functionality (replicative
potential). Taken together these results suggest that,
in some cell types, p53 independent mechanisms of
cell death initiated by DNA damage may be as
important as p53-dependent mechanisms.
We are interested in the determinants of apoptotic
death in the rapidly proliferating epithelia of the
gastrointestinal tract (reviewed by Potten, 1992). In
particular, we wish to determine what factors
promote the so-called `altruistic' apoptosis of
damaged stem cells in the small intestine, an e€ect
which we have proposed may limit cancer incidence
at this site, essentially by deleting cells with DNA
damage so as to prevent tumour initiation (Potten,
1992). The contribution that expression of p53 made
to the induction of apoptosis in the epithelia of the
small intestine was investigated using p53 homozygously null mice (7/7). The normal wave of
p53 independent cell death
AJ Merritt et al
2760
apoptotic death observed between 4 h and 8 h after g
radiation (8 Gy) was completely abrogated in the
absence of p53 (Merritt et al., 1994; Clarke et al.,
1994). This suggested that the genotypically p53 null
small intestinal epithelia were radioresistant, in the
way that thymocytes appeared to be. In the present
study we have investigated this further by analysis of
the long-term fate of the p53 null epithelial cells in
the small intestinal crypts both by a morphological
analysis and by assays of the clonogenic potential of
cells which remain viable shortly after irradiation.
Results
Induction of apoptosis in the small intestinal epithelia
Figure 1 shows the numbers of apoptotic cells at
various times post irradiation, scored at positions 1 to
7 in 200 half crypts from p53 wild type (+/+),
heterozygotes (+/7) and null (7/7) mice. After
8 Gy of g radiation, the small intestinal crypt epithelia
from the p53+/+ animals showed a rapid induction
of apoptosis which was declining by 24 h. The results
at the early timepoints (512 h) for both the p53+/+
and p537/7 animals essentially recapitulate those
reported by us (Merritt et al., 1994) and by Clarke et
al. (1994), with a highly signi®cant reduction of
radiation-induced apoptosis in the p537/7 animals.
At the lower dose of 1 Gy, there was a reduction of
apoptosis in the +/7 genotype in comparision to
wild-type +/+ animals, as he had been reported
previously for 5 Gy or irradiation (Clarke et al.,
1994). We were surprised to observe a late wave of
apoptosis in the p537/7 null animals at 24 h after
8 Gy. This late wave of apoptotic cell death was still
apparent at 40 h (Figure 1). Irradiation of animals
with 1 Gy (Figure 1) shows that the p53 null genotype
completely inhibited the apoptosis otherwise observed
in both the +/+ and +/7 mice at 4.5, with no late
apoptoses at 24 h or 40 h.
Figure 2a shows the morphology of a longitudinal
section of the crypts of murine small intestine from a
p537/7 animal 24 h after 8 Gy of radiation, stained
with haematoxylin and eosin (H&E) and incubated
with terminal deoxynucleotidyl transferase for the
detection of apoptosis-associated DNA strand
breaks. Some cells with an apoptotic morphology
had the appearance of giant cells, with distinct
fragments of condensed chromatin in their nucleus
(arrows). The cleavage of DNA to form signi®cant
numbers of strand breaks in these large cells was
con®rmed by the immunohistochemistry of DNA
fragmentation using terminal deoxynucleotidyl transferase and biotinylated dUTP (ISEL) (Figure 2a and
b). Further examples of these apoptotic cells are
shown in Figure 2c to 2i, where is it clear that not all
apoptotic cells or apoptotic bodies were ISEL
positive.
Electron microscopy con®rmed the unusual morphology of some of the late, p53-independent cell
deaths, in comparison to classically apoptotic cells
(Figure 3). Many of these giant cells (Figure 3A)
contained marginated and condensed chromatin in
nuclei which were large and, in some cases appeared to
be attempting mitosis.
Figure 1 Kinetic analysis of the appearance of apoptotic cells at
positions 1 ± 7 from the base of the crypt in the small intestine of
mice with the p53 genotypes shown. The upper panel shows the
response to 8 Gy of g-irradiation and the lower panel to 1 Gy.
Results showing signi®cant di€erences are denoted on the x-axis
DNA synthesis in small intestinal crypt epithelia after
irradiation
The incorporation of radiolabelled thymidine into
small intestinal crypt epithelial cells undergoing DNA
synthesis is shown in Figure 4. The labelling index for
p53 wild type (+/+) and homozygously null (7/7)
unirradiated animals was signi®cantly di€erent with
respect to the cell positions in the crypt at which
proliferative cells were found (positions 10 ± 17). These
are not the cell positions at which p53 is optimally
induced after irradiation (positions 4 ± 8) (Merritt et al.,
1994). Four h after irradiation with 8 Gy a signi®cantly
greater percentage of cells in small intestinal crypts
from p537/7 mice incorporated radiolabelled thymidine than those from the p53+/+ mice. This increase
in labelling index in the p537/7 crypts was at cell
positions which showed greatest expression of p53 after
irradiation (Merritt et al., 1994). At 4 h, the percentage
of proliferative cells, in both wild-type and heterozygotes, fell only very marginally despite the imposition
of considerable amounts of cell damage and loss in this
region of the crypt (see Figures 1 and 2). Minimum
labelling was observed at 14 h and there was evidence
of regenerative recovery at 24 h.
Changes in mitotic index after irradiation
Table 1 shows the mitotic indices of the small intestinal
crypt epithelia from p53 wild type (+/+), heterozygotic (+/7) and homozygously null (7/7) animals
various times after irradiation with 8 Gy of g-radiation.
p53 independent cell death
AJ Merritt et al
In all genotypes 8 Gy inhibited mitosis for at least 6 h
after irradiation, i.e. irrespective of p53 genotype, but
by 9 and 12 h the crypt cells from the p53 7/7 mice
had signicantly recovered. A second reduction of
mitotic index was observed at 32 h and was presumed
to be part of an oscillatory recovery (Potten, 1990).
Clonogenic survival of small intestinal crypt from
p53+/+ and p537/7 mice
Figure 5 shows the results from an experiment designed
to assess the survival of stem cells in the small
intestinal crypts after radiation. The regrowth of
crypts was assessed in mice from the di€erent p53
backgrounds. The data show that there was no
signi®cant di€erence in the survival of clonogenic
stem cells according to p53 status (P=0.135 by an ftest). Estimates of clonogenic cell numbers by the `split
dose' method (see Materials and methods) were made
after 8+8 Gy and 12+12 Gy of radiation. After
8+8 Gy the numbers of clonogenic cells were
estimated to be 4+2 in the p53+/+ animals and
6+2 in the p53 nulls. After 12+12 Gy these ®gures
were 13+8 and 16+15 respectively.
Figure 2 The morphology of apoptotic cells from the small intestinal crypts of a p537/7 mouse 24 h after 8 Gy of g-irradiation.
The apoptotic cells (arrows) stained by the use of terminal deoxynucleotidyl transferase (ISEL) (see Materials and methods) are
stained brown. (a) and (b) show longitudinal crypt sections with both apoptotic cells (arrows) and apoptotic fragments
(arrowheads). (M) = mitotic cells; (P) = Paneth cells. (c ± i) show a range of individual cell morphologies. In (c), the events
observed represent a possible sequence of events as apoptosis takes place in a cell attempting mitosis: A1 resembles a mitosis at a
very early stage of apoptosis where chromatin is beginning to condense and, A2, starts to fragment (ISEL+). A3 shows a fully
apoptotic morphology which is ISEL positive. (d ± i) show further examples. In these it is shown that although the majority of
morphologically apoptotic cells (arrows) or apoptotic bodies (arrowheas) were ISEL positive, there were examples with condensed
chromatin which were negative. (a,61400; b ± i,62500)
2761
p53 independent cell death
AJ Merritt et al
2762
Figure 4 The positional distribution of 3H-thymidine labelled
cells, to give a % labelling index (LI) in the small intestinal crypts
of mice with p53+/+, +/7 or 7/7 genotypes at various times
after 8 Gy of g-irradiation (cp = cell position)
Table 1 Mitotic indices of small intestinal crypt epithelia from
animals of different p53 genotypes various times after 1 or 8 Gy of girradiation
Figure 3 Electron micrographs showing: (A) the ultrastructure of
p53-independent apoptosis of small intestinal epithelial cells 24 h
after 8 Gy of g-irradiation (64300). (B), the morphology of an
apoptotic cell from a small intestinal crypt of a p53+/+ mice at
8 h. The micrograph (B) shows that there is (1) an apoptotic cell
which appears to have previously phagocytosed an apoptotic
body (2) and that both of these have been phagocytosed by a
healthy cell, whose nucleus is marked N. P = Paneth granules
(64300)
p53 genotype
+/+
+/7
7/7
0 Gy
2.7
n=4
0.1a
n=4
0a
n=4
0.3a
n=5
2.1b
n=4
2.0c
n=4
1.7c
n=4
1.4b
n=4
2.3
n=4
3.6
n=4
0.1a
n=4
0a
n=3
0.5b
n=3
1.9
n=4
ND
2.4
n=4
0.2b
n=4
0.1b
n=4
1.5
n=4
3.5
n=3
2.9
n=4
1.8#
n=4
1.9#
n=4
1.8
n=4
8 Gy 4.5 h
8 Gy 6 h
8 Gy 9 h
8 Gy 12 h
8 Gy 24 h
Discussion
It has been suggested that in some cell types the loss of
a wild-type p53 genotype renders cells resistant to the
lethal e€ects of DNA damage (Lowe et al., 1993a;
Clarke et al., 1993; 1994; Merritt et al., 1994). The loss
of p53 as the `guardian of the genome' (Lane, 1992)
might therefore be important in promoting the survival
of cells which had undergone the initiating events of
carcinogenesis, in the progression of tumours because
of increased genetic instability (reviewed by Almasan et
al., 1995), and in resistance to radiation therapy and
genotoxic chemotherapy (Lowe et al., 1993b, 1994).
Indeed, in our own previous study, which was the ®rst
to investigate the e€ects of the speci®c loss of p53 on
the response to DNA damage in vivo, utilising p53 null
mice, we suggested that epithelial cells of the
gastrointestinal tract were essentially rendered radioresistant by the deletion of the p53 gene (Merritt et al.,
1994), results con®rmed by Clarke et al. (1994) using
di€erent p537/7 mice. Clarke et al. (1994) also
showed that, in comparison to the +/+ animals,
8 Gy 32 h
8 Gy 36 h
8 Gy 40 h
1.9b
n=4
1.3a
n=4
3.3
n=4
n=number of experiments. aP40.001, bP40.02, cP40.05 compared
to controls (t-test). #, 32, 36 and 40 h signi®cantly lower than 12 h
(P40.05)
DNA synthesis, measured by bromodeoxyuridine
incorporation, continued in small intestinal crypt
epithelia after irradiation of the p537/7 animals.
However, observations of short-term radioresistance in
the absence of p53 contrast with in vitro data using
cells from p537/7 mice, where, using a clonogenic
assay, there was no di€erence in the long-term survival
of cells derived from p53+/+ or 7/7 mice
(Slichenmeyer et al., 1993, Strasser et al., 1994).
Furthermore, with respect to the intestinal tract, in
vivo studies by Clarke et al. (1995) of p537/7 mice
p53 independent cell death
AJ Merritt et al
Figure 5 Crypt survival curves for p53+/+ and 7/7 mice.
Groups of four or more mice were irradiated with 11 to 17 Gy of
g-radiation and sacri®ced 3 days later. The numbers of surviving
crypts/regenerating crypts was estimated as described (Materials
and methods). Values from individual mice were plotted. The
survival curves were not signi®cantly di€erent by an f-test
(P=0.135)
crossed with APC+/7 mice showed that there was no
increase in the incidence in the rate of adenoma
formation; it was argued cogently that the prevention
of mutation in this tissue, which might have been the
result of the engagement of a p53-dependent cell death
or the engagement of a cell cycle checkpoint, was
achieved instead by some p53 independent route.
Similar conclusions were reached with respect to the
loss of p53 on the events initiating skin papilloma
formation (Kemp et al., 1993) and a recent study of
sporadic human colon cancers has suggested a limited
role for p53 in the maintenance of genetic stability
(Kahlenberg et al., 1996).
In the light of these questions about the
importance of the role of p53 in the initiation of
some epithelial malignancies, we investigated the long
term fate of epithelial cells from the small intestinal
crypts of the p53 null mice which had not undergone
apoptosis but had sustained considerable DNA
damage after 8 Gy of g-radiation (Merritt et al.,
1994). It was considered that their continued viability
might permit DNA repair, although possibly not with
high ®delity since Clarke et al. (1994) reported that
they continued to progress into S-phase, suggesting
that this was due to the lack of any G1 checkpoint.
It is this scenario, of continued DNA synthesis and
subsequent division in the presence of DNA damage,
which is considered to be able to ®x clastogenic
damage so as to promote the emergence of malignant
cells. However, analysis of cell viability beyond 12 h
shows that there was a wave of apoptosis in the small
intestine at 24 and 40 h in the p53 null animals
irradiated with 8 Gy (Figures 1 and 2). The initial
peak of radiation-induced apoptosis induced by 8 Gy
was absent from the crypts of the p53 null animals,
as we reported previously (Merritt et al., 1994). These
cells appeared viable and, in agreement with the
studies of Clarke et al. (1994) incorporated deoxyntucleotides into DNA (Figure 4). We presume that
these, the `undead' cells, were able to complete DNA
synthesis but then reach a G2/M checkpoint, as
described in other p537/7 cells (Agarwal et al.,
1995; Strasser et al., 1994). This is supported by our
observation that mitotic ®gures were initially absent
from all epithelial cells irrespective of p53 genotype
up to 9 h after 8 Gy of irradiation, a result similar to
that reported by Clarke et al. (1994) after 5 Gy of
radiation. After accumulation in what we assume to
be the G2 phase of the cell cycle, a p53-independent
mechanism of cell death was then initiated in cells
which presumably retained signi®cant amounts of
DNA damage. G2 has been suggested to be the
common radiation-induced checkpoint for these cells
(Lesher and Bauman, 1969). A puzzling aspect of this
delayed cell death concerns those cells which were
irradiated when in late S-phase, which would shortly
reach G2. Why was death by a p53-independent
pathway not initiated within 12 h, the time it took for
mitosis to be reestablished (Table 1)? In this way it
would be expected that a continuous and steady
number of apoptotic cells would be observed from
12 h onwards, as cells move to G2, undergo a
checkpoint and die. The delay of a further 12 h,
and their presumed movement into mitosis, may be
caused by the requirement for the synthesis of some
trigger for progression into M-phase.
The morphology of some of the apoptotic cells
undergoing a p53-independent cell death was quite
unlike that of those seen 3 ± 4 h after radiation of
p53+/+ mice. These were approximately twice the
volume, and super®cially, appeared to be either
multinucleate or to possess many condensed chromatin masses (Figures 2 and 3). In addition to their
positive staining for DNA breaks using the TdT
reaction (Figure 2) electron microscopy con®rmed
that they were apoptotic, although with an unusual
morphology (Figure 3A). Electron micrographs con®rmed that these were large cells, presumably which
had reached G2, and had a pattern of condensed
chromatin which was marginated but in lobe-like
structures. This was strongly reminiscent of the
morphology of vincristine-induced apoptosis in the
small intestine (Harmon et al., 1992) suggesting some
aberration during mitosis. Indeed, we observed the
occasional apoptotic cell that appeared to be in mitosis
which was also TdT positive (Figure 2).
When animals were irradiated with 1 Gy, there was
again an early onset and peak of apoptosis between
4.5 h and 9 h in animals with wild-type p53 although,
as expected, the levels were reduced in comparison to
those given 8 Gy and was observed to be gene dosage
dependent (Figure 1). Loss of p53 completely
abrogated this early wave of cell deletion. Moreover,
although a continuing pattern of apoptosis at 24 h was
observed in the p53 +/+ animals, this was not
observed in the epithelial cells in the null animals
after 1 Gy. Measurement of the mitotic indices in all
genotypes showed that 1 Gy had no signi®cant e€ect
on the percentage of cells in mitosis at early times (data
not shown), despite the fact that considerable numbers
of cells were undergoing apoptosis (Figure 1). We have
2763
p53 independent cell death
AJ Merritt et al
2764
previously suggested that after a low dose of radiation,
such as 1 Gy, the very radiosensitive stem cells are
normally deleted but these are replaced by their
immediate daughters from within the crypt, thus reestablishing crypt hierarchy without the necessity for
regenerative or compensatory cell divisions (Potten,
1990).
The incorporation of [3H]thymidine suggests that
after radiation damage of small intestinal epithelial
crypts of wild-type (+/+) animals, p53 induced either
a checkpoint which inhibited entry to S phase or
generally slowed progress through S phase (Figure 4).
Early studies by Lesher and Bauman (1969) had
shown that irradiation of the small intestine produced
a G2 block. There was no evidence for a G1 block.
This is dicult to reconcile with the observation of a
fall in DNA synthesis. Alternatively, the higher
labelling of cells from the null animals at 4 h may
be because surviving cells enter S-phase, whilst in the
wild-type animals they have been rapidly deleted from
the crypt. We are currently investigating the expression of the cyclin-dependent kinase inhibitor waf-1
under these conditions. Preliminary data shows
expression of protein to be largely p53 dependent,
supporting the idea that a p53-regulated cell cycle
block may occur. The general pattern observed here is
similar to the changes reported in a comprehensive
study on BDF1 mice (Potten et al., 1990). In that
study, an initial regression of labelled cells and
labelling index re¯ected the shrinkage of crypt size
due to apoptosis, mitotic delay (G2 block), reproductive sterilisation and continued emigration of cells
onto the villus. This is followed by an oscillating
regenerative proliferation which originates in the
surviving clonogenic cells and becomes evident in the
labelling frequency plots by 24 h. Damaged cells
which progress into S-phase after 8 Gy then appear
to reach a G2 checkpoint and attempt to undergo
mitosis, where they undergo a mitotic catastrophe
(Figures 2 and 3). This is by a p53-independent mode
of apoptosis. This second default mechanism for the
engagement of apoptosis occurred only after 8 Gy;
after 1 Gy very little apoptosis was observed at 24 h
in the p53 null animals. This suggests that either
DNA repair had been sucient to allow escape from
the second checkpoint or that the threshold of damage
being `sensed' by this p53-independent mechanism was
greater than that of p53.
Although we have discovered that small intestinal
crypt epithelial cells which have received g-radiationinduced DNA damage may be deleted by both p53dependent and -independent mechanisms the di€erence
in the temporal onset of these two processes could be
hypothesised to be critical in determining the longterm fate of damaged cells. Failure to rapidly delete
DNA damaged cells or to impose a G1 checkpoint,
with the opportunity for repair, particularly after
lower levels of damage (1 Gy), allows damaged cells
to progress through S-phase. The survival of these
cells for a further period in G2 may also allow repair
where again a decision whether to progress through
the cell cycle or to die is made. In G2 the threshold
for the detection of, or response to damage appears to
be greater (Figure 1). If repair is pro®cient in those
cells which escape from the p53-mediated deletion, it
would be predicted that they could repopulate the
crypt. Experiments which attempted to establish
whether more clonogenic cells survived in p53 null
animals suggested that no signi®cant survival advantage was provided by a deletion of both alleles of the
p53 gene (Figure 5). Thus, in the epithelia of the
murine small intestine, the loss of p53 appears to
provide no long-term survival advantage after DNA
damage imposed by high doses of g-irradiation.
Although the methods used here to establish this
may be limited, because only high doses of radiation
can be used to destroy crypt integrity for the purpose
of measuring the emergence of viable stem cells, our
®ndings that p53 provides no clonogenic survival in
intestinal epithelia are concordant with published
studies of primary ®broblasts and lymphoid cells
(Slichenmeyer et al., 1993; Strasser et al., 1994). That
p53 may, in some settings, have a limited role as a
guardian of the genome with respect to the prevention
of events initiating intestinal carcinogenesis and in the
prevention of subsequent genetic instability is a
conclusion recently drawn by others (Fishel, 1996;
Kahlenberg, 1996).
Materials and methods
Unless otherwise stated, all general purpose chemicals were
obtained from BDH/MERCK (Poole, Dorset, UK).
p53 null mice
The p53 homozygously null mice were from a colony
derived from those generated by Donehower et al. (1992).
p53 null (7/7), heterozygous (+/7) and wildtype (+/+)
mice were generated by the appropriate crosses and the p53
null strain used had a 129/C57B16 background. All mice
used were 10 ± 12 week old males and they were maintained
and genotyped as described by us previously (Merritt et al.,
1994, 1996).
Irradiation and preparation of mouse small intestinal tissue
Groups of 4 ± 6 mice were whole body g irradiated with the
appropriate doses using a Caesium137 source at a dose rate
of 3.8 Gy/min. The mice were sacri®ced at various times.
The entire small intestine was removed, ®xed in Carnoy's
®xative and `gut-bundled' as described previously (Merritt
et al., 1994). This gave a minimum of 12 cross sections per
mouse. The tissue was processed for histology and 3 ± 5 mm
sections were cut onto uncoated microscope slides and
stained routinely with haematoxylin and eosin or processed
for staining with streptavidin-biotin-labelled dUTP after its
incorporation by terminal deoxynucleotidyltransferase
(ISEL), as described in detail by us previously (Merritt et
al., 1995, 1996).
Estimation of apoptosis
This was performed essentially as described in detail
previously (Merritt et al., 1996). Brie¯y, ®xed and stained
transverse sections of small intestine were examined and,
from haematoxylin and eosin-stained sections apoptotic
fragments and mitotic cells were scored according to their
cell position, numbering from the base of the crypt.
Between 150 and 200 half crypts were counted for each
group and in some cases apoptosis was also scored based
on the ISEL technique as described previously (Merritt et
al., 1995, 1996).
p53 independent cell death
AJ Merritt et al
Estimation of DNA synthesis and mitotic indices in small
intestinal crypts
After irradiation groups of four mice were given 25 mCi of
tritiated thymidine (6.7 Ci/mmol), intraperitoneally,
40 min prior to the time of sacri®ce. Histological samples
prepared as slides (Merritt et al., 1995), were dewaxed
overnight in xylene and immersed in four changes of
absolute alcohol for 10 min each. After washing, the slides
were then rinsed in two changes of 0.5% Photo¯ow in
distilled water (Kodak, Hemel Hempstead, Herts, UK).
The autoradiographic emulsion was prepared using a 1 : 1
solution of K-5 nuclear track emultion in distilled water
(Ilford, Cheshire, UK) and slides dipped rapidly into this
prior to air drying for 2 ± 3 h. Autoradiographs were
developed under red light conditions using Kodak-D19
developer, according to manufacturers instructions. Slides
were washed for 2 mins with 1% glacial acetic acid then
®xed with Hypan ®xative (Ilford, Cheshire, UK). After
washing with distilled water, slides were stained with
haematoxylin and eosin, dehydrated and mounted in Xam
(BDH, Poole, UK). Cells deemed to be in S-phase
contained 43 silver grains over their nuclei. The number
and cell position of labelled cells was scored in the same
way as apoptotic cells (see above).
®tted to the Standard Multitarget Model and curves were
®tted using the DRFIT programme (Roberts, 1990). The
curves were plotted as surviving fractions against dose
(Gy). In addition to generating crypt survival curves, ftests were performed to determine the signi®cance of the
di€erence between two survival curves. To estimate the
number of clonogenic cells per crypt, the `split dose'
method was used as described (Hendry, 1978; Potten and
Hendry, 1995). Groups of animals were given either a
single fraction of 8 Gy or two identical fractions of 8 Gy
plus 8 Gy separated by 4 h. A second experiment involved
using doses of 12 Gy single and 12 Gy plus 12 Gy 4 h
apart. Animals were sacri®ced 3 days later. The number of
surviving crypts were recorded in ten cross sections for
each mouse per group. The numbers were then size
corrected, as described above (Potten et al., 1981). A
mean value for the number of surviving crypts (after size
correction) was obtained for each group. Assuming that
the fractions each kill the same proportion of clonogens,
and that all clonogens have the same radiosensitivity, the
original number of clonogens, m, can be computed from
the equations:
and
S1
ˆ
1 ÿ …1 ÿ Sc †m
m
The Crypt microcolony assay
The crypt microcolony assay was performed as described
by Potten and Hendry 1995. For each mouse, ten crosssections or circumferences were chosen, excluding those
containing Peyer's patches, those badly orientated or those
damaged during sectioning. The number of surviving
crypts around the whole of each circumference was
recorded, using the criteria that a survivor consisted of at
least ten adjacent, darkly stained, healthy, non-Paneth
epithelial cells. Size correction for the regrowing crypts was
performed as described by Potten et al. (1981). This is
necessary because the size of the surviving crypts can vary
quite considerably with the time of sampling and the dose
of radiation. Brie¯y, the widths of 15 randomly chosen
crypts per mouse were measured at right angles to the line
of the lumen using an eye piece graticule and a light
microscope. The corrected number of surviving crypts for a
given treatment was then calculated using the following
equation:
Corrected Number of Survivors ˆ
crypt width
Uncorrected Number 2 Control
Treated crypt width
The size corrected numbers were then converted to
surviving fractions of the control number. The data were
S2 ˆ 1 ÿ …1 ÿ Sc 2 †
Where S1 and S2 are the crypt surviving fractions after one
and two fractions, Sc the clonogen surviving fraction and m
the original number of clonogens.
Electron microscopy
Mice were irradiated as described above and sacri®ced
either 4.5 h or 24 h later. The small intestines were dissected
out, and tissue was cut into approximately 1 mm cubes.
These were instantly ®xed in 2.5% glutaraldehyde in 0.15 M
Sorenson's phosphate bu€er pH 7.35, followed by 1%
osmium tetroxide in the same bu€er. Specimens were
dehydrated through the graded alcohol series and
embedded in Agar 100 epoxy resin (Agar Scienti®c,
Stanstead, Essex, UK). Sections were stained with uranyl
acetate and lead citrate and examined at 80 kV in a Philips
EM400 TEM (Philips Analytical, Cambridge, UK).
Acknowledgements
We thank the Cancer Research Campaign for a studentship
(to AJM) and for continued funding. We are grateful to
Caroline Chadwick for assistance with assessment of much
of the data, and Drs Mark Pritchard, Christine Chresta
and Caroline Dive for their comments on the manuscript.
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