Oncogene (1999) 18, 2901 ± 2907
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00
http://www.stockton-press.co.uk/onc
Accentuated apoptosis in normally developing p53 knockout mouse embryos
following genotoxic stress
Jene Frenkel1,3, Dan Sherman1, Amos Fein2, Dov Schwartz3, Nava Almog3, Ahuva Kapon3,
Naomi Gold®nger3 and Varda Rotter*,3
1
Department of Obstetrics and Gynecology, Assaf Harofe, Medical Center, Tel Aviv University, Tel Aviv, Israel; 2Department of
Embryology and Teratology, Sackler School of Medicine Tel Aviv University Tel Aviv, Israel; 3Department of Molecular Cell
Biology, Weizmann Institute of Science, Rehovot, Israel 76100
In order to identify the alternative pathways which may
substitute for the p53 function during embryogenesis, we
have focused our studies on p537/7 normally developing mouse embryos that survived a genotoxic stress. We
assumed that under these conditions p53-independent
pathways, which physiologically control genomic stability, are enhanced. We found that while p53+/+ mouse
embryos elicited, as expected, a p53-dependent apoptosis,
p537/7 normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53dependent apoptosis detected in p53+/+ embryos, was
an immediate reaction mostly detected in the brain,
whereas the p53-independent apoptosis was a delayed
reaction with a prominent pattern observed in epithelial
cells of most organs in the p53-de®cient mice only. These
results suggest that in the absence of p53-dependent
apoptosis, which is a fast response to damaged DNA,
p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.
Keywords: p53-dependent and independent apoptosis;
embryogenesis
Introduction
The role of p53 in normal embryonic development was
investigated by di€erent approaches. In early studies, it
was observed that p53 expression is modulated during
the various developmental stages and an evolutionary
conserved down regulation of p53 expression, con®ned
to speci®c stages of normal embryonic development,
was suggested (Mora et al., 1980; Chandrasekaran et
al., 1981; Rogel et al., 1985; Louis et al., 1988; Schmid
et al., 1991; Almog et al., 1997).
The establishment of p53 knockout mice was
expected to give clues as to the physiological function
of p53. Although it appeared that p53 is not required
for normal embryonic development (Donehower et al.,
1992), a more detailed study indicated that 8 ± 16% of
the p53-de®cient embryos developed abnormally. In
some cases, defects in neural tube development were
observed, such as exencephaly, which were more
frequent in females, thus suggesting that p53 is
*Correspondence: V Rotter
Received 6 April 1998; revised 18 September 1998; accepted 23
October 1998
associated with neural tube closure (Armstrong et al.,
1995; Sah et al., 1995).
Furthermore, p53 transgenic mice which overexpress
wild type p53, exhibited a defective di€erentiation of
the ureteric bud and abnormal kidney development
(Godley et al., 1996). Similarly, transgenic mice that
carry the human wild type p53 under the control of the
a-crystalline promoter, exhibited a defect in the
formation of ®bers, and a failure of lens cells to
undergo proper di€erentiation. These defects were
overcome by the concomitant expression of mutant
p53 (Nakamura et al., 1995). The involvement of p53
in the development of lens cells was also con®rmed in
Rb-de®cient mice (Morgenbesser et al., 1994) and in
E6-transgenic mice (Griep et al., 1993; Pan et al.,
1994).
The transcription activity of p53 during embryogenesis was studied in transgenic mice which carry a
reporter gene controlled by a promoter containing the
p53 responsive element. In such mice it was found that
under physiological conditions or following g-irradiation, transactivation by p53 varies greatly among
di€erent tissues at di€erent stages of embryonic
development, and is inversely correlated to the stage
of di€erentiation of the speci®c tissue (MacCallum et
al., 1996; Gottlieb et al., 1997; Komarova et al., 1997).
This di€erential regulation of p53 during embryogenesis supports the idea that p53 may play an important
role in the regulation of cell proliferation and
di€erentiation, and raises the possibility that p53
might be involved in controlling speci®c steps during
embryogenesis.
The conclusion that the overexpression of wild type
p53 may disrupt normal embryogenesis, is best
demonstrated in studies of mdm2 knock-out mice
where it was found that the lethal phenotype of
mdm2 null mice can be rescued in a p53 null
background (Jones et al., 1995; de Oca Luna et al.,
1995).
It is of interest to note that p53 was shown to have
an embryonic protective role, acting as a teratologic
suppressor in pregnant mice, perhaps by protecting the
embryo from DNA-damaging chemicals and developmental oxidative stress (Nicol et al., 1995).
The observation that p537/7 genotype is not lethal
and that normal mice seem to develop with a rather
low incidence of malformed embryos makes it possible
to assume that either p53 is not central to normal
embryonic development or that alternative pathways
exist that override p53-dependency. Another possibility
is that the recently observed p73, a homologue of p53
(Jost et al., 1997; Kaghad et al., 1997) may substitute
p53-dependent and p53-independent apoptosis during embryogenesis
J Frenkel et al
2902
for the lack of p53 in normal development and
di€erentiation.
In the present study we searched for possible existing
alternative pathways which enable normal embryonic
development of p53 null mice. The observation that the
most frequent embryonic malformations in p53 knockout mice were associated with neural tube development
(Armstrong et al., 1995; Sah et al., 1995), coupled with
the fact that the p53 transcription activity was mostly
con®ned to the developing brain (Komarova et al.,
1997), were the rationale for focusing our experiments
on conditions of genotoxic stress whereby neural tube
malformations were predominantly induced. We
assumed that DNA insult in normally developing
embryos which escaped malformations facilitates p53dependent and p53-independent physiological mechanisms that secure genome stability. To that end, we
compared the apoptotic patterns of normally developing p537/7 and p53+/+ mouse embryos that were
exposed to g-irradiation. Our results showed that
development of normal p537/7 mouse embryos
under stress conditions was dependent on a delayed
accentuated p53-independent apoptotic response, that
was di€erent from the immediate p53-dependent
apoptosis observed in normally developing p53+/+
embryos under the same DNA insult.
Results
Preferential induction of neural tube defects in mouse
embryos
The ®rst experiments addressed the calibration of the
optimal conditions for the induction of neural tube
defects in embryos. The experiments involved girradiation of pregnant mice under various conditions.
p53+/+ female mice at di€erent stages of
pregnancy were exposed to various doses of girradiation. Seventy-eight mice were irradiated on day
7, 8 or 9 of gestation and were sacri®ced at day 15. The
morphology of 553 implants were examined for the
appearance of external malformations. Early resorptions and intrauterine fetal deaths were also concomitantly scored. Figure 1A summarizes the pattern of
abnormalities observed under the di€erent conditions.
The highest frequency of embryos with neural tube
defects was observed in the group of pregnant mice
exposed to 200 R on day 8 of gestation. It should be
noted that under these experimental conditions the
frequency of early resorption and fetal death was
relatively low. These therefore, were the conditions
selected for the subsequent experiments.
In the next experiments p537/7 mice were
analysed. To that end p53+/7 hetrozygotic parents
were mated. Pregnant mice were g-irradiated with
200 R on day 8 and embryos were morphologically
and genotypically analysed on day 15. Figure 1B,
represent the results obtained with p537/7 embryos
as analysed in the range of 0-200 R. As can be seen
p537/7 embryos, with no g-irradiation, exhibited
neural tube defects with a higher incidence (6%) than
those observed in the p53+/+ embryos (0%). This
pattern is in agreement with previous reports
(Armstrong et al., 1995; Sah et al., 1995). Exposure
of 8 day-old p537/7 embryos to 200 R did not,
however, induce a signi®cant increase in the incidence
of malformations as compared to that observed with
the p53+/+ embryos. This could be due to the
increment observed in embryonic death following girradiation.
Analysis of p53-independent apoptotic patterns in mouse
embryos
In the next experiments we have analysed the apoptotic
patterns of normally developing embryos of p537/7
mice. Based on the above data we compared apoptotic
patterns of p537/7 and p53+/+ normally developing 15 day-old embryos exposed to 200 R on day 8 of
gestation, and the histological sections were then
exposed to Tunel analysis. Such a comparison clearly
indicated that p537/7 embryos exhibited very
pronounced patterns of apoptosis, whereas p53+/+
embryos showed hardly any apoptotic cells. Almost all
p537/7 embryos analysed presenting normal development exhibited accentuated apoptosis that was
con®ned to various organs.
Detailed analysis of the apoptotic pattern of the
p537/7 normally developing embryos (Figure 2A),
has indicated apoptosis in the di€erent bones with
enchondral ossi®cation with speci®c patterns that seem
to be con®ned to cartilage at de®ned developmental
stages. (Figure 2B). Apoptosis was very prominent in
the lungs at the alveolar epithelium developing sites
(Figure 2C), in the interdigital notches of the hand
plates (Figure 2D), and in the stomach wall (Figure
2E). In addition to the Tunel positively stained cells,
dead cells lying in front of cells undergoing apoptosis
were evident. This is clearly seen in the stomach wall
section and in the hand plate. In general, the apoptotic
response was signi®cantly evident in the epitheliaenriched regions of the various organs. It should be
stressed that no comparable apoptosis was evident in
these organs in the corresponding p53+/+ embryos.
Figure 2 depicts typical pattern of Tunel staining of
sections obtained from p53+/+ mice under the same
experimental conditions. As can be seen, no signi®cant
apoptosis is evident in sections of enchondral
ossi®cation (Figure 2H), in the developing lung
sections (Figure 2I) or in the interdigital notches of
the hand plates (Figure 2J).
The liver of p537/7 embryos represents an
interesting pattern of a substantiated amount of Tunel
positive cells that were accompanied by large sites
which accumulate dead cells (Figure 2F). However,
under the same experimental conditions, p53+/+
embryos exhibited a similar apoptotic response in the
developing liver. Both the frequency of positive cells
and the intensity of the apoptotic signal were similar to
that observed in the corresponding p537/7 embryos.
Furthermore, sections of livers obtained from nonirradiated embryos of both genotypes also exhibited a
similar frequency of Tunel positive cells. This suggests
that apoptosis in the embryonic developing liver is a
p53-independent constitutive pathway that is not
a€ected by 200 R.
Interestingly, the brain wall of the embryos exhibited
no apoptosis (Figure 2G). Both types of p53+/+ and
p537/7 embryos rarely contained any apoptotic cells.
This signal was not enhanced following g-irradiation at
day 8 of gestation.
p53-dependent and p53-independent apoptosis during embryogenesis
J Frenkel et al
2903
Figure 1 Incidence of neural tube defects in embryos following DNA insult. Pregnant mice at day 8 of gestation were subjected to
increasing doses of g-irradiation and the incidence of normally developing embryos (full bars), early resorption (shaded bars) and
malformed embryos (dotted bars) were analysed. p53+/+ (A) and p537/7 mice were compared (B)
These results suggest that in 15 day-old normally
developing mouse embryos that were exposed 7 days
earlier to 200 R, an accentuated p53-independent
apoptosis was evident in tissues of p537/7
embryos, but not in those of p53+/+. In the case
of the liver, both p53+/+ and p537/7 exhibited
similar apoptosis that was not enhanced following
exposure to 200 R.
Analysis of p53-dependent apoptotic patterns in mouse
embryos
In the following experiment we looked for the p53dependent apoptotic responses. p53-dependent apoptosis is an immediate response that can be measured in
both in vitro (Yonish-Rouach et al., 1991, 1993; Peled et
al., 1996) and in in vivo (Clarke et al., 1993, 1994; Lowe
et al., 1993b; Merritt, et al., 1994; Komarova et al.,
1997) models. To that end we have analysed the
apoptotic patterns immediately following exposure to
DNA insult. Pregnant mice at day 8 were g-irradiated
(200 R) and 3, 8 or 24 h later, the embryos were
analysed for apoptosis. To assess a possible linkage
between the developmental stage and the speci®c
apoptotic response, we also g-irradiated mice on days
11 and 14 of their pregnancy and 24 h later analysed
apoptotic patterns in the corresponding embryos. The
general trend was that p53+/+ embryos, at any de®ned
developmental stage exhibited a signi®cant apoptotic
response following g-irradiation. The p537/7 counterparts manifested hardly any enhanced apoptotic signal
that could be detected immediately following DNA
insult. Figure 3A represents a typical apoptotic pattern
in a 9 day-old p53+/+ embryo 24 h following exposure
to 200 R, while Figure 3B represents the negative
apoptotic pattern observed under the same experimental conditions in the p537/7 embryos.
In contrast to the lack of p53-independent apoptotic
response in the brain, observed above, p53-dependent
apoptosis measured several hours following g-irradiation in the p53+/+ embryos was abundant in various
regions of a 9 day-old developing brain (Figure 3A). In
a more developed brain (day 15 of gestation) the p53dependent apoptosis was con®ned to speci®c domains
within the brain. A signi®cant apoptotic signal was
evident in the telecephalon (Figure 3C) and in the
spinal cord (Figure 3D). Interestingly, however, the
rombenchephalon did not exhibit apoptosis in any of
the analysed embryos. Analysis of the corresponding
p537/7 15 day-old embryos immediately following girradiation, failed to detect speci®c apoptotic signals in
any of the brain vesicles at the various developmental
stages.
Other organs of 15-day-old p53+/+ embryos
exposed to 200 R on day 14, exhibited variable levels
of apoptotic cells in almost all organs. However, these
apoptotic patterns were signi®cantly less abundant
than the p53-independent apoptotic patterns, observed
in the 15-day-old p537/7 embryos which were
exposed to 200 R on day 8 of gestation. Figure 3E
represents a typical pattern of apoptotic cells found in
the lungs and Figure 3F represents the frequency of
apoptotic cells in the stomach of 15 day-old embryos
exposed to 200 R on day 14 of development. In the
case of the p53-dependent apoptosis no signi®cant
signals were observed in the epithelial layers that
frequently undergo p53-independent apoptosis (see
Figure 2).
Analysis of 15-day-old embryos immediately
following g-irradiation indicated that the typical
apoptotic patterns in the liver were barely modi®ed.
The fact that similar patterns of apoptosis were
detected in non-g-irradiated normally developing
embryos, with no correlation to the p53 genotype,
the onset of the p53-dependent or p53-independent
pathways again con®rms that apoptosis measured
represents an inherent developmental pathway of this
organ.
p53-dependent and p53-independent apoptosis during embryogenesis
J Frenkel et al
2904
Figure 2 p53-independent apoptotic pattern in normally developing p537/7 mouse embryos. Pregnant mice at day 8 of gestation
were exposed to 200 R and embryos were subjected to Tunel analysis on day 15. Mid-segital sections of a normally developing
p537/7 embryo (A) were analysed and the following sites are enlarged. Bone with enchondral ossi®cation in upper girdle area (B);
developing lung with the alveolar epithelium (C); interdigital notches of the hand plate (D); stomach wall including luminar
epithelium (E); developing liver (F) and the rombercephalic wall (G). Representative sections of bone with enchondral ossi®cation in
upper girdle area (H) developing lung with the alveolar epithelium; (I) and interdigital notches of the hand plate (J) of p53+/+
normally developing embryos, treated similarly, are shown
Figure 3 p53-dependent apoptotic pattern in normally developing p53+/+ mouse embryos. Pregnant mice at day 8 of gestation
were exposed to 200 R and p53+/+ (A) or p537/7 (B) embryos were subjected to Tunel analysis 24 h later. 14-day pregnant
p53+/+ mice were exposed to 200 R and embryos were subjected to Tunel analysis 24 h later. Enlarged area of mid-segital section
of rombercephalon (C); spinal cord (D); developing lung (E) stomach wall and liver (F)
p53-dependent and p53-independent apoptosis during embryogenesis
J Frenkel et al
Discussion
It is well accepted that apoptosis, one of the
hallmarks of embryonic development, serves as a
selection mechanism that controls development and
di€erentiation (Jacobson et al., 1997). Apoptosis may
occur in a p53-dependent (Yonish-Rouach et al., 1991;
Lu et al., 1993; Clarke et al., 1993, 1994; Lowe et al.,
1993a,b; Strasser et al., 1994; Symonds et al., 1994), a
p53-independent manner (Lowe et al., 1993a; Clarke
et al., 1993; Berges et al., 1993; Kelley et al., 1994;
Strasser et al., 1994; Zhuang et al., 1995) or
concomitantly utilize both pathways (Merlo et al.,
1995; Peled et al., 1996).
In our present study we found that when embryos
express p53, DNA damage will turn on the p53dependent apoptotic pathway. However in its absence,
an alternative pathway of apoptosis which is p53independent is induced. The p53-dependent apoptosis
is an immediate response while the p53-independent
one seems to linger behind. Di€erences in the kinetics
of p53-dependent and -independent apoptosis were
already observed previously in several in vitro and in
vivo experiment models. It is worth mentioning that in
most studies, p53-dependent apoptosis is measured
immediately following induction of DNA insult
(Yonish-Rouach et al., 1991, 1993; Clarke et al.,
1993, 1994; Lowe et al., 1993a; Merritt et al., 1994;
Komarova et al., 1997). We found that apoptosis
induced in a p53 non-producer cell line was
signi®cantly slower than that observed in the same
cells following reconstitution of wild type p53
expression (Peled et al., 1996). Analysis of in vivo
models where adult mice were subjected to girradiation, again p53-dependent and -independent
apoptotic responses of di€erent kinetics were evident.
For example it was observed that the apoptotic
response of intestinal cells of p53+/+ mice was
evident several hours following exposure to g-irradiation. At that early phase no apoptotic signal was
evident in the p53 de®cient mice (Merritt et al., 1994;
Clarke et al., 1994). Interestingly, high doses of girradiation seem to induce a delayed p53-independent
apoptotic e€ect in small intestinal epithelia of p53
knockout mice (Merritt et al., 1997). The fact that the
p53-dependent apoptotic pathway can be distinguished
from the p53-independent one, made it possible to
identify the individual pathways and concluded that in
the absence of the p53-dependent pathway, the p53independent one can substitute and compensate to
permit normal embryonic development.
It should be noted that Norimura et al. (1996)
claimed that p53+/+ mouse embryos exhibited an
augmented apoptotic response as compared to p537/
7 counterparts (Norimura et al., 1996). This apparent
discrepancy could be explained by the fact that they
assessed apoptosis 6 h following g-irradiation, which
triggered the p53-dependent apoptotic pathway (Norimura et al., 1996). In our experiment delayed p53independent apoptotic activity, as well as an early p53dependent apoptotic response following the same
genotoxic stress, were measured.
The observation that p53-de®cient embryos which
seem to develop normally, exhibit an accentuated
and prolonged apoptotic response following girradiation strongly suggests that this p53-indepen-
dent apoptosis is the primary alternative pathway
that causes the removal of cells containing damaged
DNA. The fact that p53+/+ normally developing
embryos do not exhibit the delayed p53-independent
apoptosis under the same DNA insult, could be
either because in these embryos the early p53dependent apoptosis eciently eradicated the cells
containing damaged DNA, or alternatively, repair of
their damaged DNA was induced. p53+/+ embryos
will preferentially utilize the p53-dependent pathway
and induce death of cells containing damaged DNA,
while p537/7 embryos are forced to use the
delayed p53-independent pathway. The p53-dependent apoptosis is probably very ecient and
successfully eliminates cells containing damaged
DNA, thus there is no requirement for the onset
of the p53-independent apoptosis to occur in the
p53+/+ embryos. It is quite possible that each
apoptotic pathway is speci®cally and eciently
triggered by unique inducers. For example growth
factor deprivation is mostly associated with p53independent pathways (Wyllie, 1995), whereas UV
(Lu and Lane, 1993) or g-irradiation (Kastan et al.,
1991; Kuerbitz et al., 1992; Lu and Lane 1993;
Nelson et al., 1994) will induce p53-dependent
pathways. In the case of p537/7 embryos,
damaged DNA, having no choice will induce the
second best apoptotic pathway available, which is
p53-independent. Being an alternative pathway it
might be less ecient in responding to g-irradiation.
Our present observation may also support the
conclusion that p53-dependent and -independent
apoptotic pathways may be di€erentially expressed
in an organ speci®c manner. For example, apoptosis
in the developing liver seems to be controlled by
p53-independent pathways. On the other hand,
apoptosis in the developing brain, seems to be
predominantly controlled by a p53-dependent apoptotic pathway. This notion that p53-dependent
apoptosis is essential for the normal development
of the embryonic brain is also supported by the
observation that p537/7 mice embryos frequently
exhibit brain malformations (Armstrong et al., 1995;
Sah et al., 1995). This is further substantiated by the
observation that p53 transcription activity is
abundant in embryo brains and that such an
activity is enhanced following exposure to girradiation (Komarova et al., 1997).
In conclusion, we would like to suggest that during
embryogenesis p53 functions in the maintenance of
genome stability by controlling apoptosis. In p53
de®cient-embryos, p53-independent apoptosis is the
alternative pathway used to secure genomic stability.
p53-independent apoptosis is probably an active
inherent process during embryogenesis. It is, therefore, not surprising that it becomes the predominant
alternative mechanism controlling DNA integrity. It is
possible that p53-independent apoptosis is turned on
more frequently in the p53 null embryos and will
eradicate cells with damaged DNA, which in p53+/+
embryos can still be repaired. In adult life however,
such an alternative p53-independent apoptotic pathway
could be less prevalent and therefore loss of the tumor
suppressor activity mediated by p53 can not be fully
replaced and thus mice will develop a high incidence of
tumors.
2905
p53-dependent and p53-independent apoptosis during embryogenesis
J Frenkel et al
2906
Materials and methods
Mouse maintenance and mating
p537/7 mice of 129SV and C57 BL strain (Donehower et
al., 1992) were bred by mating p53+/7 or p537/7
parents. Each literate was genotyped at about 1 month of
the age. In order to precisely determine the date of
pregnancy, mice were mated in conditions of darkness
and light that was altered every 12 h. Females were caged
with males for 4 h prior to termination of the dark period.
Determination of day 0 of pregnancy was based on the
presence of vaginal plugs.
Morphology analysis
The treated or untreated pregnant mice were sacri®ced.
The uteri were removed, the number of implantation sites,
resorptions, live and dead fetuses were recorded. All live
fetuses were examined for gross structural malformations.
Normal or malformed embryos were ®xed for 24 h in a
cold mixture of formaldehyde (3.7 ± 4.0%); phosphate
sodium monobasic (0.04%); sodium phosphate dibasic
(0.065%). Following ®xation embryos were washed in
water, dehydrated through a graded series of ethanol,
cleared in xylene and embedded in paran wax. Blocks
were sectioned at 7 mck thickness. The sections were
subjected to either Hematoxilin-Eosin staining or to Tunel
analysis.
PCR analysis of the p53 genotype
Genotype analysis of the mice used was performed by PCR
analysis which permits the speci®c identi®cation of
p53+/+, p53+/7 and p537/7 mice. Tissue fragments
obtained from either adult mice or embryos (tails or ears)
were analysed. Genomic DNA was prepared by incubating
tissues in 0.5 ml TE containing 0.4 mg proteinase K and
0.5% SDS over night at 378C. The samples were extracted
three times in p/CIAA (Phenol/Chloroform/Isoamyl
alcohol), and twice in CIAA. DNA was washed in
isopropanol, centrifuged, washed twice in 70% ethanol,
and dissolved in DDW. Each PCR reaction contained
0.5 mg genomic DNA. The primers used were as previously
described (Timme et al., 1994): for the wild type p53
sequences we used the 5' 5W2 primer; GTC CGC GCC
ATG GCC ATC TA and the 3' 3W2 primer: ATG GGA
GGC TGC CAG TCC TAA CCC. For the neo p537/7
speci®c marker the 5' 5ND: CAG CCC AGT GGA GTG
ACA CAC ACC T (speci®cally designed in our lab) and
sequence of the 3' 3N2: TTT ACG GAG CCC TGG CGC
TCG ATG T. The PCR program included 5 min in 948C,
36 cycles of 30 s in 948C, 30 s in 698C, and 60 s in 728C,
and ®nally 7 min in 728C. Typical patterns of DNA
fragments were obtained for the p53+/+ homozygotes
(138 bp), p537/7 homozygotes (180 bp) and p53+/7
homozygotes (138 and 180 bp).
Apoptosis
Apoptosis was analysed by the in situ Tunel staining that
was carried out as previously described (Wride et al.,
1994). Brie¯y, after deparanation of the tissue sections,
they were immersed in DDW. Sections were then incubated
for 15 min in 26SSC bu€er at 608C, washed in DDW, and
incubated with 20 mg/ml of proteinase K (Boehringer
Mannheim) for 15 min at room temperature. After a wash
with DDW, endogenous peroxidases were inactivated by
incubating the sections with 2% H2O2 in PBST (PBS with
0.05% Tween 20) for 10 min at room temperature. Sections
were then incubated in TdT bu€er (Boehringer Mannheim)
for 5 min at room temperature, and then the reaction
mixture (56TdT bu€er, 1 ml biotin-21-dUTP (Clontech,
1 mM stock), and 8 units of TdT enzyme (Boehringer
Mannheim) in total volume of 50 ml) was added. The
reactions were carried at 378C for 1.5 h in a humid
chamber. The slides were washed with 26SSC, DDW
and ®nally with PBS, and covered with 10% SM in PBST
for 15 min. After SM was removed the sections were
incubated with ABC solution from ABC kit (Vector
Laboratories, Inc.), for 30 min at room temperature,
washed with PBS and stained using the AEC procedure
(Sigma). The slides were then washed 36in DDW and
stained with Haematoxilin for 30 s and mounted with
Kaiser's glycerol gelatin (Merck).
Acknowledgements
The authors wish to thank Professor Ben-Zion Shilo of
Department of Molecular Genetics; David Wiseman of the
Department of Molecular Biology of the Weizmann
Institute for fruitful discussion and criticism and Ms
Hassida Orenstein for excellent technical assistance. Ms
Vivienne Laufer helped in the manuscript preparation. This
work was supported in part by grants from the Leo and
Julia Forchheimer Center for Molecular Genetics, the Minerva Foundation and BSF. Varda Rotter is the incumbent
of the Norman and Helen Asher Professorial Chair in
Cancer Research at the Weizmann Institute.
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