Oncogene (1997) 15, 2597 ± 2607
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Role of wild type p53 in the G2 phase: regulation of the g-irradiationinduced delay and DNA repair
Dov Schwartz, Nava Almog, Amnon Peled, Naomi Gold®nger and Varda Rotter
Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel 76100
Upregulation of the p53 protein was shown to induce cell
cycle arrest at the G1/S border and in some cases at the
G2/M border. Furthermore, it was suggested that p53 is
associated with the induction of the various DNA repair
pathways. Previously, we demonstrated that cells coexpressing endogenous wild type p53 protein, together
with dominant negative mutant p53, exhibit deregulation
of apoptosis, G1 arrest and delay in G2 following girradiation. In the present study, we investigated the role
of p53 protein in the DNA damage response at the G2
phase. Using p53-null, wild type p53 and mutant p53producer cell lines, we found that the two C-terminally
spliced p53 forms could prevent g-irradiation induced
mutagenesis prior to mitosis, at the G2/M checkpoint.
We found that at the G2 phase, p53 may facilitate repair
of DNA breaks giving rise to micronuclei, and regulate
the exit from the G2 checkpoint. At the G1 phase, only
the regularly spliced form of p53 caused growth arrest.
In contrast, both the regularly and the alternatively
spliced p53 forms directed postmitotic micronucleated
cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in normal cycling cells, as well as in cells
where p53 is up-regulated, following DNA damage.
Keywords: p53; G2 checkpoint; DNA damage; micronucleated cells
Introduction
It is well established that p53 is up regulated by various
stress conditions, characterized by DNA damage
induction (Maltzman, Czyzyc, 1984; Fritsche et al.,
1993; Nelson and Kastan, 1994). One of the major
proposed functions for p53 under these conditions, is
the maintenance of genomic stability (Livingstone et
al., 1992; Lane, 1992; Yin et al., 1992; Symonds et al.,
1994).
p53 was suggested to play a role in growth arrest at
the G1/S border prior to DNA replication, and at the
G2 phase prior to mitosis, by allowing the repair of
faulty DNA that accumulates in cells (Kastan et al.,
1991, 1992; Di Lionardo et al., 1994). p53 was also
shown to regulate the spindle checkpoint in mitotic
embryonic ®broblasts. This checkpoint prevents the
formation of aneuploid cells that are bound to harbor
an unstable genome after cytokinesis, and also prevents
ectopic DNA endoreduplication in these cells (Cross et
Correspondence: V Rotter
Received 29 April 1997; revised 18 July 1997; accepted 21 July 1997
al., 1995; Wells, 1996). We have recently found that
both the loss of wild type p53, or overexpression of
mutant p53 in myeloid cells, are associated with
polyploidity (Peled et al., 1996). This supports the
conclusion that p53 may play a role at the spindle
checkpoint.
p53 was shown to be involved in active cellular
repsonses that remove DNA damage, either by repair
of the damaged DNA, or by an active killing
(apoptosis) of cells possessing extensive, unrepairable
DNA damage (reviewed in Enoch and Norbury, 1995).
p53-dependent apoptosis is triggered by various stress
conditions, resulting in a clastrogenic e€ect on the
DNA (Yonish-Rouach et al., 1991, 1993; Lowe and
Ruley, 1993; Lowe et al., 1993; Lee et al., 1993; Clarke
et al., 1993; Symonds et al., 1994). The activity of p53
in DNA repair processes (Pan et al., 1995; Wang et al.,
1995, 1996; Ford and Hanawalt, 1995; Bogue et al.,
1996; McDonald III et al., 1996) was shown to be
controlled by the C-terminus of the molecule, that
binds several DNA repair-related proteins, including
the human Rad51 (Sturzbecher et al., 1996), RPA
(Dutta et al., 1993) and ERCC3 (Wang et al., 1994,
1995). In addition, GADD45 and the waf-1 gene, that
are transcriptionally regulated by p53, were found to
bind PCNA, another DNA repair protein (Chen et al.,
1995). Moreover, the last 100 amino acids of human
p53 contain non-speci®c single strand (Bakalkin et al.,
1994, 1995; Jayaraman and Drives, 1995) and
mismatched (Lee, et al., 1995) DNA binding activity.
The C-terminal domain of the regularly spliced p53
form (RSp53), also contains a DNA helicase activity
(Brain and Jenkins, 1994; Bakalkin et al., 1995; Wu et
al., 1995), characteristic of many DNA repair proteins.
The e€ect of p53 on DNA repair is also evident
from measuring residual DNA damage following
genotoxic stress. One of the detectable manifestations
of residual DNA damage in cells following girradiation is the appearance of micronuclei (Heddle,
Carrano, 1977). Micronuclei originate from acentric
double strand breaks in chromosomes and from
dicentric chromosomes, that give rise to chunks of
chromatin that do not segregate with the rest of the
genome after mitosis, but are often separately engulfed
by the nuclear membrane (Heddle and Carrano, 1977;
Nusse et al., 1994). Thus, the presence of micronuclei
in cells is an indicator of either chromosomal
aberrations that were not repaired until completion of
mitosis, or of an unstable karyotype. Interestingly,
reticulocytes from p53-knockout mice contain elevated
levels of micronuclei under normal growth conditions
and in cells with DNA damage (Lee et al., 1994).
Moreover, bone marrow cells from these mice exhibit a
high frequency of chromosomal abnormalities (Bou‚er
et al., 1995). Furthermore, wild type p53, in contrast to
p53 and the G2 checkpoint
D Schwartz et al
2598
mutant p53, was found to accumulate in micronuclei of
g-irradiated hepatoma cell lines (Unger et al., 1994).
Although it is clear that the onset of the G2
checkpoint is p53-independent, and involves a rapid
phosphorylation of the MPF cyclin B1-cdc2 complex,
p53 may play a role as a secondary regulator of the G2
delay (Stewart et al., 1994; Paules et al., 1995). In
several p53-null non-lymphoid cell lines, reconstitution
of wild type p53 conformation (Stewart et al., 1994), or
transcriptional induction of wild type p53 without any
DNA damage (Agarwal et al., 1995) resulted in a G2as well as a G1- arrest. The role of p53 as a second
barrier at the G2 checkpoint, is further supported by
the ®nding that annihilation of the phosphorylationdependent G2-delay by ca€eine, is reduced in wild type
p53 expressing cells (Powell et al., 1995). This agrees
with our observation that functional inactivation of
endogenous p53 in lymphoid cells, by stably expressing
dominant negative mutant p53, induces premature exit
from the g-irradiation-dependent G2 arrest (AloniGrinstein et al., 1995).
The goal of this study was to elucidate the role of
p53 in the response to DNA damage at the G2 phase.
We compared the time course of the cell cycle, the
repair of DNA damage following g-irradiation of cells
at the G2 phase and the selective apoptosis of
micronucleated postmitotic cells. Since most of the
DNA repair associated activites were ascribed to the
regularly spliced C-terminal of p53 (Shaulian et al.,
1994; Lee et al., 1995; Wu et al., 1995), we compared
the e€ect of re-introduction into p53 null cells of the
two alternatively spliced p53 forms that vary at their
C-terminal (Wolf et al., 1985; Arai et al., 1986). L12
cells that are p53-null lymphoid pre-B, stably
expressing regularly (RSp53) or alternatively-spliced
p53 (ASp53) forms and their mutant counterparts,
were analysed. Both alternatively spliced wild type p53
forms facilitated the exit from the g-irradiationdependent G2 delay. Analysis of micronuclei content
following exit from the G2 delay indicated that cell
lines expressing the two forms of wild type p53
exhibited 20% of micronuclei detected in p53-null
cells. Analysis of p53 distribution in post mitotic
diploid micronucleated cells indicated that wild type
p53 accumulates in the L12 cells expressing wild type
p53, and promotes their apoptosis. This e€ect was not
related to the spindle checkpoint, since in these cells
this checkpoint was p53-independent. While both
alternatively spliced p53 forms are involved in the
G2-related activities, the ability to arrest at the G1/S
border following g-irradiation was evident only in L12
cells reconstituted with wild type RSp53.
Results
Wild type p53 controls the kinetics of exit from G2 delay
in g-irradiated L12 cells
Previous data indicated that wild type p53 plays a role
in the G2 checkpoint. To analyse this activity,
logarithmically growing cells of p53 producer and
non-producer L12 derivatives were enriched for
tetraploid DNA constant by centrifugal elutriation
under viable conditions. The cells were collected,
replated and g-irradiated (with 300 R), or kept under
Figure 1 Wild type p53 accelerates the exit from the G2 delay after irradiation: Cell cycle patterns. L12 clones expressing regular
spliced wild type RSp53-10 (marked in gray in a, b, c, alternatively spliced wild type ASp53-22 (marked as empty in a, b, c) or
empty pSVL plasmid, clone pSVL-3 (marked in black in a, b, c) were grown at logarithmic conditions and enriched for the G2 phase
using centrifugal elutriation under viable conditions (a). Normal cell cycle distribution is depicted by dotted line in a. The cells were
either directly replated, or g-irradiated (300 R) and then replated. (b) shows the cell cycle of pattern g-irradiated cells 12 h after girradiation. (c) shows the cell cycle pattern of non-irradiated clones, 6 h after replating. (d, e, f) parallel (a, b, c) except that the
clones used in these experiments are mutant RSp53-M11-3 (marked in gray), mutant ASp53-M8-3A2 (marked as empty) or empty
pSVL plasmid pSVL-3 (marked in black)
p53 and the G2 checkpoint
D Schwartz et al
the same conditions as the g-irradiated cells, and
replated under normal growth conditions. At various
times after replating, cells were ®xed in 70% ethanol,
and analysed by FACS. To measure the distribution of
the various cell populations corresponding to the
phases of the cell cycle, standard PI DNA-content
analysis by FACS was employed (Figure 1).
The cell cycle pattern of either g-irradiated or nonirradiated G2/M enriched fractions of p53-null clones
(pSVL-3, pSVL-1) was compared to regularly spliced
wild type p53 expressors (RSp53-10 and RSp53-20),
the alternative spliced wild type p53 producers (ASp5320 and ASp53-22), and to mutant p53 expressing L12
clones (RSp53M11-3 and ASp53M8-3A2). Cell cycle
distribution of L12 clones expressing the various p53
types immediately following enrichment for G2 phase,
is shown in Figure 1a and d. Analysis of the kinetics of
progression from G2 of non-irradiated L12 cell
fractions indicated that both wild type p53 expressors
(Figure 1c) and mutant p53 expressors (Figure 1f)
exhibited the same length of the G2 phase compared to
the p53-null L12 control cells. In contrast, following girradiation, L12 cells harboring wild type p53 exited
faster from the G2 delay as compared to the p53-null
parental cell line (Figure 1bb, or cell lines that harbor
mutant p53 (Figure 1e). Furthermore, clones harboring
wild type RSp53 were more ecient than wild type
ASp53 expressors in the exit from the G2 delay (Figure
1b).
To evaluate the e€ect of p53 expression at the G1
phase in these L12 clones, G1-enriched fractions were
isolated by centrifugal elutriation (Figure 2a and d)
and analysed under the same experimental conditions
as the G2-enriched fractions. Since less cells progressed
from the diploid peak of wild type RSp53 expressing
clones, we concluded that under normal growth
conditions, wild type RSp53 confers a prolonged G1
phase, compared to the p53-null control (Figure 2b).
Mutant p53 (Figure 2e) as well as wild type ASp53
(Figure 2b) did not signi®cantly alter the length of the
G1 phase. Furthermore, following g-irradiation, only
wild type RSp53 (Figure 2c), but not mutant (Figure
2f) or wild type ASp53 (Figure 2c) altered the length of
the G1 checkpoint.
Standard cell cycle analysis using PI incorporation is
not suitable for the detection of a G2 delay, that is
manifested by the disappearance of the mitotic cells.
To determine the existence of such delay, we utilized
the FACS assay based on Acridine Orange (AO) DNA
denaturability. This assay is based on di€erential DNA
denaturability of interphase and mitotic nuclei at low
pH, thus enabling to calculate the percentage of mitotic
as well as G1, S and G2 cells in a given population. The
supercoiled DNA of mitotic cells has less re-annealing
capacity upon DNA denaturation, resulting in more
single stranded DNA (ssDNA), that can be detected
using the metachromatic DNA binding dye AO. AO
has di€erent emission wavelengths upon binding to
double stranded versus single stranded DNA. The G2/
M enriched fraction that was obtained from the
various cell lines using centrifugal elutriation, was girradiated and subjected to AO analysis. Figure 3a
shows the cell cycle distribution obtained by the AO
DNA denaturability assay. The onset of the G2 arrest
is very fast in all L12 derivatives as demonstrated by
the complete disappearance of the mitotic populations,
Figure 2 Wild type RSp53 arrests cells at G1 phase following g-irradiation: Cell cycle pattern. L12 clones expressing regular spliced
wild type RSp53-10 (marked in gray in a, b, c); alternatively spliced wild type ASp53-22 (marked as empty in a, b, c) or empty pSVL
plasmid, clone pSVL-3 (marked in black in a, b, c) were grown as baove (see Figure 1) and enriched for the G1 phase using
centrifugal elutriation at viable conditions (a). Normal cell cycle distribution is depicted by dotted line in a. The cells were either
directly replacted or g-irradiated (300 R) and then replated. (c) shows the cell cycle pattern of G1 enriched fraction 6 h after
irradiation. (b) shows the cell cycle pattern of the non-irradiated clones. (d, e, f) parallel (a, b, c) except that the clones used in these
experiments are mutant RSp53 (marked in gray), mutant ASp53 (marked as empty) or empty pSVL plasmid (marked in black)
2599
p53 and the G2 checkpoint
D Schwartz et al
2600
as early as 2 h post g-irradiation (Figure 3a, second
row). The mitotic populations of wild type p53
expressing cells, (Figure 3a, arrows) are evident
already 6 h after irradiation (third row). However,
mutant p53 expressors (Rsp53M11-3 and ASp53M83A2), or p53-null control cells (pSVL-3), were still
arrested at G2 at this time point.
The overall plot of the percentage of mitotic cells at
various time points following g-irradiation shown in
Figure 3b, con®rms that wild type p53 expressors of
both RS and AS forms, have earlier exit from the G2
arrest compared to p53-null control cells. RSp53
expressors exit the G2 delay signi®cantly faster than
ASp53 expressing cells. Mutant p53 expressors had
similar kinetics of exit from G2 delay to that of p53null pSVL control cells (data not shown).
We next analysed the same AO stained samples, by
gating on the cell populations of the G2 phase. Figure 4
shows the amount of G2 cells at various times, as
percent of the number of G2 cells at time 0. Figure 4a
shows the comparison between wild type p53
expressors and the p53-null control, while Figure 4b
compares pSVL p53 null cells to mutant p53
expressors. g-irradiation induced speci®c accumulation
at the G2 phase in all L12 clones. The exit from the G2
arrest, manifested by the reducton in the percent of the
G2-arrested cells, is much faster in wild type p53
expressing clones than in mutant p53 expressors, or in
the p53-null parental clone. Analysis of cell cycle
progression of the mutant p53 expressing clones
(Figure 4b), indicated that these p53 mutants have no
dominant negative e€ect, neither on regulation of the
a
b
Figure 3 The e€ect of irradiation on the mitotic phase of the G2 enriched L12 clones. The clones shown in the upper part were
enriched for G2 cells (the upper row), and irradiated (300 R). (a) shows the amount of mitotic and interphase cells (see markings in
the pSVL column). The enriched fractions were analysed by FACS using the Acridine Orange DNA denaturability method enabling
to distinguish between interphase and mitotic cells, evaluating the cell cycle stage in interphase cells. The second and the third rows
show the cell cycle phase 2 h and 6 h after irradiation. (b) shows the percent of mitotic cells normalized to time 0 of control. -&L12 pSVL; -&- wild type RSp53; -~- L12 wild type ASp53-22
p53 and the G2 checkpoint
D Schwartz et al
2601
Figure 4 Kinetics of the G2 phase in normal growth and after g-irradiation of L12 clones. (a) shows the percent of G2 cells in G2enriched fractions with or without irradiation: -*- L12 pSVL irradiated; -*- L12 pSVL non-irradiated; -&- L12 wild type RSp5310 irradiated; -&- L12 wild type RSp53-10 non-irradiated; -~- L12 wild type ASp53-22 irradiated; -~- L12 wild type ASp53-22
non irradiated. (b) shows the percent of G2 cells in G2-enriched fractions with or without irradiation: -&- L12 pSVL irradiated; -*L12 pSVL non-irradiated; -&- L12 mutant RSp53-M11 irradiated; -~- L12 mutant RSp53-M11 non-irradiated; -~- L12 mutant
ASp53-M8 irradiated; -*- L12 mutant ASp53-M8 non-irradiated
G2 delay upon DNA damage, nor on the length of the
G2 phase at logarithmic cell growth.
These results suggest that both RSp53 and ASp53
forms facilitate the exit from the G2 delay with
di€erent kinetics. Mutant p53 expression did not
signi®cantly a€ect the cell cycle pattern, as compared
to p53 null parental cells.
p53 is involved in the reapir of micronuclei generated by
DNA damage at the G2 phase
Accumulation of cells at the G2 phase results from
DNA damage. It was previously shown that the
amount of micronuclei in postmitotic cells induced by
DNA damage, is inversely correlated with the eciency
of the double strand break (DSB) DNA reapir at the
preceding G2 phase, and to the integrity of the G2 and
spindle checkpoints. We did not detect alterations in
the arrest at the G2 checkpoint, since all clones lacked
mitotic cells 2 h after g-irradiation. Analysis of the
integrity of the spindle checkpoint, using prolonged
cultivation in presence of nocodazole, indicated that all
L12 clones developed low super-tetraploid endoreduplicated cell populations, characteristic of a disrupted
spindle checkpont (data not shown). Thus, both G2
and the spindle checkpoints were probably intact in all
L12 clones, and could not account for the di€erent
kinetics of exit from the G2 delay. Di€erences in DNA
repair pro®ciency between wild type p53 expressors
and non-expressors at the G2 phase, could possibly
account for the di€erential exit from the G2 delay that
we observed.
We have, therefore, examined the contribution of
p53 to the elimination of DNA breaks at G2, by
determining the amount of micronuclei in cells
immediately after exit from the G2 delay. At this
point the micronucleated cells were still intact and did
not undergo apoptosis, thus enabling their quantitation.
Elutriation enriched G1 and G2/M fractions of
logarithmically growing L12 clones harboring wild
type or mutant p53 protein were replated, girradiated (at 300 R) and the amount of micronuclei
at various time points was determined by FACS.
Cells were treated with hypotonic bu€er containing
non-ionic detergent to obtain isolated nuclei, and
micronuclei. The samples were stained with PI and
analysed by FACS for DNA content, size and
granularity, that enables to distinguish micronuclei
from intact nuclei, apoptotic blebs and cell debris
(Figure 5a). Figure 5b shows percent micronuclei
normalized to the amount of divided cells in L12
clones expressing either wild type or mutant p53
protein. The ratio between micronuclei and intact
nuclei indicated that all logarithmically growing L12
clones contained up to 1.5% of micronuclei (Figure
5b, sections G1 0 h and G2 0 h). After g-irradiation
and release of the g-irradiated cells from the G2
delay, wild type RSp53 and ASp53 expressors
contained 8 ± 20% micronuclei in the postmitotic cell
population, while p53-null control cells and mutant
p53 expressors contained 38 ± 50% micronuclei
(Figure 5b, section G2 12 h IR).
The notion that the formation of micronuclei
following DNA damage is dependent on transition
through mitosis is supported by the ®nding that in
both G1 and G2 enriched cells, the time course of
micronuclei induction after DNA damage paralleled
the resumption of mitosis (Figure 5b sections, G1 12 h
IR; G2 12 h IR).
The reduced number cells with micronuclei in clones
harboring wild type p53 suggests that wild type p53
may a€ect the repair of damaged DNA at the G2
phase.
p53 and the G2 checkpoint
D Schwartz et al
2602
p53 protein is upregulated in micronucleated cells
We next studied the distribution of wild type p53 in
micronucleated cells. In all L12 derived cell lines p53
was expressed constitutively at low levels. To determine
the subcellular distribution of p53 in micronucleated
cells, cells which overexpressed wild type p53 protein
were chosen. We generated L12 derived clones
expressing high levels of the temperature sensitive
Val135 RSp53 variant. Cells were grown at 378C, girradiated (500 R) and incubated for 24 h at 328C to
allow wild type p53 conformation (Gottlieb, et al.,
1994). Cells were then ®xed and stained using the PAb421 or PAb-248 anti-p53 monoclonal antibodies, and
DNA was stained with Hoechst-33342. As seen in
Figure 6, staining (upper row) revealed that micronuclei (marked by arrows) appeared as de®ned nuclear
extensions with the same DNA pattern as the nucleus.
The apoptotic blebs exhibited a typical picnotic DNA
pattern (Figure 6a). Micronucleated cells are often
a
stained by p53 much more intensely than other cells
(Figure 6b), indicating a `speci®c activation' of p53 in
response to chromosomal aberrations or DNA breaks.
Apoptotic blebs (Figure 6a upper row), appear to loose
p53 expression. These results indicated that the p53
pathway is speci®cally activated in micronucleated
cells. Judged by the patterns of p53 expression and
the di€erence in DNA staining, the micronucleated
cells could be distinguished from apoptotic blebs.
Wild type p53 induces apoptosis in micronucleated cells
In order to examine whether wild type p53 is also able
to eliminate selectively cells containing micronuclei, the
L12 p53 non-producer cell line (pSVL), wild type
RSp53 (RSp53-10) and wild type ASp53 (ASp53-22)
were g-irradiated (600 R) and grown for 28 h when
apoptosis had reached its peak (data not shown). Each
cell line was analysed by FACS for DNA content using
the vital dye HO and for cell viability, by PI exclusion.
Figure 7Aa shows the typical HO-PI distributions
obtained by this method (represented by the pSVL
clone). Cells containing a sub-diploid population
(typical for apoptosis) also exhibit intense PI uptake,
indicating high membrane permeability (typical for
dead cells). Cellular populations containing dying cells
possessed intermediate PI incorporation that retained
normal DNA content, and living cells lacking PI. The
living and dying fractions of each clone were sorted by
the FACStar+ ¯ow cytometer using these parameters.
Such analysis revealed that these di€erent populations
in each clone were more than 96% pure. The sorted
dying cell fraction (represented in the pSVL clone) is
shown in Figure 7Ab, and the sorted living cell fraction
is shown in Figure 7Ac. The non-sorted cells (Figure
B
DNA staining
A
p35 Expression
b
Figure 5 Analysis of micronuclei by FACS. Cells were enriched
for the various cell cycle fractions by centrifugal elutriation, girradiated (300 R) and analysed by FACS for the amount of
micronucleated cells. (a) shows a typical distribution of the
micronucleated cells. (b) shows a chart with the amount of
micronuclei divided by the amount of nuclei (percent) from the
total population (in G1 and G2 0 h, and in G1 12 h IR
categories), or of the amount of post mitotic diploid cells
appearing after the release from the G2 (12 h) arrest
Figure 6 p53 distribution in micronucleated and apoptotic cells
following g-irradiation. Logarithmically growing L12 cells stably
expressing the temperature sensitive p53 mutant were g-irradiated
(500 R) and grown at 328C for 24 h. The cells were ®xed in
methanol and stained for p53 protein using monoclonal anti-p53
antibodies PAb-421 and PAb-248. The chromatin structure was
visualized using the Hoechst-33342 DNA dye. The upper row
shows the DNA pattern, and the lower row shows p53
distribution. Column A shows the comparative p53 and DNA
patterns of apoptotic versus micronucleated cells and column B
represents micronucleated versus intact cells. The micronuclei are
marked by black arrows
p53 and the G2 checkpoint
D Schwartz et al
2603
Figure 7 Wild type p53 eliminates cells harboring damaged DNA. The p53-null clone containing the empty pSVL plasmid (pSVL),
the wild type RSp53 (RSp53-10), or wild type ASp53 (ASp53-22) were irradiated (600 R), grown for 26 h, and incubated for 1 h
with 5 mg/ml of Hoechst-33342 to label DNA. Cells were also stained with 50 mg/ml of PI to label dying and dead cells. Cells were
analyzed and sorted by FACS using the FACStar+ ¯ow cytometer (A a). The sorted fractions of dying (A b) and living (A c) cells,
as well as the unsorted total cell population were subjected to micronuclei assay by FACS on a FACSort ¯ow cytometer. The ratio
of micronuclei in the dying cell fraction/micronuclei in the living fraction for each cell line is shown in section B
7Aa), as well as the two sorted fractions of each cell
line, were further examined for the amount of
micronuclei using the FACsort ¯ow cytometer as
described above (Figure 5aa. Figure 7b depicts the
relative abundance of micronuclei found in dying cells
as a fraction of micronuclei found in the living cell
population, for each clone. Wild type RSp53 and the
ASp53 producer cell lines, retained less micronucleated
living cells than the parental p53-null clones. Moreover, signi®cantly more micronucleated dying cells
were detected in wild type p53 producer clones.
These results suggest that p53 may selectively direct
cells with DNA damage that result in micronuclei
formation, towards apoptosis.
Discussion
The p53 knockout mouse model has proven unequivocally that p53 functions as a tumor supressor gene.
p53 null mice developed a high incidence of tumors
during adult life (Donehower et al., 1992; Jacks et al.,
1994). Moreover, the recent observations showing that
these mice exhibit a variety of developmental aberrations (Armstrong et al., 1995; Sah et al., 1995), support
the conclusion that p53 is also essential for normal
development. This agrees with early reports showing
that p53 expression during embryogenesis is con®ned
to proliferating and newly di€erentiating regions
(Rogel et al., 1985; Schmid et al., 1991).
p53 appears to be a key protein in the control of the
cell cycle. It is generally accepted that in addition to its
role in the G1 phase, p53 is also involved in S and G2
phases of the cell cycle (Remvikos et al., 1990; Danova
et al., 1990). p53 was shown to be upregulated
following DNA damage, at all stages of the cell cycle
(Yamaizumi, Sugano, 1994). In addition to the arrest
at the G1/S border (Kastan et al., 1991, 1992; Kuerbitz
et al., 1992; Di Leonardo et al., 1994) elicited by p53
via the waf-1 pathway (El-Deiry et al., 1994), high
levels of wild type p53 can also induce a G2 arrest
(Stewart et al., 1994; Agarwal et al., 1995). In AtaxiaTelangiectasia, where the p53 pathway is inactivated or
deregulated, the DNA damage-dependent G2 arrest is
much longer than in normal cells. This is explained by
the lower repair eciency, especially of double strand
breaks that occur in this syndrome (Hong et al., 1994;
Barlow et al., 1996; Hawley and Friend, 1996). In
agreement with this notion, we have previously
observed that inactivation of wild type p53 function
by dominant negative mutant p53, shortened the girradiation induced G2 cell arrest (Aloni-Grinstein et
al., 1995). Furthermore, ®broblasts derived from LiFraumeni patients, lost the normal G2 checkpoint
p53 and the G2 checkpoint
D Schwartz et al
2604
response following homozygotisation of the mutant
p53 allele (Paules et al., 1995; Kaufmann et al., 1995).
p53 was also shown to prevent endoreduplication of
the tetraploid genome during prolonged mitotic arrest
by regulating the spindle checkpoint (Cross et al., 1995;
Wells, 1996). In agreement with this notion, we found
that after induction of the mutant p53 conformation,
M1/2 myelomonocytic cells expressing temperature
sensitive mutant p53 had more giant polyploid cells,
and induction of the wild type p53 conformation
reduced their number (Peled et al., 1996).
The notion that a p53-dependent pathway is
involved in DNA repair in addition to the regulation
of cell cycle checkpoints and apoptosis of damaged
cells by p53 is supported by several ®ndings: For
example, p53 was shown to accelerate excision repair
(Pan et al., 1995; Wang et al., 1995; Bogue et al., 1996).
Waf-1-null human cancer cells, possess mismatch
repair de®ciency (McDonald III et al., 1996). LiFraumeni derived ®broblasts homozygous for p53
mutations, are de®cient in global DNA repair (Ford
and Hanawalt, 1995). p53 was found to bind to several
genes directly involved in excision repair, such as RPA
(Dutta et al., 1993), ERCC3 (Wang et al., 1994), and
human Rad51 protein involved in double strand break
recombination repair pathway (Sturzbecher et al.,
1996). Moreover, p53 functions as a DNA helicase
via its C-terminus, that has the ability to bind
nonspeci®cally single stranded (Bakalkin et al., 1994,
1995; Jayaraman and Prives, 1995) and mismatched, or
nicked DNA (Lee et al., 1995).
This study addressed the role of p53 in DNA repair
and cell cycle arrest at the G2 phase. The e€ect of p53
upon g-irradiation of G2 phase enriched cellular
fractions was analyzed regarding the G2 delay and
the eciency of double strand break repair in various
L12 clones. These cell lines were generated by
introducing p53 expression regulated by a weak
promoter that codes for low levels of p53, thus
mimicking physiological condition. We found that the
spindle checkpoint is inteact in all L12 clones, and
therefore, we have focused on the function of p53 at
the G2 checkpoint. We analyzed the cell cycle pattern
by standard FACS analysis of logarithmically growing
L12 clones enriched for G1 or G2/M phases by
centrifugal elutriation, before and after g-irradiation.
We found that both alternatively spliced forms of wild
type p53 protein, but not the corresponding mutant
forms, accelerated the exit, but did not a€ect the entry,
into g-irradiation-induced G2 delay when compared to
the p53-null parental clone. This implies that the onset
of the G2 checkpoint is p53-independent in L12 clones.
Non-g-irradiated wild type and mutant p53 expressing
cell lines exhibited similar kinetics of progression when
the G2 enriched fractions were analysed.
Examination of the g-irradiation reponse of G1
enriched populations showed that only the wild type
RS p53 expressing clones displayed G1 arrest, that was
evident at 6 h after g-irradiation. Constitutive expression of low levels of wild type p53 in these cells
prolonged the G1 phase, also under normal growth
conditions (Shaulsky et al., 1991). The inability of wild
type ASp53 to regulate eciently the G1 arrest is in
agreement with the notion that this form of p53 has
functions that are speci®c for the G2 phase (KuleszMartin et al., 1994).
The integrity of the G2 checkpoint could be analyzed
accurately only by measuring the percent of mitotic
cells at various times following DNA damage. We
utilized the AO DNA denaturability assay by FACS,
enabling to de®ne mitotic, as well as G1, S and G2 cells
in a given population. In agreement with previous
results, all clones arrested at the G2 phase following girradiation, did contain mitotic cells ± 2 h after treatment. Accurate analysis of the G2 fraction using this
assay con®rmed that clones expressing wild type p53 of
both alternatively spliced p53 forms existed from the
G2 checkpoint faster than the mutant p53 expressors
and p53-null cells. p53, therefore, a€ects more
signi®cantly the DNA repair at the G2 phase in these
cells, causing an accelerated exit from the G2 delay.
The analysis of the eciency of DNA repair at the G2
phase in all clones, by determining the number of
micronuclei in cells that exited the G2 delay, indicated
that wild type p53 expressors exhibited a signi®cantly
lower levels of micronuclei. Mutant p53 expressors had
the same levels of micronuclei as those in p53 nonproducers, suggesting that mutant p53 does not a€ect
these G2-related activities. Non-irradiated cells, had very
low quantities of micronuclei, and the time course of
micronuclei appearance followed that of cell division. To
prove that the number of micronuclei represents
authentically the eciency of DNA repair at the
preceding G2 phase, it was essential to determine
micronuclear number before the onset of apoptosis.
The results indicated that p53 has a role in the regulation
of double strand break repair of DNA in G2 phase, as well
as in the exit from the DNA damage dependent G2-delay.
We also analysed the possibility that wild type p53 is
activated in micronucleated cells. Immunocytochemical
analysis of p53 overproducer L12 clones expressing the
temperature sensitive RSp53 after g-irradiation, indicated that DNA damage induced the accumulation of
p53 in the micronucleated cells. Occasionally, only the
micronucleus had intense p53 staining, while the
regular nucleus showed only weak staining for p53.
This may point to the possibility that p53 has the
ability to be targeted to damaged DNA in vivo.
Finally, we have shown that wild type p53 is also
able to direct micronucleated cells towards apoptosis.
Comparison of the incidence of micronuclei in living
and dying fractions of wild type p53 producers versus
p53-null cells revealed that the former had signi®cantly
more micronucleated dying cells than the latter.
Moreover, there were less micronucleated living cells
left in the p53 producer populations. These observations suggest that wild type p53 has the ability to
speci®cally eliminate micronucleated cells.
It appears therefore, that p53 seems to exhibit two
opposite e€ects on DNA damage-dependent cell cycle
checkpoints. Based on our previous results it appears
that p53 arrests damaged cells until their DNA is
repaired, or alternatively, promotes cells with unrepairable damage towards apoptosis (Aloni-Grinstein et al.,
1995). On the other hand, our present data support the
notion that p53 facilitates the repair of damaged DNA,
thus accelerating the exit of cells from the DNA
damage-dependent cell cycle checkpoints. This implies
that the net e€ect of p53 on the cell cycle response to
DNA damage may depend on the cell type, the level of
p53 in the cell, and on the type of DNA damage, that
is repaired by the di€erent pathways.
p53 and the G2 checkpoint
D Schwartz et al
Taken together, these results can provide a
functional explanation for the cell cycle-independent
expression pattern of p53 during normal growth, and
the upregulation and nuclear targeting of p53 upon
DNA damage, in diploid and tetraploid cell cycle
stages.
Materials and methods
Cell lines and culture medium
The murine L12 cells were grown in suspension at 378C in
RPMI-1640 medium containing 10% fetal calf serum and
261075M b-mercaptoethanol. L12 clones carrying the
regularly spliced wild type p53 cDNA, L12-RSp53-10; 20
(Shaulsky et al., 1991), and alternative spliced wild type
p53, L12 ASp53-22; 23, generated by transfection with
alternatively spliced C-terminal p53 coding plasmid and the
pSV2-gpt plasmid containing the drug resistance gpt gene,
were grown in RPMI-1640 medium containing 2 mg/ml
mycophenolic acid, 150 mg/ml xantine and 15 mg/ml
hypoxantine. The L12 clones carrying mutant p53 were:
L12-M11 (Regularly spliced p53 Glu?Gly mutation at
codon 168) and L12-M8 (alternative spliced p53 Cys?Phe
mutation at codon 133). Clone L12-109 (tsRSp53) was
established by infection of the parental L12 cells by cocultivating with pLXSN-p53Va/135 gpe+ virus producing
cells (Gottlieb et al., 1994) (kindly provided by Dr M Oren,
The Weizmann Institute of Science) for 48 h and the
selection of single cell clones was achieved by limited
dilution and incubation with 2 mg/ml G-418 drug. p53
expression in G-418-resistant cells was determined by
Western blotting.
Antibodies
Monoclonal antibodies to p53 PAb-421 (Harlow et al.,
1981), PAb-242, and PAb-248 (Yewdell et al., 1986;
Gannon et al., 1990) were obtained of ascitic ¯uids of
the peritoneum of hybridoma-bearing syngeneic Balb/c
mice, that were spun to remove tissue debris, diluted in
PBS and used without further puri®cation.
Cell cycle fractionation by centrifugal elutriation and girradiation
1 ± 36108 cells were loaded into an elutriation rotor (J-6MI
centrifuge equipped with a JE-5.0 elutriation system,
including a Sanderson chamber (Beckmann Instruments
Inc.) and MasterFlex (Cole-Parmer Instruments) peristaltic
pump pre-sterilized with 5% Sodium hypochlorite. The
separation was performed at room temperature (20 ± 228C)
using RPMI medium supplemented at 2% heat inactivated
fetal calf serum with a constant centrifuge speed of 2200
r.p.m. (rmax=*470). The cells were loaded with pump
speed of 12 ml/min, and diploid (G0-G1): (at 15 ± 21 pump
speed) and tetraploid (at pump speed 29 ± 36) fractions
were collected. After the separation, cells were washed once
with DPBS, counted and replated in RPMI supplemented
with 8% fetal calf serum at 0.3 ± 0.66106 cells/ml. Part of
the cells were g-irradiated (with 300 rads) after replating,
and their fate was followed.
Measurement of cell cycle using the AO DNA denaturability
assay by FACS
Cells were harvested and ®xed for 2 ± 24 h in 70%
ethanol ± 30% HBSS (Hanks Balanced Salt Solution) v/v,
at 7208C at 16106 cells/ml. The cells were washed once
with HBSS, and resuspended in HBSS with 0.5 mg/ml
RNase A. After 1 h incubation at 378C, the cells were
washed once and resuspended in HBSS at 26106 cells/ml.
Cell suspensions (200 ml) were added to 0.5 ml of 0.1 M
HCl. After 30 s incubation at RT, 2 ml of Acridine Orange
(AO) (Molecular Probes) staining solution of pH 2.6,
containing 90% v.v. 0.1 M Na-Citrate and 10% v/v 0.2 M
Na2HPO4 and 6 mg/ml AO were added (Darzynkiewicz,
Gong, 1994). Cells were analysed by the FACS using
FACSort ¯ow cytometer and CellQuest list mode analysis
software (Beckton-Dickinson) (Darzynkiewicz, 1994).
Detection of micronuclei by ¯ow cytometry
The protocol was described before (Nusse et al., 1992,
1994; Schrieber et al., 1992). Brie¯y, the cells were washed
in PBS once. The pellet (0.1 ± 16106 cells) was resuspended
and vortexed for 20 s in 1 ml of Solution I containing
584 mg/L NaCl; 1 gr/L Na-citrate; 10 mg/L RNAse A
from bovine pancreas, preheated to inactivate the residual
DNAses (Boeringher Mannheim); 0.3 ml/L NP-40 (Sigma)
and 25 mg/ml PI (Sigma). The cell suspension was
incubated for 1 h in the dark at RT, vortexed, and 1 ml
of Solution II containing 40 mg/ml PI, 15 mg/L Na-Citrate
and 0.25 M sucrose was added. After a short vortexing, the
samples were analysed on a FACSort.
Incubation of cells with nocodazole
Logarithmically growing L12 clones were incubated with
0.05, 0.1 and 0.5 Mg/ml of Nocodazole (Sigma) for 12, 24,
48 and 96 h. The cells were ®xed in 80% ethanol (BioLab)
at 7208C for 24 h and the cell cycle pattern was analysed
at each time point using routine DNA content analysis by
FACS. Cell clumps were excluded, by live gating on single
cells, according to the area and the width of the PI (FL/2)
signal.
Immunocytochemical staining for p53 and for chromatin
Logarithmically growing L12 clones were washed once
with PBS and ®xed overnight in 80% methanol (BioLab) at
48C in suspension. The ®xed cells (1.56106) were washed
once in PBA (PBS containing 0.5% w/v BSA (fraction VII
Sigma) and 0.02% w/v sodium azide (Sigma), and
incubated for 1 h on ice in 100 MI of ascitic ¯uid
containing either PAb-421 or PAb-248 monoclonal
antibodies diluted 1 : 40 in PBA. The cells were washed
twice with PBA and incubated on ice with FluorolinkCy2TM labeled Goat anti-Mouse IgG (H&L) antibody
(Amersham Cat. PA42002) for another 1 h. After two
washes with PBA the cells were concentrated to 56106
cells/ml in PBA containing 5 mg/ml Hoechst 33342
(Molecular Probes Inc.). The cell suspension was either
directly applied on slides or centrifuged in a cytospin
centrifuge and the ¯uorescent cells were visualized and
photographed in an Axioplan ¯uorescent microscope
(Zeiss) using 1600 ASA strong BW KODAK ®lm.
Standard cell cycle analysis by FACS
Viability and DNA content analysed by HO-PI: sorting of living
and dying population
Cells were ®xed in 70% ethanol (BioLab) at 7208C, for at
least 2 h and stained with 50 mg/ml Propidium Iodide
(Sigma). The cells were analysed by a FACSort ¯ow
cytometer (Beckton Dickinson), using the CellQuest
(Beckton Dickinson) software.
Logarithmically growing L12 cells were incubated for 1 h
with 5 mg/ml of Hoechst-33342 (Molecular Probes) that
intercalates into DNA, and for 3 min with 25 mg/ml PI
(Sigma) that is incorporated in dying cells, having
ruptured membranes (Belloc et al., 1994). The cells were
2605
p53 and the G2 checkpoint
D Schwartz et al
2606
analysed on a dual laser FACStar+ cell sorter (Beckton
Dickinson Inc). The u.v. laser excited Hoechst-33342
¯uorescence was recorded on a FL/3 channel, and the
PI ¯uorescence was recorded on the FL/2 channel. Using
these two parameters, the cells were sorted into two
groups: Living-Low FL/2 ¯uorescence, Dying-Intermediate FL/2 ¯uorescence. The sorting conditions were also
selected for more than diploid DNA content according to
FL/3 ¯uorescence.
0.56106 cells were sorted from each fraction and
analysed for purity. In all fractions, the enrichment was
more than 96%. Subsequently, the sorted fractions, and
nonsorted cells were subjected to the micronucleus assay as
described.
Acknowledgements
The authors wish to thank Prof Avri Ben-Ze'ev and David
Wisemann for fruitful discussion and criticism. This work
was supported in part by grants from the Leo and Julia
Forchheimer Center for Molecular Genetics, DKFZ and
the Minerva Foundation. VR is the incumbent of the
Norman and Helen Asher Professional Chair in Cancer
Research at the Weizmann Institute.
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