1. No category

Oncogene (1997) 14, 1117 ± 1122
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
SHORT REPORT
Increase of spontaneous intrachromosomal homologous recombination in
mammalian cells expressing a mutant p53 protein
Pascale Bertrand1,4, Danielle Rouillard2, Annick Boulet2, CeÂline Levalois1, Thierry Soussi3
and Bernard S Lopez1
1
UMR 217 CNRS CEA/DSV/DRR, 60±68 Av. du GeÂneÂral Leclerc BP6, 92265, Fontenay aux Roses, France; 2Institut Curie,
Section de Recherche, 26 Rue d'Ulm, 75231 Paris cedex 05, France; 3UMR 218 CNRS, Institut Curie, Section de Recherche, 26
rue d'Ulm, 75231 Paris cedex 05, France
Homologous recombination plays an essential role in
processes involved in genome stability/instability, such
as molecular evolution, gene diversi®cation, meiotic
chromosome segregation, DNA repair and chromosomal
rearrangements. p53 devoid cells exhibit predisposition
to neoplasia, defects in G1 checkpoint and high genetic
instability but a normal rate of point mutations. We
investigated the e€ect of a p53 mutation, on spontaneous homologous recombination between intrachromosomal direct repeat sequences, in mouse L cells. In these
cells, wild type for the p53 gene, we have overexpressed
the mutant p53175(Arg4His) protein leading to a p53 mutant
phenotype, as veri®ed by the absence of a G1 arrest
after g-irradiation. We show that the rate of spontaneous recombination is increased from ®ve- to 20-fold in
the mutant p53 lines. Moreover, this increase is
observed in gene conversion as well as in deletion
events. Our results provide new insights into the
molecular mechanisms of genetic instability due to a
defect of p53.
Keywords: Homologous recombination; p53; genetic
instability; tumoral progression
Homologous recombination is a mechanism implicated
in the equilibrium between stability and variability of
the genome. It is involved in numerous fundamental
processes such as molecular evolution, gene diversification, chromosome segregation during meiosis and
DNA repair. In addition, recombination between
homologous dispersed sequences may lead to profound genome rearrangements such as inversions,
deletions, ampli®cations, translocations. Since gene
conversion may lead to the transmission of point
mutations, deletions as well as gene conversion can be
implicated in loss of heterozygosity (Xia et al., 1994).
Control of the cell cycle has been shown to be
essential to maintain genome integrity. More specifically, defects in the G1 checkpoint lead to genetic
instability and therefore may be involved in the
progressive stages of neoplasia (for reviews see
Donehover and Bradley, 1993; Smith and Fornace,
1995; Hartwell and Kastan, 1994; Hainaut, 1995; Ko
and Prives 1996; Tyler and Weinberg, 1996). In
Correspondence: BS Lopez
4
Present address: Division of Human Cancer Institute, 44 Binney
Street, Boston, MA 02115, USA
Received 20 September 1996; revised 4 November 1996; accepted 4
November 1996
mammalian cells, this conclusion is supported by
several lines of evidence. First, cells from patients
su€ering from ataxia telangiectasia, an hereditary
syndrome associated with cancer predisposition, are
hypersensitive to ionizing radiation, display genetic
instability and a high level of spontaneous homologous
recombination (Meyn, 1993; Luo et al., 1996). These
cells are also defective in G1 arrest after exposure to
ionizing radiation (Kastan et al., 1992; Lu and Lane,
1993; Beamish et al., 1994; Beamish and Lavin, 1994).
Second, numerous studies of the p53 protein indicate
that it plays a pivotal role in the G1 checkpoint (for
review see Donehover and Bradley, 1993; Smith and
Fornace, 1995; Hainaut, 1995; Ko and Prives, 1996;
Tyler and Weinberg, 1996). However, some reports
failed to ®nd any evidence of prolonged G1 arrest
induced by radiation in tumor cells expressing a wild
type p53 (Little et al., 1995; Nagasawa et al., 1995).
Nevertheless, the p53 gene is the most frequently
mutated gene in human tumors (Levine et al., 1991;
Hollstein et al., 1991). Analysis of p53 mutant cells
show that they have lost their capacity to inhibit cell
growth after exposure to DNA damaging agents.
Finally, cells devoid of p53 exhibit a higher likelihood
of gene ampli®cation (Livingstone et al., 1992; Yin et
al., 1992) and exhibit a high level of spontaneous
chromosomal abnormalities (Bou‚er et al., 1995).
It has recently been reported a physical interaction
between p53 and the human Rad51 (hRad51) proteins.
HRad51 protein shows homologies with the yeast
Rad51 and the bacterial RecA proteins (Shinohara et
al., 1993; Yoshimura et al., 1993), both involved in
recombination in their respective organisms. Although
the exact role of hRad51 in mammalian cells remains
to be established (Edelmann and Kucherlapati, 1996),
it is essential to examine the consequences of p53
mutation on homologous recombination.
We investigated here the e€ect of p53 mutation on
spontaneous intrachromosomal homologous recombination in mouse L cells. We used the pJS3-10 line which
contains a unique direct repeat sequences integrated into
the chromosomes (Liskay et al., 1984). This line allows
the direct measurement of homologous recombination
and the determination of the ratio of gene conversion
versus deletion events in the cultured cells (Figure 1). The
pJS3-10 line is wild type for p53. A p537 phenotype can
be obtained by expressing viral proteins such as large T
antigen from SV40 or the E6 protein from papillomavirus. However, because of the pleiotropic e€ects of these
proteins and also because a direct e€ect of the viral
protein cannot be ruled out, we chose to overexpress a
Stimulation of homologous recombination in p537 cells
P Bertrand et al
1118
sion was controlled by the cytomegalovirus (CMV)
promoter (Ory et al., 1994) and with pY3 plasmid
containing the hygromycin B resistant gene controlled
by Moloney sarcoma virus LTR (Blochlinder and
Diggelmann, 1984). Negative control, CDR clones,
were obtained by cotransfection of pY3 plasmid with
the pCMV vector without p53 cDNA. Clones were ®rst
selected for hygromycin resistance, then expression of
p53175(Arg-4His) protein was determined by immunocytochemical detection with a speci®c antibody for p53
proteins (Ory et al., 1994). The HDR140 clone did not
express the mutant p53 protein and was selected as a
negative control. Overexpression of a wild type p53, as
a control, is not possible since it has been shown to
suppress cell growth (Baker et al., 1990); thus isolation
of such clones is impossible.
Overexpression of the mutant p53175(Arg-4His) protein
a€ects the G1 checkpoint after g-irradiation
Figure 1 Organization of the recombination substrates in the
cells: The cell line used is the pJS3-10 and were cultured at 378C
with 5% CO2 in Dulbecco's modi®ed Eagle medium supplemented with 10% fetal bovine serum (Liskay et al., 1984). This line is
a mouse LTK7 line containing two TK genes (dashed boxes)
from Herpes Simplex type I virus, in direct repeat. Each HSV-TK
gene contains di€erent frame shift mutations created by a 8 bp
XhoI linker insertion (open !). Thus the cells are still TK7 and
sensitive to the selective medium HAT (100 mM hypoxanthine,
2 mM aminopterin, 15 mM thymidine). Recombination between the
two HSV-TK copies can recreate a functional TK gene either by
gene conversion or by deletion conferring HAT resistance to the
cell. Homologous recombination frequency can be estimated by
the frequency of HAT resistant clones and veri®ed by restriction
analysis (Liskay et al., 1984). For each clone analysed, ten
cultures were prepared by plating 50 cells in 25 cm2 ¯asks.
Cultures were maintained to con¯uence. Cells were then
trypsinized, counted and one portion was used for plating
eciency estimation. The remaining cells were plated under
HAT selection and the resulting number of TK+ clones allowed
us to calculate the recombination frequency. The rate of
recombination per cell per generation was calculated by using
the Luria and Delbruck ¯uctuation test (Luria and Delbruck,
1943; Capizzi and Jameson, 1973)
mutant p53 protein. Expression of mutant p53 protein
has been shown to override the endogenous wild type
p53 protein in a dominant negative manner by forming
complexes with wild type protein and functionally
inactivating it (for review see Donehover and Bradley,
1993; Smith and Fornace, 1995; Hainaut, 1995; Ko and
Prives, 1996; Tyler and Weinberg, 1996). We chose to
overexpress the mutant p53175(Arg-4His) protein because it
has a strong dominant negative phenotype and is one of
the most frequent mutation found in tumors (Ory et al.,
1994).
Construction of the cell lines expressing the mutant
p53175(Arg-4His) protein
The p53 mutant cell lines, HDR clones, were obtained
by cotransfection of the pJS3-10 line with the plasmid
pCMV containing p53175(Arg-4His) cDNA where expres-
We veri®ed the negative e€ect of p53175(Arg-4His) overexpression on the G1 checkpoint after g-irradiation.
The parental line pJS3-10 as well as cells transfected
with the vector lacking p53 cDNA (CDR clones) or
with the complete vector but without expression of the
mutant protein (HDR 140) show G1 arrest after girradiation. This result con®rms the wild type status of
p53 in the parental pJS3-10 line. In contrast, all the
lines expressing the mutant p53 protein are defective in
the G1 checkpoint (Figure 2).
The rate of spontaneous homologous recombination is
increased in clones expressing the mutant p53 protein
We determined the e€ect of p53 status on homologous
recombination. The rate of recombination was
measured as described (Luria and Delbruck, 1943;
Capizzi and Jameson, 1973). The rate of recombination is similar in all the p53 wild type lines, from 1.2
to 1.561076/cell/generation (Table 1). A ®ve- to 20fold incrase (6.30 to 25.00 61076/cell/generation) of
spontaneous recombination is observed in the di€erent
p53175(Arg-4His) lines (Table 1). Interestingly, the extent
of G1 block after irradiation appears associated with
the increase of the rate of recombination (compare
Figure 2 with Table 1), indicating a possible relationship between these two processes. However, the
quanti®cation of the expression of p53 protein, by
immunodetection did not give results precise enough
to certify that the extend of G1 arrest correlate with
p53 levels.
Stimulation of recombination acts on gene conversion as
well as on deletion events
To assess the underlying process, we analysed by
Southern blot the molecular structure of the HSV-TK
locus in recombinant TK+ clones as described in Figure
3. Wild type and mutant p53 lines give comparable
results: 75 to 85% of the events correspond to gene
conversion and 15 to 25% to deletion events (Table 2).
This result indicates that the e€ect of p53 mutation
acts on both gene conversion and deletion events.
In the present study, overexpression of the mutant
p53175(Arg-4His) protein results in a defect of the G1
checkpoint and in an increase in spontaneous
Stimulation of homologous recombination in p537 cells
P Bertrand et al
1119
Figure 2 Measure of the G1 arrest after g-irradiation. For each point, 36106 cells were plated in three petri dishes of 6 cm
diameter with DMEM medium. After 24 h at 378C, cells were washed in PBS bu€er and irradiated (in PBS) at a dose of ®ve grays
using a Co60 irradiator (15 grays/min). PBS was then replaced by DMEM and cells were incubated at 378C. At the indicated time,
cells were trypsinized, collected by centrifugation (5 min at 2000 g), re-suspended in 500 ml PBS and ®xed by adding 1.5 ml of cold
ethanol. DNA content was estimated by propidium iodide ¯uorescence and DNA Flow Cytometry (FACStar, Becton). (a)
Histograms of DNA content of pJS3-10 cell lines, wild type for p53 protein, at di€erent times after irradiation (indicated on the
Figure). The ®rst peak corresponds to the percentage of cells in G1 phase and the second corresponds to the cells in G2 phase.
Between these two peaks is the percentage of cell in S phase (indicated by the box). The diminution of cells in S phase indicates that
cells passing from S phase to G2 are not replaced by cells from G1 phase. Thus cells are blocked in G1 phase as expected in a wild
type p53 context. (b) One example of a clone (HDR 144) expressing mutant p53175(Arg-4His) protein. The number of cells in S phase
does not decrease, indicating the absence of blockage in G1 phase. (c and d) Percentage of cells in S phase as a function of time after
irradiation in wild type p53 (c) or mutant p53175(Arg4His) (d) lines. This result shows that the action of the endogenous wild type p53
is altered by the exogenous mutant p53175(Arg-4His). Error bars are omitted for the sake of clarity of the ®gure but they de®ne four
groups of clones with regard to the eciency of G1 arrest after irradiation (see Table 1): all the wild type p53 lines (c) in the ®rst
group; HDR208 and HDR102 in the second group; HDR211 and HDR224 in the third group; HDR144 and HDR112 in the fourth
group (d)
Stimulation of homologous recombination in p537 cells
P Bertrand et al
1120
Effect of p53175(Arg-4His on G1 arrest and on spontaneous recombination
% of cells in S
Rate of spontaneous
Number
of
phase
24h
after
Number
of
cells
recombination
Expression of
TK+ clonesb
irradiation
screened (6106)a
(610 ±6/cell/generation)
Cell line
p53175(Arg-4His)
pJS3-10
±
2 (+2)
5.20
26
1.2 (+0.3)
CDR 1
±
4 (+2)
5.70
33
1.4 (+0.2)
CDR 3
±
6 (+3)
5.00
25
1.3 (+0.4)
HDR 140
±
10 (+4)
4.10
33
1.5 (+0.3)
HDR 208
+
24 (+4)
2.25
76
6.3 (+1)
HDR 102
+
27 (+4)
2.80
92
6.3 (+0.9)
HDR 224
+
35 (+4)
4.07
271
10.6 (+1.6)
HDR 211
+
40 (+4)
2.00
139
12.5 (+2)
HDR 144
+
48 (+4)
1.13
100
16.9 (+1.9)
HDR 112
+
52 (+4)
1.44
210
25.0 (+2.5)
aMean value for the 10 independent cultures of each line. bMean value for the 10 independent cultures of each line. The numbers ofTK+clones
were corrected by the plating eciency (PE) that varies for each line. The mean PE were 55% and 80% for the mutant and for the wild type
p53 lines respectively
Table 1
Table 2
+
Frequency of Gene Conversion in the TK
Number of TK+
clones tested
clones
Number of
Gene Conversion
(GC) eventsa
Number of % of
Deletion events GC
Cell Lines
p53
pJS3-10
WT
30
23
7
77
CDR1
WT
12
10
2
83
4
4
4
4
4
4
HDR 208
175 (Arg±
His)
10
8
2
80
HDR 102
175 (Arg±
His)
13
11
2
85
HDR 224
175 (Arg±
His)
9
7
2
78
HDR 211
175 (Arg±
His)
10
8
2
80
HDR 144
175 (Arg±
His)
11
9
2
82
HDR 112
175 (Arg±
His)
14
11
3
79
a
Gene Conversion and Deletions events were detected by Southern blot as described (see Figure 1)
a
b
Kb
6
5
1
2 3 4 5 6 7 8
—
—
1.8 —
1.3 —
1
—
Figure 3 Restriction analysis of the TK+ recombinant clones.
(a) Restriction map. Dashed boxes represent the two HSV-TK
sequences. (b) an example of Southern blot analysis of one TKparental clone (1) and seven recombinant TK+ clones (2 to 8).
10 mg of genomic DNA of each clone were digested by BamHI/
HindIII/XhoI and electrophoresed through a 0.8% agarose gel.
The probe used was a puri®ed HSV-TK sequence fragment. (1):
parental TK- clone. (2) and (3) gene conversion of the TK
sequence on the right side. (4, 7, 8): gene conversion on the left
side. (5, 6): deletion event. All DNA manipulations for cell
transfection and for Southern blot analysis were performed as
described (Sambrook et al., 1989)
recombination in all the lines expressing the mutant
protein. The mean increase of spontaneous homologous recombination is about 10-fold. This value is
elevated compared to the stimulation by DNA
damaging agents in similar wild type p53 cell lines.
Indeed, treatment with u.v. or Mitomycin C stimulated
recombination two- and fourfold respectively (Wang et
al., 1988). Poly(ADP-ribose)polymerase inhibitors,
which lead to the accumulation of DNA breaks,
stimulated recombination between three- and fourfold
(Waldman and Waldman, 1991). The recombination
stimulation found here for intrachromosomal recombination, is consistent with the data showing a
stimulation of recombination between plasmids in
immortally transformed cells (Finn et al., 1989) or of
Simian Virus 40 genome intermolecular recombination
in cells with p53 protein trapped by the viral large T
antigen (Wiesmuller et al., 1996). An elevation of
recombination from ten- to 80-fold in SV40 transformed ®broblasts in which a vector expressing p53Ala143
or a human papilloma virus E6 gene was reported by
Meyn and co-workers in the proceeding of a meeting
(Meyn et al., 1994). However, this result was obtained
using SV40 transformed ®broblasts as parental lines.
The phenotype of the control lines used, in which the
endogenous protein p53 could be inactivated by the
large T antigen, was not investigated, making the
interpretation of the results dicult. Their conclusions
are nevertheless in agreement with our results
concerning the stimulation of recombination by cell
lines expressing p53 mutant protein. In addition, we
have in our study, determined the ratio gene
conversion versus deletion events. Since the
Stimulation of homologous recombination in p537 cells
P Bertrand et al
percentage of gene conversion versus deletion remains
unchanged, the stimulation due to the mutant p53
protein should act on both mechanisms.
An alteration of the G1 checkpoint would allow
replication to take place on DNA template bearing
spontaneous lesions. These lesions could block the
progression of replication forks leading to the
formation of DNA single and double-stranded breaks
which are highly recombinogenic structures (Hartwell,
1992; Hartwell and Kastan, 1994); thus, an alteration
of the cell cycle checkpoint, as is the case here in the
mutant p53 lines, could result in an increase of
recombination by providing more recombination
substrates. Additionally, wild type p53 protein
physically interacts with hRad51, a protein potentially
involved in recombination (Sturzbecher et al., 1996).
Moreover, it has been suggested that p53 could be
involved in DNA mismatch repair (Lee et al., 1995;
Jayaraman et al., 1995; Mummenbrauer et al., 1996).
Heteroduplex DNA is a common intermediate
molecule predicted by all the homologous recombination models (Holliday, 1964; Meselson and Radding,
1975; Szostak et al., 1983). In mammalian cells,
heteroduplex DNA as a recombination intermediate
has been described in in vitro reactions with human
nuclear extracts (Lopez et al., 1987) as well as in
cultured mouse L cells lines, similar to the pJS3-10 line
used here (Bollag et al., 1992). In the present
experiments, recombination between the two TK
sequences should produce a heteroduplex DNA
intermediate bearing a 8 bp long loop. One can
imagine a direct role of p53 in the control of
recombination, by a€ecting the putative activity of
hRad51. In this context, p53 may recognize the
heteroduplex created during the strand exchange
process. Mismatch repair could restore the initial
sequence status resulting in absence of gene conversion. Additionally, one can imagine that detection of
mismatched DNA by p53 would induce the apopthosis
leading to death of the recombining cell. A mutant p53
protein does not interact with hRad51 (Sturzbecher et
al., 1996); it may also be unable to recognize the
mismatches in the DNA and/or to induce apoptosis.
Consequently, a mutation in p53 should result in an
increase of the number of TK+ (recombinant) cells in
the surviving population. Alternatively, mutant p53
protein may directly act or stimulate the recombination
machinery. However cells lacking p53 proteins, thus
without mutant p53 protein, show an increase of
genetic instability. If we imagine that homologous
recombination is one of the mechanisms involved in
genome rearrangement, this latter hypothesis would be
unlikely.
The absence of increase of point mutagenesis in p53
defective cells (Nishino et al., 1995; Sands et al., 1995)
emphasizes the importance of the other pathways
responsible for genetic changes in these cells. The
results presented here demonstrate an involvement of
the p53 protein in the control of homologous
recombination. Increase of deletions such as described
here, inversions, ampli®cations, translocations can be
extremely deleterious and can account for many
pathologies. Induction of gene conversion can reveal
recessive alleles, silent in the heterozygous state but
expressed after gene conversion renders the cell
homozygous for this allele. By such a mechanism the
probability of expressing a deleterious allele is
increased with each cell generation. Thus, gene
conversion can also account for the propagation of
genetic alterations (even for point modi®cations)
observed during tumor progression.
In the experiments described here, the alteration p53
function is obtained by overexpression of a mutant p53
protein, using a vector with a strong promoter (from
CMV), in a wild type background. This raises the
question of what ratio of mutant to wild type p53
protein is required to obtain a mutant phenotype;
particularly whether p53 heterozygous cells (i.e. with an
equimolar ratio of wild type to mutant protein) show
an increase in genetic recombination. The consequence
would be that mutation of only one allele of p53 could
increase the probability of genome rearrangement.
Acknowledgements
Thanks are due to Dr M Liskay for kindly providing us the
pJS3-10 mouse cell lines. We thank Gerry Marsischky,
Philippe Noitrot, the members of J Habar Laboratory and
all the people who provided helpful discussions and
comments. This work was supported by Institut Curie
and Electricite de France (1H6284/D333). PB was supported by a fellowship from ARC.
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