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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