A novel role for the Bcl-2 protein family: specific suppression of the RAD51 recombination pathway
2001; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.1093/emboj/20.10.2596
ISSN1460-2075
Autores Tópico(s)PARP inhibition in cancer therapy
ResumoArticle15 May 2001free access A novel role for the Bcl-2 protein family: specific suppression of the RAD51 recombination pathway Yannick Saintigny Yannick Saintigny UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Anne Dumay Anne Dumay UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Sarah Lambert Sarah Lambert UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Bernard S. Lopez Corresponding Author Bernard S. Lopez UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Yannick Saintigny Yannick Saintigny UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Anne Dumay Anne Dumay UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Sarah Lambert Sarah Lambert UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Bernard S. Lopez Corresponding Author Bernard S. Lopez UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France Search for more papers by this author Author Information Yannick Saintigny1, Anne Dumay1, Sarah Lambert1 and Bernard S. Lopez 1 1UMR217 CNRS-CEA, CEA, Direction des Sciences du Vivant, Département de Radiobiologie et Radiopathologie, 60–68 Avenue du Général Leclerc, 92 265 Fontenay aux Roses, Cedex, France ‡Y.Saintigny and A.Dumay contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2596-2607https://doi.org/10.1093/emboj/20.10.2596 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The oncogenic role of Bcl-2 is generally attributed to its protective effect against apoptosis. Here, we show a novel role for Bcl-2: the specific inhibition of the conservative RAD51 recombination pathway. Bcl-2 or Bcl-XL overexpression inhibits UV-C-, γ-ray- or mutant p53-induced homologous recombination (HR). Moreover, Bcl-2 recombination inhibition is independent of the role of p53 in G1 arrest. At an acute double-strand break in the recombination substrate, Bcl-2 specifically inhibits RAD51-dependent gene conversion without affecting non-conservative recombination. Bcl-2 consistently thwarts recombination stimulated by RAD51 overexpression and alters Rad51 protein by post-translation modification. Moreover, a mutant G145ABcl-2, which is defective in Bax interaction and in apoptosis repression, also inhibits recombination, showing that the death and recombination repression functions of Bcl-2 are separable. Inhibition of error-free repair pathways by Bcl-2 results in elevated frequencies of mutagenesis. The Bcl-2 gene therefore combines two separable cancer-prone phenotypes: apoptosis repression and a genetic instability/mutator phenotype. This dual phenotype could represent a mammalian version of the bacterial SOS repair system. Introduction Genome integrity and cell proliferation/viability are commonly regulated by a network of pathways including cell cycle checkpoints, DNA repair/recombination and programmed cell death. In response to genotoxic attacks, proliferating cells temporarily stall in their cycle, allowing the repair of the injured DNA (Hartwell, 1992, Hartwell et al., 1994). Alternatively, cells activate their programmed cell death (Rich et al., 1999; Wyllie et al., 1999). p53 is the gene most frequently found to be mutated in human tumours (Hollstein et al., 1991; Levine et al., 1991). The p53 protein mediates cell cycle checkpoints and apoptosis (for review see Donehower and Bradley, 1993; Hainaut, 1995; Smith and Fornace, 1995; Ko and Prives, 1996). In addition, the status of p53 can affect homologous recombination frequency (Wiesmuller et al., 1996; Bertrand et al., 1997; Mekeel et al., 1997; Dudenhoffer et al., 1999; Saintigny et al., 1999). However, the effect of p53 on homologous recombination can be independent of G1 checkpoint alteration, suggesting that p53 acts on recombination via a pathway other than the G1 checkpoint control (Dudenhoffer et al., 1999; Saintigny et al., 1999). Since p53 protein also controls apoptosis, this raises the question, what effect does apoptosis regulation have on homologous recombination. In line with this, the mammalian recombination protein RAD51 is a target for caspase degradation during programmed cell death (Flygare et al., 1998; Huang et al., 1999). However, the relationship between apoptosis and regulation of homologous recombination is poorly understood. One efficient and classic way to repress apoptosis is to overexpress Bcl-2 oncogene family members. Indeed, DNA damage can induce two pathways to apoptosis, one p53 dependent and one p53 independent, and both pathways can be inhibited by Bcl-2 (Strasser et al., 1994). Bcl-2 becomes oncogenic when overexpressed, such as in follicular B-cell lymphomas resulting from a t(14:18) translocation (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto et al., 1985). Bcl-2, as well as other members of its family such as Bcl-XL, has anti-apoptotic activity (for review see Adams and Cory, 1998). Since cells become more resistant to genotoxic agents, this raised the question of how DNA repair pathways are regulated when protecting the cells against apoptosis. Here we address the question of whether the expression of Bcl-2 affects homologous recombination efficiency. Using a combination of different substrates and strategies to monitor recombination, the present paper focuses on the detailed characterization of the recombination pathways affected by Bcl-2 (or Bcl-XL) overexpression. We overexpressed Bcl-2 in mammalian cell lines containing the tandem repeat recombination substrates depicted in Figure 1. These substrates allow the monitoring of different intrachromosomal recombination pathways. Using a substrate containing a cleavage site for the rare-cutting endonuclease I-SceI (Figure 1B), it is possible to target a unique double-strand break (DSB) in the recombination substrate (Liang et al., 1998). This strategy has permitted the definition of two homology-directed DSB repair pathways: (i) a non-conservative recombination mechanism arising by single-strand annealing (SSA) and leading to deletion of the intervening sequence (NeoR/HygS); (ii) a conservative recombination mechanism initiated by strand invasion and leading to gene conversion with or without associated crossing over (double-resistant NeoR/HygR). In contrast to SSA, conservative recombination is RAD51 dependent in both yeast (Ivanov et al., 1996) and mammalian cells (Lambert and Lopez, 2000). Here we show that Bcl-2 expression specifically inhibits the RAD51-dependent conservative recombination pathway, independently of programmed cell death repression. We also show that the expression of Bcl-2 results in modification of post-translation regulation of Rad51 protein. Taken together, the results suggest that RAD51-dependent conservative recombination and apoptosis are controlled by two separable functions of Bcl-2 (or Bcl-XL). In addition to its anti-apoptotic phenotype, Bcl-2 also shows a mutator phenotype, both of which could contribute to a pre-cancerous predisposition. Figure 1.Recombination substrates. (A) Parental pJS3-10 is a mouse Ltk− cell line containing a tandem repeat of two inactive TK genes (black boxes) from herpes simplex virus type I (the grey square corresponds to the inactivating mutations). The cells are deficient in thymidine kinase activity (tk−) and, thus, sensitive to the HAT selective medium. Recombination between the two TK sequences restores a functional TK gene and resistance to HAT. The frequency of recombination is estimated by the frequency of HAT-resistant clones (Liskay et al., 1984). (B) Parental CHO-DRA10 is a hamster CHO K1 cell line containing a tandem repeat of two inactive neomycin (Neo) resistance genes (black boxes). In between the two Neo sequences is the hygromycin-resistant (Hyg) sequence (grey box). The parental lines are sensitive to G418 (NeoS) and resistant to hygromycin (HygR). All the recombinants become G418 resistant (NeoR). One Neo cassette contains a cleavage site (grey arrow) for the yeast rare-cutting enzyme I-SceI. Expression of I-SceI results in a DSB targeted in the recombination substrate. Conservative recombination leads to NeoR/HygR double-resistant clones. Non-conservative recombination leads to NeoR single-resistant clones (Liang et al., 1998). Non-conservative recombination mainly corresponds to single-strand annealing (SSA), an RAD51-independent process. Conservative recombination corresponds to gene conversion or intrachromatid crossing over (followed by re-integration of the excised product), two RAD51-dependent processes (Ivanov et al., 1996; Lambert and Lopez, 2000). Download figure Download PowerPoint Results Expression of Bcl-2 in cell lines carrying intrachromosomal recombination substrates We used the recipient lines and strategies described in Figure 1. The pJS3-10 line derives from mouse Ltk− cells, which are sensitive to HAT medium, and contains a copy of a duplication of inactive herpes simplex type I TK gene. Recombination between the two TK sequences restores a functional TK gene and thus HAT resistance. The frequency of recombination can be calculated from the number of HAT-resistant clones relative to the total number of viable plated cells. CHO-DRA10 contains a direct repeat of two inactive neomycin (NEO) resistance genes, separated by a hygromycin-resistant gene. Recombination restores a functional NEO gene and thus resistance to G418 (NeoR). One NEO cassette contains one I-SceI cleavage site. Expression of the I-SceI rare-cutting endonuclease produces a DSB targeted into the recombination NEO cassette (Liang et al., 1998). Repair of this DSB via a conservative gene conversion event produces a double-resistant NeoR/HygR clone, and a non-conservative event produces a NeoR but hygromycin-sensitive (HygS) clone. A few double-resistant NeoR/HygR (2 out of 11 double-resistant clones analysed) recombinant clones result from intrachromatid crossing over then random re-integration of the excised product (Lambert and Lopez, 2000). These events are also initiated by strand exchange and are RAD51 dependent. The different lines were transfected with mammalian expression vectors, to obtain derivative lines stably expressing Bcl-2 or mutant Bcl-2 proteins (Figure 2A). The wild-type Bcl-2 protein inhibits apoptosis and affects G0–G1 cell cycle entry. These two functions of Bcl-2 can be separated and the mutant Y28ABcl-2 has been shown to maintain its anti-apoptosis function but to be deficient in its role in G0–G1 cell cycle entry (Huang et al., 1997). Mutant G145ABcl-2 has been shown to be deficient in its interaction with Bax and in apoptosis repression (Yin et al., 1994). The different derivative lines are listed in Table I. Analysis by immunofluorescence shows that overexpression of Bcl-2 in our lines does not seem to modify its classical localization (data not shown). Figure 2.Bcl-2 expression and apoptosis repression. (A) Detection by western blotting of the expression of Bcl-2. pJS3-10 is the mouse parental cell line; pJSDRII.4 is a pJS3-10 line overexpressing Bcl-2. CHO-DRA10 is the hamster parental line; ADRA14 and ADRA17 are two independent clones overexpressing Y28ABcl-2. (B) Apoptotic sub-G1 population, measured by FACS, 48 h after 6 Gy irradiation. Upper panel, an example of a typical histogram (M1 corresponds to the sub-G1 population); lower panel, frequency of sub-G1 cells 48 h after irradiation. (C) Measurement of apoptosis by means of Hoechst fluorescence; the percentages correspond to the percentage of cells with fragmented nuclei. ADRA14 and ADRA17 correspond to two independent clones expressing Y28ABcl-2. (D) Effect of Bcl-2 expression on survival of CHO-DRA10, after γ-rays. Download figure Download PowerPoint Table 1. Cell lines Cell lines Origin Expression of an exogenous human protein p53 mutant protein Bcl-2 protein pJS 3.10 mouse L cell nonea none pJS DR II.4 pJS 3.10 none Bcl-2 H175 DR 211 pJS 3.10 175 (Arg→His) none H175 DR II.3 pJS 3.10 175 (Arg→His) Bcl-2 H175 DR II.5 pJS 3.10 175 (Arg→His) Bcl-2 H273 DR11 pJS 3.10 273 (Arg→His) P273 DR 4 pJS 3.10 273 (Arg→Pro) CHO-DRA10 hamster CHO-K1 noneb none Cm3c CHO-DRA10 none none A3c CHO-DRA10 none none ADRA 8 CHO-DRA10 none Y28ABcl-2 ADRA14 CHO-DRA10 none Y28ABcl-2 ADRA17 CHO-DRA10 none Y28ABcl-2 BDRA1 CHO-DRA10 none G145ABcl-2 BDRA2 CHO-DRA10 none G145ABcl-2 a The pJS 3.10 parental cell line expresses an endogenous wild-type p53 protein (see Bertrand et al., 1997; Saintigny et al., 1999). b The CHO-DRA10 parental cell line expresses an endogenous mutant p53 protein. c The Cm3 and A3 cell lines are independent clones from the CHO-DRA10 parental cell line transfected with the empty expression vector. We first verified the apoptotic cell death repression activity resulting from Y28ABcl-2 expression. The protective effect of Y28ABcl-2 in the CHO-DRA10 line, after treatment with ionizing radiation, was checked by different methods: (i) the frequency of cells with a sub-G1 DNA content, measured by fluorescence-activated cell sorting (FACS); and (ii) the nuclear fragmentation, visualized by means of Hoechst 33342, a fluorescent DNA intercalating agent. Forty-eight hours after irradiation (6 Gy), the percentage of apoptotic cells (sub-G1 cell population) was 16.5% in the control cells and 5.3 and 4.3% in two independent clones expressing Y28ABcl-2 (Figure 2B). The frequency of fragmented nuclei (monitored by Hoechst fluorescence) consistently decreased 3- and 3.5-fold in two independent irradiated lines expressing Y28ABcl-2 (Figure 2C). Thus, both methods confirmed in our cell line the well-established cell death repression activity of Y28ABcl-2. These results are in agreement with a 2- to 3-fold increased viability after γ-rays (6 Gy) of both mouse fibroblasts (pJS3-10) and CHO-DRA10 lines expressing Bcl-2 or Y28ABcl-2 (Figure 2D). Bcl-2 expression suppresses the induction of recombination by γ-rays or UV-C Both γ-rays and UV-C stimulated homologous recombination in the parental CHO-DRA10 lines (Figure 3). Y28ABcl-2 expression inhibited induction of recombination by γ-rays (Figure 3A) as well as by UV-C (Figure 3B). Since these two different genotoxic stresses produce different types of DNA damage, Bcl-2 recombination inhibition (BRI) is not specific to the type of genotoxic stress. Figure 3.Bcl-2 expression inhibits radiation-induced recombination. (A) γ-rays (6 Gy). (B) UV-C; the doses are indicated on the figure. Black bar, parental CHO-DRA10 line (control line); grey bar (ADRA14, ADRA17), two independent clones expressing Y28ABcl-2. Download figure Download PowerPoint Anti-recombination and anti-apoptosis are separable functions of Bcl-2 One important question is whether BRI requires the interaction between Bcl-2 and Bax proteins, and whether it is associated with or separable from the apoptosis repression activity. More generally, BH3-only members of the Bcl-2 family are critical initiators of apoptosis that can be repressed by Bcl-2. However, mutants in the BH1 domain of Bcl-2 are unable to inhibit the pro-apoptotic activity of such BH3-only proteins (O'Connor et al., 1998). To address this question, we expressed the mutant G145ABcl-2 (mutated in the BH1 domain of Bcl-2) in the CHO-DRA10 cell line. G145ABcl-2 is defective in the Bax interaction and the protection against apoptosis (Yin et al., 1994). We first verified the effect on apoptosis by measuring the frequency of fragmented nuclei after exposure to radiation. Y28ABcl-2 exhibited a protective effect, whereas G145ABcl-2 showed no anti-apoptotic pheno type in two independent clones (Figure 4A). We then measured the effect of G145ABcl-2 expression on radiation-induced recombination. Expression of G145ABcl-2 as well as of Y28ABcl-2 strongly impaired the induction of recombination by ionizing radiation (Figure 4B). This result shows that recombination inhibition and apoptosis repression are separable activities of Bcl-2. In addition, this result suggests that the Bcl-2–Bax interaction is not required for BRI. Figure 4.Effect of the G145ABcl-2 mutant on apoptosis (A) and radiation-induced recombination (B), 48 h after irradiation (6 Gy). (A) Frequency of fragmented nuclei measured by Hoechst fluorescence. (B) Induction of recombination by γ-rays. CHO-DRA10, parental line (control); A3, CHO-DRA10 transfected with an empty expression vector (control); ADRA8, CHO-DRA10 expressing Y28ABcl-2; BDRA1 and BDRA2, two independent clones expressing G145ABcl-2. Download figure Download PowerPoint Transient expression of Bcl-2 or Bcl-XL inhibits radiation-induced recombination Bcl-2 belongs to a family of anti-apoptosis genes (Adams and Cory, 1998). In order to determine whether BRI is specific to Bcl-2, we repeated these tests in cells expressing another member of the Bcl-2 family: Bcl-XL (Adams and Cory, 1998). We measured the impact of transient overexpression of Bcl-2, Y28ABcl-2 or Bcl-XL on radiation-induced recombination, tested 24 h after transfection in the pJS3-10 cell line and its derivatives (Figure 5). Figure 5.Transient expression of Bcl-2 family members inhibits radiation-induced recombination, independently of p53 status. Cells were irradiated at a dose of 6 Gy. The expressed transgenes and their respective symbols are indicated on the figure. (A) Parental (wild-type p53) pJS3-10. (B) pJS3-10 derivative cell lines expressing different mutant p53 with various effects on recombination (Saintigny et al., 1999). The expressed mutant p53 are reported in Table I. Download figure Download PowerPoint Transient expression of Bcl-2 as well as of Bcl-XL completely impaired radiation-induced recombination (Figure 5A). This result shows that BRI is not specific to Bcl-2 since another member of the family, such as Bcl-XL, can also suppress the induction of recombination. Furthermore, the fact that transient expression of Bcl-2 or Bcl-XL suppresses the recombination induction shows that BRI results directly from Bcl-2 or Bcl-XL expression and not from a secondary associated phenotype selected during the long-term isolation of stable transfectants (2–3 weeks of selection). Bcl-2 and Bcl-XL inhibit recombination independent of p53 status To investigate whether BRI is affected by p53 status, we used either the parental pJS3-10 (wild-type p53) or pJS3-10 expressing different mutant p53 proteins with various effects on cell cycle control and/or on recombination efficiency. His175 p53 affects the G1–S checkpoint after radiation, whereas neither His273 nor Pro273 p53 modifies the G1 block after irradiation. Moreover, His175 or Pro273 mutant p53 proteins stimulate radiation-induced recombination whereas, the His273 mutant p53 protein does not (Saintigny et al., 1999). Irradiation moderately stimulated recombination in the parental pJS3-10 and H273DR11 cell lines (expressing the His273 p53 protein) and strongly stimulated recombination in lines expressing either His175 or Pro273 p53 (Figure 5B), as previously described (Saintigny et al., 1999). Transient expression of either Bcl-2, Y28ABcl-2 or Bcl-XL inhibited radiation-induced recombination in all cell lines tested (Figure 5B). Thus, the status of p53 (for recombination as well as for the G1 checkpoint) did not affect BRI. Whatever the extent of recombination stimulation, Bcl-2 suppressed radiation-induced recombination. Effect of Bcl-2 overexpression on spontaneous recombination Expression of Bcl-2 inhibits recombination induced by profound genotoxic stresses such as UV or γ-radiation. We checked whether spontaneous recombination, i.e. without a drastic exogenous genotoxic stress, is also affected by Bcl-2 expression. Spontaneous recombination was measured by fluctuation analysis using two assays: the Luria and Delbrück or Lea and Coulson test (Luria and Delbrück, 1943; Lea and Coulson, 1948; Capizzi and Jameson, 1973). In mouse L-cells, expression of Bcl-2 has no effect on the spontaneous recombination rate in the parental pJS3-10 line (Table II). Expression of the mutant His175 p53 protein led to a 6-fold increase in spontaneous recombination in the pJS3-10 line (Table II; Bertrand et al., 1997; Saintigny et al., 1999). In these lines, expression of Bcl-2 abolished the p53 stimulation of the spontaneous recombination rate to the level of the parental wild-type p53 pJS3-10 line (Table II). The hamster CHO-DRA10 line contains an endogenous mutant (Lys211) p53 protein (Hu et al., 1999). In two independent CHO-DRA10 derivative lines, expression of Y28ABcl-2 resulted in a 4-fold decrease in the spontaneous recombination rate (Table II). Table 2. Spontaneous homologous recombination between direct repeat sequences Cell lines Expression of exogenous Bcl-2 or Y28ABcl-2 protein Number of independent cultures Recombination rate (× 10−6/cell/generation) Luria and Delbrück Lea and Coulson pJS 3.10 none 18 1.5 ± 0.8 1.9 pJS DR II.4 Bcl-2 12 2 ± 0.7 2.3 H175 DR 211 none 18 9.7 ± 0.7 11.7 H175 DR II.3 Bcl-2 6 1.8 ± 0.6 1.7 H175 DR II.5 Bcl-2 12 2.7 ± 0.6 (× 10−7/cell/generation) 3.4 CHO-DRA10 none 6 5.5 ± 0.4 6.6 ADRA14 Y28ABcl-2 6 1.3 ± 0.6 1.5 ADRA17 Y28ABcl-2 6 1.4 ± 0.6 1.6 Thus, Bcl-2 does not inhibit the basal level of spontaneous recombination, but suppresses the stimulation of recombination resulting here from the expression of mutant p53 proteins, even in the absence of profound exogenous genotoxic stress. Bcl-2 specifically inhibits conservative recombination events induced by a unique DSB Taken together, these results show that Bcl-2 expression inhibits recombination stimulation by UV-C and γ-rays as well as by mutant p53 proteins, independently of the toxicity of the treatment. In order to further the molecular characterization of the recombination pathway affected, we checked the effect of Bcl-2 expression on a unique and acute DSB, targeted to the recombination substrate. We used the I-SceI strategy in the CHO-DRA10 lines (Figure 1). Expression of I-SceI, which produces the DSB, is not toxic for the cells but strongly stimulates homology-directed recombination (Liang et al., 1998). More importantly, this strategy distinguishes between two recombination pathways: conservative recombination (double-resistant NeoR/HygR) and non-conservative recombination (single NeoR). Conservative events (double-resistant NeoR/HygR) are initiated by strand invasion and are thus RAD51-dependent, whereas non-conservative events (single NeoR) mainly correspond to SSA, an RAD51-independent process (Ivanov et al., 1996; Lambert and Lopez, 2000). SSA systematically results in the deletion of the sequence between the tandem recombination markers; it is thus an error-prone pathway. As previously shown, transfection by I-SceI stimulated the total number of recombinant (NeoR) by 100- to 1000-fold (Liang et al., 1998). These recombinants correspond to the sum of conservative and non-conservative events. The expression of Bcl-2 did not modify the overall frequency of NeoR recombinants (Figure 6A). However, the expression of Bcl-2 did modify the relative proportion of the different classes of events. Bcl-2 expression resulted in a significant 3-fold decrease in the frequency of double-resistant (NeoR/HygR) clones (Figure 6B). This result shows that the expression of Bcl-2 does not affect the non-conservative recombination events, but specifically inhibits conservative recombination events (double-resistant NeoR/HygR). The percentage of conservative events (double-resistant NeoR/HygR colonies) relative to all the recombinant colonies (NeoR alone) was examined (Figure 6C). Since this value is normalized to the frequency of NeoR colonies (representing the whole recombinant population), the calculation is based on an internal standard and is independent of transfection and cleavage efficiencies. In the control cell line, conservative events (NeoR/HygR colonies) comprised 25% of the total recombinant colonies. In the two independent lines overexpressing Bcl-2, the percentage of double-resistant NeoR/HygR colonies fell to 5.2 and 7.6%, respectively (Figure 6C). Figure 6.Effect of Bcl-2 on recombination induced by I-SceI. (A) Number of total recombinants (NeoR). (B) Number of conservative events(NeoR/HygR). (C) Distribution (percentage) of class of events: non-conservative events (grey bars); conservative events (black bars). The numbers on top of the histograms indicate the exact value of the percentage. Cm3, control corresponding to the parental CHO-DRA10 cell line transfected with the empty expression vector. ADRA14 and ADRA17 correspond to two independent clones expressing Y28ABcl-2. Download figure Download PowerPoint Taken together, these results show that global DSB healing is unaffected and that Bcl-2 does not act on all recombination pathways, but specifically on the conservative homologous recombination pathway. The fact that the frequency of NeoR is unaffected suggests that non-homologous end-joining (NHEJ) and SSA pathways are not inhibited by Bcl-2 expression. Finally, this result shows that Bcl-2 is able to exercise its effect on a unique DSB, in the absence of an extreme deleterious genotoxic stress. Bcl-2 suppresses RAD51-induced recombination Conservative recombination is RAD51 dependent, whereas NHEJ and SSA are RAD51 independent. To confirm further that Bcl-2 overexpression inhibits recombination events promoted by RAD51, we used a CHO- DRA10 derivative cell line overexpressing mammalian RAD51. We have previously shown that overexpression of the mouse MmRAD51 cDNA strongly stimulates homologous recombination after ionizing radiation (Lambert and Lopez, 2000). We tested here whether Bcl-2 expression is able to suppress the stimulation of radiation-induced recombination resulting from the expression of MmRAD51. At a dose of 6 Gy, overexpression of MmRAD51 led to a 10-fold stimulation of radiation-induced recombination compared with the control lines. In the lines transfected with the Bcl-2 expression vector, RAD51-stimulated recombination was reduced to the level of the control lines (Figure 7). This result indicates that the recombination stimulation provoked by the overexpression of MmRAD51 was thwarted by the expression of Bcl-2, and is consistent with the data showing a specific inhibition of conservative recombination events. Figure 7.Bcl-2 inhibits recombination stimulated by RAD51. Cells were irradiated at 6 Gy. Cm3, control cell line corresponding to the parental CHO-DRA10 transfected with the empty expression vector. Rm2 and Rm4 are two independent clones overexpressing mouse MmRAD51, leading to a stimulation of radiation-induced recombination (Lambert and Lopez, 2000). Black bars, transfection with an empty vector; grey bars, transfection with a Y28ABcl-2 expression vector. Download figure Download PowerPoint Bcl-2 expression affects post-translation modification of Rad51 protein The data presented above show a specific inhibition of the RAD51 recombination pathway. In order to gain some clues regarding the molecular mechanisms involved, we focused on the Rad51 protein. Rad51 protein acts in a huge protein complex, but we have previously shown that it plays a pivotal role in gene conversion regulation. Overexpression of only Rad51 protein or of a dominant-negative form is sufficient to stimulate or to inhibit gene conversion (Lambert and Lopez, 2000). These results indicate that acting on the regulation of Rad51 protein alone could be sufficient to regulate the whole gene conversion pathway. We thus checked the status of Rad51 protein in cell lines overexpressing Bcl-2. First, the amount of Rad51 protein was measured by western blotting (Figure 8A). The fact that the amount of Rad51 protein was identical in lines expressing Bcl-2 and in control lines showed that Bcl-2 did not affect transcription, RNA maturation or translation efficiencies of RAD51. Thus, if Rad51 is a target, the consequences of Bcl-2 expression should act post-translationally. Figure 8.Status of Rad51 protein in lines expressing Bcl-2. (A) Western blot. Arrows indicate Rad51 protein, actin (internal standard) and overexpression of the exogenous human Bcl-2. Cell lines are d
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