Formation of higher-order nuclear Rad51 structures is functionally linked to p21 expression and protection from DNA damage-induced apoptosis
Elke Raderschall1,
Alex Bazarov2,
Jiangping Cao3,
Rudi Lurz1,
Avril Smith1,
Wolfgang Mann1,
Hans-Hilger Ropers1,
John M. Sedivy4,
Efim I. Golub5,
Eberhard Fritz6 and
Thomas Haaf1
1 Max Planck Institute of Molecular Genetics, 14195 Berlin, Germany
2 Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94040, USA
3 Soochow University, Suzhou 215007, P.R. China
4 Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA
5 Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520, USA
6 Institute of Radiation Biology, GSF, National Research Center for Environment and Health, 85758 Neuherberg, Germany
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Fig. 1. Detection of overexpressed Rad51 protein in stably transfected cell lines by western blotting. (A) Human wild-type PPL and Rad51-overexpressing PPL928.1-2 cells. Total cell extracts were prepared from untreated and etoposide (etop.)-treated cultures. Equal amounts of total cellular protein were separated by electrophoresis and subjected sequentially to immunoblot analysis with antibodies to Rad51 and ß-actin. Antibody binding was quantified by densitometric analysis. The ß-actin signals were used to equilibrate the slightly different amounts of cell extract loaded per lane. Measurements from three independent western blot experiments were averaged. (B) Rat TGR and Rad51-overexpressing TGR928.1-9 cells. The amount of Rad51 in untreated wild-type (PPL or TGR) cells was chosen as a reference (100%).
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Fig. 2. Higher-order nuclear structures in Rad51-overexpressing cells. (A) Immunofluorescence staining of growth-arrested TGR928.1-9 cells reveals discrete nuclear foci and higher-order structures of overexpressed Rad51 protein (green) in a high percentage of cells. Nuclei are counterstained with DAPI (blue). Bar, 10 µm. (B) Ultrathin cross (left-hand side) and longitudinal sections (right) of nuclear Rad51 structures in TGR928.1-9 nuclei after pre-embedding immunolabeling with anti-Rad51 antibodies and 12 nm colloidal gold. Bar, 100 nm.
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Fig. 3. Cell cycle delay of Rad51-overexpressing cells. (A) Simultaneous staining of Rad51 protein (green) and replicating DNA (red) in exponentially growing TGR928.1-9 cells. BrdU was incorporated into DNA for two hours and detected with anti-BrdU antibody. Note that Rad51-foci-positive cells are devoid of BrdU label. (B) Reduced frequency of replicating cells in Rad51-overexpressing lines. Cells undergoing DNA replication synthesis during the last two hours of culture were scored as BrdU+ (black bars), whereas non-replicating cells were BrdU– (white bars). At least 400 cells were analyzed for each experiment.
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Fig. 4. Suppression of radiation-induced chromatid-type aberrations in Rad51-overexpressing cells. Exponentially growing TGR (black bars) and TGR928.1-9 (gray bars) cells were exposed to 0, 1, 3, 5, or 7 Gy of ionizing radiation. Metaphases were prepared 16 hours after irradiation and scored for the indicated chromosome aberrations in a double-blind manner. 200 metaphases each of two or three independent irradiation experiments were analyzed per cell line.
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Fig. 5. Anti-apoptotic effects of overexpressed Rad51 protein. (A) Simultaneous staining of etoposide-treated TGR928.1-9 cells with annexin V (green) and anti-Rad51 antibodies (red). Annexin-V-positive apoptotic cells are Rad51-foci-negative. (B) Reduced frequency of apoptotic cells in etoposide- and UV-treated Rad51-overexpressing cultures. Cells staining positively for annexin V (annexin+) were scored as apoptotic, whereas non-apoptotic cells were annexin–. Simultaneously, the cells were stained for the presence (Rad51+) or absence (Rad51–) of Rad51 foci. At least 400 cells were analyzed for each experiment.
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Fig. 6. Increased p21 protein levels in Rad51-overexpressing cells. (A) Immunoblot analysis of human wild-type PPL and Rad51-overexpressing PPL928.1-2 cells. Total cell extracts were prepared from untreated and etoposide (etop.)-treated cultures. Equal amounts of total cellular protein were loaded and analyzed by western blotting with antibodies for p21, ß-actin (loading control) and p53. p21 protein expression was calculated from three independent western blot experiments. The amount of p21 in untreated wild-type cells was chosen as a reference (100%). (B) Relative mRNA expression of selected genes in PPL928.1-2 versus PPL cells. The fluorescence ratios of three ESTs for each gene were measured in three independent chip hybridization experiments. p21 and c-Abl showed an approximately twofold increase in Rad51-overexpressing PPL928.1-2 cells. (As total RNA was reverse transcribed from oligo(dT) primers, the transfected Rad51 mRNA levels could not be determined.)
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Fig. 7. Suppression of p21 and increased etoposide (etop.)-induced apoptosis following antisense inhibition of endogenous Rad51. (A) 400 nM Rad51 antisense (AS) ODN or 400 nM scrambled (SC) ODN or no ODN was used in PPL cells. The cells were then either treated with etoposide or grown in drug-free medium. Relative expressions of p21 and Rad51 were quantified by western blotting. The ß-actin signals (not shown) were used as a loading control. The amounts of Rad51 in untreated PPL and of p21 in etoposide-treated PPL cells, respectively, were chosen as references (100%). For p21, measurements from three independent antisense experiments were averaged. (B) The number of etoposide-induced apoptotic PPL cells after treatment with 100 nM, 400 nM Rad51 antisense or scrambled ODNs, as identified by annexin V staining.
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© The Company of Biologists Ltd 2002
