doi: 10.1371/journal.pone.0055883. Epub 2013 Feb 6.
The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage
Affiliations
- PMID:23405229
- PMCID: PMC3566071
- DOI: 10.1371/journal.pone.0055883
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The level of Ets-1 protein is regulated by poly(ADP-ribose) polymerase-1 (PARP-1) in cancer cells to prevent DNA damage
Arnaud J Legrand et al. PLoS One.2013.
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Abstract
Ets-1 is a transcription factor that regulates many genes involved in cancer progression and in tumour invasion. It is a poor prognostic marker for breast, lung, colorectal and ovary carcinomas. Here, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel interaction partner of Ets-1. We show that Ets-1 activates, by direct interaction, the catalytic activity of PARP-1 and is then poly(ADP-ribosyl)ated in a DNA-independent manner. The catalytic inhibition of PARP-1 enhanced Ets-1 transcriptional activity and caused its massive accumulation in cell nuclei. Ets-1 expression was correlated with an increase in DNA damage when PARP-1 was inhibited, leading to cancer cell death. Moreover, PARP-1 inhibitors caused only Ets-1-expressing cells to accumulate DNA damage. These results provide new insight into Ets-1 regulation in cancer cells and its link with DNA repair proteins. Furthermore, our findings suggest that PARP-1 inhibitors would be useful in a new therapeutic strategy that specifically targets Ets-1-expressing tumours.
Conflict of interest statement
Figures
(A) Colloidal blue-stained gel for Ets-1-associated proteins from MDA-MB-231 nuclear extracts. Biotinylated Ets-1 proteins used for the pull-down assay were loaded alone as a control to identify the biotinylated proteins (arrowheads) and their potential breakdown products (lane 1). Pull-down proteins detected from nuclear extracts (NE) incubated with unloaded beads were considered to be non-specific products (lane 2). The colloidal blue-stained specific PARP-1 protein pulled-down by biotinylated Ets-1 is indicated by an asterisk (lane 3). (B) Identification of PARP-1 by MALDI-TOF mass spectrometry. (C) Western blot analysis confirming the Ets-1-associated protein as PARP-1. Proteins pulled-down from MDA-MB-231 nuclear extracts were analysed by Western blot with rabbit antibodies directed against PARP-1 (H-250). (D) Colloidal blue-stained gel for Ets-1-associated proteins from MDA-MB-231 nuclear extracts either untreated (lanes 1 and 2) or treated with DNase I for 40 min (lanes 3 and 4) and then either incubated with streptavidin beads loaded with biotinylated Ets-1 protein (arrowheads) (lanes 2 and 4) or with unloaded beads (lanes 1 and 3). Asterisks indicate the PARP-1 protein detected in (A). (E) and (F) Co-immunoprecipitation performed using a rabbit anti-Ets-1 (C-20) or a rabbit anti-PARP-1 (H-250) antibody-agarose conjugate (lane 2) or normal rabbit IgG as a control (lane 1) and nuclear extracts from MDA-MB-231 cells or MG-63 cells. Input (lane 3) contains 3% of nuclear extracts that underwent co-immunoprecipitation. PARP-1 and Ets-1 were analysed by Western blot using respectively H-250 and C-4 antibodies.
(A) Schematic representation of the full-length Ets-1 protein and its dominant-negative isoform Ets-1 p27. Boxes correspond to translated regions and dotted lines to untranslated regions. Threonine-38 (Thr38), Pointed domain (PNT), transactivation domain (TAD), inhibitory domain (I), translated region corresponding to the exon VII and DNA-binding domain (DBD) are indicated. (B) Streptavidin pull-down assay with recombinant Ets-1 and PARP-1 proteins. Biotinylated Ets-1 (5 µg) or Ets-1 p27 (2.5 µg) proteins were loaded on streptavidin beads and then incubated with pure recombinant PARP-1 protein (1 µg) for 1 h (lanes 2 and 3). Streptavidin beads alone incubated with PARP-1 were used as control (lane 1). PARP-1 was analysed by Western blot. (C) PARylation assay with Ets-1 and Ets-1 p27. Ets-1 (1 µg) or Ets-1 p27 (500 ng) proteins were incubated with recombinant PARP-1 protein (500 ng) in PARylation reaction buffer (see Materials and Methods) in presence (lanes 4 and 7) or in absence (lanes 5 and 8) of nicked DNA for 20 min. As a control, Ets-1 isoforms were incubated alone in reaction buffer and nicked DNA (lanes 3 and 6) and auto-activation of PARP-1 alone was checked with or without DNA (control, lanes 1 and 2) (D) PARylation of Ets-1 in stably transfected HeLa cells, obtained by retroviral infection. Ets-1 tagged with streptavidin binding peptide (SBP) was purified on stretavidin beads from HeLa cell nuclear extracts (lane 2). HeLa cells stably expressing only the SBP were used as control (lane 1). In (C) and (D), PARylation status was determined using an anti-PAR antibody.
(A) Schematic representation of the luciferase gene under the control of the human stromelysin-1 promoter. Palindromic Ets-binding sites (EBS) are shown bound with two Ets-1 molecules. (B) Effect of PARP-1 knock out (KO) on Ets-1-mediated stromelysin-1 promoter activity. pGL3 luciferase reporter constructs (100 ng) were transfected into 3T3 mouse cells wild type (WT) (lanes 1 and 3) or KO for the PARP-1 gene (lanes 2, 4 and 5) at 50–80% confluence in the absence (lanes 1 and 2) or in the presence (lanes 3, 4 and 5) of the Ets-1 expression vector (25 ng) and in the rescue experiments with PARP-1 expression vector (100 ng; lane 5). (C) Effect of PARP-1 catalytic inhibition on the Ets-1-mediated stromelysin-1 promoter activity in the presence or absence of PARP-1. pGL3 luciferase reporter constructs (100 ng) were transfected into 3T3 mouse cells WT (lanes 1- 4) or KO for the PARP-1 gene (lane 5–8) at a 50–80% confluence in the absence (lanes 1, 2, 5 and 6) or in the presence (lanes 3, 4, 7 and 8) of Ets-1 expression vector (25 ng) and in the absence (lanes 1, 3, 5 and 7) or in the presence (lanes 2, 4, 6 and 8) of PJ-34, a catalytic inhibitor of PARP-1 (10 µM). (D) Effect of PARP-1 catalytic inhibition on Ets-1-mediated stromelysin-1 promoter activity in a cancer cell model. pGL3 luciferase reporter constructs (100 ng) were transfected into HeLa cells at a 50–80% confluence in the absence (lanes 1–4) or in the presence (lanes 5–8) of Ets-1 expression vector (100 ng) and with increasing doses of PJ-34 (0–20 µM; lanes 1–4 and 5–8). In (B), (C) and (D), luciferase activity, measured 48 h after transfection and 20 h after incubation with PJ-34, is expressed as a percentage with the activity induced by Ets-1 in a PARP-1 WT context indicated as 100%. Results are the average of three experiments performed in triplicate. (E) Effect of PARP-1 catalytic inhibition on the level of Ets-1 protein. HeLa cells were grown in 6-well plates until 70% confluence, transfected with pcDNA3 (1 µg; left panel) or pcDNA3-Ets1 (1 µg; right panel) vectors for 24 h and treated with PJ-34 (1 µM) (lanes 2 and 6), 5-AIQ (1 µM) (lanes 3 and 7) or ABT-888 (1 µM) (lanes 4 and 8) for 20 h. In all experiments, cell lysates (30 µg total proteins) were analysed by Western blot using different antibodies (see Materials and Methods).
(A) Kinetics of Ets-1 accumulation observed by time-lapse imagery. HeLa cells were grown in MatTek dishes and were transfected with peGFP-C1-Ets-1 (500 ng; rows 1, 2 and 5) or peGFP-C-1-Ets-1p27 (500 ng; rows 3 and 6) or empty peGFP-C1 vectors (500 ng; row 4). 24 h after transfection, HeLa cells were treated with PJ-34 (10 µM; rows 2–4) or MG-132 (5 µM; rows 5 and 6) and observed for 12 h under a fluorescence microscope at ×20 magnification with one picture taken every hour. Bottom panel: Statistical analysis of the variation of fluorescence intensity. Conditions 1 to 6 indicated on the x-axis are the same as those described above. Mean fluorescence intensities from time-lapse experiment pictures were calculated using ImageJ software and the ratio of variation was established between the T = 12 h and T = 0 pictures. Results are the average of three experiments. (B) Visualisation of Ets-1 protein levels in MDA-MB-231 cells treated with PJ-34 by immunofluorescence. MDA-MB-231 cells were treated with PJ-34 (10 µM) for 20 h. Ets-1 is visualised in Red (Alexa Fluor® 594). Cells were examined under a fluorescence microscope at ×40 magnification. Scale bar = 20 µm. White arrows indicate cytoplasmic localisation of Ets-1 in untreated cells (lane 1). Bottom panel: Surface plot representations. Surface plots were obtained by analysing immunofluorescence pictures using ImageJ software; the x- and y-axes indicate the pixel positions and the z-axis, the intensity of fluorescence. (C) Effect of PARP-1 catalytic inhibition on the level of PARylated proteins. MDA-MB-231 cells were treated with PJ-34 (10 µM) for 20 h. Cell lysates (30 µg total proteins) were analysed by Western blot using different antibodies (see Materials and Methods) against PARP-1, p53, c-Jun and ERK-2 which are known to undergo PARylation.
(A) Time-lapse imaging experiments of HeLa cells treated with PJ-34. HeLa cells were grown in Hi-Q4 dishes until 70% confluence and transfected with empty pcDNA3 (250 µg; left panel) or pcDNA3-Ets1 (250 µg; right panel) vectors 24 h before being treated with PJ-34 (5 µM) or left untreated. Cells were stained with Hoechst 33242 (blue) and PI (red) for live-cell imaging and monitored for 20 h. Scale bar = 20 µM. (B) Graphical representation of the proportion of necrotic HeLa cells (%) at three time points (see Materials and Methods). (C) Flow cytometry cell-death detection of HeLa cells treated with PJ-34. HeLa cells were grown in 6-well plates until 70% confluence and transfected with pcDNA3 (1 µg; left panel) or pcDNA3-Ets1 (1 µg; right panel) vectors for 24 h and left untreated (dashed lines) or treated with PJ-34 (solid lines) for an additional 20 h incubation. Necrotic cell death was then determined by flow cytometry after PI staining. Numbers under the horizontal bar give the percentages of specific PJ-34-induced necrotic cell death in each condition. Flow cytometry profiles shown are representative of three replicate experiments. (D) Time-lapse imaging experiments of MDA-MB-231 cells treated with PJ-34. MDA-MB-231 cells were grown in Hi-Q4 dishes until 80% confluence and treated with PJ-34 (10 µM) or left untreated. Cells were stained with Hoechst 33242 (blue) and PI (red) for live-cell imaging and monitored for 20 h. Scale bar = 20 µM. (E) Graphical representation of the proportion of necrotic MDA-MB-231 cells (%) at three time points (see Materials and Methods).F) Flow cytometry cell-death detection of MDA-MB-231 cells treated with PJ-34. MDA-MB-231 cells were grown in 6-well plates until 80% confluence and left untreated (dashed lines) or treated with PJ-34 (solid lines) for a 20 h incubation. Necrotic cell death was then determined by flow cytometry after PI staining. Numbers under the horizontal bar represent the percentages of specific PJ-34-induced necrotic cell death in each condition. Flow cytometry profiles shown are representative of three replicate experiments.
(A) Western blot analysis of phosphorylated H2AX (γH2AX) in transfected HeLa cells treated with PJ-34. pcDNA3 expression vectors without insert (lanes 1 and 3) and encoding Ets-1 (lanes 2 and 4) were transfected in HeLa cells at 50–80% confluence. At 24 h after transfection, cells were treated for 20 h with PJ-34 (10 µM; lanes 3 and 4). Cell lysates (30 µg total proteins) were analysed by Western blot using the C-20 anti-Ets-1 antibody and the anti-γH2AX antibody. (B) Immunofluorescence of γH2AX in HeLa cells treated with PJ-34. HeLa cells were treated with PJ-34 (10 µM) for 20 h. Ets-1 is visualised in green (Alexa Fluor® 488) and γH2AX in red (Alexa Fluor® 594). (C) Immunofluorescence of γH2AX and Ets-1 in transfected HeLa cells treated with PJ-34. HeLa cells were transfected with peGFP-C1-Ets-1 expression vector (1 µg) and treated with PJ-34 for 20 h. γH2AX is visualised in red (Alexa Fluor® 594) and Ets-1 in green (eGFP). In (B) and (C), nuclei were visualised with DAPI staining. Cells were examined by fluorescence microscopy at ×40 magnification. Scale bar = 20 µm.
(A) Immunofluorescence of γH2AX in MDA-MB-231 cells after treatment with PJ-34. MDA-MB-231 cells were treated with PJ-34 (10 µM) for 20 h. Ets-1 is visualised in green (Alexa Fluor® 488) and γH2AX in red (Alexa Fluor® 594). Nuclei were visualised with DAPI staining. Cells were examined by fluorescence microscopy at ×40 magnification. Scale bar = 20 µm. Statistical analysis was performed by counting and examining after immunofluorescence more than 100 cells from three different experiments. γH2AX + indicates cells with more than 10 γH2AX foci in their nucleus (Fig. S6). All MDA-MB-231 cells are positive for Ets-1 expression (Ets-1 +). In the graph, the percentage of γH2AX + cells is given. ** p<0.01. (B) Immunofluorescence of γH2AX in MCF-7 cells after treatment with PJ-34. MCF-7 cells were treated with PJ-34 (10 µM) for 20 h. Ets-1 is visualised in green (Alexa Fluor® 488) and γH2AX in red (Alexa Fluor® 594). Statistical analysis was performed by counting and examining after immunofluorescence more than 100 cells from three different experiments. All MCF-7 cells are negative for Ets-1 expression (Ets-1 -). In the graph, the percentage of γH2AX + cells is shown. (C) Immunofluorescence of γH2AX and Ets-1 in Ets-1-transfected MCF-7 cells after treatment by PJ-34. MCF-7 cells were transfected with peGFP-C1-Ets-1 expression vector (1 µg) and treated with PJ-34 for 20 h. γH2AX is visualised in red (Alexa Fluor® 594) and Ets-1 in green (eGFP). Statistical analysis was performed as described in (A). In the graph, the percentage of γH2AX + cells is shown for the Ets-1 – and Ets-1 + cell populations. ** p<0.01. In (A) and (B), nuclei were visualised with DAPI staining. Cells were examined by fluorescence microscopy at ×40 magnification. Scale bar = 20 µm.
(A) Regulation of Ets-1 protein levels by PARP-1 in cancer cells with PARylation activity. First, PARP-1 interacts with Ets-1 and allows Ets-1 to have full transcriptional activity. Second, Ets-1 activates PARP-1 catalytic activity and is then PARylated. PARylation of Ets-1 promotes its degradation by the proteasome, thereby allowing the cancer cells to modulate Ets-1 protein levels. The stimulation of the NADPH oxidase complex by Ets-1 transcriptional activity, both directly and indirectly by the matrix metalloprotease stromelysin-1, is also represented. This complex will generate reactive oxygen species (ROS). These ROS promote genomic instability, cancer progression and processes such as cancer cell migration and invasion. (B) Deregulation of Ets-1 protein levels in cancer cells under PARylation inhibition. Ets-1 is still able to interact with PARP-1 and has full transcriptional activity. However, Ets-1 is no longer PARylated and is therefore not targeted for proteasomal degradation. Thus, Ets-1 will accumulate in cancer cells and an increase in Ets-1 results in higher Ets-1 transcriptional activity, promoting the production of more ROS. High levels of ROS will generate a substantial increase in DNA damage (indicated by γH2AX) that cannot be repaired by PARP-1 that is inhibited, nor by BRCA1/2 because Ets-1 represses their expression. DNA damage accumulation, eventually, lead to the death of cancer cells.
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