Apoptosis (2008) 13:413–422 DOI 10.1007/s10495-007-0175-4
ORIGINAL PAPER
A high-content chemical screen identifies ellipticine as a modulator of p53 nuclear localization G. Wei Xu Æ Imtiaz A. Mawji Æ Chloe J. Macrae Æ C. Anne Koch Æ Alessandro Datti Æ Jeffrey L. Wrana Æ James W. Dennis Æ Aaron D. Schimmer
Published online: 5 January 2008 Ó Springer Science+Business Media, LLC 2008
Abstract p53 regulates apoptosis and the cell cycle through actions in the nucleus and cytoplasm. Altering the subcellular localization of p53 can alter its biological function. Therefore, small molecules that change the localization of p53 would be useful chemical probes to understand the influence of subcellular localization on the function of p53. To identify such molecules, a high-content screen for compounds that increased the localization of p53 to the nucleus or cytoplasm was developed, automated, and conducted. With this image-based assay, we identified ellipticine that increased the nuclear localization of GFPmutant p53 protein but not GFP alone in Saos-2 osteosarcoma cells. In addition, ellipticine increased the nuclear localization of endogenous p53 in HCT116 colon cancer cells with a resultant increase in the transactivation of the p21 promoter. Increased nuclear p53 after ellipticine treatment was not associated with an increase in DNA double stranded breaks, indicating that ellipticine shifts p53 to the nucleus through a mechanism independent of DNA damage. Thus, a chemical biology approach has identified
G. Wei Xu and Imtiaz A. Mawji have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10495-007-0175-4) contains supplementary material, which is available to authorized users. G. W. Xu I. A. Mawji C. J. Macrae C. A. Koch A. D. Schimmer (&) Ontario Cancer Institute, Princess Margaret Hospital, 610 University Ave, M5G 2M9 Toronto, ON, Canada e-mail:
[email protected] A. Datti J. L. Wrana J. W. Dennis Samuel Lunenfeld Research Institute, Mt. Sinai Hospital, M5G 1X5 Toronto, ON, Canada
a molecule that shifts the localization of p53 and enhances its nuclear activity. Keywords
High-throughput screen p53 Ellipticine
Abbreviations HCS High-content screening SDS-PAGE SDS-polyacrylamide gel electrophoresis
Introduction In the nucleus, p53 functions as a transcription factor and increases the expression of target genes such as p21waf1, GADD45, and 14-3-3 r that induce cell-cycle arrest, as well as Bax PUMA, Pidd, Noxa, and FAS that promote apoptosis (reviewed in [1–3]). In contrast, the role of p53 in the cytoplasm is less well understood, but involves activating Bax and increasing cytochrome c release from the mitochondria [4–6]. Several mechanisms that regulate the localization of p53 to different subcellular compartments have been identified [7]. For example, p53 preferentially localizes to the nucleus in response to cell stresses such as DNA damage and hypoxia. Conversely, the amount of p53 in the cytoplasm is increased by several factors including its binding to Parc [8] or acetylation by p300 [9]. Molecules that shift the localization of p53 to different subcellular compartments could serve as useful chemical probes to better understand the role of p53 at these sites and the pathways that regulate its localization. Moreover, modifiers of p53 distribution may serve as leads for small molecule therapeutics for the treatment of malignancy that induce cell death through p53-dependent mechanisms.
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Here, we have developed an automated a high-content image-based screen for compounds that increase the localization of p53 to the nucleus or cytoplasm. With this assay, we screened a chemical library and identified ellipticine, which increased the abundance of p53 in the nucleus and enhanced transactivation of the p53-target gene p21. Thus, our findings suggest that a chemical biology approach can identify factors regulating p53 subcellular localization and function, and provide insights into the mechanism of action of ellipticine, a compound previously known to induce apoptosis in malignant cells [10, 11].
Materials and methods Reagents The Prestwick chemical library (1120 compounds) was purchased from Prestwick Chemical (Washington, DC, USA). MG132 as purchased from Calbiochem (San Diego, CA, USA). Thapsagargin was purchased from SigmaAldrich (Oakville, ON, Canada). Ellipticine was purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA).
Cell lines and cell culture MCF-7, T47D, H1299, and PPC-1 cells were maintained in RPMI 1640 media. Saos-2 cells were maintained in DMEM media. HCT116 wild type and HCT116 p53 -/(gifts from Bert Voglestein, Baltimore, MD, USA) were grown in McCoy’s 5A medium. All cell lines were grown at 37°C in a humid environment supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and antibiotics.
DNA constructs and generation of stable cell lines The wild type (WT) and V143A mutant p53 open reading frames were amplified by PCR from cDNA clones (gift from S. Benchimol, York University, Toronto, ON, Canada) using Expand PFU polymerase (Roche Diagnostics, Mississauga, ON, Canada), the sense primer p53ORF-H3-S (50 -GCA TAA GCT TCC ATG GAG GAG CCG CAG TCA GAT-30 ), and the anti-sense primer p53ORF-BH1-AS (50 -CGG TGG ATC CTC AGT CTG AGT CAG GCC CTT C-30 ). The primers p53ORF-H3-S and p53ORF-BH1-AS contain HindIII and BamHI restriction endonuclease sites, respectively, just outside p53-specific sequences to facilitate sub-cloning. PCR products were digested with HindIII
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and BamHI, gel-purified, and then sub-cloned in-frame to the C-terminus of the enhanced green fluorescent protein ORF in pEGFP-C1 (Clontech Laboratories, Mountainview, CA, USA) using standard methodologies. Clones were sequence-verified for orientation and sequence integrity using a CEQ 8000 Genetic Analysis System (Beckman, Mississauga, ON, Canada). The p21-luc reporter plasmid (gift from Sam Benchimol) contains the p53 binding site of the p21 promoter cloned upstream of the minimal adenovirus E1b promoter and firefly luciferase open reading frame. Stable cell lines expressing GFP-p53 proteins were engineered by transfecting SAOS-2 cells with pEGFP-C1 or pEGFP-p53V143A (pEGFP-p53mut) using Lipofectamine 2000 (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Cells stably expressing GFP fusion proteins were selected with 1 mg/ml G418 (Invitrogen) for 10 days. G418-resistant colonies were cloned, expanded, and then enriched for expression of GFP fusion proteins using a MoFlo fluorescent activated cell sorter (Dako, Mississauga, ON, Canada). Enriched clones were then expanded for further characterization.
High-content screening assay Plate handling was performed by a CRS Dimension4 robotics platform equipped with a Linear Plate Transport system (LPT) (Thermo Electron, Waltham, MA, USA). Plate transfer from the LPT to online peripherals was carried out by a CRS Flip Mover and Vertical Array Loader (Thermo Electron). Liquid handling steps were performed by a Biomek FX Laboratory Automation Workstation (Beckman Coulter) and ELx405 Magna cell washers (Biotek, Winooski, Vermont, USA). Robotics integration was controlled by a Polara integration software (Thermo Electron). In the automated assay, SAOS-2 cells stably overexpressing GFP-p53 mut (8.5 9 103 cells/well) were seeded in 96-well black-walled flat-bottom plates (Corning, Acton, MA, USA) and incubated in standard tissue culture conditions. Sixteen hours after seeding, aliquots from the chemical library were added to the cells at a final compound concentration of 5 lM and a final DMSO concentration of 0.2% (compound solvent). Cells were then incubated with compounds for 3.5 h and fixed. For fixation, cells were washed twice with PBS, incubated with 4% paraformaldehyde (in PBS) for 30 min, washed twice more with PBS, and then incubated with 1.2 lM DAPI dilactate (Molecular Probes, Carlsbad, CA, USA) for 15 min. DAPIstained cells were washed twice with PBS and then plates were sealed with clear plastic film. Cells were imaged on an ArrayScanII HCS imager equipped with a 20-plate
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robotic stacker and ArrayScan V3.5 software (Cellomics, Pittsburgh, PA, USA). HCS imaging was performed using a 0.5NA 109 lens and XF100 filter. At least 500 cells per well were quantified representing at least three independent imaging fields per well. Cellomics ArrayScan software was used to define the nucleus and cytoplasm and quantify the intensity of the GFP signal for each cell. The mean difference in intensity in treated cells was compared to the mean difference in intensity of buffer treated control cells on each plate and a relative localization score defined mathematically as (mean (nuclear intensity - cytoplasmic intensity) treated cells)/ (mean (nuclear intensity - cytoplasmic intensity) control cells). Hits C3 standard deviations above the mean of the population were compounds that shifted p53 to the nucleus and hits C3 standard deviations below the mean of the population were compounds that shifted p53 to the cytoplasm.
Apoptosis and viability assays Cells transfected with GFP fusion proteins were tested for apoptosis based on DAPI staining and morphology using fluorescent microscopy as previously described [12]. Briefly, cell lines were seeded into 4-well chamber slides overnight and transfected with 0.5 lg of pEGFP-C1, pEGFP-p53WT, or pEGFP-p53mut using Lipofectamine 2000 (Invitrogen,) according to the manufacturer’s protocols. The following day, transfected cells (GFP positive) were then scored for apoptotic morphology (detachment and/or nuclear fragmentation). Cells were imaged on a Zeiss Axiovert 200 M with a Zeiss A-Plan 32x/0.40NA Ph I lens using a 360 nM excitation and 460 nM emission filter set, a Coolsnap HQ camera (Roper Scientific, Tucson, AZ, USA), and Image Pro PLUS software (Media Cybernetics, Silver Spring, MD, USA). Cell viability was also measured by the MTS reduction assay as previously described [13].
Promoter transactivation assays Cells were seeded into 24-well plates and transfected as above with 0.25 lg p21-luciferase in the presence or absence of pEGFP-C1, pEGFP-p53WT, or pEGFPp53mut. Cells were lysed and analyzed for luciferase activity (Bright-Glo System, Promega) using the manufacturer’s protocols on a Lumoskan II luminometer (LabSystems, Helsinki, Finland). Lysates were also tested in parallel for protein concentration using the Protein Assay Dye Reagent (Bio-Rad, Mississauga, ON, Canada) and SpectraMax M5 spectrophotometer (Molecular
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Devices, Sunnyvale, CA, USA) using the manufacturer’s protocols. Luciferase activities were normalized to protein content. Transfection efficiency was monitored by fluorescent microscopy to detect GFP expression and was equivalent across experimental conditions and plasmids.
Immunoblot analysis Cells were lysed with RIPA buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, and 5 mM EDTA) containing Complete protease inhibitors (Roche, Indianapolis, IN, USA). Immunoblot assays were performed as described previously [14]. Briefly, protein lysates were quantified, resolved by electrophoresis through 10% SDS-polyacrylamide gels (SDSPAGE), and transferred to PVDF membranes. Membranes were incubated with monoclonal anti-human p53 (clone FL-393, Santa Cruz Biotechnology, Santa Cruz, CA, USA) monoclonal anti-human tubulin (1:30,000 v/v dilution, Sigma), on monoclonal anti-human histone H1 (1:500 v/v dilution, Santa Cruz Biotechnology). Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (goat anti-mouse IgG, Bio-Rad) and enhanced chemiluminescence (West Pico Reagent, Pierce, Rockford, IL, USA).
Subcellular fractionation Cells were harvested and resuspended in ice-cold hypotonic lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, supplemented with Complete protease inhibitors (Roche)) and immediately centrifuged at 100 9 g for 5 min. After centrifugation, the pellet was resuspended in hypotonic lysis buffer and incubated on ice for 20 min. After incubation, NP40 was added (0.6% final concentration) and the samples were vortexed for 10 s and immediately centrifuged at 12,000 9 g for 30 s. The supernatant (cytoplasmic fraction) was transferred to a fresh tube. The nuclei pellet was washed once with hypotonic lysis buffer and lysed with SDS loading buffer (100 mM Tris–Cl, pH 6.8, 2% SDS, 100 mm DTT, and 10% glycerol).
Measurement of DNA double stranded breaks Cells were seeded in 8-well chamber slides (2 9 104/well) and treated with increasing concentrations of ellipticine or etoposide. At increasing times after treatment, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS. To detect DNA double stranded
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breaks, fixed cells were incubated for 2 h at 37°C with phospho-H2AX (cH2AX) mouse monoclonal antibody (Ser139, Clone JBW301, Upstate Biotechnology Inc., Lake Placid, NY), followed by a 30 min incubation at 37°C with red fluorescent Alexa Fluor 568 dye-conjugated anti-mouse IgG secondary antibody (Invitrogen). Cells were then counterstained with DAPI (0.5 lg/ml in PBS) and mounted on slides using a fluorescent mounting medium (Dako, Mississauga, ON). Cells were viewed with a fluorescence microscope (Zeiss Axiovert 200 M with a Zeiss A-Plan 40x DIC lens) and imaged using a Coolsnap HQ camera and Image Pro PLUS software, as described above.
Results GFP-p53 fusion proteins are tools to detect changes in localization of p53 To study the nuclear and cytoplasmic localization of p53, we developed GFP-p53 fusion constructs. Wild type or V143A mutant p53 was cloned in-frame to the C-terminus of GFP in the pEGFP-C1 vector to generate plasmids that express GFP::p53WT, and GFP::p53mut proteins, respectively (Suppl. Fig. 1A). To ensure that the fusion proteins retained their biologic activity, vectors expressing GFP, GFP::p53WT, and GFP::p53mut were transfected into MCF7 and T47D breast cancer cells, Saos-2 osteosarcoma cells, H1299 lung cancer cells, and PPC-1 prostate cancer cells. After an overnight incubation, cell death in the transfected cells was quantified. Over-expression of GFP::p53WT, but not GFP or GFP-p53mut induced apoptosis (Suppl. Fig. 1B), consistent with the previously described effects of over-expressing wild type p53 and the p53V143A mutant [15]. Moreover, GFP::p53WT but not GFP nor GFP::p53 mut transactivated a p21 promoter reporter construct, demonstrating that fusion to GFP does not adversely affect p53 function (Suppl. Fig. 1C). Having validated the biologic relevance of the GFP::p53 fusion proteins, we asked whether an image-based approach could be used to study changes in the localization of p53. Since over-expression of GFP-p53WT induced apoptosis, we performed our experiments with GFP::p53mut. Saos-2 osteosarcoma cells stably over-expressing GFP::p53mut (Saos-2-GFP::p53mut) or GFP (Saos-2-GFP) were treated with increasing concentrations of Thapsigargin or MG132 that have previously been reported to increase the localization of wild type and endogenous and exogenous mutant p53 to the cytoplasm and nucleus, respectively [16–18]. Four hours after treatment, cells were fixed, stained with DAPI, and imaged by automated microscopy (Fig. 1a). Computer-based algorithms were used to define the cell’s nucleus and cytoplasm, and the difference in intensity
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between the GFP signals in the nucleus and cytoplasm was measured. The mean difference in intensity for the cells in each well was calculated and expressed as a ratio relative to buffer treated Saos-2-GFP::p53mut cells (relative localization score). Using this image-based approach, we demonstrated that Thapsigargin and MG132 increased the amount of GFP::p53mut in the cytoplasm and nucleus, respectively, more than two-fold compared to buffer treated cells (Fig. 1b, c). In contrast, neither Thapsigargin nor MG132 altered the localization of GFP alone (Fig. 1). To validate the results of this image-based approach to assess changes in the localization of p53, we measured the amount of GFP::p53mut in the nucleus and cytoplasm by immunoblotting subcellular fractions. Confirming the imagebased results, treatment of Saos-2-GFP::p53mut with Thapsigargin or MG132 increased the localization of the exogenous mutant p53 fusion protein to the cytoplasm and nucleus, respectively (data not shown). To assess changes in p53 localization over time, Saos-2-GFP::p53mut and Saos-2-GFP cells were treated with Thapsigargin or MG132 and the localization of p53 in the nucleus and cytoplasm was quantified by microscopy at increasing times after incubation. Thapsigargin and MG132 rapidly shifted the localization of p53 to the cytoplasm and nucleus, respectively, with changes detected within 3 h of incubation (Fig. 1c). Of note, no change in localization of GFP was detected. Thus, taken together, these results indicate that GFP-p53 fusion proteins retain their biologic activity and can be used in conjunction with confocal microscopy to quantify rapid changes in the localization of p53 in response to external stimuli.
High-throughput high-content image-based assay for detection of small molecules that change the localization of p53 Chemical probes that alter the localization of p53 would be useful biological tools to better understand the function and regulation of this protein. Therefore, we coupled our image-based p53 localization assay to a high-throughput chemical screen to identify small molecules that shift the localization of GFP::p53mut. In this automated assay, Saos-2-GFP::p53mut cells were seeded in 96-well plates using a robotic liquid handler and incubated for 16 h to permit the cells to adhere to the tissue culture plate. Cells were then treated with aliquots of the 1,200 compound Prestwick library at a final concentration of 5 lM and a final DMSO concentration of 0.2% for 3.5 h. After incubation with the small molecules, the cells were fixed, washed, and stained with DAPI. Stained cells were then imaged by automated microscopy and at least 500 cells
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Fig. 1 Thapsagargin and MG132 rapidly shift the localization of p53. (a) SAOS-2 cells stably expressing GFP::p53mut or GFP were seeded into a 96-well plate (8.5 9 104 cells/well). The next day, cells were treated with MG132 (10 lM) or Thapsigargin (TG) (1 lM). Four hours after treatment, cells were fixed and stained with the nuclear stain DAPI and imaged with an ArrayScan II HCS automated microscope. Representative images are shown. (b) SAOS-2 cells stably expressing GFP::p53mut or GFP were seeded into 96-well plates as above and treated with increasing concentrations of MG132 or Thapsagargin. Four hours after treatment cells were fixed, stained with DAPI, and imaged as above. The relative localization score was calculated for each well as the (mean (nuclear intensity - cytoplasmic intensity) treated cells)/mean (nuclear intensity - cytoplasmic intensity) control cells). Data represent the mean fold increase in p53
nuclear staining ± SEM for three independent experiments performed in triplicate. Values above 1 indicate increased nuclear localization and values below 1 indicate increased cytoplasmic localization. (c) SAOS-2 cells stably expressing GFP::p53mut or GFP were seeded into a 96-well plate (8.5 9 104 cells/well). The next day, cells were treated with MG132 (10 lM) or Thapsigargin (1 lM). At increasing times after treatment, cells were fixed and stained with the nuclear stain DAPI and imaged with the ArrayScan II HCS automated confocal microscope. The relative localization score was calculated for each well as in panel (b). Data represent the mean fold increase in p53 nuclear staining ± SEM for three independent experiments performed in triplicate. Values above 1 indicate increased nuclear localization and values below 1 indicate increased cytoplasmic localization
were quantified in a minimum of three independent fields and the relative localization score calculated as before (Fig. 2). To assess the robustness of the screen, we
calculated the Z factor of the assay which is defined as 1-((3SD sample + 3SD control)/(mean sample-negative mean control)) [19]. The Z factor of the assay was 0.78,
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Given the effects of ellipticine on Saos-2 cells transfected with mutant p53, we were interested in testing the effects of ellipticine on the localization of endogenous wild type 4.5
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Fig. 2 High-content screen identifies small molecules that alter the localization of p53. SAOS-2-GFP::p53mut cells were seeded into 96-well plates (8.5 9 103 cells/ well). Sixteen hours after seeding, cells were treated with aliquots of the Prestwick chemical library at a final compound concentration of *5 lM and a final DMSO concentration of 0.2%. Three and a half hours after incubation, cells were washed, fixed, stained with DAPI and imaged with an Array Scan II HCS automated microscope. The relative localization score was calculated for each well. Values above 1 indicate increased nuclear localization and values below 1 indicate increased cytoplasmic localization. * = sanguinarine, ** = ellipticine
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where a Z factor of 1 is ideal and a Z score above 0.5 denotes a very good screening quality [19]. Our high-throughput assay successfully identified wells where Thapsigargin and MG132 were spiked into the assay as positive controls for cytoplasmic and nuclear localization, respectively. In addition, the screen identified ellipticine and sanguinarine as compounds that preferentially localized GFP::p53mut to the nucleus more than 2-fold above buffer treated control (Fig. 2). Secondary inspection of the microscopic images collected in the screen demonstrated that sanguinarine was a false positive hit, as the increased nuclear signal was an artifact of cell death induced by the compound. In contrast, inspection of the images from the screen and secondary analysis using confocal microscopy demonstrated that ellipticine increased the localization of p53 to the nucleus without morphologic evidence of apoptosis (Fig. 3a). To validate the results of our screen, immunoblotting of nuclear and cytoplasmic fractions of Saos-2-GFP::p53mut cells confirmed the results of the high-content screen and demonstrated that ellipticine increased the ratio of cellular p53 in the nucleus (Fig. 3b).
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Fig. 3 Ellipticine increases the nuclear localization of p53. (a) SAOS-2-GFP::p53mut cells were treated with ellipticine (8 lM) or buffer control for 4 h. After treatment, cells were washed, fixed, stained with DAPI and imaged with a confocal microscope. Representative images are shown. (b) SAOS-2-GFP::p53mut cells were treated with MG132 (10 lM), ellipticine (5, 8 or 10 lM), or buffer control. After treatment, nuclear and cytoplasmic cellular fractions were isolated by differential lysis. Lysates were prepared and analyzed by SDS-PAGE immunoblotting using anti-p53 and antitubulin antibodies
p53. HCT116 colon cancer cells express wild type p53, and were treated with ellipticine, MG132, or buffer control. Six hours after treatment, total cell lysates as well as lysates from nuclear and cytoplasmic fractions were prepared and levels of p53 in each fraction were determined by immunoblotting. Both ellipticine and MG132 increased the total amount of p53 in the cell, however ellipticine, and to a lesser degree MG132, preferentially increased the abundance of p53 in the nucleus (Fig. 4). Of note, no change in cell viability was detected after this brief incubation with ellipticine (data not shown). To determine whether the increased levels of p53 in the nucleus after treatment with ellipticine and MG132 were functionally important, we measured the effects of these compounds on target gene activation. HCT116 cells with either wild-type p53 (WT) or lacking p53 (-/-) were transfected with p21 promoter-reporter plasmid and treated with increasing concentrations of ellipticine or MG132. After 6 h of treatment, p21 promoter transactivation by p53 was measured. Ellipticine increased the activation of p21 at concentrations that increased p53 in the nucleus,
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Fig. 4 Ellipticine increases the nuclear localization of endogenous p53. HCT116 colon cancer cells were treated with 10 lM of ellipticine or MG132 for 6 h. After treatment, total, nuclear and cytoplasmic cellular fractions were isolated by differential lysis. Lysates were prepared and analyzed by SDS-PAGE immunoblotting using anti-p53 anti-histone H1, and anti-tubulin antibodies
but failed to do so in p53 -/- cells. Interestingly, although MG132 increased the abundance of mutant and wild type p53 in the nucleus, no change in p21 activation was detected (Fig. 5).
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Fig. 5 Increased nuclear localization of p53 after ellipticine treatment is functionally important. HCT116 wild type (WT) and HCT116 p53 -/- cells were transfected with the p21 promoter-reporter construct. Twenty-four hours after transfection, cells were treated with MG132 (10 lM) or ellipticine (5, 8, or 10 lM) for 6 h. After treatment, cells were harvested, lysed, and luciferase activity measured. Data points represent the mean ± SE luciferase activity/ lg protein relative to cells transfected with p21 promoter-reporter construct but treated with buffer control
Ellipticine is a known inhibitor of the topoisomerase II enzyme [11] and inhibition of topoisomerase II can induce DNA damage thereby producing a genotoxic stress that promotes the nuclear accumulation of p53 [20]. Therefore, we tested whether the increased nuclear localization of p53 was due to ellipticine-mediated DNA damage. Saos-2 cells over-expressing GFP::p53mut were treated with ellipticine (10 lM). At increasing times after incubation, the appearance of DNA double stranded breaks as an early and sensitive marker of DNA damage was measured by staining for the c-H2AX foci by immunofluoresence. In parallel, shifts in the localization of p53 were measured by immunoblotting subcellular fractions. Ellipticine increased localization of p53 within one hour of incubation (Fig. 6a). However, up to 5 h after ellipticine treatment, no increase in c-H2AX foci was detected compared to the buffer control (Fig. 6b). Of note, 16 h after treatment with ellipticine, an increase in c-H2AX foci were observed, consistent with its known ability to induce DNA damage (data not shown). In contrast to the effects of ellipticine, the known topoisomerase II inhibitor, etoposide [11], increased c-H2AX foci within one hour after treatment and produced very intense staining for c-H2AX foci 5 h after treatment (Fig. 6b and data not shown). In these SAOS-2 cells expressing GFP::p53mut, etoposide-induced DNA damage was associated with a slight increase in nuclear localization of p53 (Fig. 6c). Interestingly, the topoisomerase II inhibitor, mitoxantrone did not shift the localization of p53 up to 5 h after treatment (data not shown). Localization of p53 to the nucleus by immunoblotting cellular fractions was confirmed by demonstrating shifts of GFP::p53mut using confocal microscopy (Fig. 3 and data not shown). Therefore, taken together, these results suggest that ellipticine increases the abundance of nuclear p53 through a mechanism unrelated to DNA damage and likely through a mechanism unrelated to its known ability to inhibit Topoisomerase II.
Discussion To better understand the different actions of p53 dependent on its localization, we developed and conducted a highcontent screen for small molecules that shifted the localization of GFP::p53mut to either the nucleus or cytoplasm. From our library of well-characterized compounds, we identified ellipticine, which increased the abundance of p53 in the nucleus. This screen also identified molecules that increased the abundance of p53 in the cytoplasm and these compounds will be explored in future studies.
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Fig. 6 Increased nuclear localization of p53 after ellipticine treatment is independent of induction of DNA damage. (a) SAOS-2GFP::p53mut cells were treated with 10 lM ellipticine. At increasing times after treatment, nuclear cellular fractions were isolated by differential lysis. Lysates were prepared and analyzed by SDS-PAGE immunoblotting using anti-p53, anti-tubulin, and anti-histone H1 antibodies. (b) SAOS-2-GFP::p53mut cells were treated with ellipticine (10 lM), etoposide (10 lM), or buffer control. After 5 h of treatment, cells were fixed, washed and stained with anti-cH2AX and
Alexa Fluor 568 dye-conjugated secondary antibodies to visualize cH2AX foci. Cells were counterstained with DAPI to visualize cell nuclei. Cells were imaged by an inverted fluorescence microscope. Representative images are shown. (c) SAOS-2-GFP::p53mut cells were treated with ellipticine (10 lM), etoposide (10 lM), or buffer control. After 5 h of treatment, nuclear and cytoplasmic cellular fractions were isolated by differential lysis. Lysates were prepared and analyzed by SDS-PAGE immunoblotting using anti-p53, anthistone H1, and anti-tubulin antibodies
Ellipticine is a naturally occurring alkaloid derived from the leaves of the evergreen tree Ochrosia ellipticia. It is known to intercalate with DNA and inhibit the enzyme topoisomerase II [10, 11]. At low micromolar concentrations, ellipticine induces apoptosis in malignant cells with activation of both the mitochondrial and death receptor pathways of caspase activation [21]. Elliptcine also increased expression of Fas and Fas ligand [22]. Thus, while ellipticine exerts multiple biological effects on the cell our study and work by others [21–23] demonstrate that ellipticine also affects p53. For example, Peng et al. [23] demonstrated that ellipticine restored transcriptional activity to several mutant p53 proteins and reverted the mutants to wild type conformations. These effects were independent of ellipticine’s ability to inhibit topoisomerase II. Here, we report the novel finding that ellipticine increased nuclear p53. Specifically we demonstrated that it increased the abundance of exogenous mutant p53 and endogenous wild type 53 in the nucleus. The shift of p53 was biologically important as the increased nuclear localization of endogenous wild type p53 enhanced transactivation of the p21 promoter. While we demonstrated that ellipticine increased the nuclear localization of exogenous mutant p53, a limitation to our study is that we
did not determine whether ellipticine altered the localization of endogenous mutant p53. Furthermore, we did not determine whether ellipticine restored biological function to mutant p53. Also, we examined increased transactivation of p21, but did not determine whether there was a resultant increase in p21 protein expression or the expression of other p53 target genes. Finally, we cannot generalize this finding beyond the cell lines tested in this proposal. Potentially, the ability of ellipticine to increase the nuclear localization of p53 may be cell-type specific. A previous report, however, demonstrated that treatment with ellipticine increased the transcriptional activity of mutant p53 and increased the transcription of the p53 target gene p21 [23]. Moreover, this study did not determine whether ellipticine increased the nuclear localization of p53. Increased nuclear localization of p53 facilitates the activation of p53-dependent target genes such as p21. In our study MG132 increased the abundance of p53 in the nucleus, but did not increase transactivation of p21. Thus, these results demonstrate that increased nuclear levels of p53 are not sufficient to increase transcription of p21. However the reasons for this phenomenon are not clear at this time. One possibility is that the transcriptional activity of p53 may require a competent ubiquitin-proteasome pathway as previously reported [24].
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The nuclear import and export of p53 is regulated by several factors. For example, nuclear import and export of p53 is enabled by nuclear localization signals and nuclear export signals, respectively, on p53 [25–28]. When imported into the nucleus, p53 undergoes tetramerization that obscures its nuclear export signal, thereby blocking nuclear export [29]. The ubiquitination pathway also influences p53 localization. Ubiquitination of p53, via the E3 ligase activity of MDM2, promotes p53 nuclear export [30, 31]. Finally, phosphorylation also regulates p53 localization as phosphorylation of p53 at serine 15 increases its nuclear localization [32, 33]. The mechanism by which ellipticine increases nuclear p53 is uncertain. Potentially, ellipticine may act to stabilize nuclear p53 by altering its phosphorylation status via its known actions as an inhibitor of Cdc2, topoisomerase II, and/or casein kinase 2 [34–36]. Alternatively, ellipticine may indirectly inhibit ubiquitination and prevent the nuclear export of p53. These possibilities will be explored in future studies. Our studies suggest that ellipticine increases the localization of p53 to the nucleus through a mechanism independent of DNA damage. In addition some of the experimental data also suggest that the shift in p53 localization may also be independent of ellipticine’s known ability to inhibit Topoisomerase II. In our system of SAOS2 cells stably expressing GFP::p53mut, etoposide produced only slight increases in nuclear p53. Potentially, longer incubation with etoposide might have demonstrated increased nuclear p53. Alternatively, the lack of increased nuclear p53 after etoposide treatment may reflect cell-type specific effects of etoposide. In HCT116 cells with wild type p53, ellipticine increased the nuclear localization of wild type p53, but also increased levels of total p53 compared to buffer treated controls. Further studies will be required to examine the mechanism by which levels of total p53 are increased. Potentially, ellipticine could have stabilized p53 in the nucleus by blockings its export and/or degradation thereby leading to increased levels of total p53. However, we cannot exclude the possibility that ellipticine also increases the transcription and synthesis of new p53. Previous studies by Kuo et al. [21, 22] demonstrated that levels of total p53 increased within 3 h after treatment with ellipticine, but the mechanism for this effect was not explored. In summary, we have developed a high-content screen that can identify small molecules that shift the localization of p53 to either the nucleus or cytoplasm. This screen identified ellipticine as a compound that increases the nuclear localization of p53 and enhanced its promoter activity. Thus, ellipticine can serve as tool to investigate mechanisms that regulate the localization of p53. In addition ellipticine may serve as a lead compound for novel therapeutic agents for the treatment of malignancy that act
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in a p53-dependent matter. Furthermore, this report is novel in that it describes a new platform for the identification of other molecules that shift the localization of p53. Acknowledgements We thank Dr. Sam Benchimol for helpful advice and discussions. This work was supported by the Canadian Institutes of Health Research (CIHR), and the Ontario Cancer Research Network through funding from the province of Ontario. I.A.M. is the recipient of the Edward Christie Stevens Fellowship in Medical Research. A.D.S. is the recipient of a CIHR Clinician Scientist Award.
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