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To whom correspondence should be addressed: Dept. of Pharmacology, The University of Michigan Medical School, 1301 Medical Science Research Bldg. III, Ann Arbor, MI 48109-0632. Tel.: 734-764-5414; Fax: 734-763-4450;
* This work was supported by National Institutes of Health Grants CA28010 (to W. B. P.) and CA82376 (to M. L.). Cell biology core services are supported in part by Michigan Diabetes Research and Training Center Grant P60DK20572 in association with the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Supported in part by Michigan Diabetes Research and Training Center Grant P60DK20572 in association with the NIDDK, National Institutes of Health.
Pifithrin-α (PFTα) was originally thought to be a specific inhibitor of signaling by the tumor suppressor protein p53. However, the laboratory that discovered pifithrin recently reported that the compound also inhibits heat shock and glucocorticoid receptor (GR) signaling, and they suggested that PFTα targets a factor common to all three signal transduction pathways, such as the hsp90/hsp70-based chaperone machinery (Komarova, E. A., Neznanov, N., Komarov, P. G., Chernov, M. V., Wang, K., and Gudkov, A. V. (2003) J. Biol. Chem. 278, 15465–15468). Because it is important for the mechanistic study of this machinery to identify unique inhibitors of chaperone action, we have examined the effect of PFTα on transcriptional activation, the hsp90 heterocomplex assembly, and hsp90-dependent nuclear translocation for both p53 and the GR. At concentrations where PFTα blocks p53-mediated induction of p21/Waf-1 in human embryonic kidney cells, we observed no inhibition of GR-mediated induction of a chloramphenicol acetyl transferase reporter in LMCAT cells. PFTα did, however, cause a left shift in the dexamethasone dose response curve by increasing intracellular dexamethasone concentration, apparently by competing for dexamethasone efflux from the cell. The assembly of p53 or GR heterocomplexes with hsp90 and immunophilins was not affected by PFTα either in vivo or in vitro and did not affect the nuclear translocation of either transcription factor. Thus, we conclude that PFTα does not inhibit GR-mediated induction or the function of the chaperone machinery, and, as originally thought, it may specifically inhibit p53 signaling by acting at a stage after p53 translocation to the nucleus.
The tumor suppressor protein p53 is a transcription factor that induces growth arrest or apoptosis in response to a variety of stress signals (
). Activation of p53-mediated apoptosis in response to a stress, such as DNA damage, results in the elimination of potential tumor cell precursors. Thus, p53 is referred to as a tumor suppressor, and p53 knock-out mice have a high incidence of spontaneous tumors (
) screened a library of 10,000 synthetic chemicals for inhibitors of p53-dependent apoptosis. A stable, water-soluble inhibitor of p53-dependent apoptosis was identified that was also shown to reduce the activation of p53-regulated genes, including cyclin G, p21/Waf-1, and mdm2 (
) recently published a report revealing that PFTα suppresses signaling through the heat shock transcription factor HSF1 and the glucocorticoid receptor (GR) but not signaling by NF-κB. This finding indicated that PFTα may not specifically target p53 but targets some unknown cellular component that is common for three major signal transduction pathways. Inasmuch as p53, HSF1, and the GR are all bound to and regulated by hsp90 (see Ref.
We have recently demonstrated that p53·hsp90 and GR·hsp90 complexes exist in identical large heterocomplexes containing dynein and one of three immunophilins, namely FKBP52, cyclophilin 40 (CyP-40), or PP5 (
). The association of either transcription factor with hsp90 is critical both for maintenance of its inactive state and for rapid translocation to the nucleus after its activation by stress or steroid (
) by examining PFTα effects on p53-mediated and GR-mediated transcriptional activation, the formation of heterocomplexes with hsp90 and immunophilins, and the nuclear movement of the two transcription factors. We find that, at concentrations where PFTα abrogates p53-dependent induction of p21/Waf-1, there is no inhibition of dexamethasone-dependent activation from a GR-dependent reporter construct. However, PFTα did cause a left shift in the dose response curve of dexamethasone, apparently by competing for dexamethasone efflux from the cell. PFTα did not affect assembly of either p53·hsp90·immunophilin or GR·hsp90·immunophilin complexes in vivo or in vitro, and it did not affect nuclear translocation of either transcription factor. Our observations indicate that PFTα may very well be a specific inhibitor of p53 signaling that does not affect the hsp90/hsp70-based chaperone machinery and that inhibits p53 function at a stage after its translocation to the nucleus.
Materials—PFTα (1-(4-methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)benzothiazolyl)ethanone hydrobromide) was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). PFTα and the cyclic PFTα analog were also purchased from Alexis Biochemicals (San Diego, CA), and similar results to those seen with PFTα from Biomol were observed. Untreated rabbit reticulocyte lysate was purchased from Green Hectares (Oregon, WI). [6,7-3H]Dexamethasone (40 Ci/mmol), [ring-3,5-3H]chloramphenicol (38 Ci/mmol), and 125I-conjugated goat anti-mouse and goat anti-rabbit IgGs were obtained from PerkinElmer Life Sciences (Boston, MA). Protein A-Sepharose, doxorubicin, nonradiolabeled dexamethasone, charcoal-stripped calf serum, pristane, mixed xylenes, and goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated antibodies were from Sigma. Dulbecco's modified Eagle's medium (DMEM) was from Bio-Whittaker (Walkersville, MD). Rhodamine-conjugated donkey anti-mouse IgG was from Jackson ImmunoResearch (West Grove, PA). The BuGR2 monoclonal IgG used to immunoblot the GR and the rabbit polyclonal antibody against cyclophilin 40 were from Affinity Bioreagents (Golden, CO). The AC88 monoclonal IgG against hsp90 was from StressGen Biotechnologies (Victoria, British Columbia, Canada). The anti-p21/Waf-1 monoclonal antibody (Ab-1) was from Oncogene Research Products (San Diego, CA). The Ab421 pan-specific mouse monoclonal antibody used to immunoadsorb p53 was kindly provided by Dr. Michael Clarke (University of Michigan Medical School). The Ab-4 mouse monoclonal IgG used for localization of p53 by indirect immunofluorescence and the Ab-7 sheep antiserum used to immunoblot p53 were purchased from Calbiochem. The FiGR monoclonal IgG used to immunoadsorb the GR was generously provided by Dr. Jack Bodwell (Dartmouth Medical School, Lebanon, NH). The UPJ56 rabbit antiserum against FKBP52 was provided by Dr. Karen Leach (Pfizer) and the rabbit antiserum against PP5 was provided by Dr. Michael Chinkers (University of South Alabama, Mobile, AL). Rhodamine-conjugated goat anti-mouse IgG was from Jackson ImmunoResearch. The mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter plasmid and the mouse fibroblast L929 cell line stably transfected with MMTV-CAT (LMCAT cells) were provided by Dr. Edwin Sanchez (Medical College of Ohio, Toledo, OH). DLD-1 human colon adenocarcinoma cells were purchased from the American Type Culture Collection (Manassas, VA). HT29-tsp53 (formerly referred to as ts29-G cells) human colorectal cancer cells overexpressing a temperature-sensitive mutant of mouse p53 were described previously (
Cell Culture and Cytosol Preparation—Cultures of mouse fibroblast L929 cells, L929 cells stably transfected with an MMTV-CAT reporter plasmid (LMCAT cells), HeLa cells, human embryonic kidney (HEK) cells, and NIH 3T3 mouse fibroblast cells, all expressing wild-type p53, were cultured in DMEM supplemented with 10% bovine calf serum. DLD-1 cells were cultured in DMEM supplemented with 10% fetal calf serum, 2 mm l-glutamine, 1.5 g/liter NaHCO3, 4.5 g/liter glucose, and 1 mm pyruvate. Cells were harvested by scraping into Hanks' buffered saline solution and centrifugation. Cell pellets were washed in Hanks' buffered saline solution, resuspended in 1.5 volumes of HEM buffer (10 mm NaOH-Hepes, 1 mm EDTA, and 20 mm sodium molybdate, pH 7.4) with 1 mm phenylmethylsulfonyl fluoride and 1 tablet of Complete-Mini protease inhibitor mixture (Roche Applied Science) per 3 ml of buffer and ruptured by Dounce homogenization. The lysate was then centrifuged at 100,000 × g for 30 min, and the supernatant, referred to as “cytosol,” was collected, aliquoted, flash-frozen, and stored at –70 °C. Mouse GR was expressed in Sf9 cells, and cytosol was prepared as described previously (
Indirect Immunofluorescence of GR and p53—NIH 3T3 cells were grown on 11 × 22 mm coverslips placed in 35-mm culture wells in 2 ml of DMEM supplemented with bovine calf serum. When the cells were ∼50% confluent, the culture medium was replaced with phenol red-free DMEM supplemented with 10% charcoal-stripped calf serum, and the cells were grown for an additional 24 h. Cells were incubated with 0.05% Me2SO or 100 μm PFTα for 2 h prior to the addition of 0.1% ethanol or 1 μm dexamethasone for 10 min to permit nuclear translocation of the GR. HT29-tsp53 cells were grown at 39 °C (non-permissive temperature) on coverslips placed in 35-mm culture wells in 2 ml of DMEM containing 10% bovine calf serum and 2 mm l-glutamine. Nuclear translocation of p53 was triggered by reducing the incubation temperature of HT29-tsp53 cells from 39 to 32 °C. Cells were fixed and permeabilized by immersion in cold (–20 °C) methanol and immunostained by inverting the coverslip on a 50-μl solution of phosphate-buffered saline with 1% bovine serum albumin containing 1 μl of the Ab-4 mouse monoclonal IgG against p53 or 1 μl of FiGR mouse monoclonal IgG overnight at 4 °C in a humid chamber. The coverslips were incubated with a washing buffer (20 mm Tris-HCl, pH 8, 630 mm NaCl, 0.05% Tween 20, and 1% bovine serum albumin) for 30 min at room temperature, and reincubated with 1:100 dilution of rhodamine-conjugated donkey anti-mouse IgG counter-antibody for 2 h. Coverslips were rinsed in washing buffer for 30 min and mounted on microscope slides using a drop of mounting medium (1 mg/ml phenylenediamine in 10% phosphate-buffered saline and 90% glycerol, pH 9).
Cells were observed with a Leitz Aristoplan epi-illumination microscope and scored for GR or p53 translocation as described previously (
) using a score of 4 for nuclear fluorescence much greater than cytoplasmic fluorescence, 3 for nuclear fluorescence greater than cytoplasmic fluorescence, 2 for nuclear fluorescence equal to cytoplasmic fluorescence, 1 for nuclear fluorescence less than cytoplasmic fluorescence, and 0 for nuclear fluorescence much less than cytoplasmic fluorescence. The translocation scores represent the mean ± S.E. of three experiments in which ≥ 50 cells per condition per experiment were counted.
Transient Transfection of MMTV-CAT Reporter—L929 or HeLa cells were grown as monolayer cultures in 35-mm culture wells to ∼50% confluency, washed, and incubated for 1 h with 1 ml of serum-free medium containing 5 μg of plasmid DNA and 15 μl of TransFast transfection reagent (Promega). The transfection medium was replaced with regular medium, and the cells were incubated for 48 h. During the incubation, cells were treated for 2 or 20 h with dexamethasone or PFTα or both as indicated in the legend of Fig. 2.
GR-mediated Transcriptional Activation—Dexamethasone-induced CAT gene expression was assayed by measuring CAT enzymatic activity in HeLa, L cell, and LMCAT cell cytosol, using a modified version of the CAT assay described in Kwok et al. (
). LMCAT cells were grown in 35-mm culture wells to ∼50% confluence and incubated with varying concentrations of dexamethasone and PFTα for up to 24 h. Cells were washed, harvested, resuspended in potassium phosphate buffer (100 mm potassium phosphate and 1 mm dithiothreitol, pH 7.8), and ruptured by exposing the cell suspensions to three freeze-thaw cycles. Cell suspensions were centrifuged at 18,000 × g for 10 min, and the protein concentration of the supernatants was measured by a Bradford assay. Aliquots of the supernatants containing 10 μg of total protein were incubated for 15 min at 70 °C in 150 mm Tris-HCl buffer, pH 7.4. The aliquots were added to a CAT reaction mixture (50 nm purified [3H]chloramphenicol, 150 mm Tris-HCl, pH 7.4, and 0.25 mm butyryl CoA) and incubated for 2 h at 37 °C. An organic phase mixture consisting of 2 parts pristane and 1 part mixed xylenes was added and samples were thoroughly subjected to a vortex. The reaction mixture was centrifuged at 20,000 × g for 10 min, and 150 μl of the organic phase was counted by liquid scintillation spectrometry.
Immunoadsorption of GR and p53—Receptors were immunoadsorbed from aliquots of 50 (for measuring steroid binding) or 100 μl (for Western blotting) of Sf9 cell cytosol by rotation for 2 h at 4 °C with 18 μl of protein A-Sepharose precoupled to 9 μl of FiGR ascites suspended in 200 μl of TEG buffer (10 mm TES, pH 7.6, 50 mm NaCl, 4 mm EDTA, and 10% glycerol). p53 was immunoadsorbed from 250-μl aliquots of DLD-1 cytosol with 10 μl of Ab421 antibody. Prior to incubation with reticulocyte lysate, immunoadsorbed p53 and GR were stripped of associated hsp90 by incubating the immunopellet for an additional 2 h at 4 °C with 350 μl of 0.5 m NaCl in TEG buffer. The pellets were then washed once with 1 ml of TEG buffer followed by a second wash with 1 ml of Hepes buffer (10 mm Hepes, pH 7.4).
GR·hsp90 and p53·hsp90 Heterocomplex Reconstitution—Immunopellets containing GR or p53 stripped of chaperones were incubated with reticulocyte lysate and 5 μl of an ATP-regenerating system (50 mm ATP, 250 mm creatine phosphate, 20 mm magnesium acetate, and 100 units/ml creatine phosphokinase). The assay mixtures were incubated for 20 min at 30 °C with suspension of the pellets by shaking the tubes every 2 min. At the end of the incubation, the pellets were washed twice with 1 ml of ice-cold TEGM buffer (TEG with 20 mm sodium molybdate) and assayed for steroid binding capacity and for GR- or p53-associated proteins.
Assay of Steroid Binding Capacity—For measuring hormone retention in intact cells, exponentially growing LMCAT cells (∼2 × 106 cells/25 cm2 flask) were incubated with 100 nm [3H]dexamethasone at 37 °C for 2 h in the absence or presence of 1,000-fold excess unlabeled dexamethasone and the presence of Me2SO or PFTα. Cells were washed, harvested, and suspended in cold phosphate-buffered saline and counted by liquid scintillation spectrometry. Specific binding activity was calculated by subtracting the radioactivity in the cells incubated in the presence of excess unlabeled dexamethasone.
Immune pellets to be assayed for steroid binding were incubated overnight at 4 °C in 50 μl of HEM buffer plus 100 nm [3H]dexamethasone. Samples were then washed three times with 1 ml of TEGM buffer and counted by liquid scintillation spectrometry. The steroid binding is expressed as counts/min of [3H]dexamethasone bound/FiGR immunopellet prepared from 50 μl of cytosol.
For cytosols to be assayed for steroid binding, a 50 μl aliquot of cytosol was incubated overnight at 4 °Cin50 μl of HEM buffer with 100 nm [3H]dexamethasone plus or minus a 1,000-fold excess of non-radioactive dexamethasone. Samples were mixed with dextran-coated charcoal, centrifuged, and counted by liquid scintillation spectrometry. The steroid binding is expressed as counts/min of bound [3H]dexamethasone/100 μl of cell cytosol.
Gel Electrophoresis and Western Blotting—Immune pellets were resolved on 12% SDS-polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were probed with 0.1% Ab-7 for p53, 0.25 μg/ml BuGR2 for GR, 1 μg/ml AC88 for hsp90, 1 μg/ml anti-p21/Waf-1, 0.1% UPJ56 for FKBP52, 0.1% anti-cyclophilin 40, or 0.1% anti-PP5. The immunoblots were then incubated a second time with the appropriate 125I-conjugated or horseradish peroxidase-conjugated counterantibody to visualize the immunoreactive bands. p53 was revealed by enhanced chemiluminescence. Because PP5, FKBP52, and p53 migrate in the same region upon gel electrophoresis, we electrophoresed replicate samples of both non-immune and immune pellets and probed replicate immunoblots with an antibody specific for each protein. Thus, the Western blots of Figs. 5 and 6 are necessarily composites prepared from two or more replicate immunoblots.
RESULTS AND DISCUSSION
PFTα Effect on p53- and GR-mediated Transcription—To confirm the activity of the PFTα preparation as an inhibitor of p53 signaling, HEK cells were treated with doxorubicin to cause DNA damage and activate p53. As shown in Fig. 1A, doxorubicin treatment causes an increase in the amount of p21/Waf-1 that is prevented by 10 μm PFTα. The PFTα inhibition of p21/Waf-1 induction is sustained over 24 h of doxorubicin treatment (Fig. 1B).
To determine the effect of PFTα on GR-mediated transcription, HeLa cells (Fig. 2A) or L929 mouse fibroblasts (Fig. 2B) were transiently transfected with an MMTV-CAT reporter plasmid and treated for 2 or 20 h with 1 μm dexamethasone or 30 μm PFTα or both prior to the assay of CAT activity. In both cell lines, there was a robust induction of CAT by dexamethasone that was not affected by PFTα. To further test any possible effect of PFTα on dexamethasone-dependent induction, we used a subline of L929 mouse fibroblasts stably transfected with an MMTV-CAT reporter plasmid (LMCAT cells) that was developed by Ning and Sanchez (
). When the LMCAT cells are treated with dexamethasone, there was an induction of CAT driven by the endogenous L cell GR. As shown in Fig. 3A, PFTα did not inhibit GR-mediated CAT induction when cells were treated with a high concentration (1 μm) of dexamethasone. At lower concentrations of dexamethasone and a short induction time, PFTα stimulated dexamethasone-dependent CAT induction (Fig. 3B).
PFTα Increases the Concentration of Dexamethasone in Cells—Potentiation of a dexamethasone response like that shown in Fig. 3B has been reported with immunosuppressant drugs (reviewed in Ref.
) showed that FK506 potentiates dexamethasone responsiveness in L cells by increasing dexamethasone accumulation without altering the hormone binding properties of the GR. Here, we show that PFTα did not affect the binding activity of the GR when it was added to cytosol (Fig. 4A), but treatment of LMCAT cells with PFTα increased the amount of [3H]dexamethasone bound to GRs in vivo (Fig. 4B). As shown in Fig. 4C, treatment with PFTα increased the amount of [3H]dexamethasone retained in the cell. This increase in intracellular dexamethasone is likely due to PFTα competition for efflux in the same manner as that seen with the immunosuppressant drugs, and it forms the basis for a potential drug interaction.
PFTα Does Not Inhibit Assembly of Heterocomplexes with hsp90 and Immunophilins—To examine the effects of PFTα on the assembly of p53·hsp90·immunophilin complexes in vivo, we selected the DLD-1 human colorectal cancer cell line, which possesses a point mutation that converts serine 241 of p53 to phenylalanine (
). Fig. 5 is an immunoblot of hsp90 and the tetratricopeptide repeat domain immunophilins co-immunoadsorbed with p53 or the GR from cytosol prepared from untreated DLD-1 cells (Fig. 5, lanes 2) or DLD-1 cells treated with PFTα (lanes 3). In neither case did PFTα affect heterocomplex formation in vivo.
Heterocomplexes with hsp90 and the tetratricopeptide repeat domain immunophilins that bind hsp90 can be assembled in vitro by incubating immunoadsorbed p53 or GR that has been stripped of endogenous bound chaperones with rabbit reticulocyte lysate. Fig. 6 presents immunoblots of hsp90 and immunophilins that associate with p53 (Fig. 6A) and the GR (Fig. 6B) during such cell-free heterocomplex assembly. It is clear that PFTα did not affect assembly in either case. When the GR is assembled into GR·hsp90 heterocomplexes, it is converted from a non-steroid binding state to a steroid binding state, and, as shown in the bar graph in Fig. 6B, the generation of steroid binding activity was not affected by pifithrin.
PFTα Does Not Inhibit Nuclear Translocation of p53 or GR— Both p53 and the GR utilize an hsp90-dependent movement system for translocation to the nucleus along microtubular tracts driven by the cytoplasmic dynein motor protein (
). In the movement system, the immunophilins bind via their tetratricopeptide repeat domain to hsp90 and via their peptidylprolyl isomerase domain to the dynamitin component of the dynein-associated dynactin complex. Thus, rapid nuclear translocation of either the GR or p53 requires dynamic assembly of heterocomplexes with hsp90, and movement is impeded by geldanamycin and radicicol, which inhibit hsp90 heterocomplex assembly (
). Nuclear translocation of p53 or the GR is also inhibited when immunophilin binding to dynein is competed by expression of a peptidylprolyl isomerase domain fragment or when dynein linkage to cargo is dissociated by the expression of dynamitin (
). This temperature-sensitive mutant of p53 is fully active and nuclear at the permissive temperature of 32 °C, but it is inactive and located in the cytoplasm at the non-permissive temperature of 39 °C (
). As shown in Fig. 7A, nuclear translocation of p53 was not affected by 100 μm PFTα. We routinely examine GR translocation in 3T3 mouse fibroblasts, where the receptor translocates to the nucleus within 10 min after the addition of steroid (
) in that we do not see PFTα inhibition of GR-dependent induction. Inasmuch as we have exposed cells to much higher concentrations of PFTα, we do not know the basis for the discrepancy. The notion of Komarova et al. (
) is intriguing and well worth testing. Here, we do not find PFTα inhibition of hsp90 heterocomplex assembly with p53 or the GR, inhibition of functional opening of the steroid binding cleft in the GR, or inhibition of the nuclear transfer of either transcription factor. The absence of PFTα inhibition shows that PFTα must be inhibiting some step in the induction mechanism after p53 transfer to the nucleus. Because, in our hands, induction by the GR is not affected, we would suggest that PFTα is not inhibiting a coactivator common to both p53-dependent and GR-dependent responses.
We thank Jack Bodwell, Michael Chinkers, Karen Leach, and Edwin Sanchez for providing reagents used in this work.