The Heat Shock Protein 90-Targeting Drug Cisplatin Selectively Inhibits Steroid Receptor Activation

Marcus C. Rosenhagen, Csaba Sõti, Ulrike Schmidt, Gabriela M. Wochnik, F. Ulrich Hartl, Florian Holsboer, Jason C. Young and Theo Rein

Max Planck Institute of Psychiatry (M.C.R., U.S., G.M.W., F.H., T.R.), D-80804 Munich, Germany; Semmelweis University School of Medicine (C.S.), Department of Medical Chemistry, H-1444 Budapest, Hungary; and Max Planck Institute of Biochemistry (F.U.H., J.C.Y.), D-82152 Martinsried, Germany

Address all correspondence and requests for reprints to: Theo Rein, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, D-80804 Munich, Germany. E-mail: theorein{at}mpipsykl.mpg.de.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cisplatin is an antineoplastic drug that binds to DNA, thereby inhibiting cell division and tumor growth. Cisplatin may also disrupt the function of some proteins, including heat shock protein 90 (Hsp90). We report that cisplatin dose-dependently inhibited transcriptional activity of the androgen receptor and the glucocorticoid receptor (GR) in transient reporter assays. A truncated, hormone-independent GR was only partially inhibited at significantly higher doses of cisplatin. Cisplatin treatment of neuroblastoma cells led to an immediate inhibition of hormone binding by GR, followed by proteasome-dependent degradation of the receptor. Other Hsp90-regulated proteins, i.e. the phosphokinases raf-1, lck, and c-src, were not affected, indicating a specific functional interference of cisplatin with the steroid receptors GR and androgen receptor. Cisplatin did not elicit a stress response, in contrast to geldanamycin. Immunoprecipitation revealed that cisplatin disrupts binding of GR to Hsp90. Moreover, cisplatin-treated Hsp90 was unable to associate with untreated ligand binding domain of GR. Reticulocyte lysate was able to restore hormone binding of GR in vitro, but not when the lysate was pretreated with geldanamycin. Our data reveal that cisplatin influences steroid receptors also independently of its DNA-mediated effects and, thus, suggest a novel modes of action for this cytostatic drug.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TOGETHER WITH ITS various derivatives, cisplatin [cis-diamminedichloroplatinum (II), CDDP] is one of the most commonly applied antineoplastic drugs today. It is a long-standing paradigm that the cytotoxic effect of cisplatin is primarily due to its well-described formation of adducts with DNA (1, 2, 3) that cause G2 arrest in the cell cycle, usually leading to apoptosis and inhibition of tumor growth (4, 5). It has been speculated that protein targets (6) may also be important for the observed effects of CDDP (5), an idea strongly supported by a recent report (7). CDDP has also been reported recently to bind to heat shock protein 90 (Hsp90) (8), thereby reducing its chaperoning activity. The CDDP-binding region has been localized near the C terminus of Hsp90, distant from the N-terminal binding site of geldanamycin (GA) (9), another antineoplastic agent that also reduces Hsp90 chaperoning activity (10). These results suggest that CDDP and GA may inhibit the chaperoning activity of Hsp90 by different mechanisms.

Hsp90 is one of the most abundant molecular chaperones in the eukaryotic cytosol and is known to regulate the stability and function of a wide spectrum of intracellular proteins. Hsp90 client proteins are involved in a variety of biological processes including development, cell cycle control and steroid hormone signaling (11, 12, 13). The two best-characterized classes in the steadily growing list of Hsp90-regulated proteins are transcription factors, e.g. steroid receptors such as the glucocorticoid (GR) and androgen receptor (AR), and protein kinases including raf1, src, and lck (11, 12). GR is an essential factor in many developmental and physiological processes (14) and has been used in numerous studies to elucidate the contribution of chaperones to proper folding and function of signal transduction molecules (15). In the absence of its ligand, the glucocorticoids, GR is part of a multiprotein complex comprising the receptor, Hsp90, and several other chaperones and cochaperones (16). This complex keeps the receptor protein in an inactive, yet ligand-activable state (17). Upon binding to hormone, GR is translocated to the nucleus and both positively and negatively regulates transcription of a variety of genes (18, 19). Similar Hsp90 heterocomplexes have been found to be important for other factors, e.g. heat shock factor 1 (HSF1) and protein kinases like raf1, lck, and c-src (20, 21, 22, 23).

There is evidence that some physiological effects of CDDP may result from its targeting of Hsp90. For example, CDDP is commonly used as antineoplastic agent for a variety of prostate cancer types which in turn are often associated with hyperactivity of the Hsp90-dependent androgen receptor (AR). Moreover, CDDP-resistance in cells transfected with the Hsp90-dependent protein kinase v-src can be overcome by the Hsp90-targeting drugs radicicol (RAD) or herbimycin A (HA) (24). Interestingly, RAD, HA, and its homolog GA originally were identified as naturally occurring antitumor antibiotics (25, 26, 27) and characterized later as tyrosine kinase inhibitors (28, 29, 30). Subsequently, it has become clear that they actually act through binding to Hsp90 (9, 31, 32, 33, 34, 35). Moreover, it is a long-standing, although unexplained, observation that glucocorticoids like dexamethasone decrease CDDP-induced emesis if added to an antiemetic pharmacological regimen (36). Thus, one can hypothesize that dexamethasone, as a potent ligand of GR, fights CDDP-induced emesis by counteracting CDDP-induced defects in GR function.

To evaluate whether important aspects of CDDP function involve Hsp90-dependent proteins, we investigated the effects of CDDP on the steroid receptors GR and AR. We demonstrate that CDDP inhibits GR- as well as AR-mediated transcriptional activation in neuroblastoma cells. Hormone binding of GR is reduced by CDDP due to perturbation of the chaperone heterocomplex and followed by proteasomal degradation of GR. However, HSF1 is not activated by CDDP and the protein levels and activation of the kinases raf1, c-src, and lck are not changed by CDDP, in contrast to GA. Impairment of GR binding to hormone by CDDP can be reversed in vitro with untreated reticulocyte lysate, but only if its Hsp90 is intact. Our results provide new insights into the molecular mechanisms of Hsp90 inhibition by CDDP, which may lead to an improvement of CDDP pharmacotherapy in the future.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dose-Dependent Inhibition of the Glucocorticoid and Androgen Receptors by Cisplatin
To test the hypothesis that cisplatin (CDDP) inhibits the activity of Hsp90-dependent steroid hormone receptors such as the glucocorticoid receptor (GR) or the androgen receptor (AR), we used a reporter gene assay in transient transfections of cultivated cells (37). Neuroblastoma SK-N-MC cells were transfected with a plasmid expressing the receptor (either GR or AR), a reporter plasmid with the mouse mammary tumor virus promoter controlling the firefly luciferase structural gene, and a ß-galactosidase encoding plasmid to monitor transfection efficiency and general transcriptional activity. Cells were incubated in medium with 10 nM cortisol for stimulation of GR or 10 nM dihydrotestosterone for stimulation of AR together with increasing concentrations of CDDP. The stimulation of the reporter gene activity in the presence of CDDP was related to the stimulation in the absence of CDDP, which was set to 100% for each titration. Both GR- and AR-dependent transcription were severely impaired (Fig. 1Go, A and B, respectively). About half-maximal activity of the receptors was observed at a concentration of CDDP of 0.1–1 µM (GR) or 1 µM (AR), and virtually complete inhibition was reached at concentrations of 100 µM CDDP. CDDP did not affect the enzymatic activity of the luciferase reporter (control not shown).



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Fig. 1. Inhibition of GR- and AR-Dependent Transactivation by CDDP

Human neuroblastoma SK-N-MC cells were transfected with a GR-dependent reporter plasmid (MTV-Luc) a control plasmid expressing ß-galactosidase and either a plasmid expressing full-length GR (A, C, D), AR (B) or GR lacking the ligand binding domain (E, "GR{Delta}LBD"). After transfection, cells were cultivated with either 10 nM cortisol (A, C, D) or 10 nM dihydrotestosterone (B), together with increasing amounts of cisplatin (CDDP) (A, B, E), transplatin (TDDP) (C), or cyclophosphamide (CYPH) (D). Luciferase activities were normalized to the ß-galactosidase activities and are presented as percent activity with hormone-stimulated cells set as 100%. Results represent mean values ± SEM of three independent experiments performed in duplicate.

 
To evaluate the specificity of the inhibitory effect on GR and AR, we tested two other reagents, the CDDP structural isomer transplatin and cyclophosphamide, which is another DNA-targeting antineoplastic drug. Transplatin and cyclophosphamide failed to affect GR-dependent activity up to a concentration of 100 µM (Fig. 1Go, C and D). We also tested whether CDDP reduced the viability of the SK-N-MC cells under our assay conditions and found no effect on the MTT assay up to 100 µM CDDP (data not shown).

The inability of other DNA targeting drugs, such as cyclophosphamide, to inhibit GR-dependent transcription from the mouse mammary tumor virus promoter does not completely rule out the possibility that the effect of CDDP is due to its interaction with DNA (38). To begin to characterize the inhibitory effect of CDDP on GR we used a truncated GR that is devoid of the ligand binding domain. This receptor is not regulated by Hsp90, does not bind to hormone, and displays constitutive transcriptional activity to a moderate extent (39). CDDP also dose-dependently inhibited this receptor (Fig. 1EGo). However, in contrast to the full-length GR (Fig. 1AGo), higher doses were needed and the effect was not as strong. We conclude that, whatever the mechanism for the inhibitory effect of CDDP on the truncated GR is, it cannot fully account for the effect on full-length GR.

A likely target for the additional effect of CDDP on full-length GR is the ability to bind to hormone, either by binding to the ligand binding domain or by binding to proteins regulating hormone binding. Hormone binding of GR was determined as a measure of GR folding. SK-N-MC cells were transfected with a GR expression vector and binding of radioactive cortisol was determined in whole cells 24 h after transfection in the presence of increasing concentrations of CDDP during a 1-h incubation. Nonspecific binding was monitored by adding 1000-fold excess of unlabeled cortisol, and specific binding was determined by subtracting the low nonspecific binding from the binding measured in the absence of unlabeled competitor. As shown in Fig. 2Go, hormone binding by GR was dose-dependently reduced by CDDP. Virtually complete inhibition was reached at 100 µM CDDP. Furthermore, the level of GR protein extracted from the cells remained unchanged throughout the titration, indicating that GR is not degraded after the 1-h incubation, but merely rendered nonfunctional. These results strongly suggest that CDDP inhibits the chaperone-dependent folding of GR, an effect independent of its interaction with DNA.



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Fig. 2. CDDP Inhibits Hormone Binding of GR in Vivo

SK-N-MC cells were transfected with a plasmid expressing GR and binding of radiolabeled cortisol was determined after 24 h in whole cells. Cortisol and CDDP (at the concentrations indicated) were added for 1 h. Hormone binding with cortisol alone was set to 100%. Results represent mean values ± SEM of three independent experiments performed in duplicate. GR levels were assessed by Western blotting.

 
The Effect of CDDP Is Specific for Steroid Receptors and Leads to Proteasome-Dependent Depletion of Cellular GR
A possible target to mediate these DNA-independent effects of CDDP is Hsp90. Other Hsp90-targeting drugs like geldanamycin (GA) and radicicol bind to the N-terminal ATP site and reduce the activity and protein levels of not only steroid receptors, but also several kinases (10, 23, 40, 41, 42, 43). Therefore, we addressed two important questions: does prolonged treatment with CDDP affect protein levels of GR and are other Hsp90-dependent proteins also affected?

Figure 3Go shows that treatment of GR-transfected SK-N-MC cells with CDDP or GA led to a dose-dependent decrease in the protein level of GR. The degradation observed here after 16 h of incubation with CDDP was clearly preceded by the inactivation of hormone binding by GR, which was observed after 1 h in the absence of degradation (Fig. 2Go). However, protein levels of the serine/threonine kinase raf-1 (Fig. 3CGo) and the tyrosine kinase c-src (Fig. 3DGo), as well as of Hsp90 itself (Fig. 3EGo), were not affected by CDDP, in contrast to GA. These data support the hypothesis that CDDP interaction with Hsp90 has specific consequences in that it impairs correct folding of GR, which leads to its degradation, but not of the protein kinases raf-1 and c-src.



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Fig. 3. CDDP Induces Proteasome-Dependent Degradation of GR, But Not of Hsp90-Dependent Kinases

SK-N-MC cells were transfected with a plasmid expressing GR (pRK7GR, 0.75 µg per plate) and treated for 16 h at 37 C with either 1 µM GA or 10 µM CDDP and the proteasome inhibitors lactacystin (100 µM, A) or MG132 (50 µM, B) where indicated. Nontransfected cells were used in C–E and treated with 1 µM GA or 10 µM CDDP (or the concentrations indicated in C and D) and lactacystin. Total cellular protein (100 µg) was fractionated by SDS-PAGE, probed with {alpha}-GR-(H-300) (A and B), {alpha}-Raf1-(C), {alpha}-c-Src-(mAB327) (D), or {alpha}-Hsp90 (H114) (E) antibody followed by chemiluminescent detection.

 
We also tested whether degradation of GR is proteasome dependent, similar to the GA-induced degradation of other Hsp90 substrate proteins (32, 44, 45). GR-transfected SK-N-MC-cells were treated with CDDP in combination with the highly specific proteasome inhibitors lactacystin (46) or MG132 (47). As a control, cell cultures were treated with GA instead of CDDP (43). After 16 h incubation in the presence of these drugs, GR levels were assessed by Western blotting of cell extracts. Treatment with lactacystin or MG132 alone had no apparent effect (Fig. 3Go, A and B), but both efficiently inhibited degradation of GR by GA, in full agreement with a previous report (32). Lactacystin and MG132 also blocked GR degradation induced by CDDP (Fig. 3Go, A and B), indicating that the same proteolytic pathway is induced.

To corroborate our conclusion that the Hsp90-targeting drug CDDP specifically affects steroid receptors, we analyzed the functions of several Hsp90-dependent proteins. GA and radicicol are known to elicit a cellular stress response by releasing HSF1 from Hsp90, and this response can be monitored by transcription from HSF1-dependent promoters (22). We therefore tested the activity of an HSF1-dependent promoter transfected into SK-N-MC cells with and without exposure to CDDP or GA. Whereas GA clearly showed induction of this promoter, CDDP had no effect (Fig. 4AGo).



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Fig. 4. CDDP Is Permissive for Activation of lck and raf Kinase and Does Not Activate HSF1-Dependent Promoters

A, SK-N-MC cells were transfected with an HSF1-dependent luciferase reporter and a ß-galactosidase control plasmid. After transfection, cells were cultivated with or without DMSO as well as with increasing amounts of CDDP or GA as indicated. Luciferase activities were normalized to the ß-galactosidase activities and are presented as fold induction with untreated cells as reference. Results represent mean values ± SEM of three independent experiments. B, After pretreatment with 1.8 µM GA or 100 µM CDDP for 16 h, Jurkat cells were stimulated with 5 µg/ml OKT3 antibody, and Raf-1 and Lck proteins were detected by immunoblotting. Shown are representative Western blots and the quantification of three independent experiments.

 
Activation of the Hsp90-regulated kinases lck, which is a member of the src kinase family, and raf-1 is accompanied by their phosphorylation (48). To analyze whether CDDP may influence the activation of these kinases, we pretreated Jurkat cells with 1.8 µM GA or with 100 µM CDDP, followed by stimulation with an anti-CD3 antibody (OKT3). Levels and phosphorylation of lck and raf-1 were detected after cell lysis and Western blotting. As shown in Fig. 4BGo, GA efficiently reduced the basal protein levels and prevented the activation of both, lck and raf-1. In contrast to GA, CDDP had only a marginal affect on the basal protein levels and phosphorylation of raf-1 and activated lck. It should be noted that GR is efficiently inhibited by CDDP also in Jurkat cells under these conditions (data not shown).

We finally compared the effect of GA and CDDP on ATP-hydrolysis of Hsp90 purified from Saccharomyces cerevisiae (Hsp82), which has previously been used to characterize the ATPase activity of Hsp90 (49, 50). CDDP did not inhibit the ATPase activity of the yeast Hsp82 up to a concentration of 1 mM (data not shown), in contrast to GA (51).

From these data, we conclude that the effect of CDDP on Hsp90-dependent processes differs from that of GA in that the effect of CDDP is much more specific, i.e. is confined to steroid receptors.

CDDP Disrupts Hsp90-GR Interaction and Prevents Hsp90-GR Complex Formation in Vitro
To shed light on the mechanism of CDDP action on Hsp90-GR interaction, we supplemented cell lysates of GR-transfected SK-N-MC cells with 10 µM CDDP. After incubation for 1 h on ice, Hsp90 was immunoprecipitated and blots were probed for Hsp90 and for co-precipitated GR. Whereas GR was readily coimmunoprecipitated with Hsp90 from untreated cell lysates, preincubation with CDDP or GA caused a marked decrease in the amounts of co-precipitated GR (Fig. 5AGo).



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Fig. 5. CDDP Interferes with Association of Hsp90 with GR

A, SK-N-MC cells were transfected with a GR expressing plasmid (pRK7GR). Cell lysates were incubated with or without 10 µM CDDP or 1 µM GA for 2 h at 4 C and then immunoprecipitated with an Hsp90-directed antibody. After SDS-PAGE and transfer to nitrocellulose, blots were probed for Hsp90 and GR. B, Hsp90 was radiolabeled using an in vitro transcription/translation system, ß-mercaptoethanol removed by gel filtration, treated with CDDP and unreacted CDDP removed by gel filtration. Association with myc-tagged GR ligand binding domain, bound to Sepharose beads via a myc antibody, was allowed for 10 min in the presence of reticulocyte lysate and bound proteins were eluted with high salt buffer. The relative amounts of bound labeled Hsp90 were assessed after gel electrophoresis (lower panel). Results represent mean values ± SEM of three independent experiments.

 
To further elucidate the molecular mechanism of CDDP action, we investigated the effect of CDDP treatment of Hsp90 on complex formation with GR. Chaperone complexes with GR can be reconstituted in reticulocyte lysate with the GR ligand binding domain alone, and immune-isolated similarly to the full-length receptor (52). We therefore analyzed complex formation between CDDP-treated Hsp90 and the ligand binding domain of GR in reticulocyte lysate. Hsp90 was radioactively labeled by in vitro translation in reticulocyte lysate, ß-mercaptoethanol was removed and the protein mixture was incubated with different concentrations of CDDP. It was important to then remove unreacted CDDP to ensure that it does not modify the ligand binding domain of GR in the following binding reaction. Labeled, CDDP-reacted Hsp90 was incubated with the myc-tagged ligand binding domain of GR, bound to myc antibodies on Sepharose beads for 10 min. As seen in Fig. 5BGo, CDDP dose-dependently interfered with complex formation between Hsp90 and GR. Thus, CDDP appears to act by both disrupting preformed Hsp90/GR complexes (Fig. 5AGo) and preventing these complexes from forming (Fig. 5BGo).

CDDP Inhibition of GR Hormone Binding Can Be Rescued in Vitro by Reticulocyte Lysates with Intact Hsp90
Whereas our data provide strong evidence that the effect of CDDP on GR involves interaction with Hsp90, the possibility remained that CDDP actually targets GR directly, which also may cause its inactivation and subsequent degradation. Moreover, CDDP is reactive toward sulfhydryl groups, and it has been previously demonstrated that sulfhydryl-targeting agents can inactivate GR (53, 54, 55). We asked whether targeting of GR or targeting of an associated chaperone like Hsp90 underlies CDDP-induced inactivation of GR. We treated cells expressing GR with CDDP as in the experiment for Fig. 2Go. After CDDP treatment, cells were lysed, unreacted CDDP was removed by gel filtration and dose-dependent inhibition of hormone binding by CDDP was confirmed (data not shown). However, when we supplemented the hormone binding reaction with reticulocyte lysate (RL) supplemented with ATP, binding was restored (Fig. 6AGo). No hormone binding was observed with RL alone. This demonstrates that the CDDP impairment of hormone binding by GR cannot be due to an interaction of CDDP with GR. Moreover, hormone binding of CDDP-treated GR was not restored, when we pretreated RL with GA before gel filtration (Fig. 6BGo). GA is established as specific inhibitor of Hsp90 used in numerous studies (56). Therefore, this result strongly suggests that it must be the intact Hsp90 from RL that restores hormone binding activity to GR by replacing the CDDP-impaired Hsp90 activity from the cell extract.



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Fig. 6. Hormone Binding of GR Can Be Restored in Vitro by Reticulocyte Lysate with Intact Hsp90

Binding of GR expressed in SK-N-MC cells and treated with CDDP (as in Fig. 2Go) to cortisol was measured. Hormone binding in the absence of CDDP was set to 100%. A, 5% reticulocyte lysate, ATP and Mg2+ was added to the extracts from cells treated with1 µM and 100 µM CDDP. B, RL was pretreated with the specific Hsp90-inhibitor geldanamycin (1 µM) before it was added to the CDDP-treated cell extracts. Results represent three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our results establish that CDDP specifically inhibits a subset of Hsp90-dependent processes, in particular the maturation of steroid hormone receptors represented by GR and AR. CDDP apparently interferes with the formation of the Hsp90 complexes required for the folding of these proteins. This discovery has a significant impact on our understanding of both the function of Hsp90 and the physiological consequences of CDDP application.

The most interesting aspect of our results with regard to Hsp90 function is that, unlike GA, CDDP disrupts some functions of Hsp90 but not others. Whereas the activity of the steroid hormone receptors GR and AR was drastically reduced by CDDP, HSF1-dependent transcription was not elicited. In contrast, GA, which targets the N-terminal ATP binding site of Hsp90, inhibits steroid receptors and activates HSF1-dependent promoters (this study and Refs. 22 and 32). Moreover, treatment of cells with CDDP leads to proteasome-dependent degradation of GR, whereas protein level and activation of the Hsp90-dependent protein kinases lck, c-src, and raf-1 were essentially unchanged. Inhibition of Hsp90 by GA, however, results in inactivation and degradation of not only GR, but also lck, c-src, and raf-1 (this study and Refs. 31 , 48 , and 57).

The inhibitory effect of CDDP on GR-dependent transcription has previously been ascribed to the well-documented interaction of CDDP with DNA (38). Moreover, in the case of the mineralocorticoid receptor induction of oxidative stress by CDDP has been suggested to cause diminished receptor activity (59). At a molecular level, oxidative stress would affect the sulfhydryl groups of proteins and sulfhydryl-targeting drugs have been shown to decrease GR function (53, 54, 55). Our observation that a truncated GR lacking the ligand binding domain is also inhibited by CDDP, albeit only at higher concentrations, can be explained by an effect on protein-DNA interactions, either by targeting the DNA or by targeting the cysteines of the zinc fingers in the DNA binding domain of GR. Modification of these cysteines has been shown before to inhibit DNA binding activity of GR (60). We assume that whatever mechanism applies to the inhibition of the truncated GR also contributes to the inhibition of full-length GR. However, our data with full-length GR strongly suggest that an additional, and possibly the major effect of CDDP on steroid receptors is due to its interaction with Hsp90. Inhibition of hormone binding of GR cannot be explained by interference of CDDP with protein-DNA interactions. Moreover, disruption of the Hsp90 heterocomplex by CDDP, as detected by immunoprecipitation, clearly shows a DNA-independent effect of CDDP. Because we are able to rescue hormone binding of GR after removal of excess CDDP and supplementing with untreated reticulocyte lysate, but not with GA-treated lysate, modification of GR resulting from oxidative stress is most likely not responsible for the effect of CDDP. Furthermore, CDDP not only disrupted existing GR-Hsp90 interactions but also prevented formation of new interactions when reacted only with Hsp90.

The differential effects of the two Hsp90-targeting agents CDDP and GA could be explained by their different interaction site with Hsp90. Crystallographic analysis revealed the ATP-binding pocket of Hsp90 as the binding site of GA (9), whereas CDDP has been reported to bind to the middle to C-terminal part of Hsp90 (8). An additional binding site for ATP in the C-terminal part of Hsp90 has been postulated (61), and CDDP has been proposed to act as a selective nucleotide competitor for this site (62). The significance of this cryptic ATP binding site remains to be elucidated. The other postulated C-terminal ATP competitor novobiocin apparently binds in this region (61) and leads to depletion of Hsp90-dependent proteins including raf1 and v-src (63). This effect is similar to that of GA but different from the effect of CDDP, as we observed no degradation of raf-1 and c-src. Also in contrast to CDDP, novobiocin has been described to inhibit allosterically the N-terminal ATP binding site (62). Thus, we suggest that CDDP and novobiocin interact with the C-terminal region of Hsp90 at different sites and/or in different manners.

With regard to the exact binding site of CDDP on Hsp90, we speculate that it reacts with cysteine residues, most likely to cysteine 596 of the human protein. The corresponding cysteine of rat Hsp90 has been reported to be particularly reactive (64). Although we have not detected significant amounts of covalent Hsp90 dimers in our assays, it is interesting to note that others did so under their experimental conditions (65). These covalent dimers could arise from subsequent reactions of platinated Hsp90.

It should be noted that inhibition of Hsp70 by the SH-targeting drug N-ethylmaleimide has been reported (66). Because Hsp70 participates in folding the GR, it is possible that it is another chaperone target for CDDP, in addition to Hsp90. Reticulocyte lysate treated with GA was unable to restore cortisol binding of GR. Because GA is established as specific inhibitor of Hsp90 (56), Hsp70 was most likely functional in this extract, arguing in favor of Hsp90 being an important, and possibly the major chaperone target of CDDP.

Considering the possible physiological relevance of our findings, we note that CDDP is typically administered at doses of 80–100 mg/m2 body surface (67, 68). As a rough estimate, the initial concentration in the blood (assuming even distribution throughout the blood) would be 140–180 µM, and about 10–12 µM assuming even distribution throughout the entire body liquid. Depending on the pharmacokinetics (distribution, uptake, degradation) there will be locally and temporally higher and lower concentrations. In any case, at least the initial concentrations are well within those needed to inhibit steroid receptors.

The effect of CDDP on steroid receptors may explain some of its clinical side effects. Delayed nausea and vomiting after application of CDDP remain a significant cause of treatment-related morbidity, because they are inadequately controlled by current therapies. Also newer therapeutics like 5-HT3 receptor antagonists are of insufficient efficacy (69). Corticosteroids make a considerable contribution toward the control of CDDP-induced acute and delayed emesis (70). Two randomized studies demonstrated that the combination of dexamethasone and ondansetron is more efficacious than ondansetron alone (71, 72); ondansetron plus dexamethasone is now considered a standard antiemetic therapy for CCDP-treated patients (67). Also, ACTH has been shown to be effective in the prevention of acute CDDP-induced vomiting (73). The mechanism by which ACTH exerts its effect against emesis are not fully understood. It stimulates the production of corticosteroids, which are known to be active against emesis, but it also acts directly on the brain and modulates behavior.

Another severe side-effect of CDDP treatment is renal failure or renal electrolyte wasting (74, 75). Because this is usually controlled by administration of mineralocorticoid and sodium, it has been suggested that renal responsiveness to mineralocorticoid may be impaired in CDDP-treated patients (59). Although we did not test the mineralocorticoid receptor, in light of our findings we would explain these clinical observations by an effect of CDDP on Hsp90, which leads to reduced function of the mineralocorticoid receptor. Further insight into the molecular mechanisms of CDDP will allow improvements in its important clinical usage.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfection
Human neuroblastoma SK-N-MC cells (American Type Culture Collection, Manassas, VA; no. HTB-10) were kindly provided by the laboratory of C. Behl (Max Planck Institute of Psychiatry, Munich, Germany). They were cultured in DMEM supplemented with 10% fetal calf serum (FCS), 36 mg/liter sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 0.25 µg/ml amphotericin (all from Life Technologies, Inc.), and 4.5 g/liter glucose at 37 C and 10% CO2.

Two days before transfection, cells were seeded into medium containing 10% charcoal-stripped, steroid-free FCS. Dextran T-70 (Pharmacia, Uppsala, Sweden) was used for charcoal-stripping of FCS (76). Jurkat (J32) cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin.

SK-N-MC cells were harvested at about 60–80% confluency and about 0.5 to 1 x 107 cells were resuspended in 400 µl of electroporation buffer [50 mM K2HPO4, 20 mM KAc (pH 7.35)]. A 1.5-µg steroid-responsive luciferase reporter plasmid MTV-Luc (77), 2.5 µg simian virus 40 promoter-driven ß-galactosidase expression vector pCH110 (Pharmacia LKB, Freiburg, Germany), and 0.75 µg pRK7GR that expresses human GR (78) from the cytomegalovirus promoter of the vector pRK7 (79) were added and transfection was performed using an electroporation system (Biotechnologies & Experimental Research, San Diego, CA) after determination of the optimal electrical field strength (80). Electroporated cells were replated and cultured for 16 h in fresh medium (containing 10% steroid-free FCS), supplemented with cortisol, dihydrotestosterone, cisplatin, transplatin, geldanamycin, and cyclophosphamide (purchased from Sigma Chemical Co., Deisenhofen, Germany) in the combinations and concentrations indicated in the text and figure legends. Cells without drug were supplemented with the solvent of the respective drug, reference cells without cortisol and drug were supplemented with the solvents of cortisol (i.e. ethanol) and the drug.

Luciferase and ß-Galactosidase Assay
Luciferase and ß-galactosidase assays were as described before (37). Briefly, cells were scraped from the plate in 1 ml of lysis buffer [0.1 M KHPO4 (pH 7.8) and 1 mM dithiothreitol] and cytosolic extracts were made by three freeze and thaw cycles and subsequent centrifugation. Fifty microliters of each supernatant (corresponding to ~1–2 x 105 cells) were transferred to a 96-well plate. One hundred fifty microliters of 33 mM KHPO4 (pH 7.8), 1.7 mM ATP, 3.3 mM MgCl2, and 13 mM Luciferin (Roche Biochemicals, Mannheim, Germany) were added to each sample by the injector of an automatic luminometer (Luminat LB 96, Wallac GmbH, Freiburg, Germany) and light emission was measured for 10 sec. To correct for variations in transfection efficiencies, values of the luciferase assay were normalized using ß-galactosidase activities that were measured as follows: 50 µl of cell extract were added to 100 µl galactosidase buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, and 50 mM ß-mercaptoethanol) on a 96-well plate. 20 µl of 2 mg/ml ONPG was added and the reaction was incubated at 37 C. After 10–30 min, absorption was measured at 405 nm in a multiphotometer (Dynatech MR5000, Billingshurst, West Sussex, UK). Correcting the luciferase activities by the ß-galactosidase activities was important, because a moderate reduction of the general transcription was observed in some experiments at the highest concentrations of cyclophosphamide and CDDP. We also found that fewer cells were attached at the highest concentrations of CDDP and cyclophosphamide. In another control experiment, we found that CDDP did not change the luciferase activity when added to cell extracts before measurement (not shown).

HSF1 Response
For analysis of HSF1-dependent transcription, SK-N-MC cells were cotransfected with 3 µg of an HSF1-driven luciferase reporter plasmid (HSE-Luc) and 3 µg of the ß-galactosidase control plasmid. Cells were incubated with or without the indicated amounts of CDDP or GA and harvested after 16 h for analysis of luciferase and ß-galactosidase activities as described above.

Hormone Binding of GR in Vivo and in Vitro
To determine hormone binding of GR in vivo, SK-N-MC cells were seeded and transfected with pRK7GR as described above. Twenty-four hours after transfection, cells were incubated with fresh medium containing 10 nM [3H]-cortisol (Amersham, Braunschweig, Germany; specific activity was 62 Ci/mmol) alone or with CDDP at the concentrations indicated. To determine nonspecific hormone binding, a 1000-fold excess of unlabeled cortisol was added in parallel samples. After 1 h, cell plates were put on ice, cells were washed three times with ice-cold PBS, and cells were scraped from the plate in 500 µl of PBS, collected by centrifugation at 2 C and resuspended in 100 µl of PBS. Ninety microliters were used for scintillation counting (Beckman LH 6500, Munich, Germany), the remaining was used to determine the cell number. Counts were divided by the cell number, and nonspecific binding was subtracted from each value.

To determine hormone binding in vitro, SK-N-MC cells were seeded out and transfected with pRK7GR and, after 24 h, treated with CDDP for 1 h as described above. Cells were lysed with RIPA buffer [10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% (vol/vol) deoxycholate, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) sodium dodecyl sulfate, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 mM aprotinin] at 4 C for 30 min. After centrifugation, residual CDDP which may be present was removed by gel filtration over biospin 6 columns (Bio-Rad, Hercules, CA), which had been preequilibrated with hormone binding buffer [5 mM Tris/HCl (pH 7.4), 1 mM EDTA, 10 mM Na2MoO6x2H2O]. At this time, the extracts were divided into aliquots.

To test hormone binding, 10-µl aliquots were either incubated (1 h, 4 C) with 100 nM radiolabeled cortisol alone, or in combination with 1000x excess (100 µM) unlabeled cortisol to determine nonspecific binding. Bound and free steroids were separated by gel filtration by using Sephadex LH-20 (Pharmacia). Radioactivity was measured in a liquid scintillation counter (Beckman LH 6500) and protein concentration was determined by the Lowry method (81) to normalize the data.

To reverse the inhibition by cisplatin, rabbit reticulocyte lysate (Green Hectares, Oregon, WI) was added (5% of the total volume) to aliquots, along with 2 mM ATP and 6 mM Mg2+ and incubated for 10 min at room temperature. Hormone binding was determined as described above. As a control, hormone binding of reticulocyte lysate alone was also determined. To inhibit Hsp90 in the reticulocyte lysate, 1 µM of the Hsp90 inhibitor geldanamycin was added to the lysate, excess was removed by gel filtration (biospin 6) before adding the lysate, together with 2 mM ATP and 6 mM Mg2+, to an aliquot of extract from CDDP-treated cells. Again, hormone binding was determined as described above.

Immunoprecipitation and Western analysis
For immunoprecipitation of Hsp90, SK-N-MC cells were transfected with 0.75 µg pRK7GR as described above and solubilized in 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 1% (vol/vol) deoxycholate, 1% (vol/vol) Triton X-100, 40 mM NaF, 1 mM Na3VO4, 0.5 mM PMSF, and 0.5 mM aprotinin. The extract was incubated for 30 min on ice. Lysates were incubated with the Hsp90-specific antibodies and protein A Sepharose (Pharmacia) for 2 h at room temperature. Immunoblot detection of GR and Hsp90 in the immunoprecipitates, as well as of proteins in total cell lysates (100 µg), was performed after SDS-PAGE and electrophoretic transfer of proteins to 0.2-µm nitrocellulose filter (Schleicher & Schuell, Keene, NH). The filters were blocked by 5% nonfat milk in Tris-buffered saline/Tween buffer, specific primary antibodies were added and agitated for 1 h at room temperature. GR and Hsp90 were detected with rabbit polyclonal antibodies (H-300, H-114, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Src1 with Mab 327, and raf1 with H-71 (Santa Cruz Biotechnology, Inc.). Washing was three times for 5 min in Tris-buffered saline/Tween buffer. Detection was achieved using appropriate peroxidase-conjugated secondary antibodies and enhanced chemiluminescent (ECL) substrate (Amersham Biosciences, Freiburg, Germany).

Activation of lck and raf
Determination of lck and raf activation was performed essentially as described (48). Jurkat T-cells at a density of 2 x 106/ml were treated with the indicated additions for 16 h. In the last 4 h, serum was deprived to decrease the basal level of growth signals. After pretreatment, T cells were stimulated with 5 µg/ml anti-CD3 antibody (OKT3, gift of Márta Szamel) for 20 min. Cells were washed twice with ice-cold PBS, then lysed using 25 mM Tris-HCl, 100 mM NaCl,1% Brij98, 4 mM EDTA, 1 mM dithiothreitol, 1 mM sodium-vanadate, 10 mM sodium-fluoride, 10 mM ß-glycerophosphate, 1 mM PMSF, 5 µg/ml leupeptin, 0.7 µg/ml pepstatin A (pH 7.4), for 30 min. at 4 C. After centrifugation (14,000 x g, 10 min), 30 µg protein of the supernatant were loaded per well on a 7.5% SDS-PAGE, transferred to nitrocellulose, then blots were probed with anti-raf-1 and anti-lck antibodies (kind gift of Attila Steták).

Binding of Hsp90 to the Ligand Binding Domain of GR in Vitro
The procedure was adapted from Young and Hartl (52). Briefly, Hsp90 was labeled using an in vitro transcription and translation system (Promega). Buffer was changed to buffer B [100 mM KOAc, 20 mM HEPES-KOH (pH 7.5), 5% glycerol] using biospin 6 columns (Bio-Rad). Incubation with or without CDDP was for 30 min, followed by another gel filtration to remove excess CDDP. Binding reaction was with myc-tagged ligand binding domain of GR, which had been bound to anti-myc antibodies on Sepharose beads (52), together with reticulocyte lysate, ATP and Mg2+ for 10 min. Unbound material was removed, beads were washed twice with buffer B and proteins were eluted with high salt buffer [20 mM Tris-Cl (pH 7.5), 500 mM NaCl, and 1 mM EDTA]. After gel electrophoresis and autoradiography, the intensity of Hsp90 bands was determined using Scion Image (Scion Corp., Frederick, MD).


    ACKNOWLEDGMENTS
 
We thank Christian Behl (University Mainz, Germany) for kindly providing SK-N-MC cells, Constanze Winklhofer (Max Planck Institute of Biochemistry, Martinsried, Germany) for the HSF1 reporter plasmid [originally a kind gift from Richard Voellmy (University of Miami, FL) (82–84)], Dietmar Spengler (Max Planck Institute of Psychiatry, Munich, Germany) for the GR- and AR-expressing plasmids, Márta Szamel (Medical School Hannover, Germany) for the OKT3 antibody, Attila Steták (Semmelweis University Budapest, Hungary) for anti-lck and anti-raf antibodies, Andrea Jarzabek (Max Planck Institute of Psychiatry) and Katalin Mihály (Semmelweis University) for excellent technical assistance, Dale Milfay (Max Planck Institute of Psychiatry) for critically reading the manuscript, and Péter Csermely and Tamás Schnaider (both Semmelweis University) for helpful discussions.


    FOOTNOTES
 
Abbreviations: AR, Androgen receptor; CDDP, cisplatin cis-diamminedichloroplatinum (II); FCS, fetal calf serum; GA, geldanamycin; GR, glucocorticoid receptor; HA, herbimycin A; HSF1, heat shock factor 1; Hsp90, heat shock protein; PMSF, phenylmethylsulfonyl fluoride; RAD, radicicol; RL, reticulocyte lysate.

Received for publication April 16, 2003. Accepted for publication July 10, 2003.


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 RESULTS
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 MATERIALS AND METHODS
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