• topoisomerase II;
  • Hsp90;
  • protein–protein interactions;
  • chemotherapy


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The modulation of DNA topology by topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and has thus become a highly attractive target for chemotherapeutic drugs. However, these drugs are highly toxic, and so new approaches are required. One such strategy is to target topoisomerase II-interacting proteins. Here we report the identification of potential topoisomerase II-associated proteins using immunoprecipitation, followed by 1-D and 2-D gel electrophoresis and MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIα-associated proteins by coimmunoprecipitation and Western blot. Six of the interacting proteins were cellular chaperones, including 3 members of the heat shock protein-90 (Hsp90) family, and so the effect of Hsp90 modulation on the antitumor activity of topoisomerase II drugs was tested using the sulforhodamine B assay, clonogenic assays and a xenograft model. The Hsp90 inhibitors geldanamycin, 17-AAG (17-allylamino-17-demethoxygeldanamycin) and radicicol significantly enhanced the activity of the topoisomerase II poisons etoposide and mitoxantrone in vitro and in vivo. Thus, our method of identifying topoisomerase II-interacting proteins appears to be effective, and at least 1 novel topoisomerase IIα-associated protein, Hsp90, may represent a valid drug target in the context of topoisomerase II-directed chemotherapy. © 2005 Wiley-Liss, Inc.

Topoisomerase II is an essential nuclear enzyme that modulates DNA topology by generating double stranded breaks, allowing the passage of 1 double strand of DNA through another via an ATP-dependent mechanism.1, 2 This enzyme is a component of DNA transcription and replication machinery, required for chromosomal segregation and maintenance of the nuclear scaffold.2 Recently, topoisomerase II has also been shown to be required for RNA polymerase II transcription.3 In humans, 2 topoisomerase II isoforms have been identified, topoisomerase IIα4 and β.5 The 2 isoforms differ in their cell cycle expression, nuclear localization and tissue specific expression.6 As type II topoisomerases play such an important role within dividing cells, they are highly attractive targets for chemotherapeutic agents, especially in human cancers.

Topoisomerase II can be inhibited via one of two mechanisms: Topoisomerase II poisons act by enhancing DNA cleavage and inhibiting DNA ligation, thus increasing the level of stabilized enzyme–DNA cleavage intermediate complexes. Poisons take advantage of the catalytic mechanism by changing the enzymatic conformation and converting it to a cellular toxin.7 Examples are etoposide (VP16) and mitoxantrone. Catalytic inhibitors, however, target other stages of the topoisomerase II catalytic cycle. The bisdioxopiperazines, e.g. ICRF-159, “lock” the ATP-operated clamp of the enzyme, causing death by deprivation of the essential activity of topoisomerase II rather than by DNA damage.8, 9 By contrast, the catalytic inhibitor merbarone prevents topoisomerase II from cleaving a DNA duplex, thus preventing the normal strand passage activity of the enzyme.9, 10

Proteins shown to have direct physical interactions with topoisomerase II represent potential chemotherapeutic targets. Topoisomerase II function has previously been demonstrated to be modulated by a number of interacting proteins: Topoisomerase II activity has been shown to increase following phosphorylation with casein kinase II11 and protein kinase C (PKC).12 Recent studies have shown that p53 interacts with topoisomerase II,13 and it has also been proposed that p53 regulates the level of topoisomerase IIα throughout the cell cycle by forming complexes that lead to the stimulation of catalytic activity,14 or alternatively by repressing the expression of the topoisomerase IIα gene.15 The topoisomerase II-sgs1 protein interaction functions to aid chromosomal segregation and it has been shown that sgs1 is required for genomic stability in S. cerevisae16 and humans.17

We hypothesized that it would be possible to enhance the cell-killing properties of current agents acting against topoisomerase II by targeting proteins that modulate topoisomerase II activity. To address this, we have used immunoprecipitation, 1-D and 2-D gel electrophoresis and subsequent MALDI-TOF mass spectrometry to identify a number of proteins that are associated with topoisomerase II. One of these, heat shock protein-90 (Hsp90), is itself a bona fide cancer target that can be modulated pharmacologically, and so was the subject of further studies. The interaction between Hsp90 and its associated “client” proteins can be destabilized with inhibitors such as geldanamycin.18 Using several structurally unrelated Hsp90 inhibitors, in combination with topoisomerase II poisons (etoposide and mitoxantrone), we observed a greater than additive cell-killing effect both in vitro and in vivo, suggesting that targeting topoisomerase II-interacting proteins may represent a new chemotherapeutic approach.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines

The isogenic pair of human colon cancer cell lines HCT116 wild type (p53 +/+) and p53 knockout (p53−/−) was a kind gift from Prof. B. Vogelstein, The John Hopkins Medical Institutions (Baltimore, MD, USA), and was maintained in McCoys 5A medium (Sigma–Aldrich Company, Poole, UK). The human colon adenocarcinoma cell line HT29-5, which has mutated p53, the human breast adenocarcinoma cell line MCF7 and the human adenosquamous cell carcinoma cell line NCI-H125 were kind gifts from Prof. H. M. Warenius, Department of Medicine, University of Liverpool. The HT29-5 and MCF-7 cell lines were maintained in DMEM (Sigma–Aldrich). The NCI-H125 was maintained in RPMI-1640 media (Sigma–Aldrich). All media were supplemented with 10% heat-inactivated (55°C for 30 min) fetal calf serum (FCS; Invitrogen), 2 mM L-glutamine (Sigma–Aldrich) and 50 units/ml penicillin/50 μg/ml streptomycin (Sigma–Aldrich), and cells were grown at 37°C in a 5% CO2 enriched humidified environment.


Athymic (nu/nu) female mice (6–8-week old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). The mice were maintained in ventilated caging with a 12 hr light/12 hr dark photoperiod. Irradiated, pelleted food (Harlan Teklad) and autoclaved water were provided ad libitum. Experiments were carried out in accordance with the Institute for Laboratory Animal Research (ILAR; NIH, Bethesda, MD) Guide for the Care and Use of Laboratory Animals.


Vepesid® (etoposide) was obtained from Bristol-Myers Squibb Pharmaceuticals (Hounslow, UK) and Novantrone® (mitoxantrone) from Cyanamid (Gosport, Hampshire, UK). 17-allylamino-17-demethoxygeldanamycin (17-AAG) and geldanamycin were kind gifts from Dr. R. J. Schultz, Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute (Rockville, MD). Geldanamycin was also obtained from Tocris Cookson (Avonmouth, UK), and radicicol was obtained from Sigma–Aldrich. 17-AAG was formulated for in vivo use by dissolving the solid at 2.5 mg/ml in a PhosphoLipon–Soy oil–Miglyol emulsion.


Rabbit anti-topoisomerase IIα, rabbit anti-Hsp90β, rabbit anti-Hsp90α, rat anti-Grp94, mouse anti-tubulin α, mouse anti-tubulin β and mouse anti-(pan)actin antibodies were all purchased from LabVision (UK) (Newmarket, UK). Mouse anti-Sti1, mouse anti-annexin I, and mouse anti-annexin II antibodies were obtained from BD Biosciences (Cowley, UK). The rabbit anti-Hsp73, mouse anti-SRp20, rabbit anti-eEF2 and the mouse anti-α-actinin-4 antibodies were obtained from Europa Bioproducts (Cambridge, UK), Cambridge Bioscience (Cambridge, UK), New England Biolabs (Hitchen, Hertfordshire, UK) and Chemicon International (Temecula, CA), respectively. The mouse anti-PDI and mouse anti-14-3-3ζ antibodies were purchased from Bioquote (York, UK). The goat anti-ezrin, rabbit anti-hnRNP B1 and rabbit anti-topoisomerase IIβ (IHIC2) antibodies were kind gifts from Prof. A. Varro (Dept. of Physiology, University of Liverpool, UK), Prof. J. Fields (The Roy Castle Lung Cancer Foundation, Liverpool, UK) and Prof. I. Hickson (Weatherall Institute of Molecular Medicine, Oxford, UK), respectively.


HCT116 whole cell extracts were prepared by lysing cells in RIPA buffer (425 mM NaCl, 1% (v/v) IGEPAL CA-630, 1 mM EDTA, 5% (w/v) deoxycholate, 50 mM Tris (pH 8.0), 0.1% (w/v) SDS, 10 mM sodium fluoride, 0.5 mM sodium orthovanadate) containing the protease inhibitor cocktail III (Merck Biosciences, Nottingham, UK). Cells were incubated on ice for 30 min and cleared by sonication and centrifugation at 13,000g for 30 min at 4°C. A 30 μl aliquot of each cell lysate was removed to load onto SDS-PAGE gels alongside the immunoprecipitations. The remaining cell lysate was preabsorbed with 25 μl 10% (w/v) protein A sepharose (Amersham Biosciences UK, Chalfont St. Giles, Buckinghamshire, UK) in PBS for 1 hr at 4°C with gentle agitation. Samples were then pelleted by centrifugation at 13,000g for 1 min at 4°C and supernatant was removed to a fresh microfuge tube. Antibodies, 2.5 μg, were added to protein extracts and incubated at 4°C overnight, followed by incubation with 50 μl 10% (w/v) protein A sepharose in PBS for 1 hr at 4°C with gentle agitation. Samples were pelleted by centrifugation at 13,000g for 1 min at 4°C and supernatant was discarded. Immunoprecipitated samples were washed with 200 μl RIPA buffer and again pelleted by centrifugation at 13,000g for 1 min at 4°C and supernatant was discarded. Immunoprecipitated samples were resuspended in either 5× SDS-PAGE loading buffer (125 mM Tris-HCl (pH 6.8), 25% (v/v) glycerol, 2.5% (w/v) SDS, 0.025% bromophenol blue, 5% (v/v) β-mercaptoethanol) or 2-DE lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 1% (w/v) DTT) for subsequent 1-D or 2-D SDS-polyacrylamide gel electrophoresis, respectively.

2-DE of immunoprecipitated samples

Immunoprecipitated samples were resuspended in 125 μl 2-DE lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 1% (w/v) DTT) and incubated overnight with nonlinear Immobiline DryStrips (7 cm; pH 3–10NL) in a reswelling chamber. The samples were subjected to IEF for 3 hr at a constant temperature of 20°C to achieve a total of 8,000 Vh. The IPG strips were then incubated with equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.025% (w/v) bromophenol blue containing 1% (w/v) DTT) for 15 min, followed by incubation in the same buffer with the DTT replaced by 2.5% (w/v) iodoacetamide for a further 15 min. The strips were applied to the surface of 12% (w/v) SDS-PAGE gels and sealed with agarose. The samples were subjected to electrophoresis at 20 mA and 12°C for ∼1½ hr. The gels were stained with colloidal Coomassie blue.


The proteins brought down by immunoprecipitation and whole cell extracts were separated by 7.5, 10 or 15% SDS-PAGE under reducing conditions and blotted onto Protran® nitrocellulose membrane (Schleider and Schuell UK, London, UK). Blots were probed with appropriate primary antibodies and the secondary antibodies conjugated with horse radish peroxidase (DAKOCytomation, Ely, UK). Detection was by Supersignal® West Dura Extended Substrate (Perbio Science UK, Tattenhall, Cheshire, UK) and imaging was done by using a Fluor-S™ bioimager (Bio-Rad Laboratories, Hemel Hempstead, UK).

MALDI-TOF mass spectrometry

Protein bands were excised from Colloidal blue (Sigma–Aldrich) stained 1-D and 2-D gels and placed into 0.5 ml microfuge tubes prerinsed with acetonitrile and methanol. Gel pieces were then washed for 15 min with gentle agitation in 50% (v/v) acetonitrile, 25 mM ammonium bicarbonate (pH 7.8), dried in a SpeedVac for 30 min and allowed to rehydrate overnight in 10 ng/μl trypsin (Promega UK, Southampton, UK), 25 mM ammonium bicarbonate solution at 37°C. Peptides were then extracted by the addition of 30% (v/v) acetonitrile, 0.1% (v/v) trifluroacetic acid (TFA) and a brief vortexing. About 0.5 μl of sample was placed onto a 96 position target and mixed with 0.5 μl of α-cyano-4-hydroxycinnamic acid (HCCA) matrix in 50% (v/v) acetonitrile/50% (v/v) ethanol/0.001% (v/v) TFA containing 0.5 fmol adrenocorticotropic hormone (ACTH) as an internal standard. Peptide mass fingerprints were obtained on a MALDI mass spectrometer (M@LDI, Micromass, Manchester, UK) and searched against a Swiss-Prot/Trembl protein database using ProteinLynx 3.4 (Micromass). The resultant mass lists were then searched against SWISS-PROT/TrEMBL, a nonredundant protein database, using ProteinLynx v3.4 software. MOWSE scores of greater than 1 × 105 were regarded as sufficient for identification, but scores of 1 × 104 were also considered if the prime identification was human and there were multiple hits against that protein (for more information see and

Growth inhibition assay

For growth inhibition studies we used the sulforhodamine B assay as described previously.19 In brief, cells were seeded into 96-welled microtiter plates, allowed to adhere overnight and then drugs were added in 6 replicate wells for a period of up to 7 days. At fixed time points (usually daily) cells were fixed with 3:1 methanol–acetic acid and stained with 0.4% (w/v) sulforhodamine B (SRB). SRB OD570 staining measures net protein and was used to determine growth inhibition. The mean OD of the treated cells was plotted against time. Six replicates per assay were used to analyze intraexperimental error. The error bars within the graphs represent the standard error.

To test for possible combination synergy, the starting drug concentration was chosen as the highest concentration of the drug (single drug) that had no inhibitory effect on cell growth. These were then used in combination SRB assays, and concentrations of the 2 drugs were decreased until no combination inhibitory effect was observed.

Isobolar analysis

The isobole method provides a simple and convenient assessment of the interaction index. The isobole method is the only generally applicable approach that does not require any assumptions about the shape of dose–response curves. It is also frequently used in an analysis wherein the drug combinations are in fixed ratio proportions, as discussed by Tallarida,21, 22 using the formula [drug A in combination]/[drug A alone] + [drug B in combination]/[drug B alone] = γ. If γ = 1 the interaction is additive, if γ < 1 it is subadditive and if γ > 1 it is super additive (synergistic).

Clonogenic assay

Cells were seeded at 1,000 cells per well in 6-welled plates and allowed to adhere overnight. The cells were then exposed to the drugs for 1 hr and incubated for 10 days to allow colony formation. Colonies were fixed in 70% methanol and stained with 0.2% (w/v) crystal violet/70% ethanol. The number of colonies formed of >50 cells each was counted. Experiments were performed on 3 separate occasions and each one contained 6 replicates. The clonogenic assay, survival rates were calculated as (mean plating efficiency of treated cells/mean plating efficiency of control cells) × 100%, where the plating efficiency is the number of colonies divided by the number of inoculated cells. Points represent the average of 6 replicates. The error bars within the graphs represent the standard error.

Xenograft model

HCT116 cells (200 μl of 2.5 × 107 cells/ml) were injected subcutaneously into the right flank of athymic mice. When the mean tumor volume reached an average of 140 mm3 as measured using callipers and the formula V = π/6(l × w2), animals were randomized into treatment groups of 6 animals per group. Etoposide (VP16) was administered intraperitoneally (i.p.) at doses of 20 and 30 mg/kg weekly. 17-AAG was formulated for in vivo use and administered i.p at doses of 40 and 80 mg/kg Monday through Friday. (The MTD is ∼100 mg/kg/day on a 5 days per week schedule for 17-AAG produced by Conforma.)

Xenograft data analysis

Mice were monitored until tumor volumes in the control group reached ∼1,000 mm3 and were killed by CO2 euthanasia. The mean tumor volumes of each group were calculated. The change in mean treated tumor volume was divided by the change in mean control tumor volume and multiplied by 100 to give the percent tumor growth for each group. The change in mean treated tumor volume was divided by the change in mean control tumor volume, subtracted from 1 and multiplied by 100 to give the percent tumor growth inhibition for each group.

Statistical analysis

Statistical analysis was performed using the standard t-test and using GraphPad Prism© Software.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Identification of topoisomerase II-associated proteins

HCT116 WT cells were lysed and proteins associated with topoisomerase IIα were isolated by immunoprecipitation using anti-human topoisomerase IIα antibody. The precipitated proteins were then separated by 1-D and 2-D gels (for an example, see Fig. 1a) SDS-PAGE and the gel stained with Coomassie brilliant blue. Bands or spots were excised, digested with trypsin and analyzed by MALDI-TOF mass spectrometry. A total of 23 proteins were identified and, of these, 17 were further validated as topoisomerase IIα associated proteins by the use of further coimmunoprecipitations and counter coimmunoprecipitations (Figs. 1b, an example of confirmation of an association; 1c, an example of a control, where the preimmune serum was available and 1d, an example of a negative result). See Table I for the identifications. The proteins of particular interest are heat shock protein 90 (Hsp90)α, Hsp90β, Hop (Sti1/p60), α-tubulin and β-tubulin.

thumbnail image

Figure 1. Examples of the gels and Western blots used in the identification and validation of potential topoisomerase IIα associated proteins. (a) An example of a 2-D SDS-polyacrylamide gel and the potential topoisomerase IIα associated protein identifications. (b) Western blots showing association of Hsp90 with topoisomerase IIα. (c) Western blots of control immunoprecipitations performed using preimmune serum. (d) A western blot as a negative example showing that SRp20 does not associate with topoisomerase IIα. TE, total cell extracts prior to immunoprecipitation (IP), WB, western blot antibody.

Download figure to PowerPoint

Table I. Identification and Validation of Potential Topoisomerase IIα Associated Proteins
ProteinAccession no.MOWSE scoreLIKELIHOODNo. of peptidesCoverage (%)MW (Da)TopoIIα IPConverse IP
  1. Proteins were identified from 1-D and 2-D SDS-polyacrylamide gels following MALDI-TOF MS analysis. The confirmation of protein association by immunoprecipitation/counter immunoprecipitation and Western blot analysis: WT, p53 wild type; KO, p53 knockout; +, band present in the immunoprecipitation (IP); −, band absent in the IP; W, band was present but weak in the IP. Lit., association of topoisomerase IIα with this protein is already documented in the literature20; NT, no antibody was commercially available against that particular protein.

Hsp90αP079002.1558849 e+0148.25 e+0042229.8284,543++++
Hsp90βP082381.4467661 e+0146.13 e+0042129.0583,163++++
Elongation factor 2 (eEF2)P136393.6301245 e+0092.35 e+0041523.0895,338++W
α-Actinin 4O437073.5849645 e+0094.01 e+0041724.5510,2269++W+
Hsp73 (Hsc70/Hsp71)P111421.6543041 e+0093.36 e+0041426.7870,898++W+
EzrinP153112.2371216 e+0084.04 e+0041827.1869,268W++
hnRNP A2/B1P226261.8623996 e+0082.05 e+0041647.3137,430WW++
Grp94P146255.8987068 e+0072.35 e+0041418.9392,469++++
Hop (Sti1/p60)P319484.7955058 e+0075.54 e+0041828.7362,639++++
Grp78P110212.5492253 e+0074.02 e+004921.972,116Lit.Lit.Lit.Lit.
β-ActinP025702.3644429 e+0074.25 e+0041136.841,737++W+
α-EnolaseP067338.7995911 e+0063.52 e+0041029.147,038NTNTNTNT
hnRNP A1P096518.6209546 e+0065.05 e+0041042.0438,715NTNTNTNT
β-TubulinP052185.0578335 e+0062.40 e+0041123.4249,671++W+
Annexin IP040834.7146864 e+0064.35 e+004941.7438,584++W+
14-3-3-protein ζ/δP293124.3356526 e+0063.02 e+004941.2227,745+++
Annexin IIP073553.9157191 e+0064.13 e+0041031.6638,473++W+
Thioredoxin peroxidase 2Q068304.8184267 e+0051.78 e+0041034.1722,110NTNTNTNT
α-TubulinP052092.4004109 e+0058.66 e+003722.8450,136++++
C-1-tetrahydrofolate synthaseP115861.1626628 e+0051.32 e+004912.7410,1428NTNTNTNT
Protein disulfide isomerase precursor (PDI)P072373.3805261 e+0041.22 e+004915.7557,116++WW
TransketolaseP294011.7350362 e+004136 e+004713.867,878NTNTNTNT
SRp20P231525.2931889 e+0041.90 e+004634.1519,330

It is known that p53 interacts with topoisomerase IIα.13 To determine whether the presence of p53 modulates the observed interaction and to further validate our topoisomerase II-interacting protein screen, we performed the counter coimmunoprecipitations using whole cell extract from both WT and p53 knockout genotypes of the HCT116 cell line with antibodies specific to Hsp90β and topoisomerase IIα. The interactions observed were found to be independent of the p53 status of the cell line.

Combined use of topoisomerase II poisons with Hsp90 inhibitors causes synergistic growth inhibition

To investigate the importance of this interaction, we investigated the effect of disrupting the association between these 2 proteins on the growth of WT HCT116 cells. We treated the cells with geldanamycin (GA), an Hsp90 inhibitor known to bind to the ATP-binding site and act as a competitive inhibitor of the Hsp90 ATPase activity. We tested the sensitivity of cells, which had been treated with Hsp90 inhibitors, to agents that target topoisomerase II. Cells were initially treated with a broad range of concentrations of etoposide (VP16), a topoisomerase II poison, and geldanamycin as single agents to determine the concentrations at which greater than 80% growth inhibition occurred. Using the SRB assay, these concentrations were found to be 2.5 μM etoposide (Fig. 2a) and 350 nM geldanamycin (Fig. 2b). We subsequently decreased the concentrations of the 2 drugs when they were used in combination in order to determine the effect of combining the 2 drugs. When used in combination, we were able to lower the concentrations of etoposide and geldanamycin to 0.5 μM and 125 nM, respectively, and still obtain a greater than 80% cell proliferation inhibition (Fig. 2c). This greater than additive effect was also observed for the p53−/− HCT116 cells (results not shown). At these concentrations these drugs have little or no effect when used in isolation. To determine whether this was a truly synergistic drug combination, the isobolar relationship was calculated and found to be 0.58, indicating synergy.

thumbnail image

Figure 2. Growth inhibition assays for HCT116 p53 +/+ cells. (a) Single VP16 dose range, (b) Single geldanamycin (GA) dose range, (c) VP16 and GA combination, (d) VP16 and 17-AAG combination, (e) VP16 and radicicol (RD) combination and (f) Mitoxantrone (MX) and GA combination. Points represent the average of 6 replicates; bars, ±SE.

Download figure to PowerPoint

We further investigated this phenomenon by combining etoposide with other Hsp90 inhibitors: 17-AAG, a derivative of geldanamycin, and radicicol, which is chemically distinct from geldanamycin and its derivatives. We also investigated the effects of combining geldanamycin with another topoisomerase II poison, mitoxantrone. As with etoposide and geldanamycin, we first determined the concentrations at which 80% growth inhibition was observed for each of these compounds. These were found to be 300 nM 17-AAG, 400 nM radicicol and 50 nM mitoxantrone (data not shown). To allow a direct comparison between the different inhibitors acting against the same protein, the concentrations of etoposide and geldanamycin remained constant (i.e. 0.5 μM etoposide and 125 nM geldanamycin). Figures 2d, 2e and 2f demonstrate that the same synergistic proliferation inhibition effect was also observed for combinations of 0.5 μM etoposide and 125 nM 17-AAG, 0.5 μM etoposide and 100 nM radicicol, and 10 nM mitoxantrone and 125 nM geldanamycin, respectively. The isobolar relationships were calculated and found to be 0.58 (mitoxantrone and geldanamycin), 0.62 (etoposide and 17-AAG) and 0.45 (etoposide and radicicol), all indicating synergy. This synergistic effect was also observed for the p53 −/− HCT116 cells in all cases (results not shown).

Similar results for the use of the combination of drugs were observed with the HT29-5, NCI-H125, and MCF-7 cell lines (Fig. 3), for example, isobolar relationship scores being 0.37 (etoposide and geldanamycin), 0.38 (mitoxantrone and geldanamycin) and 0.35 etoposide and radicicol) with HT29-5; 0.78 (etoposide and geldanamycin) and 0.39 (etoposide and radicicol) with NCI-H125; 0.36 (etoposide and geldanamycin) with MCF-7, once again demonstrating clear synergy.

thumbnail image

Figure 3. Growth inhibition assays for other cell lines. (a) Mitoxantrone (MX) and GA combination on HT29-5 cells, (b) VP16 and radicicol (RD) combination on NCI-H125 cells and (c) VP16 and GA combination on MCF-7 cells. Points represent the average of 6 replicates; bars, ± SE.

Download figure to PowerPoint

Combined use of topoisomerase II poisons with Hsp90 inhibitors causes synergistic cell killing

To assess the effect of the drugs upon cell survival, both p53 genotypes of the HCT116 cells were treated with single drugs or drug combinations for 1 hr. The media was then replaced and incubated for 10 days. The number of surviving colonies were counted following treatment and then calculated as a percentage of the number of surviving colonies in the control samples. Both drugs were used in increasing concentrations, but the ratio between the concentrations of the 2 drugs remained constant (the same ratio as determined in the SRB assay), a strategy previously proposed for rationally reducing the number of drug combinations to be tested.21 This strategy was also applied to the cell survival assays when radicicol and mitoxantrone were used. Fig. 4a, log scale for drug concentration, demonstrates that geldanamycin causes somewhat more cell death than does etoposide when used alone. However, when both were used in combination, the amount of cell death was increased at the concentrations tested. From the data obtained we were then able to determine the concentrations of the drugs, alone or in combination, needed to achieve 95% cell killing. For the WT cells these were found to be 26.5 μM etoposide and 1275 nM geldanamycin, and when used in combination these decreased to 3.92 μM etoposide and 980 nM geldanamycin. For the p53−/− cells these were found to be 19.4 μM etoposide and 1325 nM geldanamycin, and when used in combination these decreased to 3.84 μM etoposide and 960 nM geldanamycin. The aforementioned values were used to calculate an isobolar relationship giving the interaction indices, which was found to be 0.92 for both cell lines. Thus, the combination of etoposide and geldanamycin produced a synergistic cell-killing effect.

thumbnail image

Figure 4. The effect of drug combinations at fixed ratios on HCT116 clonogenic survival. (a) VP16 and geldanamycin (GA), (b) VP16 and radicicol and (c) Mitoxantrone (MX) and GA. The drug concentration is shown on a log scale. Points represent the average of 3 independent experiments; bars, ±SE.

Download figure to PowerPoint

Figure 4b shows that etoposide caused more death than did radicicol when used alone, but when used in combination, the amount of cell death was again markedly increased. The concentrations of the drugs needed to achieve 95% cell killing were determined, with the concentrations of etoposide for the WT and p53−/− cells as before (26.5 and 19.4 μM, respectively). The radicicol concentrations for the WT and p53−/− cells were found to be 16.7 and 13.5 μM, respectively. However, when used in combination, the etoposide and radicicol concentrations decreased to 9.95 μM etoposide and 1.99 μM radicicol for the WT cells, and 12.25 μM etoposide and 2.45 μM radicicol for the p53−/− cells. The isobolar relationships were calculated using these values and were found to be 0.5 and 0.81 for the WT and p53−/− cells, respectively. This demonstrates that the combination of etoposide with radicicol produces a synergistic cell-killing effect, which is greater than what is observed with the combination of etoposide and geldanamycin.

Figure 4c demonstrates that geldanamycin caused more cell death than did mitoxantrone when used alone, but again, when used in combination, the amount of cell death markedly increased. The concentrations of the drugs needed to achieve 95% cell killing were determined, with the geldanamycin concentrations as before (1275 and 1325 nM, respectively). The mitoxantrone concentrations for the WT and p53−/− cells were found to be 182 and 169 nM, respectively. However, when used in combination, the mitoxantrone and geldanamycin concentrations decreased to 39 nM mitoxantrone and 487.5 nM geldanamycin for the WT cells, and 42 nM mitoxantrone and 525 nM geldanamycin for the p53−/− cells. Again the isobolar relationships were calculated using these values and were found to be 0.59 and 0.63 for the WT and p53−/− cells, respectively. This demonstrates that the combination of geldanamycin with mitoxantrone produces a synergistic cell-killing effect, which is greater than what is observed with the combination of geldanamycin and etoposide.

Efficacy studies of 17-AGG and etoposide in colon tumor xenografts

In a study to determine whether the combination of a topoisomerase II poison and an Hsp90 inhibitor demonstrates efficacy in an animal xenograft model, we used the clinically relevant agents 17-AAG and etoposide. Two concentrations of 17-AAG (40 and 80 mg/kg) and 2 etoposide concentrations (20 and 30 mg/kg) were used. 17-AAG was administered daily on days 1–5 of a weekly regimen, and etoposide was administered as a single dose weekly. Dosing was initiated when the xenografts had reached a volume of 140 mm3. Figure 5a shows the increase in xenograft volume when the animals are treated with 40 or 80 mg/kg 17-AAG and 20 mg/kg etoposide. Figure 5b shows the increase in xenograft volume when the animals are treated with 40 or 80mg/kg 17-AAG and 30mg/kg etoposide. Tumor volumes were compared with those in animals receiving each agent alone.

thumbnail image

Figure 5. The response of HCT116 xenografts to 17-AAG and VP16 combinations. (a) 20 mg/kg VP16 with combinations of 17-AAG and (b) 30 mg/kg VP16 with combinations of 17-AAG. Points represent the average tumor volume of 6 mice; bars, ±SE.

Download figure to PowerPoint

Treatment of HCT116 tumors with 17-AAG resulted in 44% growth inhibition at 40 mg/kg and 64% growth inhibition at 80 mg/kg. Treatment with VP16 alone at 20 and 30 mg/kg gave 59 and 53% inhibition, respectively. Table II summarizes the results. Adding the treatment of 40 mg/kg 17-AAG to either 20 or 30mg/kg VP16 did not enhance the inhibitory activity. The combination of 40 mg/kg 17-AAG and 20 mg/kg VP16 resulted in 43% inhibition and the combination of 40 mg/kg 17-AAG and 30 mg/kg VP16 resulted in 65% inhibition. However, the combination of 80 mg/kg 17-AAG with 20 or 30 mg/kg VP16 did enhance tumor growth inhibition. The combination of 80 mg/kg 17-AAG with 20 mg/kg VP16 resulted in 87% tumor growth inhibition and the combination of 80 mg/kg 17-AAG with 30 mg/kg resulted in 80% tumor growth inhibition. To assess the relative effects of these drugs, the degree of synergy, we used percentage tumor growth and calculated the isobolar relationship.22 The combination of 80 mg/kg 17-AAG with 20 mg/kg VP16 had an interaction index of 0.68 when compared with the combination of 80 mg/kg 17-AAG with 30 mg/kg, which had an interaction index of 0.98. Toxicity was observed on these regimens, and as may be expected, the highest number of deaths occurs with the higher concentrations of 17-AAG and VP16.

Table II. Effect of 17-AAG and Etoposide on Growth of HCT116 Xenografts in Nude Mice
TreatmentDose (mg/kg)Route, ScheduleFinal tumor volume (mm3; Mean ± SEM1)% TG2% TGI3t-test (p-value)Deaths
  • M–F, Monday through Friday (qdx5 for 3 weeks); M, Monday (given once weekly for 3 weeks).

  • 1

    Mean tumor volume at the initiation of therapy was 140 ± 15.6 mm3.

  • 2

    % TG, % tumor growth [(change in mean treated tumor volume/change in mean control tumor volume)] × 100.

  • 3

    % TGI, % tumor growth inhibition [1 − (change in mean treated tumor volume/change in mean control tumor volume)] × 100.

Control  945 ± 181100  0/6
17-AAG40IP, M–F588 ± 1145644NS0/6
17-AAG80IP, M–F432 ± 7836640.030/6
VP1620IP, M466 ± 6741590.051/6
VP1630IP, M511 ± 6747530.050/6
17-AAG and VP1640 + 20IP, M–F + IP M594 ± 975743NS0/6
17-AAG and VP1680 + 20IP, M–F + IP M243 ± 6613870.011/6
17-AAG and VP1640 + 30IP, M–F + IP M424 ± 6535650.031/6
17-AAG and VP1680 + 30IP, M–F + IP M296 ± 5420800.022/6


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

In this article we report the first demonstration that rational identification of topoisomerase II-interacting proteins as drug targets, using an antibody pull-down strategy, can enhance the efficacy of clinical topoisomerase II poisons. Having identified a topoisomerase II–Hsp90 complex, we then show that combinations of drugs targeting both components of the complex are more potent than either drug alone.

Hsp90 is an essential and ubiquitous molecular chaperone that plays an important physiological role in the folding, activation and assembly of a broad range of “client” proteins.23 There are 2 Hsp90 isoforms, Hsp90α and β,24 and other members of the Hsp90 family include glucose-regulated protein 94 (Grp94) in the endoplasmic reticulum and the mitochondrial Hsp75/tumor necrosis factor receptor associated protein 1 (TRAP1).24 Hsp90 has engendered increasing interest as a target for cancer therapeutics because it is both overexpressed,25 and hyperactivated in most advanced human tumors.26 Inhibitors of Hsp90, such as 17-allylamino-17-demethoxygeldanamycin (17-AAG), which is currently in phase II clinical trials,27 and synthetic purine-based compounds,28 have been shown to selectively bind activated Hsp90 in cells, preferentially kill tumor cells in vitro26 and profoundly inhibit tumor growth in animals at well-tolerated doses.29, 30

The Hsp90 inhibitors used here bind competitively to the ATP-binding site and destabilize the Hsp90–client protein complex, usually resulting in the prompt ubiquitination and degradation of the client in the proteasome.23 However, certain Hsp90-interacting proteins are released in an active form following disruption of their binding to Hsp90. For instance, dissociation of heterocomplexes between Hsp90 and the transcription factor Hsf-1 (by heat or Hsp90-modulating drugs) results in the formation of active Hsf-1 trimers, which are then able to bind DNA and transactivate heat shock genes.31 Hsp90–Hsf-1 complexes do not contain cochaperones such as Hop that are present in conventional Hsp90-client complexes.32 Similarly, several other putative Hsp90 client proteins, including STAT-333 and the kinase LKB-1,34 have been found in unconventional, low Mr complexes with the chaperone. It will be interesting to determine the identity of any additional proteins in the topoisomerase II–Hsp90 complex and their role in regulation of topoisomerase II activity. We observed an increase in the acute effectiveness of topoisomerase II poisons in the presence of Hsp90 inhibitors in vitro-effective cell killing was observed in clonogenic survival assays with 1 hr of exposure to the drug combinations, followed by replacement with fresh media, as shown in Figure 3. If disruption of the interaction between topoisomerase II and Hsp90 immediately induced degradation of topoisomerase II, a decrease in the effectiveness of the topoisomerase II poison would be expected because it is required to form the drug-stabilised cleavable complex. One possible explanation is that following Hsp90 inhibition an active form of enzyme will be released, transiently increasing the formation of toxic cleavable complexes and thus enhancing the efficiency of tumor cell intoxication. Intriguingly, there appears to be further crosstalk between the stress–response and DNA repair pathways in that heat shock has been shown to activate human topoisomerase IIα transcription.35 Alternatively, the synergistic effect observed could be due to independent activities of the drugs as described recently for topoisomerase I, where Hsp90 inhibitor-induced degradation of Chk1 sensitized tumor cells to chemotherapy because of loss of DNA damage check point control.36

We tested the therapeutic implications of the drug-induced dissociation of topoisomerase II and Hsp90 by the combination of topoisomerase II poisons and Hsp90 inhibitors. The potential of this strategy was indicated by the synergistic activity observed against tumor cell proliferation, clonogenic survival and xenograft growth in vivo and was independent of p53 status. The potency of the combination was enhanced in breast, lung and colorectal carcinoma cells, suggesting that the treatment could be applied across a number of different cancer types. The xenograft studies are promising, in that the drug combinations demonstrate a synergistic trend in terms of their effect on tumor growth, but preliminary. In the present study, only a limited range of doses and a single schedule combination were employed-further studies will be required to optimize the dose and the schedule prior to clinical application of this approach.

Two previous studies have investigated the use of Hsp90 inhibitors in combination with doxorubicin.37, 38 Doxorubicin has a number of modes of action, one of which is as a topoisomerase II poison. The main objective of the first study37 was to demonstrate that the sensitivity of the chemotherapeutic agents tested was restored in Bcr-Abl expressing cells following geldanamycin treatment and where doxorubicin was used in combination little synergy was also observed. By contrast, Munster et al. reported that 17-AAG sensitized breast cancer cells to both doxorubicin and taxol, although the latter was highly schedule-dependent.38

In addition to Hsp90, several other interesting topoisomerase IIα-interacting proteins were identified in this study, including Hop, Hsp70 and tubulin (Table I). Hop is a highly abundant stress-inducible protein that forms direct associations with Hsp70 and Hsp90 and has been shown to modulate the activity of both of these proteins as well as act as a cochaperone to aid with protein folding.39 Furthermore, tubulin has also been shown to be an Hsp90-binding protein,40 raising the possibility that all 3 of these apparent interactions with topoisomerase II are indirect, occurring via Hsp90. It is possible that Hsp90 acts as a dual chaperone/bridging protein to maintain intracellular stores of cytoprotective proteins such as Hsf-1, STAT-3 and topoisomerase II immobilized by crosslinking to the tubulin cytoskeleton41 and protected from proteotoxic damage by the chaperone.42 Under conditions of stress (or in the presence of Hsp90 inhibitors), the dynamics of Hsp90 complex formation might then favor dissociation of the complex, releasing active topoisomerase IIα to bind to DNA.30

In conclusion, our work is the first identification of an Hsp90–topoisomerase II complex. This complex can be viewed as a novel, sensitive target for cancer therapy because combination treatment with an Hsp90 inhibitor and a topoisomerase II poison produced synergistic, p53-independent inhibition of tumor growth and survival in vitro and in vivo, paving the way to designing rational combination therapies using Hsp90 inhibitors and topoisomerase II poisons.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We thank Drs. M. Pritchard and Bill Greenhalf for their comments on the manuscript; Prof. B. Vogelstein, The Johns Hopkins Oncology Centre, for the HCT116 cell lines; Prof. H.M. Warenius, Department of Medicine, University of Liverpool, for the HT29-5, NCI-H125 and MCF-7 cell lines; Prof. A. Varro for the goat anti-ezrin antibody; Prof. J. Fields for the rabbit anti-hnRNP B1 antibody; Prof. I. Hickson for the rabbit anti-topoisomerase IIβ (IHIC2) antibody and Dr. R.J. Schultz, Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute (NCI; Rockville, MD, USA), for 17-AAG and geldanamycin.


  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Berger JM, Gamblin SJ, Harrison SC, Wang JC. Structure and mechanism of DNA topoisomerase II. Nature 1996; 379: 22532.
  • 2
    Wang JC. Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 2002; 3: 43040.
  • 3
    Mondal N, Parvin JD. DNA topoisomerase IIα is required for RNA polymerase II transcription on chromatin templates. Nature 2001; 413: 4358.
  • 4
    Tsai-Pflugfelder M, Liu LF, Liu AA, Tewey KM, Whang-Peng J, Knutsen T, Huebner K, Croce CM, Wang JC. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc Natl Acad Sci USA 1988; 85: 717781.
  • 5
    Jenkins JR, Ayton P, Jones T, Davies SL, Simmons DL, Harris AL, Sheer D, Hickson ID. Isolation of cDNA clones encoding the β-isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res 1992; 20: 558792.
  • 6
    Woessner RD, Mattern MR, Mirabelli CK, Johnson RK, Drake FH. Proliferation- and cell cycle-dependent differences in expression of the 170 kilodalton and 180 kilodalton forms of topoisomerase II in NIH- 3T3 cells. Cell Growth Differ 1991; 2: 20914.
  • 7
    Wilstermann AM, Osheroff N. Stabilization of eukaryotic topoisomerase II-DNA cleavage complexes. Curr Top Med Chem 2003; 3: 32138.
  • 8
    Jensen LH, Nitiss KC, Rose A, Dong J, Zhou J, Hu T, Osheroff N, Jensen PB, Sehested M, Nitiss JL. A novel mechanism of cell killing by anti-topoisomerase II bisdioxopiperazines. J Biol Chem 2000; 275: 213746.
  • 9
    Larsen AK, Escargueil AE, Skladanowski A. Catalytic topoisomerase II inhibitors in cancer therapy. Pharmacol Ther 2003; 99: 16781.
  • 10
    Fortune JM, Osheroff N. Merbarone inhibits the catalytic activity of human topoisomerase IIα by blocking DNA cleavage. J Biol Chem 1998; 273: 1764350.
  • 11
    Escargueil AE, Plisov SY, Filhol O, Cochet C, Larsen AK. Mitotic phosphorylation of DNA topoisomerase IIα by protein kinase CK2 creates the MPM-2 phosphoepitope on Ser-1469. J Biol Chem. 2000; 275: 347108.
  • 12
    Wells NJ, Fry AM, Guano F, Norbury C, Hickson ID. Cell cycle phase-specific phosphorylation of human topoisomerase IIα. Evidence of a role for protein kinase C. J Biol Chem 1995; 270: 2835763.
  • 13
    Cowell IG, Okorokov AL, Cutts SA, Padget K, Bell M, Milner J, Austin CA. Human topoisomerase IIα and IIβ interact with the C-terminal region of p53. Exp Cell Res 2000; 255: 8694.
  • 14
    Kwon Y, Shin BS, Chung IK. The p53 tumour suppressor stimulates the catalytic activity of human topoisomerase IIα by enhancing the rate of ATP hydrolysis. J Biol Chem 2000; 275: 1850310.
  • 15
    Wang Q, Zambetti GP, Suttle DP. Inhibition of DNA topoisomerase IIα gene expression by the p53 tumour suppressor. Mol Cell Biol 1997; 17: 38997.
  • 16
    Watt PM, Hickson ID, Borts RH, Louis EJ. SGS1, a homologue of the Bloom's and Werner's syndrome genes, is required for maintenance of genome stability in Saccharomyces cerevisiae. Genetics 1996; 144: 93545.
  • 17
    Yamagata K, Kato J, Shimamoto A, Goto M, Furuichi Y, Ikeda H. Bloom's and Werner's syndrome genes suppress hyperrecombination in yeast sgs1 mutant: implication for genomic stability in human diseases. Proc Natl Acad Sci USA 1998; 95: 87338.
  • 18
    Workman P. Overview: translating Hsp90 biology into Hsp90 drugs. Curr Cancer Drug Targets 2003; 3: 297300.
  • 19
    Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990; 82: 110712.
  • 20
    Kim HD, Tomida A, Ogiso Y, Tsuruo T. Glucose-regulated stresses cause degradation of DNA topoisomerase IIα by inducing nuclear proteasome during G1 cell cycle arrest in cancer cells. J Cell Physiol 1999; 180: 97104.
  • 21
    Tallarida RJ, Stone DJ,Jr, Raffa RB. Efficient designs for studying synergistic drug combinations. Life Sciences 1997; 61: 41725.
  • 22
    Tallarida RJ. The interaction index: a measure of drug synergism. Pain 2002; 98: 1638.
  • 23
    Young JC, Moarefi I, Hartl FU. Hsp90: a specialized but essential protein-folding tool. J Cell Biol 2001; 154: 26773.
  • 24
    Goetz MP, Toft DO, Ames MM, Erlichman C. The Hsp90 chaperone complex as a novel target for cancer therapy. Ann Oncol 2003; 14: 116976.
  • 25
    Ferrarini M, Heltai S, Zocchi MR, Rugarli C. Unusual expression and localisation of heat shock protein in human tumour cells. Int J Cancer 1992; 51: 61319.
  • 26
    Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, Burrows FJ. A high affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 2003; 425: 3579.
  • 27
    Neckers L, Ivy SP. Heat shock protein 90. Curr Opin Oncol 2003; 15: 41924.
  • 28
    Vilenchik M, Solit D, Basso A, Huezo H, Lucas B, He H, Rosen N, Spampinato C, Modrich P, Chiosis G. Targeting wide-range oncogenic transformation via PU24FCl, a specific inhibitor of tumour Hsp90. Chem Biol 2004; 11: 78797.
  • 29
    Isaacs JS, Xu W, Neckers L. Heat shock protein 90 as a molecular target for cancer therapeutics. Cancer Cell 2003; 3: 2137.
  • 30
    Solit DB, Zheng FF, Drobnjak M, Munster PN, Higgins B, Verbel D, Heller G, Tong W, Cordon-Cardo C, Agus DB, Scher HI, Rosen N. 17-allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res 2002; 8: 9626.
  • 31
    Zuo J, Guo Y, Guettouche T, Smith DF, Voellmy R. Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1. Cell 1998; 94: 47180.
  • 32
    Guo Y, Guettouche T, Fenna M, Boellmann F, Pratt WB, Toft DO, Smith DF, Voellmy R. Evidence for a mechanism of repression of heat shock factor 1 transcriptional activity by a multichaperone complex. J Biol Chem 2001; 276: 457919.
  • 33
    Sato N, Yamamoto T, Sekine Y, Yumioka T, Junicho A, Fuse H, Matsuda T. Involvement of heat-shock protein 90 in the interleukin-6-mediated signalling pathway through STAT3. Biochem Biophys Res Commun 2003; 300: 84752.
  • 34
    Boudeau J, Deak M, Lawler MA, Morrice NA, Alessi DR. Heat-shock protein 90 and Cdc37 interact with LKBI and regulate its stability. Biochem J 2003; 370: 84957.
  • 35
    Furukawa M, Uchiumi T, Nomoto M, Takano H, Morimoto RI, Naito S, Kuwano M, Kohno K. The role of an inverted CCAAT element in transcriptional activation of the human DNA topoisomerase IIα gene by heat shock. J Biol Chem 1998; 273: 105505.
  • 36
    Flatten K, Dai NT, Vroman BT, Loegering D, Erlichman C, Karnitz LM, Kaufmann SH. The role of checkpoint kinase 1 in sensitivity to topoisomerase I poisons. J Biol Chem 2005; 280: 1434955.
  • 37
    Blagosklonny MV, Fojo T, Bhalla KN, Kim JS, Trepel JB, Figg WD, Rivera Y, Neckers LM. The Hsp90 inhibitor geldanamycin selectively sensitizes Bcr-Abl-expressing leukaemia cells to cytotoxic chemotherapy. Leukemia 2001; 15: 153743.
  • 38
    Munster PN, Basso A, Solit D, Norton L, Rosen N. Modulation of Hsp90 function by ansamycins sensitizes breast cancer cells to chemotherapy-induced apoptosis in an RB- and schedule-dependent manner. Clin Cancer Res 2001; 7: 222836.
  • 39
    Johnson BD, Schumacher RJ, Ross ED, Toft DO. Hop modulates Hsp70/Hsp90 interactions in protein folding. J Biol Chem 1998; 273: 367986.
  • 40
    Redmond T, Sanchez ER, Bresnick EH, Schlesinger MJ, Toft DO, Pratt WB, Welsh MJ. Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells. Eur J Cell Biol 1989; 50: 6675.
  • 41
    Burrows F, Zhang H, Kamal A. Hsp90 activation and cell cycle regulation. Cell Cycle 2004; 3: 15306.
  • 42
    Shah M, Patel K, Fried VA, Sehgal PB. Interaction of STAT3 with caveolin-1 and heat shock protein 90 in plasma membrane raft and cytosolic complexes. Preservation of cytokine signalling during fever. J Biol Chem 2002; 277: 456629.