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. Author manuscript; available in PMC: 2014 Jun 30.
Published in final edited form as: Radiat Res. 2011 Jun 10;176(3):333–345. doi: 10.1667/rr2534.1

Geldanamycin Analog 17-DMAG Limits Apoptosis in Human Peripheral Blood Cells by Inhibition of p53 Activation and its Interaction with Heat-Shock Protein 90 kDa after Exposure to Ionizing Radiation

Risaku Fukumoto a, Juliann G Kiang a,b,c,1
PMCID: PMC4076157  NIHMSID: NIHMS320298  PMID: 21663398

Abstract

Exposure to ionizing radiation induces p53, and its inhibition improves mouse survival. We tested the effect of 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) on p53 expression and function after radiation exposure. 17-DMAG, a heat-shock protein 90 (Hsp90) inhibitor, protects human T cells from ionizing radiation-induced apoptosis by inhibiting inducible nitric oxide synthase (iNOS) and subsequent caspase-3 activation. Using ex vivo human peripheral blood mononuclear cells, we found that ionizing radiation increased p53 accumulation, acute p53 phosphorylation, Bax expression and caspase-3/7 activation in a radiation dose- and time postirradiation-dependent manner. 17-DMAG inhibited these increases in a concentration-dependent manner (IC50 = 0.93 ± 0.01 µM). Using in vitro models, we determined that inhibition of p53 by genetic knockout resulted in lower levels of caspase-3/7 activity 1 day after irradiation and enhanced survival at 10 days. Analysis of p53–Hsp90 interaction in ex vivo cell lysates indicated that the binding between the two molecules occurred after irradiation but 17-DMAG prevented the binding. Taken together, these results suggest the presence of p53 phosphorylation and Hsp90-dependent p53 stabilization after acute irradiation. Hsp90 inhibitors such as 17-DMAG may prove useful with radiation-based cancer therapy as well as for general radioprotection.

INTRODUCTION

More than 50% of cancer patients receive radiation therapy at least one time in their lives (1). Radiation causes DNA damage, directly or indirectly, in all living cells, which can result in cell death, tissue damage or organ dysfunction/failure (2). A poor understanding of the mechanisms of radiation injury has inhibited the development of agents that can effectively protect and/ or treat humans exposed to ionizing radiation.

p53 protein, a transcription factor encoded by the TP53 gene, plays an important role in programmed cell death, i.e., apoptosis, in response to genotoxic stressors, including ionizing radiation (3). More than half of human cancer cases involve mutations that impair p53 function; therefore, mutant p53 proteins have become targets of cancer therapy strategies (4). In healthy animals with wild-type p53, it has also been shown that the inhibition of p53 protects mice from γ radiation, suggesting the potential use of p53 inhibition in radioprotection (5).

p53 and downstream effectors that regulate apoptosis have been studied by a number of groups. It has been demonstrated that ataxia telangiectasia mutated (ATM) protein activation (6) and phosphorylation of p53 protein are early biochemical events that occur in response to DNA double-strand breaks (DSBs) caused by acute exposure to γ radiation (7). Activation of p53 requires several modifications that include phosphorylation (8), acetylation (9) and its translocation to the nucleus (10). Activated p53 within the nucleus initiates transcription of the pro-apoptotic Bax gene (11), which can be detected within several hours postirradiation. Bax protein ultimately causes caspase-3 and -7 activation and cell death at 24 h and later (3).

p53 is normally expressed at low levels in normal tissues (12). Higher levels of p53 are induced after exposure to ionizing radiation (3).

The stability of p53 plays a key role in its biological activity. Studies have shown that p53 is normally an unstable protein with a short half-life of approximately 16 min (13). Its degradation is regulated by its posttranslational modifications and its ligation with ubiquitin by MDM2, a ubiquitin ligase (8, 14). On the other hand, p53 is protected from MDM2-mediated degradation by heat-shock protein 90 (Hsp90), thereby prolonging its half-life and thus its cellular activity. Hsp90 is a ubiquitously expressed molecular chaperone that regulates, along with other chaperones, the folding, function and degradation of newly synthesized proteins through its ATPase activity (15). It is known to be activated upon heat-shock stress. It is not known whether Hsp90 plays a role in healthy human cells to stabilize and activate p53 in response to ionizing radiation.

17-Dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) is a derivative of the Hsp90 inhibitor geldanamycin (16, 17). In vitro, geldanamycin selectively kills cancer cells by chelating Hsp90 in its so-called high-affinity conformation, which exists only in cancer cells (18), and thereby prevents mutant proteins from entering the nucleus to trigger gene activation (19). Because of the roles Hsp90 plays in key processes of tumor growth and development, including induction and stabilization of growth factors and other signals in transformed cells, angiogenesis and promotion of metastasis, Hsp90 has understandably become an inviting target in the search for pharmaceutical agents to kill cancer cells (see Discussion section for more detail).

Compared to geldanamycin, 17-DMAG is more water-soluble and distributes more rapidly in animal tissues, factors that have led to superior clinical applications in humans (20, 21). 17-DMAG also has lower hepatotoxicity than geldanamycin (22). Preclinical and phase 1 clinical trials have determined the pharmacokinetics of 17-DMAG in mice, rats (21) and humans (23, 24). Human cancer studies employing a wide range of drug concentrations have shown the mean half-life of 17-DMAG after i.v. injection to be 22.3–24 h (23, 24). The drug is well tolerated over periods of months of use even when combined with other medical treatments such as radiation therapy (20).

We recently reported that 17-DMAG protects human T cells from γ radiation. This protective effect is a result of inhibition of the inducible nitric oxide synthase (iNOS)/caspase-3 cascade induced by radiation (25). Although 17-DMAG has never been tested as a radioprotector in healthy human ex vivo cells or mice, the fact that both p53 and iNOS are clients of Hsp90 (19, 26) suggests it may prove useful. In this study we used 17-DMAG to investigate the roles of (1) Hsp90 in regulation of p53 and (2) cell death in response to acute exposure to ionizing radiation. We present evidence that 17-DMAG inhibits p53 accumulation and prevents apoptosis in irradiated human ex vivo cells by blocking acute p53 phosphorylation and its interaction with Hsp90.

MATERIALS AND METHODS

Cell Culture

TK6 and NH32 cells (generously provided by Dr. J. B. Mitchell), Jurkat cells (Clone E6-1, American Type Culture Collection, Manassas, VA), and fresh normal peripheral blood mononuclear cells (PBMCs, AllCells, LLC, Emeryville, CA) were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (Invitrogen), 2 mM l-glutamine (Invitrogen), 100 U/ml penicillin and 100 µg/ml streptomycin (Quality Biological Inc., Gaithersburg, MD) and maintained in a humidified 37°C incubator with continuous 5% CO2 supply. TK6, NH32 and Jurkat cells were fed twice a week.

Drug Treatment

Cells were counted and then seeded on 6-well dishes that contained 2 ml of fresh growth medium per well and were treated with specified concentrations of 17-DMAG dissolved in water or with water alone for 24 h before irradiation.

Irradiation

Cells on 6-well dishes were exposed to 60Co γ radiation at a dose rate of 0.6 Gy/min for the time required to deliver the doses specified.

Clonogenic Assay

Five thousand cells were mixed with prewarmed, fresh RPMI 1640 medium that contained final concentrations of 40% fetal bovine serum, 2 mM l-glutamine and 0.1% low-melting-point agarose (Invitrogen) in a total volume of 3 ml. The mixture was then laid over 1 ml of solidified RPMI 1640 bottom medium (made with 1% low-melting-point agarose) in 6-well dishes. The number of colonies containing more than 50 cells was scored after 10 days of incubation.

Immunoprecipitation

Cells were disrupted by gentle agitation for 30 min at 4°C in lysis buffer, which contained 20 mM Hepes (pH 7.2–7.5) (Invitrogen), 150 mM NaCl (Sigma-Aldrich, St. Louis, MO), 0.5% Nonidet P40 (Roche; Indianapolis, IN), in the presence of protease inhibitors, phosphatase inhibitors and 10 mM sodium molybdate (Sigma-Aldrich). After removal of insoluble materials by centrifugation at 10,000g at 4°C, supernatants (total cell lysates) were precleared by the addition of 10 µl of protein G-agarose (Roche) and gentle rotation at 4°C for 1 h. Cleared lysates were collected after centrifugation at 10,000g for 10 min at 4°C and used for immunoprecipitation by incubating with 2 µg of the indicated antibodies and 30 µl of protein G-agarose overnight at 4°C with gentle rotation. Resulting precipitates were collected by centrifugation at 2,000g and then washed three times with lysis buffer.

Immunoblotting

Total cell lysates or immunoprecipitates were boiled in the presence of final concentrations of 1× LDS sample buffer (Invitrogen) and 10% β-mercaptoethanol (Invitrogen) for 5 min. Samples were briefly spun down and kept on ice until the separation by NuPAGE® 4–12% Bis-Tris gel (Invitrogen). Separated proteins in gels were transferred to 0.45-µm pore size PVDF membranes (Invitrogen) in the 1× transfer buffer (Invitrogen). Membranes were then soaked in blocking buffer, which contained 3% nonfat dry milk (Santa Cruz Biotechnology, Santa Cruz, CA) dissolved in Tris-buffered saline (50 mM Tris-HCl, pH 8.0, and 150 mM NaCl) supplemented with 0.2% Tween® 20 (TBS-T). Blocked membranes were reacted with primary and secondary antibodies against specific antigens and washed with TBS-T after each reaction. Resulting membranes were reacted with ECL reagents (Amersham, Piscataway, NJ) to identify bands using the manufacturer’s protocol and exposed to Kodak BioMax Light films (Kodak, Rochester, NY). The protein band intensities were quantified by Molecular Imaging software (Kodak).

Detection of Caspase-3/7 Activity and Analysis by Confocal Microscopy

A Magic Red® Caspase Detection Kit (MP Biomedicals, Solon, OH) was used for the detection of caspase-3/7 activity following the manufacturer’s protocol. Briefly, about 2 × 105 cells were stained in the presence of up to 300 µl of OPTI-MEM I medium (Invitrogen). Cells were seeded onto no. 1 borosilicate glass slides with 4-well chambers (Fisher Science Education, Hanover Park, IL). An LSM 5 PASCAL Zeiss laser scanning confocal microscope (Carl Zeiss MicroImaging, Thornwood, NY) with a 100×/1.3 NA Plan Apochromat oil objective was used to scan the signals. Each resulting image was provided with a simultaneous scan of differential interference contrast (DIC).

Flow Cytometry

About 105 cells were fixed in 1× fixation buffer followed by washing and antibody staining procedures using 1× permeabilization buffer following the manufacturer’s protocol (Millipore, Billerica, MA). Stained cells were analyzed using an Guava EasyCyte MiNi flow cytometer and Guava software (Millipore).

RNA Extraction and Real-Time Quantitative Reverse Transcription-PCR (qRT-PCR)

Total RNA was extracted using RNeasy (Qiagen, Valencia, CA). Synthesis of cDNAs was performed using SuperScript II reverse transcriptase (Invitrogen), and the subsequent PCR reactions were performed using iQ Supermix (Bio-Rad, Hercules, CA) following the manufacturer’s protocol. All measurements were performed on triplicate samples, each corrected by the amplification of internal control cDNA of 18S RNA.

Reagents

17-Dimethylamino-ethylamino-17-demethoxygeldanamycin (NSC 707545) was obtained from LC Laboratories (Woburn, MA). Antibodies against the following antigens were used for the analyses by immunoprecipitation, immunoblotting and flow cytometry: p53 (DO-1), Bax (B-9), Bcl-2 (C-2), Hsp70 (W-27), Hsp90 (H-114), actin (I-19), and normal mouse IgG were obtained from Santa Cruz Biotechnology. γ-H2AX and phospho-p53 on serine at position 15 were purchased from eBioscience (San Diego, CA). Primers and probes used in qRT-PCR analyses were described previously (27).

Statistical Analysis

Statistical values were obtained from triplicate samples and shown as averages with standard errors. One-way ANOVA, standardized-range test and Bonferroni inequality were used with significance levels set at 5 and 0.1%. IC50 values were determined by GraphPad Prizm® (GraphPad Software, Inc., La Jolla, CA) using average values from representative experiments.

RESULTS

17-DMAG Inhibits p53 and Bax but not Bcl-2 in Human Jurkat Cells after γ Irradiation

We used immunoblotting to test whether 17-DMAG would inhibit p53 and Bax protein levels after irradiation, since it was reported that (1) ionizing radiation-induced DNA damage activates p53 through phosphorylation of ATM and check 2 (CHK2) proteins (7) and (2) radiation upregulates transcription of the p53-dependent Bax gene (27).

To observe both radiation dose- and time-dependent changes, we irradiated Jurkat cells in the presence or absence of 17-DMAG with 4 or 8 Gy 60Co γ radiation and harvested the cells 4 and 24 h postirradiation. We found radiation dose-dependent increases in p53 protein by 4 h postirradiation that were maintained at 24 h. 17-DMAG treatment effectively inhibited those increases (Fig. 1A, B). It has been reported that p53 in Jurkat cells retains transactivation potential of the Bax gene (28). In our study, we found similar, significant radiation dose- and time postirradiation-dependent increases in Bax protein, which were significantly inhibited by 17-DMAG treatment 24 h postirradiation (Fig. 1A, C). Unlike with p53 and Bax, radiation did not increase Bcl-2, but 17-DMAG treatment led to a significant increase in this protein 24 h after irradiation (Fig. 1A, D). These data suggest the possibility that 17-DMAG exerts its radioprotective effect by inhibiting the activation of the p53-Bax pathway after radiation exposure while at the same time increasing the level of anti-apoptotic Bcl-2.

FIG. 1.

FIG. 1

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Inhibition of radiation-induced p53 pathway by 17-DMAG. Jurkat cells were counted and seeded in fresh medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated 24 h after drug treatment and returned to the incubator. Cells were harvested 4 and 24 h postirradiation. Cell lysates were prepared and immunoblotting was performed to measure p53, Bax, Bcl-2 and actin levels. Intensities of 24-h bands were quantified by optical densitometry. Panel A: Representative immunoblots. Panel B: Quantification of p53 levels relative to actin. Panel C: Quantification of Bax levels relative to actin. Panel D: Quantification of Bcl-2 levels relative to actin. ns, nonsignificant.

17-DMAG Inhibits Activation of p53 Pathway in Ex Vivo Human PBMCs after γ Irradiation

Because human Jurkat cells are cancer cells, it is not clear whether their responses to radiation and 17-DMAG are the same as those of normal cells. We therefore tested human peripheral blood mononuclear cells (PBMCs) to determine whether there is a radiation-induced increase in p53 expression and whether it is inhibited by 17-DMAG.

Cells pretreated with various concentrations of 17-DMAG were left unirradiated or were irradiated with 8 Gy (Fig. 2). We collected samples 1, 4 and 24 h postirradiation. p53 protein increased in irradiated samples at 1 h (Fig. 2A, top panel), increased markedly at 4 h (Fig. 2A, middle panel), and increased further at 24 h (Fig. 2A, bottom panel). 17-DMAG inhibited the increases in p53 protein in a concentration-dependent manner, as indicated in the samples taken 4 and 24 h postirradiation (Fig. 2A, middle and bottom panels). The inhibitory effect of 17-DMAG on radiation-induced increases in p53 protein is summarized in Fig. 2B. We determined an IC50 of 0.93 ± 0.01 µM for 17-DMAG using samples collected 4 h after 8 Gy irradiation.

FIG. 2.

FIG. 2

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Inhibition of radiation-induced p53 expression by 17-DMAG is concentration-dependent in normal human PBMCs. Freshly isolated PBMCs from healthy human donor were counted and seeded in medium containing 17-DMAG at different concentrations for 24 h. Cells were then irradiated, returned to the incubator, and harvested 1, 4 and 24 h postirradiation. Total cell lysates from each sample were prepared for immunoblot analysis to determine the levels of p53 and actin. Protein levels were measured by optical densitometry. Panel A: Representative immunoblots. Panel B: Quantification of p53 levels relative to actin. *P < 0.05 and ***P < 0.001 compared with corresponding samples with no drug treatment (0 µM).

We next tested the effects of various doses of radiation on p53 protein levels and activation in PBMCs pretreated with or without 10 µM 17-DMAG. We collected samples 24 h postirradiation. Radiation dose-dependent increases in p53 protein levels were observed, and the increases were significantly reduced by 17-DMAG pretreatment (Fig. 3A, B). 17-DMAG also induced a slight increase in the nonirradiated samples, consistent with our previous report (29); however, this basal increase was much smaller than the increase induced by radiation. As with p53 protein, radiation increased the level of Bax protein, which was also significantly inhibited by 17-DMAG treatment (Fig. 3A, C). It was previously reported that the increase in Bax is not dependent on radiation dose (30). We did not observe significant differences in Bcl-2 levels between samples (data not shown). However, we observed significant increases in p21, another p53 target gene, which 17-DMAG inhibited (Fig. 3A, D).

FIG. 3.

FIG. 3

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17-DMAGinhibits radiation-induced increases in p53/Bax in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded in medium containing 10 µM 17-DMAG or vehicle for 24 h. Cells were then irradiated, returned to the incubator, and harvested 1 h (for γ-H2AX and pp53 measurements), 4 h (for mRNA measurements), and 24 h (for p53/Bax/p21 protein) postirradiation. Panel A: Representative immunoblots showing effect of 17-DMAGon p53, Bax and p21 protein after irradiation. Total cell lysates from samples collected at 24 h were prepared for immunoblots to test p53, Bax, p21 and actin expression. Levels were measured using optical densitometry. Panel B: Quantification of p53 levels relative to actin. Panel C: Quantification of Bax levels relative to actin. Panel D: Quantification of p21 levels relative to actin. Panel E: Analysis of γ-H2AX levels 1 h postirradiation by flow cytometry. Panel F: Analysis of p53 phosphorylation levels on the serine residue at position 15; 1 h postirradiation by flow cytometry. Panel G: p53 gene expression. Total RNA was extracted from samples collected 4 h postirradiation and used for qRT-PCR analysis. Samples were tested for p53 expression levels using primers and probes specific to the human TP53 gene. Panel H: Bax gene expression. Total RNA used to test p53 gene expression was also studied by qRT-PCR using primers and probes specific to the human Bax gene. Results are presented as averages of triplicate samples from independent wells with standard errors. ns, nonsignificant.

Because ionizing radiation may activate the p53-mediated Bax pathway earlier than 24 h, we hypothesized that 17-DMAG also inhibited p53 activation in samples collected at 1 and 4 h postirradiation. To test this, we first measured phosphorylation of both H2AX (γ-H2AX) and p53 (pp53) in 1-h postirradiation samples. γ-H2AX is a marker of DNA DSBs (31), and phosphorylation of serine at position 15 on pp53 is one of the key requirements for p53 activation (8). We found that the increase in γ-H2AX levels was radiation dose-dependent, in agreement with previous reports (31), and this response was observed both in the presence and absence of 17-DMAG (Fig. 3E). In the same sets of samples, pp53 levels were significantly increased after 8 Gy and fully inhibited by 17-DMAG treatment (Fig. 3F). The increase in pp53 was transient, returning to baseline levels 4 h postirradiation (data not shown). We performed similar experiments using human PMBCs and a radiation dose of 4 Gy and did not observe significant phosphorylation. This indicates that phosphorylation at serine 15 may not be the only determinant in p53 activation.

We next analyzed p53 and Bax mRNA in samples collected 4 h postirradiation to determine if any increases were correlated with their respective protein levels observed in 24-h samples (Fig. 3 A-C). Quantitative real-time RT-PCR (qRT-PCR) analysis demonstrated very little increase in p53 mRNA in irradiated samples (Fig. 3G) but significant increases in Bax mRNA (Fig. 3H). The unexpectedly small increase in p53 mRNA at 4 h suggested that it is not likely that enhanced transcription is the main cause of the increased level of p53 protein seen at 24 h (Figs. 2, 3A and B). On the other hand, the increase in Bax mRNA at 4 h correlated well with protein accumulation at 24 h (Fig. 3A, C). Treatment with 17-DMAG significantly inhibited induction of Bax mRNA, suggesting that the radioprotection effect of 17-DMAG is mediated by inhibiting the Bax pathway (Fig. 3H). However, in the presence of 17-DMAG, the level of p53 mRNA after irradiation was significantly lower than that in vehicle-treated cells (Fig. 3G), leaving the possibility of p53 suppression at the transcriptional level in irradiated cells. Taken together, these observations support a potential role for Hsp90 in p53 protein accumulation and subsequent programmed cell death after irradiation in ex vivo human cells, because 17-DMAG, as an Hsp90 inhibitor, may impair radiation-induced p53–Hsp90 conjugation that prevents p53 from undergoing MDM2-regulated degradation (14).

17-DMAG Does Not Inhibit Hsp90 Expression but Increases Hsp70 Expression

Because 17-DMAG is known to interfere with Hsp90 function (17), we determined whether the inhibitory effects of 17-DMAG on the radiation-activated p53-Bax pathway was due to changes in Hsp90 expression. Using PBMCs, we first looked at the effect of various concentrations of 17-DMAG on Hsp90 protein levels after exposure to 8 Gy. Membranes used for the analyses reported in Fig. 2 were reprobed with antibody directed against Hsp90, and protein levels were studied by immunoblotting and densitometric analyses. Neither radiation nor the various concentrations of 17-DMAG that were used significantly changed the levels of Hsp90 24 h postirradiation (Fig. 4A, B). Similar data were observed in samples 1 and 4 h postirradiation (data not shown).

FIG. 4.

FIG. 4

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17-DMAG has no effect on Hsp90 levels but induces Hsp70 in concentration-dependent manner in normal human PBMCs. Freshly isolated PBMCs from a healthy human subject were counted and seeded for 24 h in medium containing 17-DMAG or vehicle. Cells were then irradiated, returned to the incubator, and harvested 24 h postirradiation. Total cell lysates from samples were prepared for immunoblots to test Hsp90, Hsp70 and actin levels. Levels were quantified by optical densitometry. Panel A: Representative immunoblots of Hsp90, Hsp70 and actin after pretreatment with different concentrations of 17-DMAG prior to irradiation with 8 Gy. Panel B: Quantification of Hsp90 and Hsp70 levels relative to actin shown in panel A. Panel C: Representative immunoblots of Hsp90, Hsp70 and actin in the presence or absence of 10 µM 17-DMAG prior to irradiation with 4 and 8 Gy. Panel D: Quantification of Hsp90 levels relative to actin shown in panel C. Panel E: Quantification of Hsp70 levels relative to actin shown in panel C. *P 0.05 and ***P 0.001 compared with corresponding samples with no drug treatment (0 µM). ns, nonsignificant.

We next studied the effect of various doses of radiation on Hsp90 levels in the presence or absence of a 10 µM 17-DMAG pretreatment. Membranes used for the analyses reported in Fig. 3A-D were reprobed with antibody directed against Hsp90 to quantify protein levels. In these membranes, irradiation at either 4 or 8 Gy failed to change Hsp90 levels significantly (Fig. 4C, D), suggesting that neither radiation nor 17-DMAG significantly altered the level of Hsp90 protein.

Unlike Hsp90, 17-DMAG treatment significantly increased Hsp70 protein (Fig. 4A-C, E). Ionizing radiation alone at either 4 or 8 Gy failed to alter the level of Hsp70 protein (Fig. 4A-C, E).

17-DMAG Inhibits p53–Hsp90 Conjugation after γ Irradiation

Hsp90 enhances the stability of p53 protein in cell lines by direct association with p53 after heat shock (32). Since the data in Fig. 4 show that Hsp90 protein does not increase after irradiation, we surmised the possibility of a radiation-induced increase in p53–Hsp90 complex formation that can be inhibited by 17-DMAG. To determine whether this is the case, we irradiated human PBMCs pretreated with or without 17-DMAG with 8 Gy. Cell lysates were prepared 4 h postirradiation and immunoprecipitated with anti-p53 antibody or control IgG. The precipitates were then immunoblotted with both anti-p53 and anti-Hsp90 antibodies. Analysis of immunoprecipitates indicated that Hsp90 protein from the irradiated sample lysates co-precipitated with p53 (Fig. 5). This association occurred only in irradiated samples and was reduced with 17-DMAG treatment. We also observed a significant increase in the p53 band in the irradiated samples, and the band appeared at a position of higher molecular weight, suggesting a change in conformation due to phosphorylation. 17-DMAG also reduced this apparent conformational change. All protein recovery data from immunoprecipitation experiments were normalized to actin to account for possible variations in protein content in the samples (Fig. 5).

FIG. 5.

FIG. 5

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17-DMAG inhibits radiation-induced interaction between p53 and Hsp90 in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded for 24 h in medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated with 8 Gy or left unirradiated, returned to the incubator, and harvested 4 h postirradiation. Total cell lysates were prepared and immunoprecipitated with anti-p53 antibody or control mouse IgG followed by immunoblot analysis with indicated antibodies. Total cell lysates were also tested by immunoblot analysis for protein content by probing with anti-actin antibody.

17-DMAG Inhibits Caspase-3/7 Activation after γ Irradiation

Caspase-3/7 activation is a key step in apoptosis. Based on our observation that 17-DMAG inhibited activation of the p53-Bax pathway after irradiation, we hypothesized that 17-DMAG treatment would also suppress caspase-3/7 activation. To test this possibly, we used samples of human PBMCs 24 h after irradiation and live-stained them with Magic Red® to show caspase-3/7 activities. There was a basal level of caspase-3/7 activity in all nonirradiated samples (Fig. 6 A, B, top panels). Irradiation of vehicle-treated samples at 4 or 8 Gy resulted in significant increases in caspase-3/7 activity (Fig. 6A, B, left middle and bottom panels). Samples irradiated at either dose in the presence of 17-DMAG pretreatment demonstrated significantly lower activities than those irradiated alone (Fig. 6A, B, right middle and bottom panels). These results suggest that 17-DMAG inhibits radiation-induced increases in p53-Bax pathway activation and resulting caspase-3/7 activation.

FIG. 6.

FIG. 6

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Effect of 17-DMAG treatments on radiation-induced caspase-3/7 activity in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded for 24 h in medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated with 4 or 8 Gy and returned to the incubator. Live cells were studied 24 h postirradiation for internal caspase-3/7 activities by Magic Red® staining. Panel A: Representative images. Panel B: Quantification of fluorescence intensity.

p53 is Involved in Radiation-Induced Cell Death

Our data indicate that 17-DMAG inhibits p53–Hsp90 interactions, prevents excess p53 accumulation, and reduces caspase-3/7 activation in irradiated cells, but the data do not indicate whether there is any direct role for p53 in radiation-induced cell death. To help answer this question, we sought to determine whether there is any direct correlation between p53 inhibition and cell survival using in vitro models, which allow the study of both short- and long-term effects of genotoxic stress in living cells.

To address a possible correlation between p53 inhibition and long-term cell viability after irradiation, we compared survival rates between the normal TK6 cell line and its NH32 derivative, which is p53 deficient (Fig. 7A). p53 has been shown to be strongly induced in TK6 cells after 4 Gy irradiation (33). Identical numbers of cells were irradiated at various doses and seeded into soft agar medium, and the numbers of surviving colonies were determined 10 days postirradiation. Radiation reduced TK6 cell colony-forming ability in a dose-dependent manner, indicating its sensitivity to radiation. In contrast, irradiated NH32 cells produced a significantly larger number of colonies at each radiation dose, indicating a greater resistance to radiation (Fig. 7B). TK6 also displayed higher caspase-3/7 activity (Fig. 7C, D, left panels) than NH32 cells 24 h postirradiation (Fig. 7C, D, right panels). Taken together, these data reinforce the idea that 17-DMAG improves cell survival by inhibiting p53 stabilization and accumulation.

FIG. 7.

FIG. 7

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Direct correlation between p53 and apoptosis. Panel A: Representative immunoblot of p53 in TK6 and NH32 cells. NH32 is p53 knockout cell line derived fromTK6. Total cell lysates from both untreated cell lines were tested by immunoblot analysis with antibodies against p53 and actin. Panel B: Enhanced survival of NH32 after γ irradiation. Equal numbers of TK6 and NH32 cells were irradiated at the indicated doses or left unirradiated, and 5,000 cells were assessed by clonogenic assay. Numbers of colonies were counted on day 10, and data are shown as fractions of the corresponding nonirradiated controls. Results are averages of triplicate data with standard deviations. Panel C: Representative images of caspase-3/7 activity in TK6 and NH32 cells. Live cells were studied 24 h postirradiation for internal caspase-3/7 activities by Magic Red® staining. Panel D: Quantification of fluorescence intensity. *P < 0.05 and ***P < 0.001 compared between different cell lines at each radiation dose.

DISCUSSION

The present study demonstrates that ionizing radiation increases p53 and Bax, but not Bcl-2, in a radiation dose- and time postirradiation-dependent manner. 17-DMAG treatment inhibits p53 in a drug concentration-dependent fashion and reduces Bax but increases Bcl-2. Cells demonstrate better survival when cells are low in p53 protein, such as when p53 is blocked by drug treatment or p53 gene knockout.

The radiation-induced increase in p53 is correlated with cell mortality (Fig. 7) (33). Aberrant expression of or mutation in TP53 has also been observed in many cancer onsets and progressions (34), metabolic disorders (35) and DNA damage responses (36). Because cell viability can be altered by manipulation of p53 protein levels by techniques such as miRNA, p53 inhibitors or gene depletion (37), p53 has been considered as a potentially useful target for developing therapeutic drugs.

Ionizing radiation causes DNA DSBs that lead to the phosphorylation of ATM protein, which leads to increases in γ-H2AX (Fig. 3E) and phosphorylated p53 (Fig. 3F), observations that are in agreement with the literature. Phosphorylated p53 arrests the cell cycle for DNA repair through induction of the cyclin-dependent kinase inhibitor p21/cip1 (7). When the level of p53 protein is beyond an as-yet undefined threshold, growth arrest occurs in mitotically active cells, including stem cells, and leads to senescence (38), and the affected tissues or organs undergo apoptosis. As shown by our data, radiation initiates p53 phosphorylation instead of its transcription or translation (Fig. 3G). p53 phosphorylation leads to increased p53 stability and accumulation (Figs. 1–3).

Hsp90 forms a complex with p53 after heat shock (32), which prevents p53 from being ubiquitinated and degraded (14). Using immunoprecipitation and immunoblotting techniques, we found in human ex vivo samples that ionizing radiation induced formation of the p53–Hsp90 complex (Fig. 5), an observation that is consistent with the literature.

The radiation-induced increase in Bax protein (Figs. 1 and 3) is a consequence of increases in p53 protein levels, because p53 is a transcriptional factor of the Bax gene (11), and its activation leads to an increase in Bax mRNA (Fig. 3H). The increase in p53/Bax resulted in increased mortality (Fig. 7B) that was at least partly mediated by caspase-3/7-dependent apoptosis (Figs. 6, 7C and D).

p53 levels in unstressed cells are kept low as a result of continuous degradation (12). The protein MDM2 (also called HDM2 in humans) binds to p53 (3), which blocks p53 activity as well as prevents its translocation to the nucleus. MDM2 also acts as ubiquitin ligase and covalently attaches ubiquitin to p53, marking it for degradation in proteasomes. It is suggested that p53 is protected from MDM2 after irradiation by (1) phosphorylation of the N-terminus of p53 and (2) Hsp90 binding to p53. 17-DMAG treatment inhibited both of these activities: treatment with 17-DMAG inhibited radiation-induced increases in phosphorylated p53 (Fig. 3F), binding to Hsp90 (Fig. 5), and thus total p53 protein (Figs. 1–3). It is likely that nonphosphorylated p53 that is free of Hsp90 becomes the target for MDM2-mediated degradation. 17-DMAG is known to bind within the cleft between the globular domain and the beta-sheets of Hsp90 (17), thereby leading to Hsp90 dysfunction and rapid degradation of the resulting unprotected p53.

There is an endogenous p53 antagonist in heart, namely carboxyl terminus of Hsp70-interacting protein [CHIP (39)]. 17-DMAG treatment alone significantly increased Hsp70 protein (Fig. 4A-C, E), in agreement with previous reports (23, 24, 40). The possibility that increases in Hsp70 involving the CHIP pathway or some other, as-yet unidentified p53 antagonist (41, 42) may play a role in the results we observe cannot yet be ruled out.

Our results demonstrate that radioprotection by 17-DMAG is related to the drug’s ability to bind Hsp90 and thereby help reduce the half-life of p53 stimulated by radiation exposure. Other cellular processes involving Hsp90 could also be affected by 17-DMAG, though little is currently known of their possible role in the radiation response. In tumor cells, in addition to Hsp90- mediated stabilization of mutant proteins such as v-Src and p53 that appear during cell transformation (43), Hsp90 stabilizes various proteins in tumor cells in which there is an overexpression of growth factor receptors such as EGFR (44) and signal transduction proteins such as PI3K and AKT (45, 46). Hsp90 is also required for induction of vascular endothelial growth factor (VEGF) and nitric oxide synthase (NOS) important for de novo angiogenesis (43, 47). Hsp90 is known to promote the invasion step of metastasis in cooperation with the matrix metalloproteinase MMP2 (19, 48). The effects of 17-DMAG on growth signals, stabilization of signal proteins, angiogenesis and metastasis after irradiation should be explored.

17-DMAG treatment is known to inhibit radiation-induced activation of the iNOS pathway (25) and hemorrhage-induced increases in TNF-α concentration, while it increases the anti-apoptotic protein Bcl-2 (40). It is not clear whether 17-DMAG affects FOXO protein and the PI3K/Akt pathway because UV irradiation inactivates FOXO, resulting in apoptosis (49). It would be interesting to determine the effects of 17-DMAG on these pathways since they could all be playing a role in radiation-induced mortality.

Several reports demonstrate significantly increased radiation sensitivity of tumor cells in vivo in the presence of 17-DMAG (20). A combination of drug treatment and radiation may prove to be a useful strategy in cancer therapy; 17-DMAG could sensitize tumors while normal tissues could be protected from radiation injury or ischemia-induced infarction (39, 50). Importantly, loss of p53 does not significantly increase hypermutation after irradiation, while mutant p53 does (33). Our study reveals cellular p53 level is clearly controlled by both radiation and 17-DMAG doses. Ongoing pharmacokinetics studies (21, 23, 24) may determine the optimal drug dose—and therefore p53 level—during radiation treatment in specific tissues. Our study supports the idea that 17-DMAG gives normal and cancer tissues different sensitivities to ionizing radiation, a property that could prove valuable in cancer radiotherapy in addition to the general field of radioprotection.

In summary, we have shown that ionizing radiation induces an increase in p53 and Bax in a radiation dose-and time postirradiation-dependent manner. 17-DMAG treatment lowered the radiation-induced p53 increase in a drug concentration-dependent fashion by inhibiting radiation-induced p53 phosphorylation and p53–Hsp90 complex formation, thereby reducing levels of apoptosis. p53 knockout cells demonstrated reduced apoptosis, indicating the tight link between p53 and apoptosis. The results provide mechanistic insight to support further development of 17-DMAG as a radioprotector through modulation of p53.

ACKNOWLEDGMENTS

We thank J. T. Smith, B. R. Garrison and N.G. Agravante for technical support, T. A. Baginski, Biomedical Instrumentation Center (BIC), Uniformed Services University of the Health Sciences, for the confocal microscopy analysis, and Y. S. Kim, A. P. Weaver, W. A. Melendez-Cruz and V. Nagy, AFRRI Cobalt Radiation Facility, for sample irradiation and dosimetry. We also thank D. E. McClain for his scientific discussion and editorial assistance. The views, opinions and findings contained in this report are those of the authors and do not reflect official policy or positions of the Department of the Navy, Department of Defense, NIH/NIAID, or the United States Government. This work was funded by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (Grant R21AI080553) to JGK and the Defense Threat Reduction Agency (Grant CBM.RAD.01.10.AR.010) to JGK.

REFERENCES

  • 1.Bentzen SM. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nat Rev Cancer. 2006;6:702–713. doi: 10.1038/nrc1950. [DOI] [PubMed] [Google Scholar]
  • 2.Kiang JG, Jiao W, Cary LH, Mog SR, Elliott TB, Pellmar TC, Ledney GD. Wound trauma increases radiation-induced mortality by activation of iNOS pathway and elevation of cytokine concentrations and bacterial infection. Radiat Res. 2010;173:319–332. doi: 10.1667/RR1892.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Haupt S, Berger M, Goldberg Z, Haupt Y. Apoptosis – the p53 network. J Cell Sci. 2003;116:4077–4085. doi: 10.1242/jcs.00739. [DOI] [PubMed] [Google Scholar]
  • 4.Hollstein M, Sidransky D, Vogelstein B, Harris CC. p53 mutations in human cancers. Science. 1991;253:49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  • 5.Strom E, Sathe S, Komarov PG, Chernova OB, Pavlovska I, Shyshynova I, et al. Small-molecule inhibitor of p53 binding to mitochondria protects mice from gamma radiation. Nat Chem Biol. 2006;2:474–479. doi: 10.1038/nchembio809. [DOI] [PubMed] [Google Scholar]
  • 6.Xu Y, Baltimore D. Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev. 1996;10:2401–2410. doi: 10.1101/gad.10.19.2401. [DOI] [PubMed] [Google Scholar]
  • 7.Houtgraaf JH, Versmissen J, van der Giessen WJ. A concise review of DNA damage checkpoints and repair in mammalian cells. Cardiovasc Revasc Med. 2006;7:165–172. doi: 10.1016/j.carrev.2006.02.002. [DOI] [PubMed] [Google Scholar]
  • 8.Shieh SY, Ikeda M, Taya Y, Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell. 1997;91:325–334. doi: 10.1016/s0092-8674(00)80416-x. [DOI] [PubMed] [Google Scholar]
  • 9.Tang Y, Zhao WH, Chen Y, Zhao YM, Gu W. Acetylation is indispensable for p53 activation. Cell. 2008;133:612–626. doi: 10.1016/j.cell.2008.03.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Shaulsky G, Goldfinger N, Tosky MS, Levine AJ, Rotter V. Nuclear localization is essential for the activity of p53 protein. Oncogene. 1991;6:2055–2065. [PubMed] [Google Scholar]
  • 11.Miyashita T, Reed JC. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995;80:293–299. doi: 10.1016/0092-8674(95)90412-3. [DOI] [PubMed] [Google Scholar]
  • 12.el-Deiry WS. Regulation of p53 downstream genes. Semin Cancer Biol. 1998;8:345–357. doi: 10.1006/scbi.1998.0097. [DOI] [PubMed] [Google Scholar]
  • 13.McVean M, Xiao H, Isobe K, Pelling JC. Increase in wild-type p53 stability and transactivational activity by the chemopreventive agent apigenin in keratinocytes. Carcinogenesis. 2000;21:633–639. doi: 10.1093/carcin/21.4.633. [DOI] [PubMed] [Google Scholar]
  • 14.Alarcon-Vargas D, Ronai Z. p53-Mdm2 – the affair that never ends. Carcinogenesis. 2002;23:541–547. doi: 10.1093/carcin/23.4.541. [DOI] [PubMed] [Google Scholar]
  • 15.Richter K, Hendershot LM, Freeman BC. The cellular world according to Hsp90. Nat Struct Mol Biol. 2007;14:90–94. doi: 10.1038/nsmb0207-90. [DOI] [PubMed] [Google Scholar]
  • 16.Whitesell L, Mimnaugh EG, De Costa B, Myers CE, Neckers LM. Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci USA. 1994;91:8324–8328. doi: 10.1073/pnas.91.18.8324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell. 1997;89:239–250. doi: 10.1016/s0092-8674(00)80203-2. [DOI] [PubMed] [Google Scholar]
  • 18.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;425:407–410. doi: 10.1038/nature01913. [DOI] [PubMed] [Google Scholar]
  • 19.Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer. 2005;5:761–772. doi: 10.1038/nrc1716. [DOI] [PubMed] [Google Scholar]
  • 20.Kabakov AE, Kudryavtsev VA, Gabai VL. Hsp90 inhibitors as promising agents for radiotherapy. J Mol Med. 2010;88:241–247. doi: 10.1007/s00109-009-0562-0. [DOI] [PubMed] [Google Scholar]
  • 21.Egorin MJ, Lagattuta TF, Hamburger DR, Covey JM, White KD, Musser SM, et al. Pharmacokinetics, tissue distribution, and metabolism of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (NSC 707545) in CD2F1 mice and Fischer 344 rats. Cancer Chemother Pharmacol. 2002;49:7–19. doi: 10.1007/s00280-001-0380-8. [DOI] [PubMed] [Google Scholar]
  • 22.Jez JM, Chen JC, Rastelli G, Stroud RM, Santi DV. Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90. Chem Biol. 2003;10:361–368. doi: 10.1016/s1074-5521(03)00075-9. [DOI] [PubMed] [Google Scholar]
  • 23.Ramanathan RK, Egorin MJ, Erlichman C, Remick SC, Ramalingam SS, Naret C, et al. Phase I pharmacokinetic and pharmacodynamic study of 17-dimethylaminoethylamino-17-demethoxygeldanamycin, an inhibitor of heat-shock protein 90, in patients with advanced solid tumors. J Clin Oncol. 2010;28:1520–1526. doi: 10.1200/JCO.2009.25.0415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kummar S, Gutierrez ME, Gardner ER, Chen X, Figg WD, Zajac-Kaye M, et al. Phase I trial of 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), a heat shock protein inhibitor, administered twice weekly in patients with advanced malignancies. Eur J Cancer. 2010;46:340–347. doi: 10.1016/j.ejca.2009.10.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kiang JG, Smith JT, Agravante NG. Geldanamycin analog 17-DMAG inhibits iNOS and caspases in gamma-irradiated human T cells. Radiat Res. 2009;172:321–330. doi: 10.1667/RR1585.1. [DOI] [PubMed] [Google Scholar]
  • 26.Yoshida M, Xia Y. Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase. J Biol Chem. 2003;278:36953–36958. doi: 10.1074/jbc.M305214200. [DOI] [PubMed] [Google Scholar]
  • 27.Grace MB, Blakely WF. Transcription of five p53-and Stat-3-inducible genes after ionizing radiation. Radiat Meas. 2007;42:1147–1151. [Google Scholar]
  • 28.Fimognari C, Nusse M, Cesari R, Iori R, Cantelli-Forti G, Hrelia P. Growth inhibition, cell-cycle arrest and apoptosis in human T-cell leukemia by the isothiocyanate sulforaphane. Carcinogenesis. 2002;23:581–586. doi: 10.1093/carcin/23.4.581. [DOI] [PubMed] [Google Scholar]
  • 29.Lin K, Rockliffe N, Johnson GG, Sherrington PD, Pettitt AR. Hsp90 inhibition has opposing effects on wild-type and mutant p53 and induces p21 expression and cytotoxicity irrespective of p53/ATM status in chronic lymphocytic leukaemia cells. Oncogene. 2008;27:2445–2455. doi: 10.1038/sj.onc.1210893. [DOI] [PubMed] [Google Scholar]
  • 30.Jen KY, Cheung VG. Transcriptional response of lymphoblastoid cells to ionizing radiation. Genome Res. 2003;13:2092–2100. doi: 10.1101/gr.1240103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–5868. doi: 10.1074/jbc.273.10.5858. [DOI] [PubMed] [Google Scholar]
  • 32.Wang C, Chen J. Phosphorylation and hsp90 binding mediate heat shock stabilization of p53. J Biol Chem. 2003;278:2066–2071. doi: 10.1074/jbc.M206697200. [DOI] [PubMed] [Google Scholar]
  • 33.Chuang YY, Chen Q, Brown JP, Sedivy JM, Liber HL. Radiation-induced mutations at the autosomal thymidine kinase locus are not elevated in p53-null cells. Cancer Res. 1999;59:3073–3076. [PubMed] [Google Scholar]
  • 34.Suwinski R, Jaworska M, Nikiel B, Grzegorz W, Bankowska-Wozniak M, Wojciech M, et al. Predicting the effect of accelerated fractionation in postoperative radiotherapy for head and neck cancer based on molecular marker profiles: data from a randomized clinical trial. Int J Radiat Oncol Biol Phys. 2010;77:438–446. doi: 10.1016/j.ijrobp.2009.05.021. [DOI] [PubMed] [Google Scholar]
  • 35.Nyblom HK, Bugliani M, Fung E, Boggi U, Zubarev R, Marchetti P, Bergsten P. Apoptotic, regenerative, and immunerelated signaling in human islets from type 2 diabetes individuals. J Proteome Res. 2009;8:5650–5656. doi: 10.1021/pr9006816. [DOI] [PubMed] [Google Scholar]
  • 36.Ikehata H, Okuyama R, Ogawa E, Nakamura S, Usami A, Mori T, et al. Influences of p53 deficiency on the apoptotic response, DNA damage removal and mutagenesis in UVB-exposed mouse skin. Mutagenesis. 2010;25:397–405. doi: 10.1093/mutage/geq019. [DOI] [PubMed] [Google Scholar]
  • 37.Kasinski AL, Slack FJ. Potential microRNA therapies targeting Ras, NFkappaB and p53 signaling. Curr Opin Mol Ther. 2010;12:147–157. [PubMed] [Google Scholar]
  • 38.Erol A. Systemic DNA damage response and metabolic syndrome as a premalignant state. Curr Mol Med. 2010;10:321–334. doi: 10.2174/156652410791065282. [DOI] [PubMed] [Google Scholar]
  • 39.Naito AT, Okada S, Minamino T, Iwanaga K, Liu ML, Sumida T, et al. Promotion of CHIP-mediated p53 degradation protects the heart from ischemic injury. Circ Res. 2010;106:1692–1702. doi: 10.1161/CIRCRESAHA.109.214346. [DOI] [PubMed] [Google Scholar]
  • 40.Kiang JG, Agravante NG, Smith JT, Bowman PD. 17-DMAG diminishes hemorrhage-induced small intestine injury by elevating Bcl-2 protein and inhibiting iNOS pathway, TNF-α increase, and caspase-3 activation. Cell Biosci. 2011;1:21. doi: 10.1186/2045-3701-1-21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lee SJ, Choi SA, Lee KH, Chung HY, Kim TH, Cho CK, et al. Role of inducible heat shock protein 70 in radiation-induced cell death. Cell Stress Chaperones. 2001;6:273–281. doi: 10.1379/1466-1268(2001)006<0273:roihsp>2.0.co;2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Beere HM, Wolf BB, Cain K, Mosser DD, Mahboubi A, Kuwana T, et al. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome. Nat Cell Biol. 2000;2:469–475. doi: 10.1038/35019501. [DOI] [PubMed] [Google Scholar]
  • 43.Calderwood SK, Khaleque MA, Sawyer DB, Ciocca DR. Heat shock proteins in cancer: chaperones of tumorigenesis. Trends Biochem Sci. 2006;31:164–172. doi: 10.1016/j.tibs.2006.01.006. [DOI] [PubMed] [Google Scholar]
  • 44.Lurje G, Lenz HJ. EGFR signaling and drug discovery. Oncology. 2009;77:400–410. doi: 10.1159/000279388. [DOI] [PubMed] [Google Scholar]
  • 45.Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, Pavletich NP. Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent. Cell. 1997;89:239–250. doi: 10.1016/s0092-8674(00)80203-2. [DOI] [PubMed] [Google Scholar]
  • 46.Mohsin SK, Weiss HL, Gutierrez MC, Chamness GC, Schiff R, Digiovanna MP, et al. Neoadjuvant trastuzumab induces apoptosis in primary breast cancers. J Clin Oncol. 2005;23:2460–2468. doi: 10.1200/JCO.2005.00.661. [DOI] [PubMed] [Google Scholar]
  • 47.Fontana J, Fulton D, Chen Y, Fairchild TA, McCabe TJ, Fujita N, et al. Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release. Circ Res. 2002;90:866–873. doi: 10.1161/01.res.0000016837.26733.be. [DOI] [PubMed] [Google Scholar]
  • 48.Eustace BK, Sakurai T, Stewart JK, Yimlamai D, Unger C, Zehetmeier C, et al. Functional proteomic screens reveal an essential extracellular role for hsp90 alpha in cancer cell invasiveness. Nat Cell Biol. 2004;6:507–514. doi: 10.1038/ncb1131. [DOI] [PubMed] [Google Scholar]
  • 49.Luo X, Puig O, Hyun J, Bohmann D, Jasper H. Foxo and Fos regulate the decision between cell death and survival in response to UV irradiation. EMBO J. 2007;26:380–390. doi: 10.1038/sj.emboj.7601484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Harrison EM, Sharpe E, Bellamy CO, McNally SJ, Devey L, Garden OJ, et al. Heat shock protein 90-binding agents protect renal cells from oxidative stress and reduce kidney ischemia-reperfusion injury. Am J Physiol Renal Physiol. 2008;295:F397–F405. doi: 10.1152/ajprenal.00361.2007. [DOI] [PubMed] [Google Scholar]

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