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Effects of Geldanamycin on Signaling through Activator-Protein 1 in Hypoxic HT29 Human Colon Adenocarcinoma Cells¹

Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104

	ABSTRACT

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One of the characteristic responses of HT29 human colon adenocarcinoma cells to hypoxic stress is the induction of c-jun expression and binding to the activator-protein 1 (AP-1) element. To study the mechanism of c-jun activation during hypoxia, inhibitors of signaling pathways leading to the activation of AP-1 transcription factor were used. One of them, the benzoquinone ansamycin geldanamycin (GA) M_r-90,000 heat-shock protein (hsp90)-binding antibiotic, is known to disrupt signaling pathways by inducing destabilization of the enzyme complexes and degradation of signaling intermediates involving the proteasome. In our experiments, GA inhibited both basal and hypoxia-induced c-jun expression (IC₅₀ = 75 nM). GA also abolished the hypoxia-induced increase in c-Jun NH₂-terminal kinase (JNK1) catalytic activity and demonstrated an inhibitory effect on stress-activated protein kinase/ERK kinase-1 (SEK1); other participants in the mitogen-activated protein kinase and p38 signal transduction pathways were not affected to the same degree. GA treatment led to a decrease in the nuclear content of c-Jun but not that of c-Fos or of activating transcription factor 2. Functional consequences of these effects were suggested by the inhibition of AP-1 binding in hypoxic HT29 cells in the presence of GA. Pretreatment with the proteasome inhibitor lactacystin before the addition of GA resulted in the elevation of overall c-jun level, but it was unable to restore the hypoxia-induced c-jun expression. Our results demonstrate that GA acts as a highly potent inhibitor of hypoxia-induced c-jun expression, affecting the activation of JNK and of the AP-1 transcription factor. However, the effect of GA cannot be attributed solely to the inhibition of signaling through JNK, and additional mechanisms remain to be identified.

	Introduction

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Hypoxia is a common cause of resistance to radiation and cytotoxic chemotherapy (1) . The common solid tumors, even as small as 2 mm in diameter, have variable fractions of hypoxic cells. The proportion of hypoxic cells has been shown in clinical studies to be a determinant of response to therapy (2) . Cells rendered hypoxic in vivo show similar resistance to drugs including alkylating agents and platinum compounds. To determine the basis of this resistance, we previously analyzed human colon adenocarcinoma cells and found that after hypoxic exposure elevated expression of several enzymes responsible for detoxication of these agents was induced (3) . The mechanism was found to relate principally to transcriptional induction, which was initiated during hypoxic exposure and continued thereafter in the period of reoxygenation. The peak activity and expression of DT-diaphorase and of -glutamylcysteine synthetase (-GCS) was about 3-fold that of control cells, corresponding to the level of resistance exhibited in vitro and in vivo (3 , 4) . The promoter regions of the genes encoding these enzymes have a number of transcription-factor-binding elements in common, particularly AP-1³ and nuclear factor B. Electrophoretic mobility shift assays revealed the induction of binding to AP-1 during and after hypoxic exposure and to nuclear factor B in the reoxygenation phase (5) . Thus, binding to AP-1 element was explored further as a specific hypoxia-related response.

The AP-1 transcription factor consists of homodimers of Jun family members, or heterodimers of Jun, Fos, or ATF-2 proteins and activates a broad range of genes designed to protect cells from adverse environmental conditions (6) . Activation of AP-1 occurs primarily through signaling pathways, terminating in a group of serine-threonine kinases, MAP kinases that act separately on its component proteins (7 , 8) . MAP kinase (MAPK/ERK) phosphorylates Elk-1, which is part of the ternary complex factor that binds to the serum response element in the fos promotor; JNK phosphorylates c-Jun, Elk-1, and ATF-2; and p38 kinase phosphorylates both ternary-complex-factor /Elk-1 and ATF-2. To determine the relative contributions of these pathways to hypoxia signaling in HT29 cells, we carried out studies involving inhibitors that act on components of the pathways, starting with GA.

The benzoquinone ansamycin GA (homologous to herbimycin A) binds to hsp90, interfering with its chaperoning action and, thereby, affects multiple events in mammalian cells (9) . GA interferes with signal transduction pathways by destabilizing kinase complexes, rendering some of the kinases (Raf-1, for example) susceptible to degradation (10) . It has been shown that, by this mechanism, GA is able to disrupt signaling through Raf-1-MEK1-MAPK in PMA-stimulated NIH 3T3 cells (11) and to reduce overall EGF signaling in HeLa cells (12) . The binding of GA to hsp90 inhibits the function of glucocorticoid receptors and steroid-mediated transcription activation by reducing hormone-binding affinity, enhancing degradation of receptors through the ubiquitin-proteasome mechanism, and interfering with steroid-dependent transport of the receptors from cytoplasm to nucleus (13) . Study of the crystal structure of the hsp90-GA complex (14) suggests that GA acts by blocking the binding of ATP to hsp90. Although the molecular basis for the disruption of the signaling pathways by GA in particular cell types has not been completely elucidated, polyubiquitination and proteasomal degradation that is induced and/or mediated by GA was demonstrated for Raf-1, mutant p53, and p185^c-erbB-2 receptor protein-tyrosine kinase (Her2/Neu; Refs. 15, 16, 17 ).

In this report, we demonstrate that signaling through the JNK pathway is critical for the elevation of c-jun expression during prolonged hypoxia. We show for the first time that the hsp90-binding antibiotic GA: (a) reduces both basal and hypoxia-induced c-jun expression in a dose-dependent manner; (b) inhibits the hypoxia-induced activity of JNK; and (c) leads to depletion of c-Jun from nuclei and the loss of AP-1 binding in HT29 cells subjected to hypoxia. The inability of Lc to restore the specific hypoxia-induced c-jun expression in HT29 treated with GA suggests that an additional mechanism(s) besides the disruption of JNK signaling is involved in the inhibition of hypoxic response through AP-1.

	Materials and Methods

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Cells and Hypoxic Treatment.
The human colon adenocarcinoma cell line HT29 was from American Type Culture Collection (Rockville, MD). Cells were grown in MEM (Life Technologies, Inc., Grand Island, NY) supplemented with penicillin (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum. Cultures were maintained in a humidified incubator at 37°C in 5% CO₂-95% air. Stock solutions of 1 mM GA (Life Technologies, Inc.) and 5 mM Lc (Biomol, Plymouth Meeting, PA) were prepared in DMSO (Sigma Chemical Co., St. Louis, MO).

The exposure of cells to hypoxia was essentially performed as described previously (18) . Cells were plated in 160-ml glass-milk-dilution bottles to a density of 1–1.5 x 10⁶ cells per bottle and subjected to hypoxia within 2 days, when the cells had reached 70–80% confluence. The bottles were gassed for 2 h with a mixture of 5%CO₂ and 95% NO₂, sealed tightly, and placed into a CO₂ incubator. The cells were harvested at various time points for further isolation of RNA or for the preparation of cellular and nuclear extracts. Cells usually were treated with 750 nM GA overnight before hypoxia; control HT29 cells were incubated in medium containing 0.01% DMSO. Proteasome inhibitor Lc (5 µM) was added 1 h before GA.

Isolation and Analysis of RNA.
Total cellular RNA was isolated using TRIzol Reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. Total RNA (10–15 µg) was subjected to electrophoresis in 1% agarose-2.2 M formaldehyde gel, transferred onto nitrocellulose membranes (NitroPure, MSI, Westboro, MA), and hybridized to ³²P-DNA probes. The DNA probe for c-jun was purified from plasmid as described previously (18) , and a human ß-actin cDNA probe was from Clontech (Palo Alto, CA). Blots were exposed to BIOMAX MR film (Kodak, Rochester, NY) at -70°C, and results were quantified with a laser densitometer (Molecular Dynamic, Sunnyvale, CA).

Cell Extract Preparation and Kinase Assay.
After hypoxia, cells were washed twice with ice-cold PBS and lysed in 10 mM sodium phosphate (pH 7.0) buffer supplemented with 150 mM NaCl, 2 mM EDTA, 1% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM NaF, 1 mM ß-glycerophosphate, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na₃VO₄, 1 mM sodium PP_i, 5 µg/ml aprotinin, and 5 µg/ml leupeptin. Lysates were centrifuged for 10 min at 12,000 rpm (4°C) and the protein concentration of cleared cellular extracts was measured using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Extracts were diluted with lysis buffer to obtain equal protein concentration, aliquoted and stored at -70°C.

For the immunocomplex kinase assays, protein extracts were incubated with appropriate antibodies for 2 h at 4°C followed by the addition of Protein A Agarose (Life Technologies, Inc.) and further immunoprecipitation for 1 h. Agarose beads were washed three times with lysis buffer, once with 50 mM Tris-HCl (pH 7.5), and once with buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl₂, and 1 mM DTT. After removal of the final washing solution, 30 µl of assay buffer were added to each sample. The kinase assay buffer contained 12.5 mM HEPES (pH 7.5), 12.5 mM ß-glycerophosphate, 7.5 mM MgCl₂, 0.5 mM EGTA, 0.6 mM NaF, 1 mM DTT, 0.5 mM Na₃VO₄, 1 µg of leupeptin, 1 µg of aprotinin, 1 µl of [-³²P]ATP (4500 Ci/mmol; ICN, Costa Mesa, CA), 50 µM ATP, and 1 µg of appropriate exogenous substrate (per reaction). Myelin basic protein (Upstate Biotechnology, Lake Placid, NY), c-Jun-GST fusion protein, or ATF-2 protein (Santa Cruz Biotechnology) were used as substrates for MAPK, JNK, and p38, respectively. Assays for Raf-1, MEKK1, MEK1, and SEK1 were carried out using corresponding antibodies and the appropriate exogenous substrates (Santa Cruz Biotechnology). After incubation for 20 min at 30°C, the reactions were stopped by the addition of 20 µl of 3x SDS and boiled for 5 min; 20 µl from each reaction were subjected to electrophoresis in SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and exposed to Kodak BIOMAX MR film. The activation of MKK3/MKK6 was determined by Western blotting with antibodies raised against phosphorylated forms of the kinases (New England Biolabs, Beverly, MA).

Nuclear Extract Preparation.
Nuclear extract preparation was carried out as described previously (19) . Nuclei were resuspended in 100 µl of nuclear resuspension buffer consisting of 250 mM Tris-HCl (pH 7.8), 60 mM KCl, 50 mM NaF, 1 mM Na₃VO₄, 250 mM okadaic acid, 1 mM DTT, and 1 mM phenylmethanesulfonyl fluoride. After three rounds of freezing/thawing, samples were centrifuged at 7000 rpm for 15 min at 4°C. After the protein content of supernatants was measured, samples were diluted to equal concentrations with nuclear resuspension buffer, aliquoted, and stored at -70°C.

Western Blotting.
For protein electrophoresis, total protein extracts and nuclear extracts were used in amounts of 50 and 20 µg per lane, respectively. Samples were subjected to electrophoresis in 10% SDS-polyacrylamide gels and transferred onto Hybond-P membrane (Amersham, Arlington Heights, IL). Western blotting was carried out using the following antibodies: rabbit polyclonal antibodies raised against c-Jun (AP-1), ERK2, JNK1, MEKK1, MEK1, SEK1, p38, and ATF-2 (from Santa Cruz), c-Fos mouse monoclonal antibodies (from Oncogene Science, Cambridge, MA), and rabbit polyclonal antibodies against phosphorylated SEK1, MKK3/6, JNK and p38 (from New England Biolabs).

Electrophoretic Mobility Shift Assay.
The AP-1 consensus and mutant oligonucleotides were purchased from Santa Cruz Biotechnology, Inc. Double-stranded oligonucleotides containing the binding site for AP-1 c-Jun homodimer or Jun/Fos heterodimer were of the following sequence: 5'-CGCTTGATGACTCAGCCGGAA-3', whereas the mutant oligonucleotide had "CA"-"TG" substitution in the AP-1 binding motif. Oligonucleotides were labeled with [-³²P]ATP (ICN), and nuclear extracts were analyzed for AP-1 binding activity as described previously (18) . The binding reaction mixture contained 10 µg of nuclear extract, 1 µg of poly(dI-dC), 20 mM HEPES (pH 7.5), 40 mM KCl, 1 mM MgCl₂, 0.1 mM EGTA, 0.5 mM DTT, 1 ng of labeled AP-1 oligonucleotides, and 5% glycerol. After incubation for 30 min at room temperature and the addition of loading buffer, samples were resolved in a 4% polyacrylamide gel in 0.5x TBE buffer. Dried gels were exposed to Kodak film at -70°C.

	Results

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GA Inhibits Basal and Hypoxia-induced c-jun Expression in Dose-dependent Manner.
Previous data obtained in our laboratory during studies of hypoxia in HT29 cells showed a significant induction of c-jun by the end of hypoxic exposure and a subsequent fall of mRNA levels during reoxygenation (18) . To investigate the role of the major signal transduction pathways in the c-jun/AP-1 mediated response to hypoxia, we used GA. HT29 cells were treated overnight with 750 nM GA and subjected to hypoxia. Northern blot analysis of total RNA isolated at several time points during hypoxia revealed that GA strongly inhibited both the basal RNA level and hypoxia-induced c-jun expression (Fig. 1A) . Numerous independent experiments showed hypoxic induction of c-jun expression by 6.7 ± 1.3-fold after an 8-h exposure, whereas in the presence of GA, it was effectively inhibited (1.14 ± 0.55-fold). The inhibitory effect of GA was concentration-dependent (Fig. 1B) , with an IC₅₀ of 75 nM. Incubation with 300 nM GA completely inhibited the hypoxia-induced increase in c-jun expression (Fig. 1C) .

View larger version (35K): [in this window] [in a new window]   Fig. 1. Hypoxia-induced expression of c-jun is inhibited by GA in concentration-dependent manner. A, Northern analysis of total RNA (15 µg per lane) from control cells and cells treated with 750 nM GA overnight before hypoxia was carried out according to standard procedure using 32P-labeled c-jun as a probe. Hybridization to ß-actin was used as control of RNA content. B, Northern analysis of total RNA isolated from HT29 treated with various concentrations of GA before hypoxia (8 h). In C, results were quantified using a laser densitometer, and c-jun expression relative to ß-actin signal was plotted. The diagram represents an average of the values from two independent experiments; bars, SD.

View larger version (57K): [in this window] [in a new window]   Fig. 2. GA treatment selectively diminishes c-Jun content in hypoxic HT29 cells and inhibits AP-1 binding. Cells were treated with GA (750 nM), exposed to hypoxia (6 h) followed by nuclear extract isolation. A, Western blot of nuclear extracts demonstrating the depletion of c-Jun but not that of c-Fos and ATF-2. In B, electrophoretic mobility shift assay was performed as described in "Materials and Methods." Arrow, hypoxia-inducible DNA-protein complexes.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Effects of GA treatment on the MAPK pathways in HT29 cells. Cells were harvested at 2 and 6 h of hypoxia, and protein extracts were prepared as described in "Materials and Methods." In vitro immunocomplex kinase assay or Western blotting with phospho-antibodies determined kinase activities. Western blotting of cell lysates with appropriate antibodies was done to monitor the protein content of each kinase.

View larger version (49K): [in this window] [in a new window]   Fig. 4. Inhibition by GA of signaling through stress-activated pathways in HT29 correlates with depletion of SEK1 and MKK3/MKK6 proteins and can be restored by proteasome inhibitor Lc. In A, HT29 cells were treated with GA overnight before the addition of anisomycin for 30 min. Cells were lysed directly in SDS-PAGE loading buffer and subjected to electrophoresis and Western blotting. In B, cells were incubated with 5 µM Lc for 1 h before the addition of 500 nM GA and then were treated as above. Western blotting was carried out with antibodies against total and phosphorylated SEK1, MKK3/MKK6, JNK1, and p38.

Proteasome Inhibitor Lc Does Not Restore Hypoxia-induced c-jun Expression in the Presence of GA.
If signaling through JNK is the major determinant of hypoxic induction of c-jun activation, then restoration of the JNK pathway in GA-treated HT29 cells should restore hypoxic induction of c-jun expression. To investigate this hypothesis, we treated cells with 5 µM Lc for 1 h before the addition of GA, followed by exposure to hypoxia. Northern analysis of RNA isolated after 8 h of hypoxia revealed that treatment with Lc alone increased c-jun RNA level in both oxic and hypoxic cells proportionally (Fig. 5A) . However, when Lc treatment was followed by the addition of GA, the specific hypoxia-induced 6- to 8-fold increase in c-jun expression was not observed despite the higher overall c-jun RNA level in both oxic and hypoxic cells (Fig. 5B) . We obtained similar results with various concentrations of Lc (2.5–10 µM) and GA (500–750 nM) and with different durations of hypoxic treatment (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 5. Lc does not restore hypoxia-induced increase in c-jun expression in GA-treated HT29 cells. A, Northern analysis of total RNA isolated from cells treated with Lc for 1 h before the addition of GA followed by hypoxia. In B, autoradiograms were quantified with a laser densitometer, and c-jun expression relative to ß-actin signal was plotted. The diagram represents an average of the values from three independent experiments. Fold increase in c-jun expression during hypoxia, below the diagram, is given in comparison with oxic cells for each pretreatment protocol.

	Discussion

Top ABSTRACT Introduction Materials and Methods Results Discussion REFERENCES

In this study, we decided to evaluate the impact of hypoxic treatment on the major candidate proteins participating in kinase cascades leading to AP-1 activation and to the induction of c-jun expression. Previous studies of upstream signaling events that occur early during hypoxia show rapid and transient activation of MAPK, JNK, and p38 pathways. Seko et al. (23 , 24) working with cultured rat cardiac myocytes demonstrated that hypoxia and hypoxia/reoxygenation activate Raf-1, MEK1, and MAPK with maximum activity by 5–10 min of hypoxic treatment, as well as JNK and p38 kinases by 2–5 min. Muller et al. (21) showed, that in HeLa cells hypoxia induces transient c-fos expression (with a peak at 30 min)because of phosphorylation of Elk-1 by MAP kinase. On the other hand, hypoxia was not capable of activating MAPK activity in CCL39 hamster fibroblasts (25) . In the above studies, cells were rendered hypoxic by the addition of the medium equilibrated with hypoxic atmosphere overnight, which allowed essentially instant establishment of hypoxic conditions. Our experimental design results in the development of hypoxia over 2 h, and we did not, therefore, investigate those early events. In our experiments, kinases of the Raf-1/MEK1/MAPK pathway always demonstrated significant basal activity, which did not change during prolonged hypoxia. Stress pathways leading to AP-1 activation and c-jun induction, on the other hand, were induced by hypoxia, with the largest time-dependent increase in the activity of JNK and the less significant transient activation of p38. These results are in accord with current views that the AP-1 transcription factor binding to the AP-1 site on the c-jun promoter consists predominantly of c-Jun/ATF-2 dimers (26, 27, 28) . Because ATF-2 is constitutively expressed and uninfluenced by hypoxia (Fig. 3A) , although c-Jun is an inducible transcription factor and a central component of the majority of the AP-1 dimers, we focused on c-Jun as the primary mediator of hypoxia signaling through AP-1 in HT29 cells. Our decision to use GA as an inhibitor of signal transduction was prompted by reports (29) that GA destabilizes Raf-1, interrupts the PMA-induced Raf-1-MEK-MAPK signaling pathway in NIH 3T3 cells, and inhibits proliferation. More importantly, GA blocked PMA-stimulated transactivation of a TRE-Luc reporter construct, implying possible disregulation of AP-1 function. We used GA to define further the role of major signaling pathways leading to hypoxic induction of c-jun expression because the elucidation of hypoxia-specific signaling mechanisms has the potential to reveal novel targets to reverse its deleterious effects. Our data show a profound inhibitory effect of GA on JNK activation by hypoxia, on c-Jun content, and on AP-1 binding activity, leading to a decrease of basal and hypoxia-induced c-jun expression (Figs. 1 and 3 ).

An inhibitory effect of GA on gene expression was demonstrated thus far only for c-myc, in murine lymphoblastoma cells (30) , and in two colon cell lines, SW837 and HCT8 (31) . Heruth et al. (31) used GA and genistein as tyrosine kinase inhibitors and showed inhibition of c-myc expression. The authors also tried to establish which step in control of c-myc expression was altered by genistein. They demonstrated that genistein did not alter either the transcriptional rate or the RNA half-life of c-myc, thus, attributing the effect of this drug on c-myc expression and cell proliferation to the inhibition of protein tyrosine kinase(s). However, because genistein and GA are mechanistically distinct protein kinase inhibitors, it cannot be assumed that GA does not affect the transcription rate or half-life of c-jun RNA in HT29. This possible aspect of GA effect on c-jun expression needs to be examined further. As for the proteins depleted from cells in the presence of benzoquinone ansamycins such as focal adhesion kinase and p185^c-erbB-2 kinase, both GA and herbimycin A induce their degradation without altering mRNA levels in several cell lines (32 , 33) .

Through its interference with the chaperoning function of hsp90, GA affects the stability of multiple proteins and is unlikely to be a specific inhibitor of any particular kinase (13) . Moreover, it has been shown that GA does not inhibit purified kinases in vitro (29 , 34) , exerting its inhibitory effect on signaling indirectly. In our system, GA clearly demonstrated a strong inhibitory effect on hypoxia-induced JNK activity, whereas MAPK and p38 kinase were affected less. The fact that GA did not alter the protein level or the activity of MEKK1, suggests that GA interferes with hypoxic signaling downstream of this kinase. Indeed, model experiments involving treatment of HT29 with different concentrations of GA before the addition of anisomycin showed inhibition of both SEK1 and MKK3/MKK6, which correlated with the depletion of their protein levels in GA-treated cells. It is also possible that kinase(s) other than MEKK1 activate SEK1/JNK in HT29 cells during hypoxia. A large number of kinases capable of activating JNK pathways have been described to date, and several are able to activate SEK1 directly (35) . It would be informative to investigate the effect of GA treatment on the activities of those kinases.

GA interferes with signal transduction by inducing ubiquitination and degradation of signaling molecules chaperoned by hsp90 through a mechanism involving the 20S proteasome (15, 16, 17) . Proteasome inhibitors such as Lc have been shown to reverse the effect of GA treatment and to restore signaling through Raf1-MEK-MAPK pathway in NIH 3T3 cells (29) . We were interested in determining whether this is also the case for stress-induced pathways in HT29 cells. The fact that Lc restored signaling through stress-activated pathways but was unable to restore specific hypoxia-induced increase in c-jun expression in the presence of GA suggests that, besides disruption of the signaling through JNK, additional mechanisms of GA action are involved in its inhibition of the hypoxic response through AP-1. Further investigation is needed to evaluate the effect of GA on modulation of c-Jun stability against ubiquitination and degradation. In addition to c-Jun phosphorylation, redox regulation also plays a major role in the AP-1 activation during hypoxia. Thus, it is important to determine whether GA is able to affect the redox status of AP-1 proteins. Also, GA was shown to prevent nuclear translocation of mutant p53 (36) and to inhibit steroid-mediated transcription activation by interfering with steroid-dependent transport of the receptors from cytoplasm to nucleus (13) . If significant amounts of newly synthesized c-Jun are necessary for the formation of additional AP-1 complexes (and an increase in c-jun mRNA and c-Jun protein in response to hypoxia would suggest that they are necessary), inhibition of its translocation to the nucleus by GA could well be the reason for the inability of proteasome inhibitors alone to sustain the massive increase in c-jun expression during hypoxia.

Hypoxia is an important regulator of tumor cell biology because many solid tumors contain regions of very low to undetectable O₂ levels. Hypoxia activates the hypoxia-inducible-factor-1-mediated pathway that results in the expression of various dependent genes including the hematopoietic growth factor erithropoietin and the potent angiogenic growth factor VEGF, which stimulate endothelial cell migration and proliferation (37) . Strategies to inhibit hypoxia signaling through hypoxia-inducible factor 1 and VEGF have pronounced antitumor effects in preclinical models and are being developed in the clinic (38 , 39) . The inhibition of hypoxia signaling through AP-1 is extremely important, also, because it can facilitate the basal expression of VEGF and amplify the response to hypoxia (40) . Profound hypoxia also activates the expression of genes that confer resistance to cytotoxic drugs, in particular, alkylating agents and platinum compounds. Our work implicates a JNK/c-jun/AP-1 based mechanism for the effects on detoxication genes, and provides novel targets for resistance reversal. It has been shown that the JNK pathway plays an important role in cell survival after treatment with cisplatin in T98G glioblastoma cells (41) . In a different model of cisplatin-resistant cell lines, we have shown the potential of c-jun targeting with antisense oligodeoxynucleotides to reverse resistance.⁴ The current data suggest a possible role for GA in reversing hypoxic cell resistance. Although the parental compound is likely to be prohibitively nonspecific, analogues such as 17-allylamino-17-demethoxyGA (17-AAG) have been found to have selective antitumor activity in vivo (42) . We are currently investigating the potential of these compounds to reverse hypoxic resistance in tumor cell lines.

	ACKNOWLEDGMENTS

 
We thank Drs. Trevor Penning, Jeffrey Field, Steven Johnson, and Kang-Shen Yao for critically reviewing the manuscript and Amy Hummel for assistance in the preparation of the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by NIH Grant RO1-CA49820.

² To whom requests for reprints should be addressed, at University of Pennsylvania, 303G Abramson Building, Civic Center Boulevard at Osler Circle, Philadelphia, PA 19104-4318. Phone: (215) 573-7300; Fax: (215) 573-9889; E-mail: vasilevs{at}mail.med.upenn.edu

³ The abbreviations used are: AP-1, activator-protein 1; JNK, c-Jun NH₂-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase (same as MAPK); ATF-2, activating transcription factor 2; GA, geldanamycin; Lc, lactacystin; MEK1, MAPK kinase; SEK1, stress-activated protein kinase/ERK kinase-1 (same as MKK4); MEKK1, SEK1 kinase; MKK3/6, p38 kinase kinases; hsp90, M_r-90,000 heat-shock protein; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor; PMA, 4-phorbol-12-myristate-13-acetate (same as TPA).

⁴ Bin Pan and P. J. O’Dwyer, unpublished data.

Received 5/18/99. Accepted 6/29/99.

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