Cancer Research Meeting Calendar Genetics and Biology of Brain Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Comerford, K. M.
Right arrow Articles by Taylor, C. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Comerford, K. M.
Right arrow Articles by Taylor, C. T.
[Cancer Research 64, 9057-9061, December 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

c-Jun NH2-Terminal Kinase Activation Contributes to Hypoxia-Inducible Factor 1{alpha}–Dependent P-Glycoprotein Expression in Hypoxia

Katrina M. Comerford, Eoin P. Cummins and Cormac T. Taylor

Department of Medicine and Therapeutics, The Conway Institute for Biomolecular and Biomedical Research and the Dublin Molecular Medicine Centre, University College, Dublin, Ireland


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously have shown that hypoxia increases the expression of P-glycoprotein, which in turn increases tumor cell capacity to actively extrude chemotherapeutic agents and may contribute to tumor drug resistance. This event is mediated through the hypoxia-inducible factor (HIF-1). Here, we investigated the role of the stress-activated protein kinase c-Jun NH2-terminal kinase (JNK) in the signaling mechanisms underlying these events. Hypoxia activates JNK activity in vitro and in vivo. Overexpression of mitogen-activated protein kinase (MAPK) kinase kinase (MEKK-1), which preferentially activates JNK, mimics, in a nonadditive way, hypoxia-induced activity of the MDR1 promoter and expression of MDR1 mRNA and P-glycoprotein. Furthermore, the JNK inhibitor SP600125 selectively and specifically inhibits hypoxia- and MEKK-1–induced MDR1 promoter activity in a dose-dependent manner. JNK inhibition also reversed hypoxia- and MEKK-1–induced activity of an HIF-1–dependent reporter gene. MEKK-1–induced MDR1 expression depends on a functional HIF-1 binding site (hypoxia-responsive element). Hypoxia- but not cobalt chloride–dependent HIF-1–DNA binding and transcriptional activation was inhibited by SP600125, indicating that hypoxia-induced signaling to HIF-1 depends on JNK activation. Because it has been reported that reactive oxygen species are increased in hypoxia and related to JNK activation, we investigated their role in signaling this response. Whereas exogenous addition of H2O2 was sufficient to activate JNK, reactive oxygen species scavengers were without effect on hypoxia-induced JNK or HIF-1 activation. Thus, hypoxia-elicited MDR1 expression, which depends on HIF-1 activation, depends at least in part on signaling via activation of JNK. Furthermore, these events are independent of the generation of reactive oxygen intermediates. Thus, JNK may represent a therapeutic target in the prevention of tumor resistance to chemotherapeutic treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Within the microenvironment of a developing tumor, decreased vascular supply and increased energy demand to maintain proliferation result in the formation of regions of hypoxia (1) . This results in the activation of a rapid and specific maladaptive molecular response, which results in the up-regulation of genes that promote tumor perfusion, survival, and growth (2) . Hypoxia-inducible factor 1 (HIF-1) is the master regulator of this adaptive response to hypoxia (3) . In normoxia, HIF-1{alpha} is generated at a high level and degraded in an oxygen-dependent manner (2) . During conditions of hypoxia, HIF-1{alpha} accumulates, dimerizes with HIF-1ß to form HIF-1, and translocates to the nucleus where it activates the expression of an array of adaptive genes, such as vascular endothelial growth factor (2) . Genes under the regulation of HIF-1 generally support tumor survival through increased angiogenesis, vasodilation, and glycolytic capacity (2) .

A number of studies have shown a link between tumor hypoxia and chemotherapeutic resistance. Chemotherapeutic agents are broadly less effective in the induction of cell death in hypoxia in studies using drugs, including cisplatin, etoposide, bleomycin, and mitomycin C (4) . Hypoxia-elicited chemotherapeutic resistance has been reported in a number of cell types, including fibroblasts, breast cancer cells, glioma cells, and testicular germ cells (4, 5, 6) . Thus, hypoxia-associated chemotherapeutic resistance is a broad phenomenon. Among the genes increased in response to hypoxia, which may contribute to drug resistance, is the multidrug resistance (MDR1) gene, which encodes P-glycoprotein. We previously have shown that MDR1 gene expression and subsequent functional P-glycoprotein expression is dramatically up-regulated in a HIF-1–dependent manner in response to hypoxia (7) . Confirmation of this work by Wartenberg et al. (8) implicated an inverse role for reactive oxygen species (ROS) in the regulation of HIF-1 and subsequent MDR1 expression.

The c-Jun NH2-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) signaling cascade that typically is activated in response to cellular stress (9) . JNK activation has been observed in hypoxia associated in vivo in melanoma metastases (10) , pulmonary arterial hypertension (11) , cerebral ischemia (12 , 13) , and mouse embryo fibroblasts (14) , as well as in vitro in a number of cell lines (15, 16, 17, 18) . Osborn et al. (19) also have shown a link between JNK activity and MDR1 gene expression. The role of ROS production in the activation of JNK remains incompletely understood. Although it appears that JNK is sensitive to activation in response to oxidative stress, it is not clear whether this is the mechanism of induction in hypoxia. In this study, we confirm that JNK activation occurs in response to hypoxia in a time-dependent manner in vitro and in colonic mucosal tissues from mice exposed to whole animal hypoxia. Because activation of JNK through overexpression of the upstream kinase MAPK kinase kinase (MEKK-1) in normoxia is capable of mimicking hypoxia-induced HIF-1 activation and subsequent MDR1 up-regulation, we hypothesized a link between hypoxia-elicited JNK activation and increased MDR1 gene expression through HIF.

A number of studies have investigated functional interactions between increased JNK activation and increased HIF-1 activity. Indirect effects of JNK on HIF-1 have been reported through c-Jun (20) and activator protein–1 (21) . Furthermore, hypoxia-independent activation of HIF-1 in response to paracrine stimuli, including hepatocyte growth factor (22 , 23) and insulin-like growth factor (21) , has been shown to involve a role for JNK. However, the role of JNK in modulating hypoxia-elicited HIF-1 stability and transactivation potential is not known. In this study, we show that hypoxia-elicited HIF-1 activity depends on the activation of JNK. Furthermore, these are pivotal signaling events in mediating the hypoxic up-regulation of drug resistance–associated genes such as MDR1. Thus, JNK activation may represent a promising therapeutic target to combat hypoxia-dependent chemotherapeutic resistance.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
HeLa cells were cultured in minimum essential medium supplemented with nonessential amino acids, penicillin/streptomycin, and 10% (v/v) fetal calf serum as described previously (24) . Cells were cultured at 37°C in a humidified atmosphere under hypoxic (1% O2, measured cell-free medium PO2 = 15 torr) or normoxic (21% O2, measured cell-free medium PO2 = 136 torr) conditions with an atmospheric balance of 95%N2/5%CO2. Medium PO2 measurements were made using an Oxylite PO2 measurement system (Oxford Optronix, Oxford, United Kingdom).

Animal Model of Hypoxia.
Mice were exposed to hypoxia (8% atmospheric oxygen) for 0 to 6 hours as described previously (25) . This protocol was in accordance with NIH guidelines for the use of live animals and was approved by the institutional animal care and use committee at Brigham and Women’s Hospital (Boston, MA). Following exposure, colonic mucosal tissue was removed, dissected along the mesenteric border, washed, and snap frozen for further analysis.

Western Blot Analysis.
To examine JNK activation, whole cell lysates were prepared as described previously (26) . Lysates normalized to protein levels were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-JNK (Cell Signaling Technology, Inc., Beverly, MA), anti–phospho-JNK (Cell Signaling Technology, Inc.), P-glycoprotein (Calbiochem, La Jolla, CA) or anti–HIF-1{alpha} (Affinity BioReagents, Golden, CO) antibodies. Following washing, a species-matched, peroxidase-conjugated secondary antibody was added (Cell Signaling Technologies, Inc.). Labeled bands were detected by enhanced chemiluminescence (Bio-Rad, Hercules, CA).

Reverse-Transcription PCR.
MDR1expression was assessed by reverse transcription-PCR from cDNA prepared from total cell RNA using the following primers (Sigma Chemical Co., St. Louis, MO): MDR1sense, 5'-CCCATCATTGCAATAGCAGG-3' and MDR1antisense, 5'-GTTCAAACTTCTGCTCCTGA-3'. Control primers for 18S RNA were sense, 5'-GTGGAGCGATTTGTCTGGTT-3' and antisense, 5'-CGCTGAGCCAGTCAGTGTAG-3'. cDNA was amplified by an initial incubation at 95°C for 5 minutes, followed by 25 (18S) or 39 (MDR1) cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds, and a final extension at 72°C for 5 minutes. PCR products were separated by electrophoresis on a 2% agarose gel.

Transfections.
An HIF-1– or cyclic AMP-responsive element binding protein (CREB)–dependent luciferase reporter assay was used to investigate the impact of hypoxia or MEKK-1 overexpression on transcriptional events as described previously (27) . Furthermore, reporter genes under the control of wild-type or hypoxia-response element (HRE)–mutated MDR promoter regions were used as described previously (7) . MDR promoter mutations involved a three nucleotide mutation in the HRE (7) . Briefly, HeLa cells were grown to ~60% confluence and transfected with a luciferase reporter plasmid under the control of a basic promoter element (TATA) plus a defined inducible cis-enhancer element containing HRE or cyclic AMP response-element (CRE) motifs. Luciferase activity was measured by luminometry.

Hypoxia-Inducible Factor 1/DNA Binding.
To investigate HIF-1 binding to the consensus HRE sequence, we used the TransAM HIF-1 kit according to manufacturer’s instructions (Active Motif, Carlsbad, CA). Briefly, cells were exposed to various times of hypoxia or CoCl2 (0 to 100 µmol/L), and nuclear lysates were made. Lysates were put on a 96-well plate that had immobilized double-stranded DNA sequences containing the HIF-1 binding HRE. HIF-1 binding was detected using a primary mouse anti–HIF-1 antibody and a secondary horseradish peroxidase–labeled antimouse antibody.

Statistical Analysis.
Statistical analysis was carried out using one-way ANOVA or either paired or unpaired Student t test with P < 0.05 for n independent samples deemed statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxia Activates JNK Phosphorylation In vitro and In vivo.
HeLa cells were exposed to increasing periods of instantaneous hypoxia by exposing cells in a hypoxia chamber to preconditioned hypoxic medium for 0 to 60 minutes. Western blot analysis reveals that hypoxia causes a rapid and time-dependent increase in JNK phosphorylation, which is maximal at 30 minutes (Fig. 1A)Citation . Similarly, in colonic mucosal tissue taken from mice exposed to whole animal hypoxia (0 to 6 hours), phospho-JNK levels increase, whereas total JNK levels remain stable (Fig. 1B)Citation .



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Hypoxia activates JNK in vitro and in vivo. A. Confluent monolayers of HeLa cells were exposed to preconditioned hypoxic medium in a hypoxia chamber (0.5% atmospheric O2) for 0 to 60 minutes. Whole cell lysates were separated by SDS-PAGE and immunoblotted for the activated form of JNK using a specific anti–phospho-JNK antibody. Hypoxia resulted in a time-dependent and transient JNK phosphorylation. B. Mice were exposed to whole animal hypoxia (8%) for 0 to 6 hours. Colonic tissue was removed, homogenized, and tissue lysates were prepared. Phospho-JNK (top) but not total JNK (bottom) was increased in a time-dependent manner in colonic tissue from mice exposed 0 to 6 hours to whole animal hypoxia.

 
Role of JNK in the Activation of MDR1Promoter Activity and P-Glycoprotein Expression.
Cells transiently transfected with the MDR1 promoter linked to a luciferase reporter gene showed an increase in transcriptional activity in response to hypoxia (4.49- ± 0.89-fold; P < 0.005; n = 12) and MEKK-1 (4.515- ± 0.08-fold; P < 0.05; n = 12) overexpression. Interestingly, the combination of hypoxia and MEKK-1 was less than additive (6.714- ± 1.67-fold; n = 12), indicating at least some degree of commonality in the pathways activated (Fig. 2A)Citation . Preincubation of cells with the specific JNK inhibitor SP600125 (0 to 10 µmol/L) resulted in a significant inhibition of hypoxia-elicited MDR1promoter activity (1.47- ± 0.031-fold versus 3.87- ± 0.76-fold increase for 10 µmol/L SP600125), implicating a role for JNK in signaling this response (Fig. 2B)Citation . Importantly, JNK inhibition with SP600125 (5 µmol/L) also inhibited MEKK-1–induced MDR1activity (8.25-fold over basal increase in activity) as compared with control (19.6-fold over basal increase in activity). Western blot analysis of whole cell lysates from HeLa cells overexpressing MEKK-1 shows up-regulation of P-glycoprotein expression (Fig. 2C)Citation . Similarly, MDR1mRNA was increased in cells overexpressing MEKK-1 as shown by reverse transcription-PCR (Fig. 2D)Citation .



View larger version (24K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. JNK inhibition prevents hypoxia-elicited MDR promoter activity. HeLa cells were transfected with a luciferase reporter construct under the control of the MDR promoter. A, Hypoxia (24 hours) and MEKK-1 overexpression increased MDR promoter activity. However, the effects of hypoxia and MEKK overexpression together were not additive (*P < 0.05; n = 3 to 12). B. Hypoxia-induced MDR promoter activity is inhibited in a dose-dependent manner by the JNK inhibitor SP600125 (0 to 10 µmol/L; *P < 0.05; **P < 0.01; n = 3 to 12). C, HeLa cells overexpressing MEKK-1, which selectively activates JNK, increase P-glycoprotein expression. D, HeLa cells overexpressing MEKK-1, which selectively activates JNK, increase MDR1 mRNA expression.

 
JNK Inhibition Specifically Inhibits Hypoxia-Inducible Factor 1–Dependent Transcriptional Activity.
HeLa cells transiently transfected with an HRE-dependent luciferase reporter construct showed a significant induction when exposed to hypoxia (20.43- ± 3.24-fold; P < 0.05) or MEKK-1 overexpression (10.51- ± 4.37-fold; P < 0.05; Fig. 3ACitation ). In separate experiments, hypoxia-induced HRE-luciferase activity was inhibited in a dose-dependent manner by 1 µmol/L and 10 µmol/L SP600125 by 62% and 81%, respectively (Fig. 3BCitation ; P < 0.05 in each case). Pretreatment with SP600125 also significantly inhibited HRE activity induced through overexpression of MEKK-1 (data not shown). CREB-dependent transcriptional activity also is induced during exposure of cells to hypoxia, albeit to a lesser extent than HIF-1–dependent activity. CRE-dependent reporter activity was increased by 90.2% in hypoxia in a manner that was not significantly altered by SP600125 (118.7% increase over normoxia; Fig. 3CCitation ). Furthermore, PD98059 and SB203850, which inhibit extracellular signal-regulated kinase 1/2 MAPKs and p38, respectively, were without effect on hypoxia-induced HRE-luciferase activity (Fig. 3D)Citation . Mutation of the HRE present in the MDR1promoter significantly attenuates MEKK-1–induced expression (Fig. 4)Citation . Collectively, these data indicate that HIF-1–dependent MDR1up-regulation in hypoxia specifically depends on JNK activation.



View larger version (40K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Hypoxia-elicited JNK HIF-1 activity is specifically inhibited by SP600125. HeLa cells were transfected with a luciferase reporter construct under the control of multiple HREs before exposure to hypoxia or MEKK overexpression. Hypoxia and MEKK overexpression significantly induced the activity of the HIF-1–dependent reporter gene. B. Hypoxia-activated HIF-1 reporter gene activity was inhibited in a dose-dependent manner by SP600125 (0 to 10 µmol/L; *P < 0.05). C. HeLa cells were transfected with a luciferase reporter construct under the control of multiple CREs before exposure to hypoxia. Inhibition of JNK with SP600125 was without effect on hypoxia-induced CREB activity. D. In cells transfected with the HRE-luciferase reporter gene, PD98059 and SB203580 were without effect on hypoxia-induced HRE-luciferase activity (*P < 0.05; **P < 0.01; n = 6 to 14).

 


View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. SP600125 is without effect on cobalt-induced HIF-1 activity. HeLa cells were transfected with a luciferase reporter gene under the control of the wild-type MDR promoter or the MDR promoter mutated at the HRE. Mutation of the HRE resulted in significant attenuation of the responsiveness of this promoter to increased JNK activity induced by overexpression of MEKK-1 (n = 4; **P < 0.005).

 
JNK Inhibition Reverses Hypoxia but not Cobalt-Induced Hypoxia-Inducible Factor 1 Transcriptional Activity.
HeLa cells were transiently transfected with HRE-luciferase before exposure to CoCl2 (0 to 100 µmol/L; 24 hours). CoCl2 caused a dose-dependent increase in HIF-1 transcriptional activity (9.90- ± 5.14-fold increase for 100 µmol/L), which was not significantly inhibited by the presence of SP600125 (6.7- ± 3.8-fold). Hypoxia-induced HIF-1 binding to the HRE (463 ± 141% increase over normoxia) is significantly inhibited by SP600125 (147 ± 46% increase over normoxia; Fig. 5ACitation ), whereas CoCl2-induced HIF-1 DNA binding (100 µmol/L; 458 ± 22% increase over normoxia) is unaltered by the presence of SP600125 (430 ± 18% increase over normoxia; Fig. 5ACitation ). Furthermore, HIF-1 stabilization induced by hypoxia is partially inhibited by the presence of SP600125 (Fig. 5B)Citation . Collectively, these data support a role for JNK in hypoxia-dependent HIF-1 stabilization.



View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. JNK inhibition inhibits hypoxia- but not cobalt-induced HIF-1 DNA binding activity. A. HeLa cells were exposed to hypoxia (24 hours) or CoCl2 (100 µmol/L; 24 hours) in the presence or absence of SP600125. Following exposure to stimuli, nuclear extracts were generated, and nuclear HIF-1/HRE binding was measured using an ELISA-based method to measure HIF-1 binding to an immobilized HRE sequence. The JNK inhibitor SP600125 reversed hypoxia-induced HIF-1 DNA binding but was without effect on cobalt-induced HIF-1 DNA binding (n = 3). B. HeLa cells were exposed to hypoxia in the presence and absence of the JNK inhibitor SP600125. SP600125 inhibited HIF-1 protein accumulation in response to hypoxia as determined by SDS-PAGE (n = 2).

 
Antioxidants Do Not Inhibit JNK or Hypoxia-Inducible Factor Activation in Hypoxia.
To investigate the possible role of ROS in mediating the effects of hypoxia on JNK activation, we investigated the impact of ROS scavengers N-acetylcysteine (NAC) and glutathione (GTH) on hypoxia-elicited JNK phosphorylation. Although hypoxia and addition of exogenous H2O2 caused an increase in JNK phosphorylation, hypoxia-induced activity was unaltered in cells pretreated with the ROS scavengers NAC (5 mmol/L) or GTH (5 mmol/L; Fig. 6ACitation ). Furthermore, NAC and GTH were without effect on hypoxia-induced HRE activity (Fig. 6B)Citation . These data suggest that although JNK activation occurs in response to increased ROS production, this is not the signaling mechanism involved in mediating the hypoxic response.



View larger version (37K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. ROSs are not involved in hypoxia-induced JNK or HIF-1 activation. A. HeLa cells were exposed to normoxia, hypoxia, or hydrogen peroxide (H2O2) in the absence or hypoxia in the presence of the antioxidants NAC or GTH. Cells exposed to hypoxia or H2O2 (0 to 100 µmol/L) showed an increased JNK phosphorylation (Lanes 2, 5, and 6). The response to hypoxia was unaltered by the presence of NAC or GTH (5 mmol/L each; Lanes 3 and 4). B. HeLa cells were transfected with a luciferase reporter construct under the control of multiple HREs before exposure to hypoxia in the presence or absence of the antioxidants NAC or GTH. Hypoxia-elicited HRE-luciferase activity was not significantly altered in the presence of NAC or GTH (5 mmol/L; n = 3; P = NS).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Increased P-glycoprotein expression in hypoxia may contribute significantly to chemotherapeutic resistance in developing tumors. We have shown previously that functional up-regulation of P-glycoprotein depends on HIF-1 activation; however, the underlying molecular mechanisms remain unknown. Because hypoxia activates the stress-activated protein kinase JNK and because JNK has been associated previously with growth factor-mediated HIF-1 activation and increased expression of MDR1 (19) , we investigated whether JNK has a role in hypoxia-induced HIF-1 and subsequent MDR1 activation.

Initially, we showed in vitro and in vivo that hypoxia increases JNK phosphorylation and subsequent activation. This observation is consistent with previous studies that document increased JNK activity in hypoxia (10, 11, 12, 13, 14, 15, 16, 17, 18) . JNK has been implicated in hypoxia-induced apoptosis (10 , 17) and ischemic brain injury (12) . Although the molecular mechanism(s) through which JNK mediates its effects in hypoxia remains to be fully elucidated, it appears that the transcription factors activator protein–1 and c-Jun are important (18) . Recent data also have implicated a positive role for nitric oxide (NO) in mediating JNK phosphorylation in hypoxia (13) . We recently have implicated NO as a pivotal regulator of the cellular hypoxic HIF-1 response through the regulation of intracellular oxygen availability (28) . Thus, hypoxia-elicited JNK activation may be under the control of endogenous levels of NO.

ROS have been implicated in the activation of JNK and MDR1 (29 , 30) . Furthermore, it has been reported that mitochondrial ROS production is increased in hypoxia (31) . Thus, we investigated whether ROS were responsible for hypoxic activation of JNK. Our findings support the concept that JNK is ROS sensitive because the addition of exogenous H2O2 resulted in JNK activity; however, ROS scavengers did not alter hypoxia-elicited JNK or HIF-1 activation, indicating that ROS production is not a primary signaling mechanism to JNK in hypoxia.

The mechanism by which JNK activation leads to altered HIF-1 accumulation and activity remains to be fully elucidated. Previous work has implicated a direct effect of activated JNK on HIF (22) or an indirect effect mediated through c-Jun activation. In each case, however, JNK activation correlated positively with HIF-1–dependent transcription.

Interestingly, the inhibitory effects of SP600125 were specific for hypoxia-induced HIF-1 activation and were not effective in cobalt-induced activation. It is likely that although the signaling mechanisms activated by hypoxia and cobalt result in the same endpoint (HIF-1{alpha} stabilization), the initial sensing mechanisms are different. Although hypoxia-induced HIF-1 activation is likely to involve a mitochondrial component (because this is the primary consumer of oxygen in the cell; ref. 32 ), cobalt directly inhibits the prolyl hydroxylase enzymes that direct HIF-1{alpha} to degradation in the presence of oxygen (2) . Thus, it is likely that role of JNK in HIF-1 activation occurs at a site upstream of prolyl hydroxylase activity. Future work will determine whether JNK specifically alters mitochondrial oxygen consumption through the regulation of oxidative phosphorylation.

Finally, we and others have shown previously that a range of genes are under the control of the transcriptional regulator HIF-1 in response to hypoxia (24) . These genes are generally directed toward the induction of a protective or adaptive response to hypoxia and support tissue survival. In developing tumors, this may represent a maladaptive response promoting tumor vascularization and survival. Increased P-glycoprotein expression may exacerbate this phenotype through the induction of chemotherapeutic resistance.


   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.

Requests for reprints: Cormac T. Taylor, Department of Medicine and Therapeutics, The Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland. Phone: 353-1-716-6732; Fax: 353-1-716-6701; E-mail: cormac.taylor{at}ucd.ie

Received 6/ 3/04. Revised 10/ 4/04. Accepted 10/12/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Semenza GL Targeting HIF-1 for cancer therapy. Nat Rev Cancer 2003;3:721-32.[CrossRef][Medline]
  2. Schofeild CJ, Ratcliffe PJ Oxygen sensing by HIF hydroxylases. Nat Rev Mol Cell Biol 2004;5:343-54.[CrossRef][Medline]
  3. Semenza GL Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev 1998;8:558-94.
  4. Koch S, Mayer F, Honecker F, Schittenhelm M, Bokemeyer C Efficacy of cytotoxic agents used in the treatment of testicular germ cell tumors under normoxic and hypoxic conditions in vitro. Br J Cancer 2003;89:2133-9.[CrossRef][Medline]
  5. Kalr R, Jones AM, Kirk J, Adams GE, Stratford IJ The effect of hypoxia on acquired drug resistance and response to epidermal growth factor in Chinese hamster lung fibroblasts and human breast cancer cells in vitro. Int J Cancer 1993;54:650-5.[Medline]
  6. Liang BC Effects of hypoxia on drug resistance phenotype and genotype in human glioma cell lines. J Neurooncol 1996;29:149-55.[Medline]
  7. Comerford KM, Wallace TJ, Karhausen J, Louis NA, Montalto MC, Colgan SP Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene. Cancer Res 2002;62:3387-94.[Abstract/Free Full Text]
  8. Wartenberg M, Ling FC, Muschen M, et al Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) and reactive oxygen species. FASEB J 2003;17:503-5.[Abstract/Free Full Text]
  9. Davis RJ Signal transduction by the c-Jun N-terminal kinase. Biochem Soc Symp 1999;64:1-12.[Medline]
  10. Kunz M, Ibrahim S, Koczan D, et al Activation of c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) is critical for hypoxia-induced apoptosis of human malignant melanoma. Cell Growth Differ 2001;12:137-45.[Abstract/Free Full Text]
  11. Jin N, Hatton N, Swartz DR, et al Hypoxia activates jun-N-terminal kinase, extracellular signal-regulated protein kinase, and p38 kinase in pulmonary arteries. Am J Respir Cell Mol Biol 2000;23:593-601.[Abstract/Free Full Text]
  12. Kuan CY, Whitmarsh AJ, Yang DD, et al A critical role of neural-specific JNK3 for ischemic apoptosis. Proc Natl Acad Sci USA 2003;100:15184-9.[Abstract/Free Full Text]
  13. Mishra OP, Zubrow AB, Ashraf QM Nitric oxide-mediated activation of extracellular signal-regulated kinase (ERK) and c-jun N terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of newborn piglets. Neuroscience 2004;123:179-86.[CrossRef][Medline]
  14. Laderoute KR, Calaoagan JM, Knapp M, Johnson RS Glucose utilization is essential for hypoxia-inducible factor 1 {alpha}-dependent phosphorylation of c-jun. Mol Cell Biol 2004;24:4128-37.[Abstract/Free Full Text]
  15. Seko Y, Takahashi N, Tobe K, Kadowaki T, Yazaki Y Hypoxia and hypoxia/reoxygenation activate p65PAK, p38 mitogen activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) in cultured rat cardiac myocytes. Biochem Biophys Res Comm 1997;239:804-44.[CrossRef][Medline]
  16. Le YJ, Corry PM Hypoxia-inducible bFGF gene expression is mediated through the JNK signal transduction pathway. Mol Cell Biochem 1999;202:1-8.[CrossRef][Medline]
  17. Garay M, Gaarde W, Monia BP, Nero P, Cioffi CL Inhibition of hypoxia/reoxygenation-induced apoptosis by an antisense oligonucleotide targeted to JNK1 in human kidney cells. Biochem Pharmacol 2000;59:1033-43.[CrossRef][Medline]
  18. Minet E, Michel G, Mottet D, et al c-JUN gene induction and AP-1 activity is regulated by a JNK-dependent pathway in hypoxic HepG2 cells. Exp Cell Res 2001;265:114-24.[CrossRef][Medline]
  19. Osborn MT, Chambers TC Role of the stress-activated/c-Jun NH2-trminal kinase pathway in the cellular response to adriamycin and other chemotherapeutic drugs. J Biol Chem 1996;271:30950-5.[Abstract/Free Full Text]
  20. Alfranca A, Gutierrez MD, Vara A, Aragones J, Vidal F, Landazuri MO c-jun and hypoxia-inducible factor 1 functionally cooperate in hypoxia-induced gene transcription. Mol Cell Biol 2002;22:12-22.[Abstract/Free Full Text]
  21. Poulaki V, Mitsiades CS, McMullan C, et al Regulation of vascular endothelial growth factor expression by insulin-like growth factor I in thyroid carcinomas. J Clin Endocrinol Metab 2003;88:5392-8.[Abstract/Free Full Text]
  22. Tacchini L, Matteucci E, De Ponti C, Desiderio MA Hepatocyte growth factor signaling regulates transactivation of genes belonging to the plasminogen activation system via hypoxia inducible factor 1. Exp Cell Res 2003;290:391-401.[CrossRef][Medline]
  23. Tacchini L, Dansi P, Matteucci E, Desiderio MA Hepatocyte growth factor signaling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells. Carcinogenesis 2001;22:1363-71.[Abstract/Free Full Text]
  24. Leonard MO, Cottell DC, Godson C, Brady HR, Taylor CT The role of HIF-1{alpha} in transcriptional regulation of the proximal tubular epithelial cell response to hypoxia. J Biol Chem 2003;278:40296-304.[Abstract/Free Full Text]
  25. Taylor CT, Furuta GT, Synnestvedt K, Colgan SP Phosphorylation-dependent targeting of cAMP response element binding protein to the ubiquitin/proteasome pathway in hypoxia. Proc Natl Acad Sci USA 2000;97:12091-6.[Abstract/Free Full Text]
  26. Taylor CT, Fueki N, Agah A, Hershberg RM, Colgan SP Critical role of the cAMP response element binding protein in hypoxia-elicited induction of epithelial tumor necrosis factor-{alpha}. J Biol Chem 1999;274:19447-54.[Abstract/Free Full Text]
  27. Comerford KM, Leonard MO, Karhausen J, Carey R, Colgan SP, Taylor CT Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia. Proc Natl Acad Sci USA 2003;100:986-91.[Abstract/Free Full Text]
  28. Hagen T, Taylor CT, Lam F, Moncada S Redistribution of intracellular oxygen in hypoxia by nitric oxide: effect on HIF-1{alpha}. Science 2003;302:1975-8.[Abstract/Free Full Text]
  29. Felix RA, Barrand MA P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress. J Neurochem 2002;80:64-72.[CrossRef][Medline]
  30. Dougherty CJ, Kubasiak LA, Prentice H, et al Activation of c-jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress. Biochem J 2002;362:561-71.[CrossRef][Medline]
  31. Chandel NS, Schumacker PT Cellular oxygen sensing by mitochondria: old questions, new insight. J Appl Physiol 2000;88:1880-9.[Abstract/Free Full Text]
  32. Rolfe DE, Brown GC Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Phys Rev 1997;77:731-58.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Clin. Cancer Res.Home page
C. K. Lau, Z. F. Yang, D. W. Ho, M. N. Ng, G. C.T. Yeoh, R. T.P. Poon, and S. T. Fan
An Akt/Hypoxia-Inducible Factor-1{alpha}/Platelet-Derived Growth Factor-BB Autocrine Loop Mediates Hypoxia-Induced Chemoresistance in Liver Cancer Cells and Tumorigenic Hepatic Progenitor Cells
Clin. Cancer Res., May 15, 2009; 15(10): 3462 - 3471.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Wang, J. Gao, W. Dai, and L. Lu
Activation of Polo-like Kinase 3 by Hypoxic Stresses
J. Biol. Chem., September 19, 2008; 283(38): 25928 - 25935.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S.-N. Jung, W. K. Yang, J. Kim, H. S. Kim, E. J. Kim, H. Yun, H. Park, S. S. Kim, W. Choe, I. Kang, et al.
Reactive oxygen species stabilize hypoxia-inducible factor-1 alpha protein and stimulate transcriptional activity via AMP-activated protein kinase in DU145 human prostate cancer cells
Carcinogenesis, April 1, 2008; 29(4): 713 - 721.
[Abstract] [Full Text] [PDF]

Home page
 J Clin PharmacolHome page
R. Callaghan, E. Crowley, S. Potter, and I. D. Kerr
P-glycoprotein: So Many Ways to Turn It On
J. Clin. Pharmacol., March 1, 2008; 48(3): 365 - 378.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Blanco, C. Santos, and P. A. Lazo
Vaccinia-Related Kinase 2 Modulates the Stress Response to Hypoxia Mediated by TAK1
Mol. Cell. Biol., October 15, 2007; 27(20): 7273 - 7283.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Liu, D. Li, R. Aneja, H. C. Joshi, S. Xie, C. Zhang, and J. Zhou
PO2-dependent Differential Regulation of Multidrug Resistance 1 Gene Expression by the c-Jun NH2-terminal Kinase Pathway
J. Biol. Chem., June 15, 2007; 282(24): 17581 - 17586.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. R. Rezvani, S. Dedieu, S. North, F. Belloc, R. Rossignol, T. Letellier, H. de Verneuil, A. Taieb, and F. Mazurier
Hypoxia-inducible Factor-1{alpha}, a Key Factor in the Keratinocyte Response to UVB Exposure
J. Biol. Chem., June 1, 2007; 282(22): 16413 - 16422.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
C. Fradette, J. Batonga, S. Teng, M. Piquette-Miller, and P. du Souich
Animal Models of Acute Moderate Hypoxia Are Associated with a Down-Regulation of CYP1A1, 1A2, 2B4, 2C5, and 2C16 and Up-Regulation of CYP3A6 and P-glycoprotein in Liver
Drug Metab. Dispos., May 1, 2007; 35(5): 765 - 771.
[Abstract] [Full Text] [PDF]

Home page
 Antimicrob. Agents Chemother.Home page
T. Prasad, A. Chandra, C. K. Mukhopadhyay, and R. Prasad
Unexpected Link between Iron and Drug Resistance of Candida spp.: Iron Depletion Enhances Membrane Fluidity and Drug Diffusion, Leading to Drug-Susceptible Cells
Antimicrob. Agents Chemother., November 1, 2006; 50(11): 3597 - 3606.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
B. Sarkadi, L. Homolya, G. Szakacs, and A. Varadi
Human Multidrug Resistance ABCB and ABCG Transporters: Participation in a Chemoimmunity Defense System.
Physiol Rev, October 1, 2006; 86(4): 1179 - 1236.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
S. M. Mense, A. Sengupta, M. Zhou, C. Lan, G. Bentsman, D. J. Volsky, and L. Zhang
Gene expression profiling reveals the profound upregulation of hypoxia-responsive genes in primary human astrocytes
Physiol Genomics, May 16, 2006; 25(3): 435 - 449.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
J. Zhou, M. Liu, R. Aneja, R. Chandra, H. Lage, and H. C. Joshi
Reversal of P-glycoprotein-Mediated Multidrug Resistance in Cancer Cells by the c-Jun NH2-Terminal Kinase
Cancer Res., January 1, 2006; 66(1): 445 - 452.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Comerford, K. M.
Right arrow Articles by Taylor, C. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Comerford, K. M.
Right arrow Articles by Taylor, C. T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online