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[Cancer Research 61, 3682-3688, May 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Intersection of Interferon and Hypoxia Signal Transduction Pathways in Nitric Oxide-induced Tumor Apoptosis1

Drory S. Tendler, Clare Bao, Tianhong Wang, Eric L. Huang, Edward A. Ratovitski, Drew A. Pardoll and Charles J. Lowenstein2

Division of Cardiology, Department of Medicine [D. S. T., C. B., C. J. L.], and Departments of Oncology [T. W., D. A. P.], and Dermatology [E. A. R.], The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and Division of Hematology and Oncology [E. L. H.], Brigham and Women‘s Hospital, Boston, Massachusetts 02115


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated macrophages play a central role in antitumor immunity. However, the stimuli that activate macrophages to kill tumor cells are not completely understood. Because the center of solid tumors can be hypoxic, we hypothesized that hypoxia may be an important signal in activating macrophages to kill tumor cells. Hypoxia stimulates IFN-primed macrophages to express the inducible nitric oxide synthase (NOS2) and to synthesize nitric oxide (NO). We show that this synergy between IFN and hypoxia is mediated by the direct interaction of the hypoxia inducible factor-1 (HIF-1) and IFN regulatory factor-1 (IRF-1), which are both required for the hypoxic transcription of NOS2. This interaction between HIF-1 and IRF-1 may explain the mechanism by which macrophages infiltrating into tumors are activated to express NOS2 and to produce NO, a mediator of tumor apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages play a critical role in antitumor immunity (1, 2, 3, 4) . Activated macrophages can infiltrate into tumors, express the inducible NOS23 (5, 6, 7) , and produce NO, which kills tumor cells (1 , 4 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27) . CD4+ lymphocytes regulate this antitumor response, in part, by differentiating into Th1 cells and secreting IFN-{gamma} (28 , 29) . However, IFN-{gamma} alone activates macrophages to express minimal levels of NOS2 (30 , 31) .

Previous studies have shown that two signals are necessary to activate maximal transcription of NOS2: IFN-{gamma} and one other stimulus such as endotoxin, TNF-{alpha}, or IL-1ß (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) . These two signals in turn activate at least two transcription factors that interact with the NOS2 5' regulatory region (36 , 37) . Stimuli such as TNF-{alpha} or IL-1ß stimulate NOS2 transcription by activating the transcription factor NF-{kappa}B, which binds to a {kappa}B element in the NOS promoter (31) . IFN-{gamma} synergistically increases NOS2 transcription by activating the transcription factor IRF-1, which binds to elements in the NOS2 promoter (30 , 38) . Synergism of NF-{kappa}B and IRF-1 may be achieved in part by an interaction between these two transcription factors while bound to the NOS2 promoter, thereby altering the physical structure of the NOS2 5' flanking region (51) .

Although both IFN-{gamma} and another stimulus together are necessary for maximal NOS2 transcription, endotoxin or TNF-{alpha} or IL-ß are usually absent from tumor cells. Hypoxia, on the other hand, is often present in the center of tumors that are not well vascularized (52, 53, 54) . Hypoxia cannot by itself induce NOS2 expression in macrophages, but hypoxia and IFN-{gamma} together synergistically induce NOS2 in macrophages (55, 56, 57) . We now demonstrate that the molecular mechanism underlying this synergy is the physical interaction between two transcription factors, HIF-1 and IRF-1. Hypoxia and IFN can induce NOS2 expression in macrophages, leading to NO-mediated apoptosis of tumor cells. This intersection of the hypoxia and IFN signaling pathways may, therefore, promote antitumor immunity within solid tumor masses.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
RAW 264.7 cells were obtained from the American Type Culture Collection. Primary macrophages were prepared as described previously from C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) that had been given injections of thioglycolate (58) . Antibodies to HIF-1{alpha} were purchased from Novus Biologicals (Littleton, CO). Antibodies to IRF-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The cDNA for murine NOS2 was isolated by us (6) . All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).


   Northern Analysis.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Steady-state RNA levels were assayed by Northern analysis from total RNA prepared from RAW 264.7 cells or primary macrophages (51) .


   Cell Culture.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages were stimulated with media alone, 10 ng/ml lipopolysaccharide (Sigma Chemical Co.), 50–100 units/ml IFN-{gamma} (generous gift of American Cancer Society), 75–125 µM CoCl2, 75–125 µM desferrioxamine, 3 mM L-NAME, or a combination of these factors. Cells were cultured under normoxia or hypoxia (5% CO2 and 95% N2) in an Incubator Chamber (Billups-Rothenberg, Del Mar, CA), and were harvested after incubation for up to 24 h.


   Cell Transfection.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
A reporter vector was prepared that contained the murine NOS2 5' regulatory region driving expression of the Photinus luciferase gene (36) . Macrophages were transiently transfected with this reporter construct using Lipofectamine Plus (Life Technologies, Inc., Rockville, MD). A total of 4 µg of plasmid DNA and 12 µl of Lipofectamine was prepared in a 200-µl reaction that was subsequently added to cells in 6-well dishes at a confluence of 60–80%. Cells were cotransfected with a constitutively expressed Renilla luciferase cDNA as an internal standard. Cells were incubated for 6 h at 37°C, fed with DMEM, and incubated for 16 h. The cells were then stimulated with medium alone, 10 ng/ml lipopolysaccharide (Sigma Chemical Co.), 100 units/ml IFN-{gamma} (a generous gift of American Cancer Society), 125 µM CoCl2, 125 µM desferrioxamine, 3 mM L-NAME, or a combination of these factors. Some cells were immediately placed into normoxia or hypoxia (5% CO2 and 95% N2) in an Incubator Chamber (Billups-Rothenberg, Del Mar, CA). Macrophages were harvested after incubation for 6–24 h. Total cell lysates were assayed for Firefly and Renilla luciferase activity using luciferin reagent (Promega) in a luminometer (Turner model 20e; Turner). Hypoxia results were calculated as the ratio of hypoxia:normoxia, corrected for Renilla luciferase transfection efficiency and total protein concentration.


   EMSAs.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Whole-cell extracts prepared from either normoxic or hypoxic RAW cells and subsequent analysis for HIF-1 DNA-binding were performed essentially as described previously (59) . Oligonucleotides derived from the human NOS2 promoter contain a HIF-1 binding site and consist of a sense strand (5'-GATCGTGACTACGTGCTGCCTAGA-3'). The mutant NOS2 oligonucleotides were synthesized by mutating the HIF-1 binding site (underlined above) to TAAATGCT. For oligonucleotide competition, a 100-fold (2-pmol) excess of unlabeled oligonucleotides was used. In supershift of HIF-1 DNA complexes, anti-HIF-1{alpha} (1:20) or anti-ARNT antibodies (Novus Biological) were incubated with cell extracts for 1 h prior to addition of the probes.


   Immunoblotting and Immunoprecipitation.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells were stimulated with media alone or with the combination of 125 µM CoCl2 and 50 units/ml IFN-{gamma} for 4 h at 37°C. Nuclear extracts were prepared as described previously (51) . Some of the nuclear extracts were immunoprecipitated with a primary mouse monoclonal antibody to HIF-1{alpha} (Novus Biologicals). Total nuclear extracts or immunoprecipitated nuclear extracts were fractionated by SDS-PAGE, transferred to a membrane, and then probed with rabbit polyclonal antibody to IRF-1 (Transduction Laboratories, Lexington, KY).


   Yeast Two-Hybrid Analysis.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
Saccharomyces cerevisiae (strain SFY526) was cotransformed with various combinations of bait plasmids (pGal4-BD expressing a fusion protein of the Gal4 binding domain and IRF-1) and prey plasmid (pGal4-AD expressing a fusion protein of the Gal4 activation domain and HIF-1{alpha}). Yeast were grown on agar plates lacking tryptophan and leucine, or lacking tryptophan and leucine and histidine, and then were assayed for ß-galactosidase activity by liquid culture assay method using OPNG as a substrate (nmol OPNG cleaved/min/mg protein measured at A420 nm). Assays were performed in triplicate.


   Apoptosis Assay.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 
RAW 264.7 macrophages were stimulated in 24-well plates (Costar) for 8 h with media alone or with 10 ng/ml lipopolysaccharide (Sigma Chemical Co.), 100 units/ml IFN-{gamma}, 125 µM CoCl2, 3 mM L-NAME, or a combination of these factors. P815 murine mastocytoma cells were plated on inserts (Transwell 24-well plate insert system, Costar) with a membrane diameter of 6.5 mm and a pore size of 0.4 µM; these P815 cells on inserts were then placed into the 24-well plates containing stimulated macrophages. Each well contained 9 x 105 macrophages and 3.75 x 104 P815 cells for a ratio of killer:target cells of 24:1. Cells were cocultured for 12, 18, and 24 h. Tumor cells were then removed and analyzed by FACS for Annexin V staining and propidium iodide staining: early apoptotic cells were defined as Annexin V positive (upper right quadrant), and late apoptotic cells were defined as Annexin V positive and propidium iodide positive (lower right quadrant). The amount of nitrite produced by stimulated macrophages was measured by the Griess assay.


   In Vivo Tumor Staining.
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
RESULTS
 DISCUSSION
 REFERENCES
 
Female C57BL/6 mice, 6–8 weeks old, were given injections of 106 B16 melanoma cells transduced with the GM-CSF gene (B16-GM-CSF) s.c. in two upper arms and the left flank; they were then irradiated (50 Gy). Five x 107 nontransduced B16 cells (B16WT) were injected in the right flank 1 week later. This tumor was removed 4 days later and flash-frozen in O.C.T. compound. Tissue sections cut at 5 µm were fixed, quenched with 0.05% H2O2, and then blocked with milk and 1% normal goat serum and goat antibody to mouse IgG in Tris-buffered saline at 22°C for 1 h. The sections were then incubated with murine antibody to HIF-1{alpha} (Novus Biologicals), rabbit antibody to NOS2 (Transduction Laboratories), or rabbit antibody to mac-3. Sections were then incubated with goat antibody to mouse IgG conjugated to FITC for HIF-1 localization, or goat antibody to rabbit IgG conjugated to Cy 3 for NOS2 localization (Jackson ImmunoResearch Laboratories, West Grove, PA). Images of these sections were acquired using a laser scanning confocal microscope (Zeiss LSM 410 confocal microscope).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
DISCUSSION
 REFERENCES
 
Hypoxic Induction of NOS2 mRNA Requires IFN Priming of Macrophages.
To confirm that hypoxic induction of NOS2 in macrophages requires IFN-{gamma}, RAW macrophages were incubated with medium alone, IFN-{gamma} alone, hypoxia alone, or both IFN-{gamma} and hypoxia. RNA from stimulated cells was analyzed by Northern analysis with a radiolabeled NOS2 cDNA probe. Hypoxia or IFN-{gamma} alone did not induce NOS2 in macrophages, but both together led to a major increase in macrophages of NOS2 steady-state mRNA levels, comparable with the levels seen in the positive control of LPS and IFN-{gamma} (Fig. 1Citation ; Refs. 55, 56, 57 ).



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  		Fig. 1. IFN-{gamma} and hypoxia regulation of NOS2 mRNA in macrophages. Induction of NOS2 mRNA in macrophages by IFN-{gamma} and hypoxia. Macrophages were treated with IFN-{gamma}, or hypoxia, or both, and total RNA was analyzed by Northern blotting for NOS2.

 
Hypoxic Transactivation of NOS2 Regulatory Region Requires Both IRF and HIF-1 Binding Elements.
We next showed that hypoxic activation of NOS2 transcription requires binding sites for IRF-1 and HIF-1 on the NOS2 promoter region (30 , 38) We transfected macrophages with vectors expressing luciferase driven by (a) the wild-type NOS2 promoter, or (b) by a mutant NOS2 promoter lacking an IRE binding site for IRF-1, or (c) by a mutant NOS2 promoter lacking a HRE binding site for HIF-1. Transfected macrophages were stimulated with hypoxia, IFN-{gamma}, or both. Only hypoxia and IFN-{gamma} together activated the wild-type NOS2 promoter (data not shown). However, hypoxia and IFN-{gamma} together did not activate mutant NOS2 promoters lacking either the HRE or the IRE (data not shown). These data confirm that HIF-1 and IRF can regulate NOS2 transcription (55, 56, 57) .

HIF-1 Interacts with IRF-1 in Macrophages.
Our results above show that both hypoxia and IFN-{gamma} synergistically induce NOS2. Because hypoxia and IFN-{gamma} activate the transcription factors HIF-1 and IRF-1, respectively, and because binding sites for both transcription factors are necessary for transactivation of the NOS2 regulatory region, we hypothesized that HIF-1 and IRF-1 interact with each other.

Accordingly, we immunoprecipitated nuclear extracts from both RAW macrophages and primary peritoneal macrophages with antibody to HIF-1{alpha}, and then immunoblotted the immunoprecipitants with antibody to IRF-1 (Fig. 2A)Citation . IRF-1 is present in protein complexes immunoprecipitated by antibody to HIF-1, which suggests that HIF-1 and IRF-1 interact in stimulated cells (Fig. 2A, Lanes 4 and 8)Citation but not in resting cells (Fig. 2A, Lanes 3 and 7)Citation . [As others have shown, IRF-1 is phosphorylated at several different sites, which may explain the presence of two bands in the IRF-1 immunoblot (60) .]



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  		Fig. 2. Interaction of HIF-1 and IRF-1 in macrophages. A, interaction of HIF-1 and IRF-1 by immunoprecipitation. RAW macrophages or primary macrophages (1° M{Phi}) were stimulated with CoCl2 and IFN-{gamma}, and nuclear extracts were isolated, immunoprecipitated with antibody to HIF-1{alpha}, and then immunoblotted with antibody to IRF-1. B, interaction of HIF-1 with HRE by EMSA. Nuclear extracts from RAW macrophages stimulated with hypoxia (H) and IFN-{gamma} (I) were incubated with a radiolabeled HRE oligonucleotide, fractionated, and exposed to film. Some reactions were also incubated with a nonlabeled wild-type (wt) HRE or mutant (mut) HRE oligonucleotide. Some reactions were incubated with antibody to HIF-1-{alpha} or its binding partner ARNT. C, interaction of HIF-1 and IRF-1 by EMSA. Nuclear extracts from RAW macrophages that were stimulated with hypoxia and IFN-{gamma} (H+I) were incubated with a radiolabeled HRE oligonucleotide, fractionated, and exposed to film. Some reactions were also incubated with an antibody to IRF-1, C/EBP, or the p65 subunit of NF-{kappa}B.

 
To further explore the interaction of HIF-1 and IRF-1, we repeated the EMSA as above, adding various antibodies to nuclear extracts and the radiolabeled HRE oligonucleotide of the NOS2 5' regulatory region. Nuclear extracts from macrophages stimulated with hypoxia and IFN-{gamma} contained HRE oligonucleotide binding activity (Fig. 2B, Lane 2)Citation . Because HIF-1 is a heterodimer composed of HIF-1{alpha} and ARNT, we next examined whether or not antibodies to each component of HIF-1 could alter the mobility of the HIF-1/HRE oligonucleotide complex. The antibody to HIF-1{alpha} shifted the mobility of this HRE oligonucleotide, confirming that HIF-1{alpha} binds to its HRE site (Fig. 2B, Lane 5)Citation . The antibody to ARNT, the binding partner of HIF-1{alpha}, did not produce a supershift, but did abolish the original binding of HIF-1{alpha} to the HRE oligonucleotide (Fig. 2B, Lane 6)Citation . Finally, we examined whether or not IRF-1 would interact with the HIF-1/HRE oligonucleotide complex. The antibody to IRF-1 did not alter the mobility of the HRE oligonucleotide but did reduce the intensity of the binding of HIF-1 and the HRE oligonucleotide, which suggested that IRF-1 might be a component of the HIF-1 complex (Fig. 2C, Lane 3)Citation . These data suggest that HIF-1 interacts with the HRE site in the NOS2 5' regulatory region, and that IRF-1 interacts with the HIF-1/HRE nucleoprotein complex.

It has been shown that an assembly of transcription factors, coactivators, and scaffolding proteins forms on the 5' regulatory region of certain genes, forming a nucleoprotein complex called an "enhanceosome" (61, 62, 63, 64, 65, 66, 67) . This enhanceosome increases the transactivation of genes such as ß-IFN or ICAM-1. Nathan and others have shown that both NF-{kappa}B and IRF-1 are critical factors in the activation of NOS2 transcription (30 , 31 , 38) . We and others have shown that an enhanceosome involving these and other proteins may form on the 5' NOS2 regulatory region as well (36 , 51 , 68, 69, 70, 71) .

To explore the presence of additional proteins in the HIF-1/IRF-1 complex, we repeated the EMSA studies with a radiolabeled 18-bp probe containing the HRE of the NOS2 5' regulatory region and nuclear extracts from RAW macrophage cells exposed to hypoxia. We added various antibodies to the nuclear extracts, including antibodies to the p65 subunit of NF-{kappa}B and to C/EBP. None of these antibodies affected the mobility of the HRE oligonucleotide (Fig. 2C)Citation . Antibodies to NF-IL6, AP-1, and HMG-YI also do not affect the mobility of the HRE oligonucleotide (data not shown). Although HIF-1 has been shown by others (59 , 72, 73, 74, 75) to interact with p300, the antibody to p300 does not bind to proteins coimmunoprecipitated with HIF-1 (data not shown).

HIF-1 Interacts with IRF-1 in Yeast.
To explore whether or not IRF-1 directly interacts with HIF-1, we next used the yeast two-hybrid system. Yeast were cotransfected with one plasmid encoding a fusion protein of the Gal4 binding domain and IRF-1, and with another plasmid encoding a fusion protein of the Gal4 activation domain and HIF-1{alpha}. Yeast grew only on histidine-deficient media if it was cotransfected with both plasmids expressing IRF-1 and HIF-1{alpha}, but not if it was transfected either with plasmid alone or with the negative control plasmids (Fig. 3)Citation . Quantitation of ß-galactosidase activity shows that IRF-1 and HIF-1{alpha} can also interact in yeast (Table 1)Citation .



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  		Fig. 3. Interaction of HIF-1 and IRF-1 in yeast. Yeast were cotransfected with a plasmid expressing a fusion protein consisting of Gal4 binding domain and IRF-1, and also a plasmid expressing a fusion protein consisting of Gal4 activation domain and HIF-1. Yeast was plated out on Trp/Leu/His-deficient media and photographed after 3 days. (Various other plasmids were used as negative controls, expressing Gal4BD alone or Gal4AD alone; for positive controls, yeast were cotransfected with plasmids expressing Gal4BD-p53 and Gal4AD-SV40T.)

 

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  		Table 1 Interaction between IRF-1 and HIF-1{alpha} in yeast

 
HIF-1 Interacts with IRF-1 in Vitro.
To further demonstrate that HIF-1{alpha} directly interacts with IRF-1, we examined the interaction between recombinant HIF-1{alpha} and IRF-1. HIF-1{alpha} was expressed in an in vitro transcription and translation system supplemented with [35S]methionine. Nonlabeled IRF-1 was also expressed in an in vitro transcription and translation system. IRF-1 (or a control transcription/translation solution) was incubated with [35S]HIF-1{alpha} (or with a control transcription/translation solution), and then immunoprecipitated with antibody to IRF-1. Precipitants were fractionated by SDS-PAGE and autoradiographed. IRF-1 interacts with HIF-1{alpha} in vitro (Fig. 4, Lane 8)Citation .



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  		Fig. 4. Interaction of recombinant HIF-1 and IRF-1 in vitro. [35S]HIF-1{alpha} and nonlabeled IRF-1 were synthesized by an in vitro transcription and translation system. HIF-1{alpha} (or control mixture) and IRF-1 (or control mixture) were incubated together, and then IRF-1 along with any interacting proteins were immunoprecipitated with antibody to IRF-1, and agarose beads coupled to antibody to IgG. Precipitants were then fractionated on SDS-PAGE and autoradiographed. Lanes 1–5, controls that were loaded directly from the transcription/translation reaction without immunoprecipitation: Lane 1, no addition; Lane 2, input of [35S]HIF-1{alpha}; Lane 3, input of IRF-1; Lane 4, input of empty HIF-1 expression vector; Lane 5, input of empty IRF-1 expression vector. Lanes 6–10, precipitants of [35S]HIF-1{alpha} alone (Lane 6); IRF-1 alone (Lane 7); [35S]HIF-1{alpha} and IRF-1 (Lane 8); empty HIF-1 expression vector and IRF-1 (Lane 9); and [35S]HIF-1{alpha} and empty IRF-1 expression vector (Lane 10).

 
These data in vitro, in yeast, and in macrophages together suggest that IRF-1 and HIF-1 interact directly with each other.

Hypoxic Induction of RAW Macrophages Mediates NOS2-induced Tumor Apoptosis.
Although NO that is derived from stimulated macrophages can kill tumor cells, the ability of macrophages to kill tumor cells in a hypoxic environment is unknown (4 , 8, 9, 10, 11, 12, 13) . Furthermore, under certain conditions, NO can protect cells and inhibit apoptosis (76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87) . To establish the physiological relevance of the HIF-1 and IRF-1 interaction in antitumor immunity, stimulated macrophages were cocultured with tumor cells. Macrophages were stimulated with IFN-{gamma}, CoCl2, or both. [CoCl2 induces the expression of hypoxia-inducible genes such as erythropoietin (88, 89, 90, 91, 92, 93, 94) . CoCl2 activates HIF-1 and HIF-1-dependent gene expression (95, 96, 97) . The precise mechanism by which CoCl2 activates HIF-1 is unknown.] Then P815 mastocytoma cells were added to porous inserts overlaying the stimulated macrophages. This system ensured the absence of direct cell-to-cell contact between the macrophages and tumor cells, although permitting the diffusion of soluble molecules across the porous membrane. The tumor cells were removed after 24-h exposure to macrophages and were analyzed by FACS for Annexin V and propidium iodine staining, markers for early and late apoptosis, respectively. Medium from the stimulated macrophages was analyzed for NO2-, a metabolite of NO. Only macrophages stimulated with both CoCl2 and IFN-{gamma} induce tumor apoptosis (Fig. 5)Citation . NO (or a derivative of NO) is the mediator responsible for macrophage killing of tumor cells, because L-NAME, an inhibitor of NOS, inhibits tumor apoptosis.



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  		Fig. 5. Synergistic induction by IFN-{gamma} and hypoxia of tumoricidal NOS activity. Macrophages were stimulated for 8 h with IFN-{gamma}, CoCl2, or both. P815 mastocytoma cells were added to porous inserts overlaying the stimulated macrophages and cocultured for 24 h. The NOS inhibitor L-NAME was added to some cultures. NO2- was measured in the media of stimulated macrophages by the Griess reaction. Apoptosis in P815 cells was then measured by FACS analysis after Annexin V and propidium iodine staining. (n = 3 ± SD; *, P < 0.03 for CoCl2 + IFN compared with medium alone.)

 
In Vivo Evidence for HIF-1 Activation of NOS2 in Tumor-infiltrating Macrophages.
If hypoxia regulates NOS2 expression in tumoricidal macrophages, then HIF-1 and NOS2 should colocalize in macrophages infiltrating into tumors. We have previously shown that immunization of mice with GM-CSF-transduced tumor vaccines induces Th1-dependent immune responses, manifest by expression of NOS2 in macrophages that infiltrate into sites of metastatic disease (2) . In this vaccine system, NOS2 expression by macrophages was demonstrated to be dependent on IFN-{gamma}, because infiltrating macrophages in vaccinated IFN-{gamma}-/- animals failed to express NOS2. A direct role for NO in tumor killing was confirmed by the partial abrogation of vaccine-induced antitumor immunity in NOS2-/- mice (2) . We, therefore, used this vaccination system to evaluate whether or not tumor-infiltrating macrophages in which NOS2 was induced display evidence of HIF-1 expression. Mice, vaccinated with B16 melanoma cells constitutively expressing GM-CSF, were challenged 1 week later at a distant site with wild-type B16 tumor cells. The tumor was excised after 4 days, embedded, sectioned, and analyzed by laser scanning confocal microscopy for the presence of both HIF-1 and NOS2 in the same tissue section.

NOS2 and HIF-1 are colocalized in cells within the tumor (Fig. 6)Citation . Indeed, colocalization studies demonstrated that every infiltrating cell that was NOS2 positive also expressed high levels of HIF-1 (Fig. 6)Citation . Furthermore, the cells that express both NOS2 and HIF-1 contain NOS2 in the cytoplasm and HIF-1 in the nucleus. This pattern of expression matches prior observations of others that NOS2 is located in the cytoplasm, low levels of HIF-1 are present in the cytoplasm of normoxic cells, and higher levels of HIF-1 are located in the nucleus of hypoxic cells.



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  		Fig. 6. Localization of HIF-1, IRF-1, and NOS2 in murine tumors. Mice were given injections of B16 tumors expressing GM-CSF and, after 1 week, injections of 5 x 107 B16 cells. Tumors were excised after 4 days, fixed, and sectioned. Each section was analyzed by laser scanning confocal microscopy after hybridization with antibodies for (A) HIF-1 or (B) NOS2. C, the two images of the same section were then superimposed to reveal colocalization of HIF-1 and NOS2. Controls that showed no fluorescence included serial sections hybridized with secondary antibodies but no primary antibodies (not shown).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
REFERENCES
 
Recent reports have demonstrated that antitumor activity is critically dependent on the CD4+ T cell, which coordinates the antitumor response of multiple effector cells, including macrophages. The importance of NOS2 expression and NO production by tumor-infiltrating macrophages is now appreciated as a mechanism of immune mediated tumor killing (1 , 2) . Our data suggest a novel mechanism for activating the tumoricidal activity of macrophages. Once primed by IFN-{gamma}-producing CD4+ T cells, macrophages are stimulated by intratumor hypoxia to express NOS2 and to produce NO. Our demonstration of a direct protein-protein interaction between HIF-1 and IRF-1 reveals a mechanism for this synergism.

Although the partial pressure of oxygen within tumors is heterogeneous and difficult to assess, hypoxia can activate HIF-1-dependent gene transcription in tumors (98 , 99) . Recent work has shown that HIF-1 is expressed in a variety of human tumors, including prostate, brain, bladder, breast, colon, ovarian, pancreatic, and renal carcinomas (100, 101, 102, 103) . HIF-1 is not only expressed in the neoplastic cells within tumors but is also found in macrophages infiltrating into the tumor (100) . These observations support our findings that HIF-1 mediates hypoxic activation of NOS2 expression in tumors in vivo.

The intersection of the oxygen-sensing and IFN-{gamma} signal pathways may restrict production of NO by primed macrophages to hypoxic regions of the tumor. This mechanism emphasizes the potential role of NOS2 as an antitumor effector molecule within hypoxic regions of tumor in which other components of the immune system such as CTLs may display diminished function. Cooperation between hypoxia and IFN-{gamma} signaling pathways may also be of potential importance in the Th1-dependent killing of intracellular pathogens such as mycobacteria, which often reside in poorly vascularized, hypoxic granulomas. Furthermore, it is intriguing to speculate that the inflammatory response at sites of infarction might result in IFN production which, together with hypoxia, would enhance the production of NO, a mediator of compensatory vasodilation.


   ACKNOWLEDGMENTS
 
We thank Dr. H. F. Bunn for the HIF-1 cDNA constructs, and Dr. Paula Pitha for the IRF-1 cDNA.


   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 Supported in part by an America Heart Association grant (to D. S. T.), by NIH Grants P50 HL52315 (to C. J. L.), NIH R01 HL53615 (to C. J. L.), by the Ciccarone Center for the Prevention of Heart Disease (to C. J. L.), and by the Cora and John H. Davis Foundation (to C. J. L). This work was also supported by a National Cancer Institute Prostate SPORE (Specialized Program of Research Excellence in Prostate Cancer, Grant CA58236), and gifts by the Topercer and Needle families. Back

2 To whom requests for reprints should be addressed, Division of Cardiology, The Johns Hopkins University School of Medicine, 950 Ross Building, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 955-1530; Fax: (410) 614-5129; E-mail: clowenst{at}jhmi.edu Back

3 The abbreviations used are: NOS2, NO synthase; NO, nitric oxide; HIF-1, hypoxia inducible factor-1; IRF-1, IFN regulatory factor-1; TNF-{alpha}, tumor necrosis factor-{alpha}; NF-{kappa}B, nuclear factor {kappa}B; NAME, nitro-arginine methyl ester; FACS, fluorescence-activated cell sorting; EMSA, electrophoretic mobility shift assay; HRE, HIF-1 response element; ARNT, aryl hydrocarbon receptor nuclear translocator; LPS, lipopolysaccharide; IRE, interferon response element; C/EBP, CCAAT/ element binding protein; GM-CSF, granulocyte monocyte colony stimulating factor. Back

Received 11/ 6/00. Accepted 2/27/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 Northern Analysis.
 Cell Culture.
 Cell Transfection.
 EMSAs.
 Immunoblotting and...
 Yeast Two-Hybrid Analysis.
 Apoptosis Assay.
 In Vivo Tumor Staining.
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. MacMicking J., Xie Q. W., Nathan C. Nitric oxide and macrophage function. Annu. Rev. Immunol., 15: 323-350, 1997.[Medline]
  2. Hung K., Hayashi R., Lafond-Walker A., Lowenstein C., Pardoll D., Levitsky H. The central role of CD4(+) T cells in the antitumor immune response. J. Exp. Med., 188: 2357-2368, 1998.[Abstract/Free Full Text]
  3. Pardoll D. M., Topalian S. L. The role of CD4+ T cell responses in antitumor immunity. Curr. Opin. Immunol., 10: 588-594, 1998.[Medline]
  4. Keller R. The macrophage response to infectious agents: mechanisms of macrophage activation and tumour cell killing. Res. Immunol., 144: 271-273, 1993.[Medline]
  5. Xie Q. W., Cho H. J., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Ding A., Troso T., Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science (Wash. DC), 256: 225-228, 1992.[Abstract/Free Full Text]
  6. Geller D. A., Lowenstein C. J., Shapiro R. A., Nussler A. K., Di Silvio M., Wang S. C., Nakayama D. K., Simmons R. L., Snyder S. H., Billiar T. R. Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes. Proc. Natl. Acad. Sci. USA, 90: 3491-3495, 1993.[Abstract/Free Full Text]
  7. Lowenstein C. J., Glatt C. S., Bredt D. S., Snyder S. H. Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme. Proc. Natl. Acad. Sci. USA, 89: 6711-6715, 1992.[Abstract/Free Full Text]
  8. Hibbs J. B., Jr., Taintor R. R., Vavrin Z., Rachlin E. M. Nitric oxide: a cytotoxic activated macrophage effector molecule[published erratum appears in Biochem. Biophys. Res. Commun., 158: 624, 1989]. Biochem. Biophys. Res. Commun., 157: 87-94, 1988.[Medline]
  9. Hibbs J. B., Jr., Taintor R. R., Vavrin Z. Macrophage cytotoxicity: role for L-arginine deiminase and imino nitrogen oxidation to nitrite. Science (Wash. DC)., 235: 473-476, 1987.[Abstract/Free Full Text]
  10. Stuehr D. J., Nathan C. F. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med., 169: 1543-1555, 1989.[Abstract/Free Full Text]
  11. Maeda H., Akaike T. Nitric oxide and oxygen radicals in infection, inflammation, and cancer. Biochemistry (Mosc.), 63: 854-865, 1998.[Medline]
  12. Brune B., von Knethen A., Sandau K. B. Nitric oxide and its role in apoptosis. Eur. J. Pharmacol., 351: 261-272, 1998.[Medline]
  13. Wink D. A., Vodovotz Y., Laval J., Laval F., Dewhirst M. W., Mitchell J. B. The multifaceted roles of nitric oxide in cancer. Carcinogenesis (Lond.), 19: 711-721, 1998.[Abstract/Free Full Text]
  14. Dong Z., Staroselsky A. H., Qi X., Xie K., Fidler I. J. Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells. Cancer Res., 54: 789-793, 1994.[Abstract/Free Full Text]
  15. Kumar R., Yoneda J., Fidler I. J., Dong Z. GM-CSF-transduced B16 melanoma cells are highly susceptible to lysis by normal murine macrophages and poorly tumorigenic in immune-compromised mice. J. Leukoc. Biol., 65: 102-108, 1999.[Abstract]
  16. Xie K., Huang S., Dong Z., Juang S. H., Gutman M., Xie Q. W., Nathan C., Fidler I. J. Transfection with the inducible nitric oxide synthase gene suppresses tumorigenicity and abrogates metastasis by K-1735 murine melanoma cells. J. Exp. Med., 181: 1333-1343, 1995.[Abstract/Free Full Text]
  17. Cui S., Reichner J. S., Mateo R. B., Albina J. E. Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms. Cancer Res., 54: 2462-2467, 1994.[Abstract/Free Full Text]
  18. Yim C. Y., McGregor J. R., Kwon O. D., Bastian N. R., Rees M., Mori M., Hibbs J. B., Jr., Samlowski W. E. Nitric oxide synthesis contributes to IL-2-induced antitumor responses against intraperitoneal Meth A tumor. J. Immunol., 155: 4382-4390, 1995.[Abstract]
  19. Bastian N. R., Yim C. Y., Hibbs J. B., Jr., Samlowski W. E. Induction of iron-derived EPR signals in murine cancers by nitric oxide. Evidence for multiple intracellular targets. J. Biol. Chem., 269: 5127-5131, 1994.[Abstract/Free Full Text]
  20. Xie K., Bielenberg D., Huang S., Xu L., Salas T., Juang S. H., Dong Z., Fidler I. J. Abrogation of tumorigenicity and metastasis of murine and human tumor cells by transfection with the murine IFN-ß gene: possible role of nitric oxide. Clin. Cancer Res., 3: 2283-2294, 1997.[Abstract/Free Full Text]
  21. Xie K., Huang S., Dong Z., Juang S. H., Wang Y., Fidler I. J. Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene. J. Natl. Cancer Inst., 89: 421-427, 1997.[Abstract/Free Full Text]
  22. Xie K., Huang S., Dong Z., Gutman M., Fidler I. J. Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362. Cancer Res., 55: 3123-3131, 1995.[Abstract/Free Full Text]
  23. Yim C. Y., Bastian N. R., Smith J. C., Hibbs J. B., Jr., Samlowski W. E. Macrophage nitric oxide synthesis delays progression of ultraviolet light-induced murine skin cancers. Cancer Res., 53: 5507-5511, 1993.[Abstract/Free Full Text]
  24. Kwon N. S., Stuehr D. J., Nathan C. F. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J. Exp. Med., 174: 761-767, 1991.[Abstract/Free Full Text]
  25. Li L. M., Kilbourn R. G., Adams J., Fidler I. J. Role of nitric oxide in lysis of tumor cells by cytokine-activated endothelial cells. Cancer Res., 51: 2531-2535, 1991.[Abstract/Free Full Text]
  26. Stamler J. S. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell, 78: 931-936, 1994.[Medline]
  27. Stamler J. S., Singel D. J., Loscalzo J. Biochemistry of nitric oxide and its redox-activated forms. Science (Wash. DC), 258: 1898-1902, 1992.[Abstract/Free Full Text]
  28. Del Prete G. The concept of type-1 and type-2 helper T cells and their cytokines in humans. Int. Rev. Immunol., 16: 427-455, 1998.[Medline]
  29. Toes R. E., Ossendorp F., Offringa R., Melief C. J. CD4 T cells and their role in antitumor immune responses. J. Exp. Med., 189: 753-756, 1999.[Free Full Text]
  30. Martin E., Nathan C., Xie Q. W. Role of interferon regulatory factor 1 in induction of nitric oxide synthase. J. Exp. Med., 180: 977-984, 1994.[Abstract/Free Full Text]
  31. Xie Q. W., Kashiwabara Y., Nathan C. Role of transcription factor NF-{kappa}B/Rel in induction of nitric oxide synthase. J. Biol. Chem., 269: 4705-4708, 1994.[Abstract/Free Full Text]
  32. Nathan C., Xie Q. W. Nitric oxide synthases: roles, tolls, and controls. Cell, 78: 915-918, 1994.[Medline]
  33. Nathan C. Inducible nitric oxide synthase: what difference does it make?. J. Clin. Investig., 100: 2417-2423, 1997.[Medline]
  34. Nathan C. Natural resistance and nitric oxide. Cell, 82: 873-876, 1995.[Medline]
  35. de Vera M. E., Shapiro R. A., Nussler A. K., Mudgett J. S., Simmons R. L., Morris S. M., Jr., Billiar T. R., Geller D. A. Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc. Natl. Acad. Sci. USA, 93: 1054-1059, 1996.[Abstract/Free Full Text]
  36. Lowenstein C. J., Alley E. W., Raval P., Snowman A. M., Snyder S. H., Russell S. W., Murphy W. J. Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon {gamma} and lipopolysaccharide. Proc. Natl. Acad. Sci. USA, 90: 9730-9734, 1993.[Abstract/Free Full Text]
  37. Xie Q. W., Whisnant R., Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon {gamma} and bacterial lipopolysaccharide. J. Exp. Med., 177: 1779-1784, 1993.[Abstract/Free Full Text]
  38. Kamijo R., Harada H., Matsuyama T., Bosland M., Gerecitano J., Shapiro D., Le J., Koh S. I., Kimura T., Green S. J. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science (Wash. DC), 263: 1612-1615, 1994.[Abstract/Free Full Text]
  39. Gao J., Morrison D. C., Parmely T. J., Russell S. W., Murphy W. J. An interferon-{gamma}-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-{gamma} and lipopolysaccharide. J. Biol. Chem., 272: 1226-1230, 1997.[Abstract/Free Full Text]
  40. Harada H., Taniguchi T., Tanaka N. The role of interferon regulatory factors in the interferon system and cell growth control. Biochimie (Paris), 80: 641-650, 1998.[Medline]
  41. Taniguchi T., Tanaka N., Taki S. Regulation of the interferon system, immune response and oncogenesis by the transcription factor interferon regulatory factor-1. Eur. Cytokine Netw., 9: 43-48, 1998.[Medline]
  42. Nguyen H., Hiscott J., Pitha P. M. The growing family of interferon regulatory factors. Cytokine Growth Factor Rev., 8: 293-312, 1997.[Medline]
  43. Taniguchi T. IRF-1 and IRF-2 as regulators of the interferon system and cell growth. Indian J. Biochem. Biophys., 32: 235-239, 1995.[Medline]
  44. Vilcek J., Oliveira I. C. Recent progress in the elucidation of interferon-{gamma} actions: molecular biology and biological functions. Int. Arch. Allergy Immunol., 104: 311-316, 1994.[Medline]
  45. Taniguchi T. Regulation of interferon-ß gene: structure and function of cis-elements and trans-acting factors. J. Interferon Res., 9: 633-640, 1989.[Medline]
  46. Mamane Y., Heylbroeck C., Genin P., Algarte M., Servant M. J., LePage C., DeLuca C., Kwon H., Lin R., Hiscott J. Interferon regulatory factors: the next generation. Gene, 237: 1-14, 1999.[Medline]
  47. Pitha P. M., Au W. C., Lowther W., Juang Y. T., Schafer S. L., Burysek L., Hiscott J., Moore P. A. Role of the interferon regulatory factors (IRFs) in virus-mediated signaling and regulation of cell growth. Biochimie (Paris), 80: 651-658, 1998.[Medline]
  48. Taniguchi T. Transcription factors IRF-1 and IRF-2: linking the immune responses and tumor suppression. J. Cell Physiol., 173: 128-130, 1997.[Medline]
  49. Karin M. Signal transduction and gene control. Curr. Opin. Cell Biol., 3: 467-473, 1991.[Medline]
  50. Taylor B. S., de Vera M. E., Ganster R. W., Wang Q., Shapiro R. A., Morris S. M., Jr., Billiar T. R., Geller D. A. Multiple NF-{kappa}B enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene. J. Biol. Chem., 273: 15148-15156, 1998.[Abstract/Free Full Text]
  51. Saura M., Zaragoza C., Bao C., McMillan A., Lowenstein C. J. Interaction of interferon regulatory factor-1 and nuclear factor {kappa}B during activation of inducible nitric oxide synthase transcription. J. Mol. Biol., 289: 459-471, 1999.[Medline]
  52. Rockwell S. Oxygen delivery: implications for the biology and therapy of solid tumors. Oncol. Res., 9: 383-390, 1997.[Medline]
  53. Chapman J. D. Measurement of tumor hypoxia by invasive and non-invasive procedures: a review of recent clinical studies. Radiother. Oncol., 20 (Suppl. 1): 13-19, 1991.
  54. Rockwell S., Moulder J. E. Hypoxic fractions of human tumors xenografted into mice: a review. Int. J. Radiat. Oncol. Biol. Phys., 19: 197-202, 1990.[Medline]
  55. Melillo G., Taylor L. S., Brooks A., Musso T., Cox G. W., Varesio L. Functional requirement of the hypoxia-responsive element in the activation of the inducible nitric oxide synthase promoter by the iron chelator desferrioxamine. J. Biol. Chem., 272: 12236-12243, 1997.[Abstract/Free Full Text]
  56. Melillo G., Taylor L. S., Brooks A., Cox G. W., Varesio L. Regulation of inducible nitric oxide synthase expression in IFN-{gamma}-treated murine macrophages cultured under hypoxic conditions. J. Immunol., 157: 2638-2644, 1996.[Abstract]
  57. Melillo G., Musso T., Sica A., Taylor L. S., Cox G. W., Varesio L. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J. Exp. Med., 182: 1683-1693, 1995.[Abstract/Free Full Text]
  58. Hodge-Dufour J., Noble P. W., Horton M. R., Bao C., Wysoka M., Burdick M. D., Strieter R. M., Trinchieri G., Pure E. Induction of IL-12 and chemokines by hyaluronan requires adhesion- dependent priming of resident but not elicited macrophages. J. Immunol., 159: 2492-2500, 1997.[Abstract/Free Full Text]
  59. Arany Z., Huang L. E., Eckner R., Bhattacharya S., Jiang C., Goldberg M. A., Bunn H. F., Livingston D. M. An essential role for p300/CBP in the cellular response to hypoxia. Proc. Natl. Acad. Sci. USA, 93: 12969-12973, 1996.[Abstract/Free Full Text]
  60. Pine R., Decker T., Kessler D. S., Levy D. E., Darnell J. E., Jr. Purification and cloning of interferon-stimulated gene factor 2 (ISGF2): ISGF2 (IRF-1) can bind to the promoters of both ß interferon- and interferon-stimulated genes but is not a primary transcriptional activator of either. Mol. Cell. Biol., 10: 2448-2457, 1990.[Abstract/Free Full Text]
  61. Merika M., Williams A. J., Chen G., Collins T., Thanos D. Recruitment of CBP/p300 by the IFN ß enhanceosome is required for synergistic activation of transcription. Mol. Cell, 1: 277-287, 1998.[Medline]
  62. Maniatis T., Falvo J. V., Kim T. H., Kim T. K., Lin C. H., Parekh B. S., Wathelet M. G. Structure and function of the interferon-ß enhanceosome. Cold Spring Harbor Symp. Quant. Biol., 63: 609-620, 1998.
  63. Yie J., Merika M., Munshi N., Chen G., Thanos D. The role of HMG I(Y) in the assembly and function of the IFN-ß enhanceosome. EMBO J., 18: 3074-3089, 1999.[Medline]
  64. Carey M. The enhanceosome and transcriptional synergy. Cell, 92: 5-8, 1998.[Medline]
  65. Falvo J. V., Thanos D., Maniatis T. Reversal of intrinsic DNA bends in the IFN ß gene enhancer by transcription factors and the architectural protein HMG I(Y). Cell, 83: 1101-1111, 1995.[Medline]
  66. Thanos D., Maniatis T. Virus induction of human IFN ß gene expression requires the assembly of an enhanceosome. Cell, 83: 1091-1100, 1995.[Medline]
  67. Kim T. K., Maniatis T. The mechanism of transcriptional synergy of an in vitro assembled interferon-ß enhanceosome. Mol. Cell, 1: 119-129, 1997.[Medline]
  68. Spink J., Evans T. Binding of the transcription factor interferon regulatory factor-1 to the inducible nitric-oxide synthase promoter. J. Biol. Chem., 272: 24417-24425, 1997.[Abstract/Free Full Text]
  69. Alley E. W., Murphy W. J., Russell S. W. A classical enhancer element responsive to both lipopolysaccharide and interferon-{gamma} augments induction of the iNOS gene in mouse macrophages. Gene, 158: 247-251, 1995.[Medline]
  70. Henderson S. A., Lee P. H., Aeberhard E. E., Adams J. W., Ignarro L. J., Murphy W. J., Sherman M. P. Nitric oxide reduces early growth response-1 gene expression in rat lung macrophages treated with interferon-{gamma} and lipopolysaccharide. J. Biol. Chem., 269: 25239-25242, 1994.[Abstract/Free Full Text]
  71. Lowenstein C. J., Dinerman J. L., Snyder S. H. Nitric oxide: a physiologic messenger. Ann. Intern. Med., 120: 227-237, 1994.[Abstract/Free Full Text]
  72. Huang L. E., Ho V., Arany Z., Krainc D., Galson D., Tendler D., Livingston D. M., Bunn H. F. Erythropoietin gene regulation depends on heme-dependent oxygen sensing and assembly of interacting transcription factors. Kidney Int., 51: 548-552, 1997.[Medline]
  73. Ema M., Hirota K., Mimura J., Abe H., Yodoi J., Sogawa K., Poellinger L., Fujii-Kuriyama Y. Molecular mechanisms of transcription activation by HLF and HIF-1{alpha} in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBO J., 18: 1905-1914, 1999.[Medline]
  74. Kallio P. J., Okamoto K., O‘Brien S., Carrero P., Makino Y., Tanaka H., Poellinger L. Signal transduction in hypoxic cells: inducible nuclear translocation and recruitment of the CBP/p300 coactivator by the hypoxia-inducible factor-1{alpha}. EMBO J., 17: 6573-6586, 1998.[Medline]
  75. Ebert B. L., Bunn H. F. Regulation of transcription by hypoxia requires a multiprotein complex that includes hypoxia-inducible factor 1, an adjacent transcription factor, and p300/CREB binding protein. Mol. Cell. Biol., 18: 4089-4096, 1998.[Abstract/Free Full Text]
  76. Liu L., Stamler J. S. NO: an inhibitor of cell death. Cell Death Differ., 6: 937-942, 1999.[Medline]
  77. Mannick J. B., Hausladen A., Liu L., Hess D. T., Zeng M., Miao Q. X., Kane L. S., Gow A. J., Stamler J. S. Fas-induced caspase denitrosylation. Science (Wash. DC), 284: 651-654, 1999.[Abstract/Free Full Text]
  78. Mannick J. B., Miao X. Q., Stamler J. S. Nitric oxide inhibits Fas-induced apoptosis. J. Biol. Chem., 272: 24125-24128, 1997.[Abstract/Free Full Text]
  79. Mannick J. B., Asano K., Izumi K., Kieff E., Stamler J. S. Nitric oxide produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus reactivation. Cell, 79: 1137-1146, 1994.[Medline]
  80. Li J., Billiar T. R., Talanian R. V., Kim Y. M. Nitric oxide reversibly inhibits seven members of the caspase family via S-nitrosylation. Biochem. Biophys. Res. Commun., 240: 419-424, 1997.[Medline]
  81. Mohr S., Zech B., Lapetina E. G., Brune B. Inhibition of caspase-3 by S-nitrosation and oxidation caused by nitric oxide. Biochem. Biophys. Res. Commun., 238: 387-391, 1997.[Medline]
  82. Dimmeler S., Haendeler J., Nehls M., Zeiher A. M. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1ß-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J. Exp. Med., 185: 601-607, 1997.[Abstract/Free Full Text]
  83. Kim Y. M., Talanian R. V., Billiar T. R. Nitric oxide inhibits apoptosis by preventing increases in caspase-3- like activity via two distinct mechanisms. J. Biol. Chem., 272: 31138-31148, 1997.[Abstract/Free Full Text]
  84. Li J., Bombeck C. A., Yang S., Kim Y. M., Billiar T. R. Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. J. Biol. Chem., 274: 17325-17333, 1999.[Abstract/Free Full Text]
  85. Kim Y. M., Kim T. H., Seol D. W., Talanian R. V., Billiar T. R. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome c release. J. Biol. Chem., 273: 31437-31441, 1998.[Abstract/Free Full Text]
  86. Tzeng E., Billiar T. R., Williams D. L., Li J., Lizonova A., Kovesdi I., Kim Y. M. Adenovirus-mediated inducible nitric oxide synthase gene transfer inhibits hepatocyte apoptosis. Surgery, 124: 278-283, 1998.[Medline]
  87. Kim Y. M., de Vera M. E., Watkins S. C., Billiar T. R. Nitric oxide protects cultured rat hepatocytes from tumor necrosis factor-{alpha}-induced apoptosis by inducing heat shock protein 70 expression. J. Biol. Chem., 272: 1402-1411, 1997.[Abstract/Free Full Text]
  88. Minchenko A., Caro J. Regulation of endothelin-1 gene expression in human microvascular endothelial cells by hypoxia and cobalt: role of hypoxia responsive element. Mol. Cell. Biochem., 208: 53-62, 2001.
  89. Tsuchiya T., Ueda M., Ochiai H., Imajoh-Ohmi S., Kanegasaki S. Erythropoietin 5'-flanking sequence-binding protein induced during hypoxia and cobalt exposure. J. Biochem. (Tokyo), 113: 395-400, 1993.[Abstract/Free Full Text]
  90. Schuster S. J., Badiavas E. V., Costa-Giomi P., Weinmann R., Erslev A. J., Caro J. Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure. Blood, 73: 13-16, 1989.[Abstract/Free Full Text]
  91. Katsuoka Y., Beckman B., George W. J., Fisher J. W. Increased levels of erythropoietin in kidney extracts of rats treated with cobalt and hypoxia. Am. J. Physiol, 244: F129-F133, 1983.
  92. Fisher J. W., Langston J. W. Effects of testosterone, cobalt and hypoxia on erythropoietin production in the isolated perfused dog kidney. Ann. NY Acad. Sci., 149: 75-87, 1968.[Medline]
  93. Fisher J. W., Langston J. W. The influence of hypoxemia and cobalt on erythropoietin production in the isolated perfused dog kidney. Blood, 29: 114-125, 1967.[Abstract/Free Full Text]
  94. Thorling E. B., Jensen K. The lactate dehydrogenase isoenzymes in various organs of the rabbit in anaemia, hypoxia, and after cobalt administration. Acta Pathol. Microbiol. Scand., 66: 426-436, 1966.[Medline]
  95. Semenza G. L. Perspectives on oxygen sensing. Cell, 98: 281-284, 1999.[Medline]
  96. Wang G. L., Jiang B. H., Rue E. A., Semenza G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. USA, 92: 5510-5514, 1995.[Abstract/Free Full Text]
  97. Guillemin K., Krasnow M. A. The hypoxic response: huffing and HIFing. Cell, 89: 9-12, 1997.[Medline]
  98. Helmlinger G., Yuan F., Dellian M., Jain R. K. Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nat. Med., 3: 177-182, 1997.[Medline]
  99. Dachs G. U., Patterson A. V., Firth J. D., Ratcliffe P. J., Townsend K. M., Stratford I. J., Harris A. L. Targeting gene expression to hypoxic tumor cells. Nat. Med., 3: 515-520, 1997.[Medline]
  100. Talks K. L., Turley H., Gatter K. C., Maxwell P. H., Pugh C. W., Ratcliffe P. J., Harris A. L. The expression and distribution of the hypoxia-inducible factors HIF-1{alpha} and HIF-2{alpha} in normal human tissues, cancers, and tumor-associated macrophages. Am. J. Pathol., 157: 411-421, 2001.[Abstract/Free Full Text]
  101. Zagzag D., Zhong H., Scalzitti J. M., Laughner E., Simons J. W., Semenza G. L. Expression of hypoxia-inducible factor 1{alpha} in brain tumors: association with angiogenesis, invasion, and progression. Cancer (Phila.), 88: 2606-2218, 2001.
  102. Zhong H., De Marzo A. M., Laughner E., Lim M., Hilton D. A., Zagzag D., Buechler P., Isaacs W. B., Semenza G. L., Simons J. W. Overexpression of hypoxia-inducible factor 1{alpha} in common human cancers and their metastases. Cancer Res., 59: 5830-5835, 1999.[Abstract/Free Full Text]
  103. Zhong H., Agani F., Baccala A. A., Laughner E., Rioseco-Camacho N., Isaacs W. B., Simons J. W., Semenza G. L. Increased expression of hypoxia inducible factor-1{alpha} in rat and human prostate cancer. Cancer Res., 58: 5280-5284, 1998.[Abstract/Free Full Text]



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Mol. Cancer Ther., May 1, 2009; 8(5): 1261 - 1269.
[Abstract] [Full Text] [PDF]

Home page
 J Am Coll CardiolHome page
M. Kido, L. Du, C. C. Sullivan, X. Li, R. Deutsch, S. W. Jamieson, and P. A. Thistlethwaite
Hypoxia-Inducible Factor 1-Alpha Reduces Infarction and Attenuates Progression of Cardiac Dysfunction After Myocardial Infarction in the Mouse
J. Am. Coll. Cardiol., December 6, 2005; 46(11): 2116 - 2124.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
C. L. Galindo, A. A. Fadl, J. Sha, and A. K. Chopra
Microarray Analysis of Aeromonas hydrophila Cytotoxic Enterotoxin-Treated Murine Primary Macrophages
Infect. Immun., September 1, 2004; 72(9): 5439 - 5445.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-I Chao, P.-C. Kuo, and T.-S. Hsu
Down-regulation of Survivin in Nitric Oxide-induced Cell Growth Inhibition and Apoptosis of the Human Lung Carcinoma Cells
J. Biol. Chem., May 7, 2004; 279(19): 20267 - 20276.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Hoshiya, V. Gupta, H. Kawakubo, E. Brachtel, J. L. Carey, L. Sasur, A. Scott, P. K. Donahoe, and S. Maheswaran
Mullerian Inhibiting Substance Promotes Interferon {gamma}-induced Gene Expression and Apoptosis in Breast Cancer Cells
J. Biol. Chem., December 19, 2003; 278(51): 51703 - 51712.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Daniliuc, H. Bitterman, M. A. Rahat, A. Kinarty, D. Rosenzweig, and L. Nitza
Hypoxia Inactivates Inducible Nitric Oxide Synthase in Mouse Macrophages by Disrupting Its Interaction with {alpha}-Actinin 4
J. Immunol., September 15, 2003; 171(6): 3225 - 3232.
[Abstract] [Full Text] [PDF]

Home page
 Exp. Biol. Med.Home page
M. Ramanathan, A. Giladi, and S. J. Leibovich
Regulation of Vascular Endothelial Growth Factor Gene Expression in Murine Macrophages by Nitric Oxide and Hypoxia
Experimental Biology and Medicine, June 1, 2003; 228(6): 697 - 705.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. Alfranca, M. D. Gutierrez, A. Vara, J. Aragones, F. Vidal, and M. O. Landazuri
c-Jun and Hypoxia-Inducible Factor 1 Functionally Cooperate in Hypoxia-Induced Gene Transcription
Mol. Cell. Biol., January 1, 2002; 22(1): 12 - 22.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
B. R. Pitt and C. M. St. Croix
Complex Regulation of iNOS in Lung
Am. J. Respir. Cell Mol. Biol., January 1, 2002; 26(1): 6 - 9.
[Full Text] [PDF]

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