Mechanisms of Hypoxic Gene Regulation of Angiogenesis Factor Cyr61 in Melanoma Cells
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m301373200
ISSN1083-351X
AutoresManfred Kunz, Steffen Moeller, Dirk Koczan, Peter Lorenz, Roland H. Wenger, Michael O. Glocker, Hans‐Juergen Thiesen, Gerd Gross, Saleh Ibrahim,
Tópico(s)Fibroblast Growth Factor Research
ResumoHypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors. A commercially available Affymetrix® gene chip system was used to analyze five melanoma cell lines of different aggressiveness. A total of 160 hypoxia-inducible genes were identified, clustering in four different functional clusters. In search of putative angiogenesis and tumor progression factors within these clusters, Cyr61, a recently discovered angiogenesis factor, was identified. Cyr61 was hypoxia-inducible in low aggressive melanoma cells; however, it showed constitutive high expression in highly aggressive melanoma cells. Further analyses of transcriptional mechanisms underlying Cyr61 gene expression under hypoxia demonstrated that an AP-1 binding motif within the Cyr61 promoter plays a central role in the hypoxic regulation of Cyr61. It could be shown by use of in vitro luciferase assays, electrophoretic mobility shift assays, and immunoprecipitation that hypoxia-inducible factor-1α interacts with c-Jun/AP-1 and may thereby contribute to Cyr61 transcriptional regulation under hypoxia. Taken together, the presented data show that Cyr61 is a hypoxia-inducible angiogenesis factor in malignant melanoma with tumor stage-dependent expression. This may argue for a hypoxia-induced selection process during tumor progression toward melanoma cells with constitutive high Cyr61 expression. Hypoxia has a profound influence on progression and metastasis of malignant tumors. In the present report, we used the oligonucleotide microarray technique to identify new hypoxia-inducible genes in malignant melanoma with a special emphasis on angiogenesis factors. A commercially available Affymetrix® gene chip system was used to analyze five melanoma cell lines of different aggressiveness. A total of 160 hypoxia-inducible genes were identified, clustering in four different functional clusters. In search of putative angiogenesis and tumor progression factors within these clusters, Cyr61, a recently discovered angiogenesis factor, was identified. Cyr61 was hypoxia-inducible in low aggressive melanoma cells; however, it showed constitutive high expression in highly aggressive melanoma cells. Further analyses of transcriptional mechanisms underlying Cyr61 gene expression under hypoxia demonstrated that an AP-1 binding motif within the Cyr61 promoter plays a central role in the hypoxic regulation of Cyr61. It could be shown by use of in vitro luciferase assays, electrophoretic mobility shift assays, and immunoprecipitation that hypoxia-inducible factor-1α interacts with c-Jun/AP-1 and may thereby contribute to Cyr61 transcriptional regulation under hypoxia. Taken together, the presented data show that Cyr61 is a hypoxia-inducible angiogenesis factor in malignant melanoma with tumor stage-dependent expression. This may argue for a hypoxia-induced selection process during tumor progression toward melanoma cells with constitutive high Cyr61 expression. It is well accepted that local growth and metastasis of a large variety of malignant tumors are dependent on neoangiogenesis (1Folkman J. J. Natl. Cancer Inst. 1990; 82: 4-6Crossref PubMed Scopus (4376) Google Scholar, 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6016) Google Scholar, 3Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7350) Google Scholar). The process of tumor angiogenesis is largely based on the production and secretion of so-called angiogenesis factors such as vascular endothelial growth factor (VEGF), 1The abbreviations used are: VEGF, vascular endothelial growth factor; ECL, enhanced chemiluminescence; DMRIE-C™, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide; EMSA, electrophoretic mobility shift assay; HIF-1α, hypoxia-inducible factor-1α; AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; CTGF, connective tissue growth factor; STAT-1, signal transducer and activator of transcription-1; Ab, antibody; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; MEKK, mitogen-activated extracellular signal-regulated protein kinase kinase; GST, glutathione S-transferase; CMV, cytomegalovirus; MOPS, 4-morpholinepropane-sulfonic acid; MES, 4-morpholineethanesulfonic acid; FCS, fetal calf serum.1The abbreviations used are: VEGF, vascular endothelial growth factor; ECL, enhanced chemiluminescence; DMRIE-C™, 1,2-dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide; EMSA, electrophoretic mobility shift assay; HIF-1α, hypoxia-inducible factor-1α; AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; CTGF, connective tissue growth factor; STAT-1, signal transducer and activator of transcription-1; Ab, antibody; IL, interleukin; PMA, phorbol 12-myristate 13-acetate; MEKK, mitogen-activated extracellular signal-regulated protein kinase kinase; GST, glutathione S-transferase; CMV, cytomegalovirus; MOPS, 4-morpholinepropane-sulfonic acid; MES, 4-morpholineethanesulfonic acid; FCS, fetal calf serum. fibroblast growth factor, and interleukin (IL)-8 (3Carmeliet P. 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This constitutive high expression in tumor cells may derive from the so-called “angiogenic switch,” which is supposed to happen very early during tumor development initiating a selection process (for review, see Ref. 2Hanahan D. Folkman J. Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (6016) Google Scholar). In accordance with this, it has been shown that a constitutive high expression of IL-8 promotes tumor growth of melanoma cells in vivo (7Singh R.K. Gutman M. Radinsky R. Bucana C.D. Fiedler I.J. Cancer Res. 1994; 54: 3242-3247PubMed Google Scholar, 8Luca M. Huang S. Gershenwald J.E. Singh R.K. Reich R. Bar-Eli M. Am. J. Pathol. 1997; 151: 1105-1113PubMed Google Scholar). However, the molecular mechanisms underlying the induction of the angiogenic switch in tumors have not been defined so far.More recent investigations have emphasized that the specific conditions of the tumor microenvironment may have a strong impact on the secretion of angiogenesis factors. In particular, tissue hypoxia has been shown to play a key role for the induction of these factors (9Folkman J. Shing Y. J. Biol. Chem. 1992; 267: 10931-10934Abstract Full Text PDF PubMed Google Scholar, 10Bouck N. Stellmach V. Hsu S.C. Adv. Cancer Res. 1996; 69: 135-174Crossref PubMed Google Scholar, 11Harris A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4204) Google Scholar). Interestingly, even after neovascularization, tumor areas may remain under low oxygen tension, because of inadequate vascularization after neoangiogenesis (12Helmlinger G. Yuan F. Dellian M. Jain R.K. Nat. Med. 1997; 3: 177-182Crossref PubMed Scopus (1326) Google Scholar). Thus, hypoxic areas remain a constant feature of malignant tumors and metastases. Up to now a large series of angiogenesis factors have been identified to be inducible by hypoxia. Among these are fibroblast growth factor, VEGF, platelet-derived growth factor, IL-8, and angiogenin (13Shweiki D. Itin A. Soffer D. Keshet E. 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Biochem. 1995; 234: 632-640Crossref PubMed Scopus (161) Google Scholar, 24Semenza G.L. J. Appl. Physiol. 2000; 88: 1474-1480Crossref PubMed Scopus (1470) Google Scholar, 25Wenger R.H. J. Exp. Biol. 2000; 203: 1253-1263Crossref PubMed Google Scholar, 26Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar, 27Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1004) Google Scholar). In search for further upstream activators, signal transduction cascades interfering with transcription factors were analyzed in more detail. It could be shown that protein kinase C and the stress signaling pathway of c-Jun N-terminal kinase might play a central role in hypoxic signaling (28Goldberg M. Zhang H.L. Steinberg S.F. J. Clin. Invest. 1997; 99: 55-61Crossref PubMed Scopus (128) Google Scholar, 29Laderoute K.R. Webster K.A. Circ. Res. 1997; 80: 336-344Crossref PubMed Scopus (163) Google Scholar, 30Laderoute K.R. Mendonca H.L. Calaoagan J.M. Knapp A.M. Giaccia A.J. Stork P.J.S. J. Biol. Chem. 1999; 274: 12890-12897Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, the mechanisms underlying hypoxic signal transduction are still poorly defined.In the present report we used the oligonucleotide microarray technique to analyze the gene expression pattern of malignant melanoma cells in search for new hypoxia-inducible genes involved in tumor angiogenesis. It could be shown that Cyr61, a recently discovered angiogenesis factor, is hypoxia-inducible in malignant melanoma cells. Moreover, Cyr61 showed a tumor stage-dependent expression with a constitutive strong expression in highly aggressive melanoma cell lines. The underlying mechanisms of its transcriptional regulation under hypoxia were further studied. We demonstrated that AP-1 and HIF-1α may both contribute to hypoxia-induced gene expression of Cyr61.MATERIALS AND METHODSPlasmids—The following plasmid constructs were used for co-transfection studies in in vitro luciferase assays: pRc/CMV, pRc/CMV-HIF-1α, and pARNT/CMV4. The pRc/CMV plasmid (empty control vector) was purchased from Invitrogen (Leek, The Netherlands); the pRc/CMV-HIF-1α vector was generated by cloning of HIF-1α cDNA into the pRc/CMV vector. The pARNT/CMV4 plasmid (31Lindebro M.C. Poellinger L. Whitelaw M.L. EMBO J. 1995; 14: 3528-3539Crossref PubMed Scopus (161) Google Scholar, 32Wenger R.H. Camenisch G. Desbaillets I. Chilov D. Gassmann M. Cancer Res. 1998; 58: 5678-5680PubMed Google Scholar) carrying the ARNT cDNA was kindly provided by L. Poellinger (Karolinska Institute, Stockholm, Sweden).Cell Lines and Culture Conditions—The human melanoma cell lines 1F6, 530, Mel57, BLM, and MV3 were kindly provided by G. van Muijen (Institute of Pathology, University of Nijmegen, Nijmegen, The Netherlands). The metastatic potential of these cell lines has been investigated in nude mice. The cell lines 530 and 1F6 represent low aggressive, non-metastatic cell lines; the cell line Mel57 has been characterized as intermediate to highly aggressive; and BLM and MV3 are both highly aggressive, metastatic cell lines (33Van Muijen G.N. Cornelissen L.M. Jansen C.F. Figdor C.G. Johnson J.P. Bröcker E.B. Ruiter D.J. Clin. Exp. Metastasis. 1991; 9: 259-272Crossref PubMed Scopus (116) Google Scholar, 34Van Muijen G.N. Jansen C.F. Cornelissen L.M. Beck J.L.M. Ruiter D.J. Int. J. Cancer. 1991; 48: 85-91Crossref PubMed Scopus (137) Google Scholar, 35Westphal J.R. van't Hullenaar R.G. van der Laak J.A. Cornelissen I.M. Schalkwijk L.J. van Muijen G.N. Wesseling P. de Wilde P.C. Ruiter D.J. de Waal R.M. Br. J. Cancer. 1997; 76: 561-570Crossref PubMed Scopus (67) Google Scholar). Melanoma cell lines and Jurkat cells (which were used in control experiments) were maintained in RPMI 1640 medium (Linaris, Bettingen, Darmstadt, Germany), supplemented with 10% fetal calf serum (FCS; Linaris), 2 mm l-glutamine, 100 units/ml penicillin/streptomycin, and 1% nonessential amino acids. Cells were cultured in a humidified incubator (37 °C in 5% CO2, 95% air). For hypoxic treatment cultures were transferred for different time periods to hypoxic culture conditions (1% O2; mentioned as hypoxia) in an hypoxic incubator as described previously (32Wenger R.H. Camenisch G. Desbaillets I. Chilov D. Gassmann M. Cancer Res. 1998; 58: 5678-5680PubMed Google Scholar). Harvesting of cells was also performed under hypoxic conditions to avoid reoxygenation artifacts. Parallel cultures were constantly kept under normal oxygen conditions (mentioned as normoxia). H-Ras-transformed wild type mouse embryonic fibroblasts (MEF-HIF+/+) and HIF-1α-null mouse embryonic fibroblasts (MEF-HIF-/-) (36Ryan H.E. Poloni M. McNulty W. Elson D. Gassmann M. Arbeit J.M. Johnson R.S. Cancer Res. 2000; 60: 4010-4015PubMed Google Scholar) were kept in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units/ml penicillin/streptomycin.RNA Extraction and Gene Chip Hybridization—Melanoma cell lines 530, 1F6, Mel57, BLM, and MV3, respectively, were kept under normoxic conditions or exposed to 24 h of hypoxia (1% oxygen). Total RNA was isolated using the total RNeasy kit (Qiagen, Hilden, Germany). RNA concentrations were determined spectrophotometrically at 260 nm. Probes for chip hybridization derived from isolated RNA samples were generated according to the instructions from the supplier (Affymetrix, Santa Clara, CA). The HuGeneFL Array™ (Affymetrix) with a capacity of 5600 human full-length characterized genes was used for mRNA expression profiling. Hybridization and washing of gene chips were performed according to the instructions from the supplier and as described earlier by Lockhart and co-workers (37Lockhart D.J. Dong H. Byrne M.C. Follettie M.T Gallo M.V. Chee M.S. Mittmann M. Wang C. Kobayashi M. Horton H. Brown E.L. Nat. Biotechnol. 1996; 14: 1675-1680Crossref PubMed Scopus (2795) Google Scholar). A laser scanner (Hewlett-Packard Gene Scanner™) was used for analysis of gene chips, and the expression levels were calculated using a commercially available software provided by Affymetrix (Microarray Suite®).Biostatistical Analysis of Gene Chip Data—Before cluster analysis genes were pre-selected for those that showed increased expression under hypoxia in at least one out of five cell lines to encompass all hypoxia-inducible genes of our experiments. Pre-selection resulted in a list of 1349 candidate genes. These were subjected to hierarchical clustering using Eisen software as recently described (38Eisen M.B. Spellman P.T. Brown P.O. Botstein D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14863-14868Crossref PubMed Scopus (13134) Google Scholar). Clustering tools were downloaded from www.rana.lbl.gov/EisenSoftware.htm. Hierarchical clustering was performed using average linkage differences. The “-fold change” of gene expression under hypoxia was used as clustering parameter. By this means four different clusters (cluster I–IV) were generated overall covering 160 genes. Cluster IV was re-clustered to analyze absolute gene expression with the average difference (representing the absolute value of gene expression) as clustering parameter. In the latter analyses, gene expression values were normalized to the mean value of all genes in this experiment.Generation of a Cyr61 cDNA Probe—A Cyr61 cDNA probe for Northern hybridization was generated by reverse transcriptase-PCR. For this purpose total RNA was extracted from BLM melanoma cells using the total RNeasy kit (Qiagen). One μ g of total RNA was reverse transcribed by superscript reverse transcriptase (Invitrogen, Eggenstein, Germany) using hexamer priming. Primers for PCR amplification were purchased from ARK Scientific Biosystems (Darmstadt, Germany). These had the following recently published sequences (39Tsai M.S. Hornby A.E. Lakins J. Lupu R. Cancer Res. 2000; 60: 5603-5607PubMed Google Scholar): 5′ primer, 5′-TGTGGAACTGGTATCTCCACACGA-3′; 3′ primer, TCTTTTCACTGAATATAAAATTAAAA-3′. PCR amplification generated a cDNA probe of 1039 bp length. PCR conditions: an initial 5-min denaturation step was followed by 10 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 58 °C, 1 min of primer extension at 72 °C. After that, 20 cycles of 30 s of denaturation at 94 °C, 30 s of annealing at 60 °C, and 1 min of primer extension at 72 °C were carried out. A terminal primer extension step of 10 min at 72 °C was added. The PCR products were separated by agarose gel electrophoresis. The specific fragment of 1039 bp was cut out, purified using the QIAExII gel extraction kit (Qiagen), cloned into the pGEM-T vector (Promega, Heidelberg, Germany), and sequenced using an automated capillary sequencer (PerkinElmer, Weiterstadt, Germany). For Northern hybridization the cDNA probe was cut out from the pGEM-T vector by restriction enzyme digest and radioactively labeled.RNA Extraction and Northern Blot Analysis—Total cytoplasmic RNA was isolated using the total RNeasy kit (Qiagen). RNA concentration was determined spectrophotometrically at 260 nm. Fifteen micrograms of RNA/sample were denatured in 50% formamide in gel running buffer (0.1 m MOPS, pH 7.0, 40 mm sodium acetate, 5 mm EDTA, pH 8.0) for 15 min at 65 °C, fractionated on a 1% agarose gel in formaldehyde buffer, and subsequently transferred to a nylon membrane (Hybond N+, Amersham Biosciences) in 20× SSC. As a probe for Cyr61 mRNA, a specific 1039 cDNA fragment was used. For Northern hybridization the purified fragment was labeled to high specific activity with [32P]dATP using a random primer labeling system (Roche Molecular Biochemicals, Mannheim, Germany). Membranes were cross-linked by ultraviolet light irradiation and prehybridized in a dextran sulfate buffer (100 g/liter dextran sulfate, 0.6 m NaCl, 0.2 m Na2HPO4, 6 mm EDTA, 1.75% lauroylsarcosinate, 50 μg/ml salmon sperm DNA, pH 6.2) for 1 h at 65 °C. Hybridization was carried out in the same prehybridization solution containing 5 × 106 cpm/ml of labeled probe. After hybridization for 16 h at 65 °C and 6 h at 60 °C, membranes were washed twice with 2× SSPE, 0.1% sodium dodecyl sulfate (SDS) at room temperature; once with 1× SSPE, 0.1% SDS at 60 °C; and once again with 0.2× SSPE, 0.1% SDS. Membranes were then exposed to Hyperfilm™ (Amersham Biosciences) with intensifying screens at -80 °C for 3 days. Northern blots for ribosomal L28 RNA were performed, which served as control for equal loading (40Wenger R.H. Rolfs A. Marti H.H. Bauer C. Gassmann M. J. Biol. Chem. 1995; 270: 27865-27870Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).Generation of Cyr61 Promoter Luciferase Constructs—A 883-bp and a 605-bp fragment, respectively, of the recently published promoter region of the Cyr61 gene (Ref. 41Schütze N. Rücker N. Müller J. Adamski J. Jakob F. Mol. Pathol. 2001; 54: 170-175Crossref PubMed Scopus (31) Google Scholar; EMBL GenBank™, accession no. HSA249826) were generated by PCR amplification. The first fragment covers almost the complete Cyr61 promoter region of 935 bp beginning at the transcription start site. Further 5′ sequences (beyond position -883) carry no binding motifs for known transcription factors. The second fragment (605 bp) covers the promoter region (also beginning at the transcription start site) ending immediately downstream of an AP-1 binding motif (TGACTCA) at position -624. For fragment generation of the Cyr61 promoter, genomic DNA was isolated from BLM melanoma cells using a commercially available DNA isolation kit (Qiagen). PCR amplification was carried out using the following primers: 883-bp fragment: 5′ primer (5′-CGGGTACCTAAAGTGGGAACCTCCA-3′) and 3′ primer (5′-CCGCTCGAGTCTCGCTCGCGGTCTGCC-3′); 605-bp fragment: 5′ primer (5′-CGGGGTACCTCTTCCCCGTTCTACTC-3′) and 3′ primer (5′-CCGCTCGAGTCTCGCTCGCGGTCTGCC-3′). Both pairs of primers generated a KpnI restriction site at the 5′ end and an XhoI restriction site at the 3′ end of the amplified fragments. These sites were used for further cloning of the fragments into the pGL3 vector (Promega, Heidelberg, Germany). The pGL3 promoter constructs were sequenced using an automated capillary sequencer (PerkinElmer Life Sciences). The promoter construct carrying the 883-bp fragment was termed cyr-900-luc, and the promoter construct with the 605-bp construct was termed cyr-600-luc.In Vitro Mutagenesis of Cyr61 Promoter—In vitro mutagenesis was performed to generate mutated cyr-900-luc constructs. A promoter construct with a mutated AP-1 binding motif was termed cyr-900APmut-luc. A promoter construct where all four HRE-like motifs (position -68, -371, -400, -653) mutated was termed cyr-900HIFmut-luc. For mutagenesis of the AP-1 binding motif, TGACTCA, this motif was changed to TGACTAC, which results in an inhibition of transcription factor binding (42Wenig H. Choi S.Y. Faller D.V. J. Biol. Chem. 1995; 270: 13637-13644Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). For mutagenesis of the four HRE-like binding motifs, present on the promoter as CACG, this motif was changed to CTTT. A commercially available mutagenesis kit was used (QuikChange™ site-directed mutagenesis kit, Stratagene, La Jolla, CA), and mutagenesis was performed according to the specifications from the manufacturer. The mutagenesis primers used were as follows.For AP-1 motif mutagenesis, primers were 5′-GAAGTCCACAAATATTCCTGACTACGAGACACACTCCTC-3′ and 5′-GAGGAGTGTGTCTCGTAGTCAGGAATATTTGTGGACTTC-3′. For mutagenesis of HRE-like motifs at position -68, primers were 5′-ACGTCACTGCAACTTTCGGCGCCTCCGC-3′ and 5′-GCGGAGGCGCCGAAAGTTGCAGTGACGT-3′; at position -371, 5′-CATCACCACCATCTTTCCCAAAGAACC-3′ and 5′-GGTTCTTTGGGGAAAGATGGTGGTGATG-3′; at position -400, 5′-CCCCTCGCCCCTCTTTACCCTCCAACTA-3′ and 5′-TAGTTGGAGGGTAAAAGGGGCGAGGGG-3′; and at position -653, 5′-ACTTGTTCCGAACTTTCCTCTTTGAAGT-3′ and 5′-ACTTCAAAGAGGAAAGTTCGGAACAAGT-3′.Successful mutagenesis was confirmed by sequencing using an automated capillary sequencer (PerkinElmer Life Sciences).In Vitro Luciferase Assays—Cyr61 promoter luciferase constructs were used for in vitro luciferase assays. For this purpose the melanoma cell line 1F6 was transfected with 2 μg of plasmid DNA (of wild type and mutated promoter constructs) using the DMRIE-C™ (1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide) reagent (Invitrogen) according to the specifications from the manufacturer. Briefly, cells were kept under serum-free medium for at least 16 h. 2 μg of plasmid DNA and DMRIE-C™ reagent, respectively, were diluted in 500 μl of OptiMEM (Invitrogen). Both mixtures were put together and incubated at room temperature for 30 min. Culture medium was removed and the lipid-DNA overlaid onto cells and incubated overnight. Subsequently, medium was replaced by RPMI medium (10% FCS) and after additional 24 h changed to low serum conditions (1% FCS) for another 24 h. Subsequently, cells were exposed to hypoxic conditions for indicated time points.For luciferase assays, total cell extracts were prepared. Briefly, cells were harvested in 100 μl of lysis buffer (50 mm NaMES, pH 7.8, 50 mm Tris-HCl, pH 7.8, 10 mm dithiothreitol, 2% Triton X-100). The crude cell lysates were cleared by centrifugation, 50 μl of cleared cell extracts were added to 50 μl of luciferase assay buffer (125 mm NaMES, pH 7.8, 25 mm magnesium acetate, 2 mg/ml ATP), and activity was measured after injection of 50 μl of 1 mm d-luciferin (AppliChem, Darmstadt, Germany) in a Berthold luminometer (Berthold, Bad Wildbach, Germany). Total protein concentration was measured by the Bradford technique (Bio-Rad, München, Germany). The luciferase activities were normalized on the basis of protein content as well as on β-galactosidase activity of cotransfected Rous sarcoma virus-β-gal vector. The β-galactosidase assay was performed with 20 μl of precleared cell lysate according to a standard protocol, as described earlier (43Hoffmeyer A. Grosse-Wilde A. Flory E. Neufeld B. Kunz M. Rapp U.R. Ludwig S. J. Biol. Chem. 1999; 274: 4319-4327Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). For calculation of the -fold induction of luciferase activities, parallel cultures under normoxia and hypoxia were analyzed. Mean ± S.D. of four independent experiments are shown.To analyze the effect of HIF-1α/ARNT on c-Jun-dependent transcription, a commercially available trans-reporting system (PathDetect®, Stratagene, Heidelberg, Germany) was used. This system analyses the effects of upstream molecules (e.g. signaling kinases) on the transcriptional activity of c-Jun in an in vitro luciferase assay system. In our experiments, 1F6 melanoma cell were transfected with the fusion transactivator plasmid, pFA2-c-Jun, which contains the activation domain of c-Jun fused with the yeast GAL4 binding domain. The co-transfected pFR-Luc reporter plasmid carries five tandem repeats of a GAL4 binding site linked to the firefly luciferase gene. Co-transfection was performed with a combination of both HIF-1α/ARNT-carrying plasmids or an appropriate control plasmid, which was used for mock transfection (pFC-dbd). Parallel experiments were performed by co-transfection of mitogen-activated extracellular signal-regulated protein kinase kinase (MEKK), which served as a positive control. Total cell extracts were prepared for luciferase assays as described above.Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)—Melanoma cells (1F6 cell line) were exposed to hypoxic conditions for 1, 2, 4, 8, and 12 h, respectively. Parallel cultures were kept under normoxia. Nuclear extracts were prepared as previously described (17Kunz M. Hartmann A. Flory E. Toksoy A. Koczan D. Thiesen H.J. Mukaida N. Neumann M. Rapp U.R. Bröcker E.B. Gillitzer R. Am. J. Pathol. 1999; 155: 753-763Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, cells were washed with ice-cold phosphate-buffered saline and pelleted. Supernatants were removed, and cells were resuspended in 500 μl of buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) and allowed to swell for 10 min. Cells were pulled 10–15 times through a 26-gauge 3/8 needle for cell membrane disruption, and nuclei were pelleted in a microcentrifuge. Nuclei were washed twice in buffer A and resuspended in 50 μl of buffer C (20 mm Hepes, 20% glycerol, 0.4 m NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride) and incubated on ice for 45 min with occasional shaking. After centrifugation, supernatants were harvested, frozen, and stored at -70 °C. The following double-stranded oligonucleotide was used as probe (derived from Cyr61 promoter region including the AP-1 binding sequence (underlined)): 5′-AATATTCCTGACTCAGAGACACA-3′ and 5′-TGTGTCTCTGAGTCAGGAATATT-3′. In control experiments a commercially available STAT-1 probe (sc-2573; Santa Cruz Biotechnology, Heidelberg, Germany) with the following sequence was used (STAT-1 binding sequence underlined
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