Artigo Acesso aberto Revisado por pares

Evidence for a Role of p38 Kinase in Hypoxia-inducible Factor 1-independent Induction of Vascular Endothelial Growth Factor Expression by Sodium Arsenite

2003; Elsevier BV; Volume: 278; Issue: 9 Linguagem: Inglês

10.1074/jbc.m206320200

ISSN

1083-351X

Autores

Monique C.A. Duyndam, Saskia Hulscher, Elsken van der Wall, Herbert M. Pinedo, Epie Boven,

Tópico(s)

Fibroblast Growth Factor Research

Resumo

Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1α (HIF-1α) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1α and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1α protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1α−/− mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite. Recently we have demonstrated that sodium arsenite induces the expression of hypoxia-inducible factor 1α (HIF-1α) protein and vascular endothelial growth factor (VEGF) in OVCAR-3 human ovarian cancer cells. We now show that arsenic trioxide, an experimental anticancer drug, exerts the same effects. The involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase (MAPK) pathways in the effects of sodium arsenite was investigated. By using kinase inhibitors in OVCAR-3 cells, both effects of sodium arsenite were found to be independent of phosphatidylinositol 3-kinase and p44/p42 MAPKS but were attenuated by inhibition of p38 MAPK. A role for p38 in the regulation of HIF-1α and VEGF expression was supported further by analysis of activation kinetics. Experiments in mouse fibroblast cell lines, lacking expression of c-Jun N-terminal kinases 1 and 2, suggested that these kinases are not required for induction of HIF-1α protein and VEGF mRNA. Unexpectedly, sodium arsenite did not activate a HIF-1-dependent reporter gene in OVCAR-3 cells, indicating that functional HIF-1 was not induced. In agreement with this hypothesis, up-regulation of VEGF mRNA was not reduced in HIF-1α−/− mouse fibroblast cell lines. Altogether, these data suggest that not HIF-1, but rather p38, mediates induction of VEGF mRNA expression by sodium arsenite. vascular endothelial growth factor buthionine-sulfoximine trans-1,2-diaminocyclohexane-N,N,N′N′-tetraacetic acid cytomegalovirus dominant-negative enzyme-linked immunosorbent assay extracellular signal-regulated kinase hypoxia-inducible factor 1 hypoxia-responsive element c-Jun N-terminal kinase mitogen-activated protein kinase MAPK/ERK kinase N-acetylcysteine phosphatidylinositol 3-kinase reactive oxygen species stress-activated protein kinases von Hippel Lindau time point Vasular Endothelial Growth Factor (VEGF)1 plays a key role in tumor angiogenesis. VEGF occurs in at least six isoforms of 121, 145, 165, 183, 189, and 206 amino acids, which are generated from a single gene by alternative splicing (1Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Crossref PubMed Google Scholar). We have reported that VEGF165 overexpression stimulates angiogenesis in human ovarian cancer xenografts (2Duyndam M.C.A. Hilhorst M.C.G.W. Schluper H.M.M. Verheul H.M.W. van Diest P.J. Kraal G. Pinedo H.M. Boven E. Am. J. Pathol. 2002; 160: 537-548Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Elevated expression of VEGF in tumor cells can be the result of environmental factors, such as hypoxia, or mutations in oncogenes or tumor suppressor genes that regulate growth factor signal transduction pathways (3Shweiki D. Itin A. Soffer D. Keshet E. Nature. 1992; 359: 843-845Crossref PubMed Scopus (4144) Google Scholar, 4Mazure N.M. Chen E.Y. Yeh P. Laderoute K.R. Giaccia A.J. Cancer Res. 1996; 56: 3436-3440PubMed Google Scholar, 5Jiang B.H. Agani F. Passaniti A. Semenza G.L. Cancer Res. 1997; 57: 5328-5335PubMed Google Scholar, 6Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 7Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 8Blancher C. Moore J.W. Robertson N. Harris A.L. Cancer Res. 2001; 61: 7349-7355PubMed Google Scholar, 9Jiang B.H. Jiang G. Zheng J.Z. Lu Z. Hunter T. Vogt P.K. Cell Growth Differ. 2001; 12: 363-369PubMed Google Scholar). Many stimuli, including hypoxia, growth factors, hormones, and oxidative stressors, can increase VEGF expression in tumor cells in vitro (6Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 10Gleadle J.M. Ebert B.L. Firth J.D. Ratcliffe P.J. Am. J. Physiol. 1995; 268: C1362-C1368Crossref PubMed Google Scholar, 11Forsythe J.A. Jiang B.H. Iyer N.V. Agani F. Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. 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Leung S.W. Koos R.D. Semenza G.L. Mol. Cell. Biol. 1996; 16: 4604-4613Crossref PubMed Scopus (3178) Google Scholar). HIF-1 is composed of two subunits, HIF-1α and HIF-1β. The activity of HIF-1 is regulated mainly by the expression and activity of the HIF-1α subunit. Although HIF-1β protein is rather stable and readily detected in the nucleus of most normoxic cells, HIF-1α protein is often hardly detectable because of rapid degradation by the ubiquitin-proteasome system (16Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1394) Google Scholar, 17Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1836) Google Scholar, 18Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). Hypoxia increases the level of HIF-1α protein by inhibiting its ubiquitination and degradation (19Semenza G.L. Curr. Opin. Cell Biol. 2001; 13: 167-171Crossref PubMed Scopus (881) Google Scholar). Accumulation of HIF-1α protein can also be observed in stimulated normoxic cells (5Jiang B.H. Agani F. Passaniti A. Semenza G.L. Cancer Res. 1997; 57: 5328-5335PubMed Google Scholar, 8Blancher C. Moore J.W. Robertson N. Harris A.L. Cancer Res. 2001; 61: 7349-7355PubMed Google Scholar, 9Jiang B.H. Jiang G. Zheng J.Z. Lu Z. Hunter T. Vogt P.K. Cell Growth Differ. 2001; 12: 363-369PubMed Google Scholar, 13Kimura H. Weisz A. Kurashima Y. Hashimoto K. Ogura T. D'Acquisto F. Addeo R. Makuuchi M. Esumi H. Blood. 2000; 95: 189-197Crossref PubMed Google Scholar, 14Richard D.E. Berra E. Pouyssegur J. J. Biol. Chem. 2000; 275: 26765-26771Abstract Full Text Full Text PDF PubMed Google Scholar, 15Jiang B.H. Zheng J.Z. Leung S.W. Roe R. Semenza G.L. J. Biol. Chem. 1997; 272: 19253-19260Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). HIF-1α is subsequently translocated to the nucleus, where it can dimerize with HIF-1β to form the HIF-1 complex (20Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar). To be fully active, HIF-1α requires interaction(s) with coactivators or/and transcription factor(s) (20Kallio P.J. Okamoto K. O'Brien S. Carrero P. Makino Y. Tanaka H. Poellinger L. EMBO J. 1998; 17: 6573-6586Crossref PubMed Google Scholar, 21Ema M. Hirota K. Mimura J. Abe H. Yodoi J. Sogawa K. Poellinger L. Fujii-Kuriyama Y. EMBO J. 1999; 18: 1905-1914Crossref PubMed Google Scholar, 22Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (322) Google Scholar, 23Alfranca A. Gutierrez M.D. Vara A. Aragones J. Vidal F. Landazuri M.O. Mol. Cell. Biol. 2002; 22: 12-22Crossref PubMed Scopus (100) Google Scholar). The activated protein-1 transcription factor family member c-Jun interacts with HIF-1α and is suggested to cooperate with HIF-1 in the induction of VEGF expression by hypoxia (23Alfranca A. Gutierrez M.D. Vara A. Aragones J. Vidal F. Landazuri M.O. Mol. Cell. Biol. 2002; 22: 12-22Crossref PubMed Scopus (100) Google Scholar). The stabilization and transcriptional activation of the HIF-1α protein involve changes in its phosphorylation state. Activation of the lipid kinase phosphatidylinositol 3-kinase (PI3K), and/or its downstream target the protein-serine/threonine kinase Akt, can result in the phosphorylation and stabilization of HIF-1α under hypoxic and normoxic conditions (8Blancher C. Moore J.W. Robertson N. Harris A.L. Cancer Res. 2001; 61: 7349-7355PubMed Google Scholar, 24Minet E. Michel G. Mottet D. Raes M. Michiels C. Free Radic. Biol. Med. 2001; 31: 847-855Crossref PubMed Scopus (152) Google Scholar). In addition, inhibition of PI3K activity has been shown to reduce the transactivation function of HIF-1α in hypoxic cells (25Hirota K. Semenza G.L. J. Biol. Chem. 2001; 276: 21166-21172Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). So far, there is no evidence that PI3K and Akt can phosphorylate HIF-1α directly. Other pathways that regulate HIF-1α phosphorylation involve members of the mitogen-activated protein kinase (MAPK) family (26Widmann C. Gibson S. Jarpe M.B. Johnson G.L. Physiol. Rev. 1999; 79: 143-180Crossref PubMed Scopus (2258) Google Scholar). In hypoxic and in stimulated normoxic cells, p44/p42 MAPK (extracellular signal-regulated kinase (ERK)-1 and ERK-2) and p38 MAPK enhance the transactivation function of HIF-1α (24Minet E. Michel G. Mottet D. Raes M. Michiels C. Free Radic. Biol. Med. 2001; 31: 847-855Crossref PubMed Scopus (152) Google Scholar, 25Hirota K. Semenza G.L. J. Biol. 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The stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs) do not phosphorylate HIF-1α in vitro, but may indirectly regulate HIF-1-mediated transcription of VEGF under hypoxia through phosphorylating the transcription factor c-Jun (18Kallio P.J. Wilson W.J. O'Brien S. Makino Y. Poellinger L. J. Biol. Chem. 1999; 274: 6519-6525Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 23Alfranca A. Gutierrez M.D. Vara A. Aragones J. Vidal F. Landazuri M.O. Mol. Cell. Biol. 2002; 22: 12-22Crossref PubMed Scopus (100) Google Scholar). The stabilization and transcriptional activation of HIF-1α may also involve alterations in its redox state. Evidence is provide that changes in the levels of reactive oxygen species (ROS) may play a role in HIF-1α protein induction and HIF-1 transactivation (14Richard D.E. Berra E. Pouyssegur J. J. Biol. Chem. 2000; 275: 26765-26771Abstract Full Text Full Text PDF PubMed Google Scholar, 30Gorlach A. Diebold I. Schini-Kerth V.B. Berchner-Pfannschmidt U. Roth U. Brandes R.P. Kietzmann T. Busse R. Circ. Res. 2001; 89: 47-54Crossref PubMed Scopus (353) Google Scholar, 31Fandrey J. Frede S. Jelkmann W. Biochem. J. 1994; 303: 507-510Crossref PubMed Scopus (224) Google Scholar, 32Chandel N.S. McClintock D.S. Feliciano C.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1532) Google Scholar). Increased levels of ROS are suggested to mediate PI3K activation under hypoxia as well as under normoxia (32Chandel N.S. McClintock D.S. Feliciano C.E. Wood T.M. Melendez J.A. Rodriguez A.M. Schumacker P.T. J. Biol. Chem. 2000; 275: 25130-25138Abstract Full Text Full Text PDF PubMed Scopus (1532) Google Scholar, 33Sandau K.B. Faus H.G. Brune B. Biochem. Biophys. Res. Commun. 2000; 278: 263-267Crossref PubMed Scopus (115) Google Scholar). The levels of ROS may also directly or indirectly influence the redox status of cysteine residues in the transactivation domains of HIF-1α, which can affect interactions with transcriptional coactivators (21Ema M. Hirota K. Mimura J. Abe H. Yodoi J. Sogawa K. Poellinger L. Fujii-Kuriyama Y. EMBO J. 1999; 18: 1905-1914Crossref PubMed Google Scholar, 22Carrero P. Okamoto K. Coumailleau P. O'Brien S. Tanaka H. Poellinger L. Mol. Cell. Biol. 2000; 20: 402-415Crossref PubMed Scopus (322) Google Scholar). In a previous study on the role of oxidative stress in the regulation of VEGF, we showed that sodium arsenite (NaAsO2) induces HIF-1α protein and VEGF mRNA and protein levels in the human ovarian cancer cell lines OVCAR-3 and H134 (34Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Arsenite induces oxidative stress by binding to thiol groups of cellular proteins and by increasing the production of ROS. Because the effects of sodium arsenite on HIF-1α protein and VEGF expression are independent of increased ROS production, we hypothesized that they may be mediated through binding of arsenite to thiol (SH) groups of the HIF-1α protein itself or of components of signal transduction pathways involved in HIF-1 or VEGF regulation (34Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Our findings with sodium arsenite may be of clinical relevance. Several cytotoxic agents in cancer treatment are susceptible of interacting with thiol groups of cellular proteins. Moreover, arsenic trioxide (As2O3), another trivalent arsenic compound, has potential as an anticancer agent (35Murgo A.J. Oncologist. 2001; 6, (Suppl. 2): 22-28Crossref PubMed Scopus (194) Google Scholar). At low dosages (1–10 μm), arsenic trioxide has a significant cytotoxic effect on human ovarian cancer cell lines and is suggested to be a useful agent for the treatment of ovarian cancer (36Uslu R. Sanli U.A. Sezgin C. Karabulut B. Terzioglu E. Omay S.B. Goker E. Clin. Cancer Res. 2000; 6: 4957-4964PubMed Google Scholar). Therefore, we now compared the potency of arsenic trioxide with that of sodium arsenite to induce HIF-1α protein and VEGF mRNA and protein levels in the OVCAR-3 human ovarian cancer cell line. We also investigated the role of the PI3K/Akt pathway and of MAPK family members in sodium arsenite-induced HIF-1α protein accumulation and VEGF expression. Furthermore, we examined whether up-regulation of VEGF mRNA expression by sodium arsenite was mediated by HIF-1. Sodium arsenite, arsenic trioxide, wortmannin, glutathione (GSH), N-acetylcysteine (NAc), and buthionine-sulfoximine (BSO) were purchased from Sigma. PD98059, SB203580 and SB202190 were purchased from Calbiochem. OVCAR-3 human ovarian cancer cells and Jnk1 +/− Jnk2 −/−,Jnk1 −/− Jnk2 −/−,HIF-1α +/+, andHIF-1α −/− mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Sanbio, Uden, The Netherlands), 50 units/ml penicillin (ICN Biochemicals, Zoetermeer, The Netherlands), and 50 μg/ml streptomycin (ICN Biochemicals). Cells were routinely cultured in 95% air and 5% CO2 at 37 °C. Hypoxic conditions were performed by incubation of cells in a tightly sealed chamber maintained at 1% oxygen, 94% N2, and 5% CO2 at 37 °C. Immortalized Jnk1 + − Jnk2 −/− andJnk1 −/− Jnk2 −/− cell lines were kindly provided by Dr. E. F. Wagner (Research Institute of Molecular Pathology, Vienna, Austria). These cell lines have been established independently from individual primary mouse embryo fibroblasts that were each isolated from a single mouse embryo following the 3T3 protocol (37Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1998) Google Scholar, 38Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (301) Google Scholar). ImmortalizedHIF-1α +/+ andHIF-1α −/− cell lines were kindly provided by Dr. G. L. Semenza (Johns Hopkins University School of Medicine, Baltimore, MD). Both cell lines were established by transfection of primary mouse embryo fibroblast cultures with an expression vector encoding simian virus 40 T-antigen as described elsewhere (39Feldser D. Agani F. Iyer N.V. Pak B. Ferreira G. Semenza G.L. Cancer Res. 1999; 59: 3915-3918PubMed Google Scholar). For treatment of cells with sodium arsenite or arsenic trioxide, cells were seeded in culture dishes in medium and grown overnight. Thereafter, sodium arsenite or arsenic trioxide was added to the conditioned medium, and cells were incubated further for the time periods as indicated in each experiment. Pretreatment of cells with wortmannin, PD98059, SB203580, SB202190, GSH, and NAc was performed for 1 h before the addition of sodium arsenite; pretreatment with BSO was performed for 16 h. Rabbit polyclonal antisera directed against phospho-Akt-1 (Ser473), phospho-p44/p42 MAPK (Thr202/Tyr204), phospho-SAPK/JNK (Thr183/Tyr185), phospho-p38 MAPK (Thr180/Tyr182), phospho-c-Jun (Ser73), p44/p42 MAPK, SAPK/JNK, p38 MAPK, and a horseradish peroxidase-coupled anti-rabbit antiserum were purchased from New England Biolabs. Mouse monoclonal antisera to HIF-1α were purchased from Novus Biologicals/AbCam (Cambridge, U. K.) and BD Transduction Laboratories (Alphen a/d Rijn, The Netherlands). The sheep polyclonal antiserum directed against Akt-1 was from Upstate Biotechnology Inc. Rabbit polyclonal antisera directed against c-Jun (H-79) and Raf-1 (C12) were purchased from Santa Cruz Biotechnology. The mouse monoclonal antiserum against β-actin (C4) was purchased from Roche Molecular Biochemicals. The polyclonal human antiserum against topoisomerase I was purchased from TopoGEN (Columbus, OH). The mouse monoclonal antiserum against human p53 (DO-7) and the horseradish peroxidase-coupled anti-mouse serum were purchased from DAKO (Glostrup, Denmark). The horseradish peroxidase-coupled anti-sheep serum was purchased from Calbiochem. Cells were washed once with ice-cold phosphate-buffered saline and lysed by scraping with a rubber policeman in 250 μl of radioimmune precipitation assay buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, and 1% sodium deoxycholate) for Western blotting or in 450 μl of E1A buffer (50 mm Tris-HCl, pH 7.5, 250 mm NaCl, 5 mm EDTA, and 0.1% Nonidet P-40) for ELISA. Both lysis buffers were supplemented with 50 mm NaF, 1 mmNa3VO4, 1.0 mm phenylmethylsulfonyl fluoride, 0.5 mm trypsin inhibitor, and 0.5 μg/ml leupeptin. After a 15-min incubation period on ice, the extracts were clarified by centrifugation at 14,000 rpm for 15 min at 4 °C and stored at −70 °C. Isolation of nuclear and cytoplasmic protein fractions from OVCAR-3 cells in the subcellular fractionation experiment was performed as described previously (40De Rooij K.E. Dorsman J.C. Smoor M.A. Den Dunnen J.T. Van Ommen G.J. Hum. Mol. Genet. 1996; 5: 1093-1099Crossref PubMed Scopus (96) Google Scholar). Protein concentrations were determined by the Coomassie Plus Protein assay (Pierce). Equal amounts of protein cell extracts were resolved in SDS-polyacrylamide gels and transferred electrophoretically onto a polyvinylidene difluoride membrane (Immobilon). Membranes were blocked for 1 h in TBST (10 mm Tris, pH 8.0, 150 mm NaCl, and 0.025% Tween 20) and 5% milk and incubated overnight with antiserum directed against phospho-Akt-1(1:3,000), phospho-p44/p42 MAPK (1:1,000), phospho-SAPK/JNK (1:1,000), phospho-p38 (1:1,000), phospho-c-Jun (1:1,000), HIF-1α (1:500), β-actin (1:2,000), Raf-1 (1:500), topoisomerase I (1:5,000) or p53 (1:1,000). After washing with TBST, the membranes were incubated for 1 h with horseradish peroxidase-linked anti-rabbit or anti-mouse antiserum in TBST and 5% milk. The membranes were washed again with TBST, and proteins were visualized by enhanced chemiluminescence. To detect both nonphosphorylated and phosphorylated forms of Akt-1, c-Jun, and the MAPK family members, membranes were stripped for 15 min in strip buffer (10 mm Tris, pH 8.0, 2% SDS, 10 mmβ-mercaptoethanol) at 42 °C and washed with TBST. Subsequently, the blots were blocked again and incubated for 2 h with Akt-1, p44/p42 MAPK, SAPK/JNK, or p38-specific antiserum (1:1,000 dilution) or with a c-Jun-specific antiserum (1:500 dilution) in TBST and 5% milk. The incubations with horseradish peroxidase-linked anti-rabbit, anti-mouse, and anti-sheep antisera and the detection of the proteins were performed as described above. Equal numbers of cells were plated on 9.6-mm culture dishes and grown overnight. The conditioned medium of all dishes was collected, pooled, and 450 μl was sampled (time-point (T) = 0 medium sample). Cells of one dish were washed and lysed in 450 μl of lysis buffer as described above (T = 0 lysate sample). The conditioned medium was divided into 5 volumes. Sodium arsenite was added to 1 volume at a concentration of 100 μm, and arsenic trioxide was added to 3 volumes at concentrations of 100, 50, and 10 μm. Subsequently, conditioned medium with or without sodium arsenite or arsenic trioxide was again added to culture dishes with cells. Thus, atT = 0 the amount of VEGF protein in the medium was the same in each culture dish. Cells were incubated further for the time periods indicated in each experiment. Thereafter, 450 μl of conditioned medium was sampled, and cells were lysed. VEGF concentrations in nondiluted media samples and lysates were determined in duplicate by ELISA using the reagents and the protocol supplied with the Quantikine Human VEGF Immunoassay kit (R&D Systems). Differences in VEGF concentrations in medium and lysates of nontreatedversus sodium arsenite- or arsenic trioxide-treated cells were evaluated using Student's t test for two groups.p values <0.05 were considered to be significant. Generation of human γ-actin and VEGF165 antisense probes has been described elsewhere (34Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). pcDNA3 vectors (Invitrogen) containing a fragment of murine VEGF164 or γ-actin cDNA were used as templates for the synthesis of murine VEGF164 and γ-actin antisense probes. The templates were generated as follows. Total RNA fromJnk1 +/− Jnk2 −/− mouse fibroblasts was reversed transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Murine VEGF164cDNA (nucleotides 454–648) was amplified with the forward primer 5′-ATAACAAGCTT AGCACAGCAGATGTGAATGC-3′ and the reversed primer 5′-GCAACCTCGA GCTTGTCACATCTGCAAGTAC-3′. Murine γ-actin cDNA (nucleotides 630–780) was amplified with the forward primer 5′-ATAACAAGCT TGCTATGTTGCCCTGGATTTTGAG-3′ and the reversed primer 5′-GCAACCTCGAG GGA AGGAAGGCTGGAAGAGT-3′. Amplification of VEGF164and γ-actin cDNA fragments was performed for 36 cycles at 94 °C, 56 °C, and 72 °C. In addition to VEGF164 or γ-actin sequences (underlined), the forward and reverse primers contain restriction sites (bold) for HindIII andXhoI, respectively. The 216-nucleotide VEGF164and 172-nucleotide γ-actin amplified fragments were extracted with phenol/chloroform, precipitated, dissolved in water, and digested withHindIII and XhoI. The digestion mixtures were subjected to gel electrophoresis, and the VEGF164 and γ-actin cDNA fragments were isolated from the agarose gel by use of the QIAquick gel extraction kit (Qiagen). The isolated VEGF164 and γ-actin cDNA fragments were cloned into the HindIII and XhoI restriction sites of pcDNA3, and the sequence of the VEGF164 and γ-actin cDNA fragments was verified by sequencing. pcDNA3VEGF164 and pcDNA3γ-actin were linearized with HindIII, and 228-nucleotide VEGF164 and 185-nucleotide γ-actin antisense probes were generated with SP6 polymerase. The RNase protection assay was carried out as described (34Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Hybridization of total RNA to a 136-nucleotide human γ-actin antisense probe gives rise to a protected fragment of 130 nucleotide, whereas hybridization to the 185-nucleotide murine γ-actin antisense probe is expected to result in the protection of a fragment of 151 nucleotides. It should be noted that hybridization of total RNA to the 301-nucleotide human VEGF165 antisense probe and the 228-nucleotide murine VEGF164 can give rise to fragments of different sizes because of protection by VEGF mRNAs of different isoforms (1Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Crossref PubMed Google Scholar, 34Duyndam M.C. Hulscher T.M. Fontijn D. Pinedo H.M. Boven E. J. Biol. Chem. 2001; 276: 48066-48076Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 41Shima D.T. Kuroki M. Deutsch U. Ng Y.S. Adamis A.P. D'Amore P.A. J. Biol. Chem. 1996; 271: 3877-3883Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 42Sugihara T. Wadhwa R. Kaul S.C. Mitsui Y. J. Biol. Chem. 1998; 273: 3033-3038Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Protection by human VEGF165mRNA and murine VEGF164 mRNA is most efficient, giving rise to protected fragments of 252 and 195 nucleotides, respectively. Therefore, effects on VEGF mRNA levels were monitored by assessing the mRNA levels of human VEGF165 and murine VEGF164. The pGL3 promoter vector was purchased from Promega. This vector contains the SV40 basal promoter upstream of luciferase coding sequences and the SV40 late polyadenylation signal. 5xHREpGL3 contains five copies of a HIF-1 consensus sequence of the human VEGF promoter and was cloned from the reporter plasmid 5HRE/hCMVmp (43Shibata T. Giaccia A.J. Brown J.M. Gene Ther. 2000; 7: 493-498Crossref PubMed Scopus (238) Google Scholar). 5HRE/hCMVmp was digested with KpnI andBglII, and the KpnI-BglII fragment was ligated into the KpnI-BglII-digested pGL3-promoter vector. 5xjun2pGL3 contains five copies of thejun2 element of the human c-jun promoter (44van Dam H. Duyndam M. Rottier R. Bosch A. de Vries-Smits L. Herrlich P. Zantema A. Angel P. van der Eb A.J. EMBO J. 1993; 12: 479-487Crossref PubMed Scopus (342) Google Scholar) and was constructed by ligation of the PvuII-BglII fragment of the 5xjun2-tata-luciferase reporter construct (45van Dam H. Huguier S. Kooistra K. Baguet J. Vial E. van der Eb A.J. Herrlich P. Angel P. Castellazzi M. Genes Dev. 1998; 12: 1227-1239Crossref PubMed Scopus (100) Google Scholar) in theSmaI-BglII-digested pGL3 promoter vector. OVCAR-3 cells were seeded on six-well culture plates and grown overnight in medium. Transfection of OVCAR-3 cells with luci

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