Dual effect of echinomycin on hypoxia-inducible factor-1 activity under normoxic and hypoxic conditions Benoit Vlaminck, Sebastien Toffoli, Benjamin Ghislain, Catherine Demazy, Martine Raes and Carine Michiels Laboratory of Biochemistry and Cellular Biology, FUNDP-University of Namur, Belgium A low oxygen level is a characteristic feature of solid tumours and a negative prognostic factor for the sur- vival of cancer patients. The response of cancer cells to hypoxia not only drives neo-angiogenesis, but also enhances cancer cell survival and malignant phenotype. Hypoxia-inducible factor-1 (HIF-1) is the major regu- lator of the adaptive responses of cells to hypoxia [1]. It is a Bcl2 ⁄ adenovirus E1B 19 kDa interacting protein (bHLH-PAS) transcription factor composed of two subunits: aryl hydrocarbon receptor nuclear transloca- tor (ARNT), which is constitutively expressed in the nucleus, and HIF-1a, whose level and activity are reg- ulated by the oxygen level. In the presence of oxygen, HIF-1a is post-translationally modified by prolyl hydroxylases, targeting the protein for proteasomal degradation, and by an asparagine hydroxylase, pre- venting its interactions with transcription coactivators [2]. Limiting oxygen availability prevents these modifi- cations, leading to HIF-1a accumulation, translocation into the nucleus and interaction with coactivators. On activation, the active dimer binds to target gene pro- moters containing the core recognition sequence 5¢-RCGTC-3¢ (HRE, hypoxia response element), lead- ing to overexpression of the genes involved in glucose metabolism, angiogenesis and cell survival [1]. This transcriptional response mediates cell adaptation to low oxygen level, but also contributes to tumour progression, neo-angiogenesis and metastasis [3,4]. Keywords HIF-1; hypoxia; hypoxia-inducible factor-1; inhibitors; transcription Correspondence C. Michiels, Laboratory of Biochemistry and Cellular Biology, FUNDP-University of Namur, 61 Rue de Bruxelles, 5000 Namur, Belgium Fax: +32 81 724135 Tel: +32 81 724131 E-mail: carine.michiels@fundp.ac.be (Received 31 May 2007, revised 26 July 2007, accepted 29 August 2007) doi:10.1111/j.1742-4658.2007.06072.x Hypoxia-inducible factor-1 (HIF-1) is now recognized as a possible target for cancer treatment. This transcription factor is responsible for the overex- pression of several genes favouring cancer cell survival and inducing neo- angiogenesis. Echinomycin has recently been described to inhibit HIF-1 DNA binding and transcriptional activity. In this work, it is shown that echinomycin strongly inhibits the activity of HIF-1 under hypoxic condi- tions, and also interferes with the activity of other transcription factors. These results demonstrate the lack of specificity of this molecule. More- over, it is demonstrated that echinomycin induces an increase in HIF-1 activity under normoxic conditions, parallel to an increase in the expression of HIF-1 target genes. This effect is caused by an increase in HIF-1a pro- tein level, resulting from an increase in the transcription of the HIF-1A gene in the presence of a low concentration of echinomycin. Transfection experiments with HIF-1a promoter constructs revealed the presence of an Sp1 binding element responsive to echinomycin. Furthermore, echinomycin enhanced Sp1 activity, as measured by the use of a specific reporter system. These findings show, for the first time, that echinomycin has a dual effect on HIF-1 activity under normoxic and hypoxic conditions, demonstrating that this molecule cannot be used in cancer treatment. Abbreviations AP-1, activator protein-1; ARNT, aryl hydrocarbon receptor nuclear translocator; DHG, DMEM high glucose; HB, hypotonic buffer; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia response element; Hsp90, heat shock protein 90; IGF, insulin-like growth factor; IOP1, iron-only hydrogenase-like protein 1; 2ME-2, 2-methoxyestradiol; PMA, 4b-phorbol 12-myristate 13-acetate; ROS–NF-jB, reactive oxygen species– nuclear factor-jB; YC-1, 3-(5¢-hydroxymethyl-2¢-furyl)-1-benzyl indazole. FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 5533 Moreover, increased levels of HIF-1a are frequently observed in human primary tumours [5,6]. Significant associations between HIF-1a overexpression and patient mortality have been shown in different types of cancer [7]. The identification of HIF-1 involvement in tumour progression and angiogenesis led to the concept of HIF-1 as a promising molecular target for the develop- ment of cancer therapeutics. Different approaches have been developed to inhibit HIF-1 activity [8]. Major efforts have been made to identify small molecules that are selective HIF-1 inhibitors. Most molecules inhibit HIF-1 by altering the signal transduction pathways that are associated with HIF-1, such as 2-methoxyest- radiol (2ME-2), which interacts with microtubules [9], camptothecin derivatives, which target topoisomerase II [10], and geldanamycin, which inhibits heat shock protein 90 (Hsp90) [11,12]. Only a few examples target selective pathways associated with HIF-1 activation, such as chetomin, which blocks HIF-1 interaction with coactivators [13], and echinomycin, which prevents HIF-1 DNA binding [14]. 3-(5¢-Hydroxymethyl-2¢- furyl)-1-benzyl indazole (YC-1) has also been described to specifically inhibit HIF-1 via the suppression of HIF-1a expression through a mechanism that is not yet clear [15]. Echinomycin, a cyclic peptide of the family of qui- noxaline antibiotics derived from Streptomyces echinatus [16], was originally discovered as a sequence- specific DNA binding agent: the strong binding site for this molecule is 5¢-A ⁄ TCGT-3¢ [17]. This sequence is contained in the core binding site (E box, 5¢-CAC- GTG-3¢) of the bHLH family of transcription factors, and hence of HIF-1. A recent study from Kong et al. [14] showed that this molecule is able to inhibit HIF-1 DNA binding activity, and thus the expression of cor- responding target genes, raising the attractive possibil- ity of the use of this small molecule in cancer therapy. In an effort to extend these results to other cancer cell lines, it was observed that, although echinomycin can inhibit HIF-1 activity under hypoxic conditions in HepG2 cells, its effects are not specific to this tran- scription factor, as activator protein-1 (AP-1) and c-myc activities are also affected. Moreover, a dual effect of this molecule was demonstrated, as it appeared to enhance HIF-1 activity under normoxia. Results and Discussion Echinomycin inhibits HIF-1 activity Echinomycin was described by Kong et al. [14] to be a strong HIF-1 inhibitor by inhibiting its DNA binding capacity and hence transcriptional activity. These experiments were performed using MCF-7 and U251 human glioma cells. Similar results were obtained using HepG2 cells. Figure 1A shows the concentra- tion-dependent inhibition of HIF-1 transcriptional activity under hypoxic conditions, measured using a reporter system with 6HRE upstream of the firefly luciferase gene: no inhibition was observed at 2 nm, 50% inhibition at 5 nm and 100% inhibition at 10 nm. It was, however, surprising to observe a significant and reproducible increase in HIF-1 activity at 2 nm under normoxic conditions. Similar results were obtained in HeLa cells (Fig. 1B). To investigate the effects of echi- nomycin on endogenous gene expression, mRNA expression of two HIF-1 target genes (BNIP3 and aldolase) was quantified by real-time RT-PCR. Echino- mycin, at 10 nm, significantly decreased BNIP3 and aldolase overexpression induced by 16 h of incubation under hypoxic conditions. Again, a slight increase in HIF-1 activity was observed in the presence of 2 nm echinomycin under normoxic conditions, as measured by a 1.23-fold increase in BNIP3 mRNA level and a 1.3-fold increase in aldolase mRNA level (Fig. 1C). As echinomycin was described as a DNA binding inhibit- ing agent, we tested this effect using a DNA binding ELISA with an HRE double-strand DNA probe covalently bound to the bottom of multiwell plates (TransAM assay, Carlsbad, CA, USA). Hypoxia markedly increased HIF-1 DNA binding activity. The incubation of cells with echinomycin during normoxia or hypoxia had a minimal effect on the HIF-1 DNA binding activity detected in the nuclear extract (Fig. 1D). This is probably a result of the loss of the molecule during the extraction procedure: indeed, only the nuclear proteins are recovered and the DNA is dis- carded. By contrast, when echinomycin was added to the nuclear extract from hypoxic cells just before the assay, a clear inhibition of HIF-1 DNA binding activ- ity to the HRE probe was observed (Fig. 1D), indicat- ing that echinomycin can prevent HIF-1 binding to the HRE sequence. The core sequence to which echinomycin binds is not only present in HRE, but also in other E-boxes recognized by other members of the bHLH family, such as c-myc. This raises the possibility that echino- mycin may also inhibit the DNA binding of this type of transcription factor. To test this possibility, reporter system experiments and TransAM assays were per- formed for c-myc, and also for another transcription factor that does not recognize such a sequence, AP-1. As shown in Fig. 2, echinomycin, when added to nuclear extracts from hypoxic cells just before the assay, inhibited the DNA binding activity of c-myc by Dual effect of echinomycin on HIF-1 activity B. Vlaminck et al. 5534 FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 30%, and that of AP-1 by 50% (Fig. 2A,B). Echino- mycin also inhibited the activity of both transcription factors measured using a reporter system. Basal c-myc activity was very low in HepG2 cells and was even lower under hypoxic conditions. Gordan et al. [18] and Zhang et al. [19] have shown that HIF-1 ⁄ HIF-1a inhibits c-myc activity. This is in accordance with our results, as we detected a lower c-myc DNA binding activity and a lower c-myc transcriptional activity under hypoxia (i.e. when HIF-1a is more abundant) relative to normoxia. However, the activity was mark- edly enhanced when cells were stimulated with 100 nm 4b-phorbol 12-myristate 13-acetate (PMA). Echinomy- cin markedly inhibited both basal and PMA-stimulated c-myc transcriptional activity (Fig. 2C). AP-1 activity was also low in unstimulated HepG2 cells. PMA enhanced this activity and, under normoxic and hypoxic conditions, echinomycin inhibited PMA-induced AP-1 activity (Fig. 2D). Together, these results, summarized in Fig. 2E, indi- cate that echinomycin strongly inhibits HIF-1 DNA binding activity, and hence HIF-1 transcriptional activity (between 80 and 100% inhibition). However, this effect is far from specific, because inhibition was also observed for c-myc, which binds to a similar DNA sequence (between 30 and 80% inhibition according to the type of assay), and AP-1, which binds to a totally different DNA sequence. Our results contrast with those described by Kong et al. [14], as we observed the inhibition of AP-1 activity, whereas they did not. The reasons for this discrepancy are not clear: we used a different cell type and stimulated the cells with PMA to activate c-myc and AP-1, because their basal activity was low. It is possible that inhibition 10 8 Relative reporter activity Echinomycin concentration (n M ) Echinomycin concentration (n M ) 6 4 2 0 8 6 Relative reporter activity 4 2 0 4 3.5 2.5 Absorbance Relative mRNA induction 1.5 0.5 0 N HN + E 5n M H + E 5n M H + EA320n M Conditions N HN + E 2n M H + E 10n M Conditions BNIP3 aldolase 3 2 1 4 3.5 2.5 1.5 0.5 0 3 2 1 012510 012510 Normoxia Hypoxia Normoxia Hypoxia (***) *** (*) (***) *** (*) (**) * * ** ** *** (***) HeLa HeLa * (**) HepG2 * * ** AB DC Fig. 1. Effect of echinomycin on HIF-1 activity. HepG2 and HeLa cells were incubated for 5 or 16 h under hypoxia or normoxia in the pres- ence or absence of increasing concentrations of echinomycin. HepG2 cells (A) and HeLa cells (B) were transfected with the pGL3- SV40 ⁄ 6HRE reporter plasmid and the pCMVb normalization vector. After incubation (16 h), luciferase and b-galactosidase activities were assayed. The results are expressed as the ratio between the luciferase activity and the b-galactosidase activity, as means ± 1SD (n ¼ 3). (C) After incubation (16 h), total RNA was extracted from HepG2 cells, retrotranscribed into cDNA and submitted to real-time PCR for BNIP3 and aldolase. RPL13 was used as the housekeeping gene. The results are expressed as fold induction, as means ± 1SD (n ¼ 3). (D) After incubation (5 h), nuclear extracts were recovered from HeLa cells. The DNA binding activity was quantified using the TransAM assay. An assay was also performed by adding echinomycin directly to the extracts from control hypoxic cells at 320 n M (EA320nM). The results are expressed as means ± 1SD (n ¼ 3). *, ** and ***, P < 0.05, 0.01 and 0.001 versus normoxia. (*), (**) and (***), P < 0.05, 0.01 and 0.001 versus hypoxia. B. Vlaminck et al. Dual effect of echinomycin on HIF-1 activity FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 5535 can only be observed when these factors are fully activated. Echinomycin increases HIF-1 a protein level under normoxia The results in Fig. 1 revealed a surprising observation: HIF-1 activity was increased when the cells were incubated in the presence of low concentrations of echinomycin (1–2 nm) under normoxia. In order to investigate the mechanism for this increased activity, the HIF-1a protein level was assessed by western blot- ting and immunofluorescence. Figure 3 shows that the HIF-1a protein was almost undetectable by western blotting in extracts from normoxic control cells. Hypoxia induced a strong stabilization of the protein. Echinomycin did not influence HIF-1a stabilization under hypoxic conditions, as already observed by Kong et al. [14]. However, this molecule induced an increase in the HIF-1a protein level under normoxic conditions; this effect was optimal at 2 nm, which cor- responds to the concentration leading to the maximal A B C D E Trans-AM reporter system HIF-1 70% 100% c-myc 30% 80% AP-1 50% 40% ** * ** (*) [*] HepG2 Relative reporter activity 5 Normoxia Hypoxia 4 3 2 1 0 CTL E 10 n M PMA Conditions PMA + E * *** (***) [*] [**] HepG2 Relative reporter activity 16 14 12 10 8 6 4 2 0 CTL Normoxia Hypoxia E 10 n M PMA Conditions PMA + E ** * (*) HeLa 1.4 1.2 Absorbance Conditions 1 0.8 0.6 0.4 0.2 0 NN + EHH + EH + EA 320n M ** * (**) HeLa Absorbance 2 1.5 0 1 0.5 Conditions N N + E H H + E H + EA 320n M Fig. 2. Effect of echinomycin on c-myc (A, C) and AP-1 (B, D) activity. HepG2 and HeLa cells were incubated for 5 or 16 h under hypoxia or normoxia in the presence or absence of echinomycin at 10 n M. (A, B) After incubation (5 h), nuclear extracts were recovered from HeLa cells. The DNA binding activity was quantified using the TransAM assay. An assay was also performed by adding echinomycin directly to the extracts from control hypoxic cells at 320 n M (EA320nM). The results are expressed as means ± 1SD (n ¼ 3). (C, D) HepG2 cells were transfected with the pGL2-M4-Luc (C) or pAP-1-Luc (D) reporter plasmid and the pCMVb normalization vector. PMA at 100 n M was used as a positive control. After incubation (16 h), luciferase and b-galactosidase activities were assayed. The results are expressed as the ratio between the luciferase activity and the b-galactosidase activity, as means ± 1SD (n ¼ 3). *, ** and ***, P < 0.05, 0.01 and 0.001 versus normoxia. (*), (**) and (***), P < 0.05, 0.01 and 0.001 versus hypoxia. [*] and [**], P < 0.05 and 0.01 versus PMA alone. (E) The table sum- marizes the inhibition percentage of HIF-1, c-myc and AP-1 DNA binding activity (TransAM) when echinomycin was added to the nuclear extracts before the assay, and of HIF-1, c-myc and AP-1 transcriptional activity (reporter system) in the presence of 10 n M echinomycin. Dual effect of echinomycin on HIF-1 activity B. Vlaminck et al. 5536 FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS increase in HIF-1 activity in the previous experiments. This effect was observed in both HepG2 and HeLa cells (Fig. 3A,B). Similar results were obtained when the HIF-1a protein level was assessed by immunofluo- rescence labelling and confocal observation (Fig. 3D). Again, echinomycin did not influence hypoxia-induced HIF-1a accumulation, but led to an increase in the HIF-1a protein level under normoxic conditions. In these conditions, as under hypoxia, HIF-1a was local- ized in the nucleus. We also tested whether the effect of echinomycin on the HIF-1a protein level under normoxia was revers- ible. Cells were incubated in the presence or absence of echinomycin at 2 nm for 16 h under normoxia; the medium was then changed to medium without echino- mycin and the cells were lysed directly (as a positive control) or after 4 h or 24 h of recovery. The results showed that there was still an increase in HIF-1a pro- tein level after 4 h of recovery, but to a lower extent than directly after incubation in the presence of echi- nomycin. After 24 h of recovery, the HIF-1a protein level had returned to the basal level (Fig. 3C). Echinomycin increases HIF-1 a mRNA expression under normoxia Several mechanisms have been described in the litera- ture to account for an increase in the HIF-1a protein level: (a) under hypoxia, HIF-1 a is no longer modified by the prolyl hydroxylases; it therefore escapes recog- nition by the E3 ubiquitin ligase pVHL and degrada- tion via the proteasome [20,21]; (b) on stimulation by cytokines or growth factors, such as insulin and insu- lin-like growth factor (IGF), in normoxia, HIF-1a A B D C Fig. 3. Effect of echinomycin on HIF-1a protein level. HepG2 and HeLa cells were incubated for 5 or 16 h under hypoxia or normoxia in the presence or absence of increasing concentrations of echinomycin. (A) After incubation (5 h), protein extracts were recovered from HepG2 cells for western blot analysis using HIF-1a-specific antibodies. a-Tubulin was used to assess the total amount of proteins loaded on the gel. (B) After incubation (16 h), protein extracts were recovered from HeLa cells for western blot analysis using HIF-1a-specific antibodies. a-Tubulin was used to assess the total amount of proteins loaded on the gel. (C) After incubation (16 h), the medium was changed to med- ium without echinomycin and, after 0, 4 and 24 h of recovery, protein extracts were recovered from HepG2 cells for western blot analysis using HIF-1a-specific antibodies. a-Tubulin was used to assess the total amount of proteins loaded on the gel. (D) After incubation (5 h), cells were fixed, permeabilized and labelled with anti-HIF-1a-specific IgG. Observations were made using a confocal microscope with a constant photomultiplier tube. B. Vlaminck et al. Dual effect of echinomycin on HIF-1 activity FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 5537 mRNA translation is increased through a phosphatidyl inositol 3-kinase–Akt-dependent pathway, leading to the production of more HIF-1a proteins that saturate the prolyl hydroxylase-dependent degradation pathway [22,23]; (c) recently, a third mechanism has been described in pulmonary smooth muscle cells stimulated by thrombin, which leads to an increase in HIF-1A gene transcription through a reactive oxygen species (ROS)–nuclear factor-jB (NF-jB)-dependent pathway [24]. HIF-1A gene transcription is also modulated by iron-only hydrogenase-like protein 1 (IOP1), a novel hydrogenase-like protein, through an as yet unidenti- fied mechanism [25]. Echinomycin induced a significant increase in HIF- 1a mRNA level under normoxic conditions (Fig. 4A). This observation suggests that HIF-1A gene transcrip- tion may be increased by this molecule. As we have previously cloned the HIF-1A promoter in a reporter system upstream of the luciferase gene (pH800) [26], we used this construct to investigate whether echino- mycin is able to increase HIF-1A transcription. An increase in luciferase activity was observed in the pres- ence of echinomycin under normoxic conditions, but not under hypoxia (Fig. 4B). These results are similar to those obtained when measuring the HIF-1a protein level and HIF-1 activity. Progressive deletions of the promoter were then generated in order to delineate the sequence responsive to echinomycin. An increase in luciferase activity was still observed in the presence of echinomycin with the plasmid spanning from )41 to +287 (pD4), but not with the plasmid spanning from )30 to +287 (p15C) (Fig. 4B). These results indicate that the sequence from )41 to )31 is responsible for the increased transcription in the presence of this mole- cule. This sequence contains a putative Sp1 binding site (5¢-CCGCCC-3¢) [26]. In order to investigate whether echinomycin could increase Sp1 activity, a reporter vector containing three consensus Sp1 binding sites was used. Figure 5 shows that echinomycin at 2 nm was capable of increasing luciferase activity under normoxia, but had no effect under hypoxia, indicating that this molecule may enhance Sp1 activity in these conditions. This effect was no longer observed at higher concentrations. The protein level of Sp1 was checked in the different conditions: the results showed that echinomycin did not influence the Sp1 protein level under normoxic and hypoxic conditions (Fig. 5B). These results suggest that echinomycin may increase Sp1 activity. The mechanism responsible for this effect remains to be investigated. Activated Sp1 is then A B ** * * Luc+ +1 -541 -201 -41 -30 +243 Relative reporter activity N + E 2nM Fig. 4. Effect of echinomycin on HIF-1A mRNA level and promoter activity. HepG2 cells were incubated for 16 h under hypoxia or normoxia in the presence or absence of 2n M echinomycin. (A) After incubation (16 h), total RNA was extracted, retrotran- scribed into cDNA and submitted to real- time PCR for HIF-1a. RPL13 was used as the housekeeping gene. The results are expressed as fold induction, as means ± 1SD (n ¼ 3). (B) Cells were trans- fected with different constructs containing sequences of the HIF-1A promoter and the pCMVb normalization vector. After incuba- tion (16 h), luciferase and b-galactosidase activities were assayed. The results are expressed as the ratio between the lucifer- ase activity and the b-galactosidase activity, as means ± 1SD (n ¼ 3). *, P < 0.05 versus normoxia. Dual effect of echinomycin on HIF-1 activity B. Vlaminck et al. 5538 FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS responsible for an increase in HIF-1A gene transcrip- tion, resulting in a higher level of HIF-1a protein and higher expression of HIF-1 target genes. In conclusion, our results demonstrate a lack of spec- ificity of echinomycin towards HIF-1, as it also inhibits the activity of several other transcription factors. More- over, these findings show, for the first time, that echino- mycin has a dual effect on HIF-1 activity under normoxic and hypoxic conditions, demonstrating that this molecule cannot be used in cancer treatment. In the context of cancer treatment, the use of this molecule would lead to an increase in expression of pro-survival and pro-angiogenic genes in normoxic conditions, a factor that would promote tumour growth. Experimental procedures Cell culture Human hepatoma cell lines HepG2 were grown in Dul- becco’s modified Eagle’s medium (DMEM, Invitrogen, Paisley, UK), supplemented with 10% fetal bovine serum (Invitrogen). HeLa cells (wt p53) were cultured in DMEM high glucose (DHG), supplemented with 10% fetal bovine serum. The cells were kept at 37 °C in a humidified atmo- sphere of 5% CO 2 and 95% air. For hypoxia experiments (1% O 2 ), the cells were incubated in serum-free CO 2 -inde- pendent medium (Invitrogen), supplemented with 1 mm l-glutamine (Sigma, St Louis, MO, USA) with different concentrations of echinomycin (Sigma). PMA (Sigma), at 100 nm, was used as a positive control in some experiments. Immunofluorescence 10 5 cells were seeded in a 24-well culture plate containing a glass coverslip. After 24 h of incubation in standard condi- tions, the cells were incubated for 5 h under normoxia or hypoxia; thereafter, the medium was removed and the cells were fixed for 10 min with NaCl ⁄ Pi containing 4% parafor- maldehyde (Merck, Darmstadt, Germany). Fixed cells were then washed three times with NaCl ⁄ P i and permeabilized with NaCl ⁄ P i –Triton X-100 (Merck) 1% for 4 min. After three washings with NaCl ⁄ P i –BSA 2%, the cells were incu- bated at 4 °C overnight with the primary antibody (anti- HIF-1a, BD Bioscience, San Diego, CA, USA). The cells were washed three times as described above and the second- ary antibody conjugated to Alexa fluorochrome (488 nm, dilution 1 : 500) was added. After 1 h of incubation, the cells were washed three times with NaCl ⁄ P i . For the labelling of nuclei, the cells were incubated for 30 min with TOPRO-3 (Molecular Probes, Eugene, OR, USA, dilution 1 : 80 v ⁄ v) in the presence of 2 mgÆmL )1 RNase, and then washed three times with NaCl ⁄ P i . Finally, glass coverslips were mounted in Mowiol for observation in confocal microscopy (Leica, Solms, Germany). Semiquantitative observations were per- formed with a constant photomultiplier tube value. Western blot analysis Total cell extracts were prepared from HepG2 cells grown to subconfluence in T25 cm 2 flasks. After incubation, the cells were scraped in lysis buffer [Tris 20 mm pH 7.5 (Merck), KCl 150 mm (Merck), EDTA 1 mm (Merck), Triton X-100 1% (Merck), protease inhibitors (Complete, Boehringer ⁄ Roche, Mannheim, Germany) and phosphatase inhibitors (25 mm Na 2 VO 4 ,10mm para-nitrophenyl phosphate, 10 mm b-glycero-phosphate and 5 mm NaF)]. The lysate was centrifuged for 5 min at 15 000 g at 4 °C and the superna- tant was kept frozen. The protein concentration of each sam- ple was determined by the Bradford method. Samples were applied to 10% NuPAGE Bis-Tris gels (Invitrogen), accord- ing to the manufacturer’s instructions, and then transferred to Hybond-poly(vinylidene difluoride) membrane (Amer- sham, Chalfont St Giles, UK). The membranes were blocked overnight at 4 °C in NaCl ⁄ Tris-T solution containing 20 mm Tris, 140 mm NaCl, 0.1% Tween 20, pH 7.6, containing 5% nonfat dry milk. Then, the membranes were probed with * (*) (*) B 0 1 2 5 10 0 1 2 5 10 normoxia hypoxia Sp1 - tubulin echinomycin (n M) A Relative reporter activity 52 CTL 0.5 0 1.5 2.5 1 2 10 Normoxia Hypoxia Echinomycin concentration (n M) Fig. 5. Effect of echinomycin on Sp1 activity and protein level. HepG2 cells were incubated for 16 h under hypoxia or normoxia in the presence or absence of increasing concentrations of echinomy- cin. (A) Cells were transfected with the pSp1-Luc reporter vector and the pCMVb normalization vector. After incubation, luciferase and b-galactosidase activities were assayed. The results are expressed as the ratio between the luciferase activity and the b-galactosidase activity, as means ± 1SD (n ¼ 4). *, P < 0.05 ver- sus normoxia. (*), P < 0.05 versus hypoxia. (B) After incubation (16 h), protein extracts were recovered from HepG2 cells for wes- tern blot analysis using Sp1-specific antibodies. a-Tubulin was used to assess the total amount of proteins loaded on the gel. B. Vlaminck et al. Dual effect of echinomycin on HIF-1 activity FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 5539 monoclonal anti-HIF-1a IgG (BD Bioscience) at a final dilu- tion of 1 : 2000 for 2 h, or with polyclonal anti-Sp1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature. After three 15 min washes in NaCl ⁄ Tris-T with 5% nonfat dry milk, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies at a final dilution of 1 : 50 000, and washed twice for 15 min in NaCl ⁄ Tris-T with 5% nonfat dry milk and twice for 5 min in NaCl ⁄ Tris-T. The proteins were visualized by enhanced chemiluminescence (Amersham) according to the manufacturer’s instructions. The mem- branes were reprobed with a-tubulin antibody (Sigma, final dilution 1 : 50 000) for normalization. Preparation of nuclear extracts Nuclear protein extracts in high salt buffer were prepared as described previously [27]. HeLa cells were grown to sub- confluence in T75 cm 2 flasks and incubated under normoxia or hypoxia for 5 h before cell lysis. The cells were washed with ice-cold NaCl ⁄ P i containing 1 mm Na 2 MoO 4 and 5mm NaF. Then, the cells were incubated for 3 min with ice-cold hypotonic buffer (HB) containing 20 mm Hepes pH 7.9, 5 mm NaF, 1 mm Na 2 MoO 4 and 0.1 mm EDTA. The cells were harvested in lysis buffer containing HB, sup- plemented with 5% Nonidet P40 (Sigma). The lysates were centrifuged for 30 s at 13 000 g and the supernatants were discarded. The pellets were dissolved in 50 lL of RE buffer, composed of HB supplemented with 20% glycerol and pro- tease inhibitors (Complete, Roche) and phosphatase inhibi- tors (25 mm Na 2 VO 4 ,10mm para-nitrophenyl phosphate, 10 mm b-glycero-phosphate and 5 mm NaF). Then, 50 lL of SA buffer, composed of RE buffer, supplemented with 800 mm NaCl, was added. The samples were placed at 4 °C for 30 min under gentle rotation for nuclear protein extrac- tion under high salt concentration. Then, the samples were centrifuged for 10 min at 13 000 g at 4 °C, and the super- natants containing the nuclear proteins were stored at )70 °C. The pellets containing the DNA were discarded. Colorimetric assay for HIF-1, c-myc or AP-1 DNA binding HIF-1, c-myc and AP-1 DNA binding activity was mea- sured using a colorimetric assay (Trans-AM) developed in our laboratory [28], and sold by Active Motif (Carlsbad, CA, USA). Assays were performed according to the manu- facturer’s instructions. Transient transfection and luciferase activity measurement HepG2 transfections were performed in 24–well plates (50 000 cells per well) with SuperFect reagent (Qiagen, Hilden, Germany); 1846 ng of the reporter plasmid contain- ing binding sites for the transcription factor to be assayed, or the HIF-1A promoter sequences upstream of the firefly luciferase gene, was cotransfected with 1154 ng of normali- zation vector (pCMVb vector coding for b-galactosidase, Promega, Madison, WI, USA) in DMEM without serum for 7 h. The reporter plasmids were the pGL3-SV40 ⁄ 6HRE vector containing six HRE binding sites upstream of the firefly luciferase gene [29], pAP1-Luc (Stratagene, La Jolla, CA, USA), pGL2-M4-luciferase containing four c-myc binding sites upstream of the firefly luciferase gene [30] and pSp1-Luc containing three Sp1 binding sites upstream of the firefly luciferase gene [31]. The different constructs for the HIF-1A promoter are described in [26]. The cells were then directly incubated under hypoxia for 16 h. After incu- bation under hypoxia, b-galactosidase was assayed in paral- lel with the firefly luciferase activity in a luminometer using the Luciferase Reporter Assay System (Promega). Experi- ments were performed in triplicate. The results are expressed as means of the ratio between the firefly luciferase activity and the b-galactosidase activity. Real-time PCR analysis The levels of HIF-1a, BNIP3 and aldolase transcripts were determined by real-time RT-PCR using SYBR Green (Invi- trogen). To normalize for the input load of cDNA between samples, human RPL13 was used as an endogenous stan- dard. Specific primers were used: HIF-1a forward, 5¢-TCAAGCAGTAGCGAATTGGAACATTATT-3¢; HIF- 1a reverse, 5¢-TTTACACGTTTCCAAGAAAGTGATG TA-3¢; BNIP3 forward, 5¢-TTTGCTGGCCATCGGATT- 3¢; BNIP3 reverse, 5¢-ACCAAGTCAGACTCCAGTTCTT CA-3¢; aldolase forward, 5¢-GAATTGGATGAAAGATA AAGCCCTTA-3¢; aldolase reverse, 5¢-TTGCCAGACC ATCCGTACTG-3¢; RPL13A forward, 5¢-CTCAAGGTC GTGCGTCTGAA,-3¢: RPL13 reverse, 5¢-TGGCTGTCAC TGCCTGGTACT-3¢. cDNA was added to SYBR Green Master Mix PCR (300 nm of each specific primer). PCRs were performed in a total volume of 25 lL. PCRs were car- ried out in a real time PCR cycler (ABI Prism 7700 Sequence Detector, Applied Biosystems, Branchburg, NJ, USA). The thermal cycling conditions were as follows: ini- tial incubation of 10 min at 95 °C, followed by 40 cycles of 30 s at 95 °C, 1 min at an annealing temperature of 57 °C and 30 s at 72 °C. All cDNA samples were tested in dupli- cate and analysed with ABI Prism Sequence Detection Soft- ware version 1.7 (PE Applied Biosystems). Samples were compared using the relative Ct method. The Ct value, which is inversely proportional to the initial template copy num- ber, is the calculated cycle number for which the fluores- cence signal is significantly above background levels. Fold induction or repression was measured relative to controls, and was calculated after adjusting for a-tubulin using Dual effect of echinomycin on HIF-1 activity B. Vlaminck et al. 5540 FEBS Journal 274 (2007) 5533–5542 ª 2007 The Authors Journal compilation ª 2007 FEBS 2 –[DDCt] , where DCt ¼ Ct(tested gene) ) Ct(a-tubulin) and DDCt ¼ DCt(control) ) DCt(treatment). Statistical analysis Statistical analyses were performed using Student’s t-test. For each set of data, real triplicates were performed in one experiment. Each experiment was repeated at least twice. Acknowledgements B. Vlaminck and B. 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