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..
Trang 1activity 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.
Trang 2Moreover, 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 usinhibit-ing a DNA bindinhibit-ing 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 bindindicat-ing 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
Trang 330%, 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
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N N + E 5n M H H + E 5n M H +
EA320n M
Conditions
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Conditions
BNIP3 aldolase
3
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Normoxia Hypoxia
Normoxia Hypoxia
(***)
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(*)
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HeLa
HeLa
* (**)
HepG2
*
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D C
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 abspres-ence 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 (EA320n M ) 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.
Trang 4can only be observed when these factors are fully
activated
Echinomycin increases HIF-1a 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
**
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16 14 12 10 8 6 4 2 0 CTL
Normoxia Hypoxia
Conditions
PMA + E
(*)
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1.2
Conditions
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Conditions
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 (EA320n M ) 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.
Trang 5increase 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-1a 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-1a 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 stimuladegrada-tion 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.
Trang 6mRNA 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
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+1
-541
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-41
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+243
Relative reporter activity
N + E 2n M
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
2 n 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-galactosidlucifer-ase activity,
as means ± 1SD (n ¼ 3) *, P < 0.05 versus normoxia.
Trang 7responsible 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 37C in a humidified atmo-sphere of 5% CO2and 95% air For hypoxia experiments (1% O2), the cells were incubated in serum-free CO2 -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
105cells 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⁄ Pi and permeabilized with NaCl⁄ Pi–Triton X-100 (Merck) 1% for 4 min After three washings with NaCl⁄ Pi–BSA 2%, the cells were incu-bated at 4C 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⁄ Pi 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)1RNase, and then washed three times with NaCl⁄ Pi 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 cm2flasks 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 Na2VO4, 10 mm para-nitrophenyl phosphate,
10 mm b-glycero-phosphate and 5 mm NaF)] The lysate was centrifuged for 5 min at 15 000 g at 4C and the superna-tant was kept frozen The protein concentration of each sam-ple was determined by the Bradford method Samsam-ples 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 4C 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
*
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Sp1
-tubulin
echinomycin (n M )
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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 verver-sus 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.
Trang 8monoclonal 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 cm2flasks and incubated under normoxia
or hypoxia for 5 h before cell lysis The cells were washed
with ice-cold NaCl⁄ Pi containing 1 mm Na2MoO4 and
5 mm 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 Na2MoO4 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 Na2VO4, 10 mm 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 4C
for 30 min under gentle rotation for nuclear protein
extrac-tion under high salt concentraextrac-tion Then, the samples were
centrifuged for 10 min at 13 000 g at 4C, 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 bindcontain-ing 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 95C, followed by 40 cycles of
30 s at 95C, 1 min at an annealing temperature of 57 C and 30 s at 72C 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
Trang 92–[DDCt], where DCt¼ Ct(tested gene)) Ct(a-tubulin) and
DDCt¼ 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 Ghislain are recipients of a grant
from Fonds de la Recherche en Industrie et pour
l’Agriculture (Brussels, Belgium) S Toffoli is a
recipi-ent of a Televie grant (FNRS, National Funds for
Sci-entific Research, Belgium) and Carine Michiels is a
senior research associate of FNRS
We thank Professor R.N Eisenman (Fred
Hutchin-son Cancer Research Center, Seattle, WA, USA) for the
pGL2-M4-luciferase plasmid and Professor H Nomura
(Chugai Research Institute for Molecular Medicine,
Ibaraki, Japan) for the pSp1-luc plasmid This article
presents results of the Belgian Programme on
Inter-university Poles of Attraction initiated by the Belgian
State, Prime Minister’s Office, Science Policy
Program-ming Responsibility is assumed by the authors
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