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Department of Cell and Cancer Biology (M.G., A.M., S.L., R.P.,
W.G.A., L.N., J.T., F.C.) National Cancer Institute National
Institutes of Health Bethesda, Maryland 20892
Department
of Histology and Pathology (L.M.M.) University of Navarra 31080
Pamplona, Spain
Department of Biology (H.R., R.J.)
University of California San Diego La Jolla, California 92093
Institute of Physiology (M.G.) University of
Zürich-Irchel CH-8057, Switzerland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and HIF-1ß knockout mouse cell lines had an ablated or
greatly reduced hypoxia AM mRNA induction. Similarly, inhibition or
enhancement of HIF-1 activity in human tumor cells showed an analogous
modulation of AM mRNA. Under hypoxic conditions, immunohistochemical
analysis of tumor cell lines revealed elevated levels of AM and
HIF-1 as compared with normoxia, and we also found an increase
of immunoreactive AM in the conditioned medium of tumor cells analyzed
by RIA. AM mRNA stabilization was shown to be partially responsible for
the hypoxic up-regulated expression of AM. In addition, we have
identified several putative hypoxia response elements (HREs) in the
human AM gene, and reporter studies with selected HREs were capable of
enhancing luciferase expression after exposure to DFX. Furthermore,
transient coexpression of HIF-1 resulted in an augmented
transactivation of the reporter gene after DFX treatment. Given that
most solid human tumors have focal hypoxic areas and that AM functions
as a mitogen, angiogenic factor, and apoptosis-survival factor, our
findings implicate the HIF-1/AM link as a possible promotion mechanism
of carcinogenesis. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Several prior reports have demonstrated AM and its corresponding receptor (AM-R) to be ubiquitously expressed during embryogenesis and carcinogenesis. Early in both mouse and rat fetal development AM/AM-R are first detected in the heart, and then they appear progressively in other anatomical sites during organogenesis (7, 8). Maternal decidual cells and embryonic cells (fetal cytotrophoblast giant cells) of the ectoplacental cone, a site that mimics the invasion process of carcinogenesis, also show abundant expression of AM/AM-R (8, 9). After its initial identification in a human pheochromocytoma, further studies have demonstrated increased AM expression in tumor tissue of ganglioneuroblastoma, neuroblastoma, and adrenocortical carcinomas (10, 11). In addition, AM plasma levels are elevated in patients with gastrointestinal or lung cancer (12). Our group has shown that AM and AM-R are expressed in human tumor cell lines of the lung, breast, colon, ovary, prostate, brain, cartilage, and blood (13). In several of these lines, AM functioned as an autocrine proliferation factor whose effect could be inhibited by a neutralizing monoclonal antibody (MoAb-G6) causing growth cessation in vitro (13). Recently, it has been shown that hypoxic conditions or exposure to CoCl2 (a transition metal that mimics hypoxia) induces an increase in AM mRNA expression and protein production in a human colorectal carcinoma cell line, DLD-1 (14).
Focal areas of low oxygen tension (
2.0% O2)
are inherent to the biological processes of embryogenesis, wound
repair, and carcinogenesis (15, 16, 17). A state of diminished free oxygen
availability results when regional growth demands exceed the oxygen
supply of the capillary bed (15). Under such conditions, an
oxygen-sensing mechanism activates a transcription factor known as
hypoxia-inducible factor-1 (HIF-1), which in turn up-regulates a series
of genes that support the cell to compensate for the potentially lethal
microenvironment (18). HIF-1 is a heterodimer composed of HIF-1 and
HIF-1ß/ARNT (aryl hydrocarbon receptor nuclear translocator)
subunits, both representing members of the PAS (Per, ARNT, Sim)
basic-helix-loop-helix family (18). Transcription/translation products
of HIF-1 and HIF-1ß are constitutively expressed; however, the
HIF-1 protein contains an oxygen-dependent degradation domain
that is rapidly cleaved by the ubiquitin-proteasome pathway under
normoxic conditions, thus enabling the modulation of HIF-1 activity in
an oxygen-dependent manner (19). Genes transactivated by HIF-1 include
aldolase A, enolase 1, erythropoietin (Epo), glucose transporter 1,
heme oxygenase 1, inducible nitric oxide synthase, lactate
dehydrogenase A, phosphofructokinase L, phosphoglycerate kinase
1, transferrin (Tf), vascular endothelial growth factor (VEGF), and
endothelin-1 (ET-1) (18, 20). Low oxygen tension is known to play a
critical role in embryonic development, causes the emergence of
drug/radiation-resistant tumor cells, enhances mutagenesis of
neoplastic lesions, and elevates metastatic potential of the tumor (15, 21, 22, 23, 24).
The way AM gene expression is regulated in human tumors is not yet known, but a decrease in oxygen tension could be a major cause for the induction of this molecule. In our present study we actually demonstrate the ability of hypoxia and hypoxia mimetics to up-regulate the AM message and protein expression in a variety of human tumor cell lines. We also made use of both molecular and biochemical characterization approaches to support that this induction is mediated by transactivation of the AM promoter by HIF-1 transcription factor as well as posttranscriptional mRNA stabilization.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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illustrates a representative example of
the observed responses to our test conditions (6 h exposure to 100
µM CoCl2, 6 h exposure to 260
µM DFX or 12 h exposure to 1%
O2). Interestingly, although there is variability
of expression in the basal AM mRNA levels between cell lines [the two
pulmonary cancer cell lines NCI-H1264 (adenocarcinoma) and NCI-H157
(squamous cell carcinoma) being the opposing extremes], all tumor cell
lines show increases in AM message expression on exposure to our
hypoxic test conditions, with calculated test/basal ratios ranging from
1.3- to 25-fold.
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, A–C, demonstrates the
induction of AM message at different time increments over a 24–48 h
exposure series. Note that although CoCl2
exposure causes AM mRNA levels to reach a maximum at 8 h, both DFX
and 1% O2 induced an AM message zenith at
12 h, indicating a potential mechanistic difference between test
reagents. Induced maximum levels of AM message are maintained with all
the hypoxia treatments tested at 24 h of exposure, and even
elevated levels are still observed at 48 h of exposure to 1%
O2. Of the three treatments, exposure to 1%
O2 is the one that shows a steeper induction of
AM mRNA over time, and also more dramatic increases between the basal
and the maximum induction are observed (>25-fold increase between
maximum induction and baseline levels).
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mRNA in MCF7
cells throughout the hypoxic studies. HIF-1 transcript levels
remained constant or showed a slight decline over time in the
CoCl2 and 1% O2 time
course studies (Fig. 2
, A and C). This observation is in accordance
with other reports indicating that HIF-1 is mainly regulated not at
the transcriptional level but by protein stabilization in hypoxia,
whereas the protein is rapidly degraded by the
ubiquitin-proteasome pathway during normoxic conditions (19, 25).
Under Hypoxic Conditions, AM Is Also Induced at the Protein
Level
To address whether the induction of AM mRNA was accompanied by an
increase in the production of AM protein, the presence and cellular
localization of AM and HIF-1 in tumor cell lines at normoxic or
hypoxic conditions were studied using double immunofluorescence
followed by confocal microscopy. A typical image is shown in Fig. 3, in which prostate carcinoma DU 145
cells are immunostained for both AM (green fluorescence) and
HIF-1 (red fluorescence). At normoxic conditions (Fig. 3, A and B), both AM and HIF-1 are moderately expressed in the
cytoplasm and nucleus of the cells. After 12 h exposure to 260
µM DFX, the cells showed a marked increase of
AM staining in the cytoplasm and nucleus (Fig. 3C); in agreement with
our previous results (26) HIF-1 immunoreactivity is primarily
elevated in the nucleus of the cells (Fig. 3D). Similar patterns of
staining were obtained with H157, MCF7, and SNUC-1 cell lines (data not
shown).
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, a significant increase in
immunoreactive AM (IR-AM) was observed for MCF7 cells treated with 1%
O2 at various times as compared with the cells
maintained in normoxic conditions. Increasing values of accumulated
IR-AM in the conditioned media of this cell line were also observed for
the CoCl2 or DFX treatments (data not shown).
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or HIF-1ß
knockout mice to evaluate AM induction capabilities as compared with
those of their wild-type counterparts under hypoxic treatments. Figure 5A illustrates an experiment designed to
compare AM mRNA levels in these cell lines when subjected to 6 h
treatment of CoCl2 or DFX. It is shown that
HIF-1 null (-/-) mouse fibroblast cells failed to induce AM mRNA
expression in these conditions, while the wild-type HIF-1 (+/+)
fibroblasts showed an inducible response. Similarly, normal HIF-1ß
(+/+) mouse embryonic stem (ES) cells demonstrated an inducible
response, whereas HIF-1ß null (-/-) ES cells showed a 32% and a
60% reduced induction when compared with the wild-type control,
depending on the test reagent examined.
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+/+ and HIF-1 -/- cell lines were
exposed to a hypoxia (1% O2) time course study
(Fig. 5B), the hypoxic induction of AM mRNA was not detectable on the
cells with the null mutation, although it was present in the wild-type
cells. We also analyzed the levels of HIF-1 message expression in
this experiment. Note that HIF-1 mRNA remained constant through the
hypoxia treatment; however, HIF-1 transcripts in HIF-1 -/-
cells present a higher molecular size than in the corresponding
wild-type cells, since the null mutation was obtained by replacement of
the helix-loop-helix domain of HIF-1 with a neomycin resistance
cassette (28).
Furthermore, artificial manipulation of HIF-1 activity with appropriate
biochemical reagents was also used to elucidate the role of this
transcription factor in AM message regulation under hypoxia. In this
sense, the nitric oxide donor sodium nitroprusside (SNP) as well as
genistein (a tyrosine kinase inhibitor), are known to inhibit HIF-1
activity by blocking the synthesis of HIF-1 subunits and/or interfering
with HIF-1 DNA binding activity in hypoxia (29, 30). MCF7 cells were
cultured for 12 h in normoxic or hypoxic conditions, with or
without the appropriate biochemical reagents, and AM mRNA expression
was analyzed comparing the treated vs. the nontreated cells
(control). As is shown in Fig. 6, addition of 100 µM SNP completely inhibited the
induced expression of AM mRNA after 12 h of hypoxia (1%
O2) treatment, while genistein at 100
µM was a less potent suppressor of the AM mRNA
induction mediated by low oxygen tension. Thus, the inhibited activity
of HIF-1 with SNP and genistein is correlated with a suppressive effect
on the AM mRNA hypoxic induction. Conversely, it has been reported that
the CO scavenger hemoglobin (Hb) enhances HIF-1 activity by increasing
HIF-1 DNA binding (31). Treatment of MCF7 cells with 50
µM Hb was shown to further increase AM mRNA
expression under hypoxic conditions by approximately 1.7-fold (Fig. 6).
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, AM mRNA clearly decayed
more rapidly under normoxic than under hypoxic conditions, thus
indicating a stabilization process in hypoxia.
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Identification of Putative Hypoxia-Response Element (HRE) Sites in
the Human AM Gene
Since it is known that HIF-1 mediated gene transactivation
involves its binding to distinct nucleic acid motifs, namely HREs, we
used GCG computer software from Genetics Computer Group
(Madison, WI) to analyze the human and mouse AM genes (GenBank
accession nos. D43639 and D78349, respectively) for appropriate HRE
sequences. In this analysis we followed the HRE consensus motif
proposed by Wenger and Gassmann (18, 32): (T,G,C) (A,G)
CGTG (C,G,A) (G,T,C) (G,T,C) (C,T,G), which has been
constructed from the nucleotide sequence of HIF-1 binding sites of 13
oxygen-dependent genes, and allowed for no more than a single base
mismatch outside the CGTG core sequence. We have analyzed
not only the 5'-promoter region but also the 3'-flanking region,
introns/exons, and looked for consensus motifs in both sense and
antisense strands, since these genomic areas have been previously shown
to have functional HRE sites in other HIF-1-inducible genes (18, 20, 33). With these premises, eight putative HRE sites at the 5'- and six
sites at the 3'-untranslated flanking sequences of the human AM gene
were found, together with putative HRE sequences in the first intron
and in exon 3 and exon 4 (see Fig. 8).
Similar analysis of the mouse AM gene identified three putative HRE
sites in the antisense strand of the 5'-promoter region (positions:
-1143, [AACTCACGgA]; -98, [CAAGCACGCt];
and -62, [tGACCACGCC]), and another three on the sense
orientation: one in intron 1 (position 533,
[CGCGTGCTGa]), one in intron 2 (position 727,
[GcCGTGCTTT]), and finally one at exon 4 (position: 1960,
[GACGTGAaTG]).
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). A cotransfected ß-galactosidase
expression vector served as internal control for transfection
efficiency and extract preparation. After the transfection, cells were
left under normoxic conditions or exposed to 260 µM DFX
for 24 h, and luciferase activity was determined. No significant
increase for the luciferase activity in the DFX-treated cells/normoxic
cells was obtained when transfections were performed with constructs
containing the one or two putative HREs closest to the TATA box (Fig. 9A). However, when cells were transfected with constructs containing
four or eight putative HREs sites at the 5'-end of the TATA box, a
significant (P < 0.01) 1.5- and 1.7-fold increase in
luciferase activity was observed when comparing DFX-treated
vs. normoxic cells.
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expression vector pCMVß-HA-HIF-1
(34). This expression vector was cotransfected into MCF7 cells together
with the empty parental vector or plasmid constructs containing four or
eight HREs from the AM promoter (pGL2basic, pGL2b-4, or pGL2b-8), and
the normalization ß-galactosidase plasmid. A representative
experiment is shown in Fig. 9B, in which the transient overexpression
of HIF-1 augmented the luciferase reporter activity after DFX
treatment up to 2.9-fold when four HREs were present, and up to
4.8-fold with eight HREs as compared with the values of transfected
cells in normoxic conditions; no significant increase was observed when
the HIF-1 expression vector was cotransfected with the pGL2basic
empty vector. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Our initial analysis by Northern blot in a variety of human tumor cell
lines clearly demonstrates that hypoxia increases mRNA levels in these
cells as compared with the untreated controls. Interestingly, there is
a considerable variation in the basal levels of AM mRNA observed in
normoxic conditions. High levels of basal AM mRNA may arise from the
constitutive expression of HIF-1 protein in certain cells, a
condition that has been previously reported for primary cultures of
human pulmonary arterial smooth muscle cells (37). In the time course
experiments using MCF7 cells subjected to an hypoxic environment, we
also demonstrate that the AM mRNA induction is paralleled by a somewhat
delayed increased secretion of AM peptide from the cells to the
conditioned media. Given our prior finding that endogenous AM functions
as an autocrine growth factor for MCF7 (13), the demonstration that
hypoxia augments bioactive AM production and secretion in these cells
suggests that a similar scenario may take place in tissue neoplasms.
The production and secretion of AM at the hypoxic areas present in
tumors (15) could establish an autocrine/paracrine-mediated
proliferation event leading to tumor growth. In addition, since AM has
angiogenic and vasodilator capabilities (1, 6), the secreted AM could
induce neovascularization and facilitate nutritional supplementation to
the tumor cells. Finally, AM also has been shown to protect cells from
apoptosis (5), and this feature could selectively rescue tumor cells
from programmed cell death and might even predispose tumors to a more
malignant phenotype (15).
The data obtained with the HIF-1 and HIF-1ß knockout mouse cell
lines, together with those from the HIF-1 biochemical modulation
studies performed on MCF7 cells, provide consistent evidence supporting
the major involvement of HIF-1 in the transcriptional activation of AM
by hypoxia and suggest that AM could be considered a new member of the
growing family of HIF-1-targeted genes. The absence of inducible AM
mRNA expression in HIF-1 null mouse fibroblast cell line under
hypoxia seems to highlight the critical importance of the HIF-1
subunit in the transactivation of AM mRNA. Recent studies with HIF-1
knockout mice have shown this genetic deletion to be an embryonic
lethal event in the later stages of fetal development (28, 38). In
addition, tumors derived from mouse ES cells with HIF-1 null
genotype have retarded growth, reduced VEGF expression, and less
angiogenesis than their wild-type counterparts (28). Given that AM is
also highly expressed during embryogenesis (8), it would be interesting
to evaluate whether there are modifications in the AM distribution
patterns in early HIF-1 null embryos. HIF-2, a hypoxia-inducible
transcription factor sharing homology with HIF-1, has recently been
shown to be essential in embryonic vascularization and catecholamine
production (39); we cannot exclude a possible role of HIF-2 in AM
transactivation in endothelial and catecholamines-producing cells of
embryos as well as catecholamines-producing tumors. In contrast to
HIF-1 knockout cell lines, ES HIF-1ß null cells under hypoxia
showed a diminished AM mRNA induction as compared with their wild-type
counterparts, which may reflect the ability of other basic
helix-loop-helix family members (e.g. ARNT 2 and ARNT 3) to
compensate for the loss of HIF-1ß in the formation of a functional
heterodimer with HIF-1, albeit at lower efficiency (40, 41).
Prior studies have demonstrated the ability of reduced oxygen tension to mediate elevations in AM message/protein expression in several animal and cell systems. In this sense, hypoxia was shown to induce AM gene expression and secretion in cultured human umbilical vein endothelial cells (42); focal ischemic regions of the rat brain show high AM mRNA expression (43), and patients with chronic obstructive pulmonary disease involving tissue hypoxia have elevated AM plasma levels (44). Nakayama and colleagues (14) have demonstrated that hypoxia can elevate AM mRNA and protein expression in a single human colorectal carcinoma cell line, DLD-1; however, these investigators did not identify any HRE motifs in the 5'-upstream flanking region of the human AM gene and suggested the possible involvement of AP-1 in the hypoxia elevation they observed. Given that the AM promoter has several AP-1 binding motifs and that hypoxia can elevate c-fos, which in turn can activate AP-1 expression (45, 46), their supposition had a logical basis and may in fact work in concert with our observed HIF-1-driven AM expression. Recently, it was also reported that the hypoxic expression of AM in rodent cardiomyocytes is under HIF-1 control (47); although Cormier-Regard and colleagues clearly demonstrate the induction of AM mRNA under hypoxia, no experimental data confirming a similar relationship for AM peptide were shown.
In our luciferase reporter studies, MCF7 cells were transfected with
reporter plasmid constructs containing one, two, four, or eight of the
putative HRE consensus sequences that we identified in the human AM
promoter. Only when four or eight potential HREs were present, a
statistically significant fold increase in luciferase expression was
observed on exposure to DFX over that of untreated controls. Although
the luciferase induction observed in our test conditions is modest, it
is within the range observed for the mouse AM gene (47) and human Tf
(48). For the human VEGF gene, it has been reported that the increase
in transcription rate cannot account for all the observed increase in
the steady-state VEGF mRNA levels induced by hypoxia (49), and
important posttranscriptional regulation events mediated by mRNA
stabilization do, in fact, take place (50). A similar situation could
also be depicted for the human AM gene, since not only transcriptional
activation but also, as we have shown, RNA stabilization events
contribute to the AM mRNA up-regulation under hypoxic conditions. We
cannot exclude, however, the possibility of major functional HRE sites
at the 3'-flanking region of the human AM gene, a location that has
been shown for HREs in the human and mouse Epo gene (51, 52).
Furthermore, when MCF7 cells were cotransfected with a HIF-1
expression vector and plasmid constructs containing four or eight HREs
of the 5'-flanking region of the human AM gene, the increase in
luciferase activity was remarkably potentiated as compared with the
increase obtained with the pGL2–4 or pGL2–8 plasmids alone. This
stimulated luciferase reporter activity with the transient
overexpression of HIF-1 has also been reported for the human Tf,
VEGF, and Epo genes (48, 53). These data further support that the
transcriptional activation of AM in hypoxic conditions is driven by
HIF-1 and not through other transcriptional factors activated by
hypoxia; additionally, they give stronger evidence that at least some
of the selected potential HRE sites in the 5'- flanking region of the
human AM gene are functional in the up-regulation of AM transcription
under hypoxia.
Based on our actinomycin D studies, we clearly demonstrate AM mRNA
stabilization mediated by hypoxic conditions. Recent studies on RNA
degradation mechanisms have identified hypoxia-inducible proteins that
bind to adenylate-uridylate (AU)-rich elements of the 3'-untranslated
region (3'-UTR) of short half-life RNA (i.e. VEGF, c-Myc,
c-fos) and suppress ribonuclease degradation (54, 55). One
such stabilizing RNA-binding protein, HuR, has been shown to interact
with AUUUA or AUUUUA base sequences in the 3'-UTR of the VEGF mRNA and
to extend its half-life under hypoxic conditions (55). Interestingly,
the 3'-UTR of both human and mouse AM mRNA have AUUUA and AUUUUA
sequences that could possibly augment message survival during reduced
oxygen tension through a similar HuR or HuR-like interaction. In
addition, tumor cell lines that have a mutated von Hippel-Lindau (VHL)
tumor suppressor gene contain constitutively stabilized VEGF mRNA and
also have constitutively expressed stabilizing RNA-binding proteins
(54). Considering these reports and the recent discovery by Ratcliffe
and co-workers that the VHL gene product controls the degradation of
HIF-1 protein (25), it will be interesting to determine the status
of the VHL suppressor gene in those human tumor cell lines that have an
elevated basal expression of AM message under normoxic conditions
(i.e. H157), to determine whether this feature relates to an
increased half-life of AM mRNA mediated by constitutive expression of
stabilizing RNA-binding proteins.
In conclusion, we have shown evidence in favor of hypoxia as an inducer of AM mRNA and protein expression in human tumor cell lines. The data obtained from HIF-1 knockout mouse cell lines, biochemical modulation of HIF-1 activity, and transfection experiments give solid proof for the involvement of HIF-1 in the up-regulation of AM mRNA under hypoxic conditions. In addition to HIF-1 transcriptional activation, increased hypoxic mRNA stability also accounts for AM induction based on our actinomycin D assays. Our collective data, taken together with previous reports that most solid human tumors have common hypoxic regions (15) and that AM can function as a mitogen/angiogenic factor/apoptotic survival factor (5, 6, 13), implicate HIF-1/AM as members of a potential promotion mechanism of carcinogenesis and identify a possible biological target for intervention strategies against malignant disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(-/-) fibroblast cell line was generated from HIF-1 null
mice (28) via SV-40 transformation of embryonic fibroblast cells;
HIF-1 (-/-), and (+/+) fibroblast cell lines were maintained with
the same media as specified for the human tumor cell lines. Cells were cultured at 37 C in 20% O2, 5% CO2, 75% N2 for normoxic conditions, and new media were added 12 h before the beginning of each hypoxia experiment. The hypoxia induction was achieved either by hypoxia mimetics: 100 µM CoCl2, 260 µM DFX mesylate (both from Sigma, St. Louis, MO), or by culturing cells in a hypoxia chamber at 37 C with 1% O2, 5% CO2, 94% N2 atmosphere. The hypoxia chamber was fabricated from a Labconco seamless fiberglass vacuum desiccator (Fisher Scientific, Pittsburgh, PA) fitted with two stainless steel angle ball valves having serrated hose connectors (Washington Valve & Fitting Co., Frederick, MD), which allowed for chamber equilibration to hypoxic environment via venting with gas mixture (Roberts Oxygen Co., Gaithersburg, MD). The chamber was tested for leaks under positive pressure using a bubble-forming agent (SNOOP, Nupro Co., Willouhby, OH) and was shown to hold a 3 psi charge for 48 h.
In the HIF-1 modulating studies, different agents were added to the culture media, and then the cells were incubated under normoxic conditions or in the hypoxia chamber for 12 h at 37 C. SNP (a NO donor), and genistein (a tyrosine kinase inhibitor), were used at 100 µM to inhibit HIF-1 activity (29, 30). Hemoglobin (Hb), which acts as a CO scavenger, was used at a final concentration of 50 µM to up-regulate HIF-1 activity (31); Hb was prepared by treatment with excess reducing agent sodium dithionite (57). All reagents were purchased from Sigma.
For the AM mRNA stabilization studies, cells were initially exposed to 12 h hypoxia, after which actinomycin D was added at a final concentration of 4 µg/ml (Sigma); cells were subsequently maintained from 1 h to 4 h under normoxic or hypoxic conditions.
Northern Blot Analysis
Immediately after treatment for the indicated times, cells were
washed once in PBS, and total RNA was extracted using the guanidine
isothiocyanate and cesium chloride method (58). Fifteen micrograms of
RNA were loaded per lane, run in 1% agarose gels containing 2.2
M formaldehyde, blotted by capillarity onto nitrocellulose
membranes (Schleicher & Schuell, Inc., Keene, NH), and
baked for 2 h at 80 C. Equal loading and integrity of RNA were
monitored by ethidium bromide staining of the 28 S subunit of rRNA.
The human AM cDNA probe used in this study was generated as an
RT-PCR product (831 bp) obtained using oligonucleotide primers:
5'-TACCTGGGTTCGCTCGCCTTCCTA-3' (sense, bp 184–207) and
5'-CTCCGGGGGTCTCAGCATTCATTT-3' (antisense, bp 991-1014). The human
HIF-1 cDNA probe was also produced by RT-PCR (1308-bp product) using
oligonucleotide primers: 5'-CGGCGCGAACGACAAGAAAAAGAT-3' (sense, bp
43–66) and 5'-TCGTTGGGTGAGGGGAGCATTACA-3' (antisense, bp 1327–1350).
Numbering of the nucleotide base positioning was taken from the GenBank
profile accession no. D14874 (human AM mRNA) and U22431 (human HIF-1
mRNA). All RT-PCR products were sequenced to validate base integrity of
the probes. The mouse AM cDNA 550-bp probe was a gift from Dr. Sonia
Jakowlew (59).
Probes were labeled with [-32P]dCTP (3000
Ci/mmol; NEN Life Science Products, Boston, MA) by random
priming, and unincorporated nucleotides were removed by ProbeQuant G-50
Micro Columns (Amersham Pharmacia Biotech, Piscataway,
NJ). Hybridization was carried out overnight at 42 C in a hybridization
buffer containing 40% formamide (58). After stringency washes, blots
were exposed to XAR film (Eastman Kodak Co., Rochester,
NY) at -80 C for varying times. Densitometry of the autoradiograms was
performed using a ChemiImager 4000 (Alpha Innotech Corp., San Leandro,
CA). The half-life of the endogenous AM mRNA was calculated using Prism
3.0 software.
Confocal Immunofluorescence for AM and HIF-1
Cells were grown on glass slides, treated with 260
µM DFX for 12 h, and fixed in Bouin’s fluid
(Sigma) for 10 min at room temperature. Slides were
blocked with normal goat serum (1:30 in PBS) for 30 min and then
incubated overnight at 4 C in a mixture of both antibodies: mgc3
antihuman HIF-1 monoclonal antibody (60) at 1:500 dilution and
rabbit antihuman AM 22–52 antibody (13) at 1:1,000 dilution. The
second layer consisted on a mixture of Rhodamine-antimouse and
Bodipy-antirabbit IgGs (Molecular Probes, Inc., Eugene,
OR) at a final concentration of 1:200 each. The cells were observed
with a Carl Zeiss Laser Scanning Microscope 510, equipped
with four lasers. Images from cells subjected to DFX treatment and
normoxic controls were taken with exactly the same microscope settings
and exposures, to compare expression of AM and HIF-1 in both
conditions.
RIA of Immunoreactive AM
Concentrations of AM in culture media of MCF7 cells under
hypoxia or mimetics treatment were measured by double antibody RIA.
Samples of culture media (1 ml) were mixed with an equal volume of
0.1% alkali-treated casein in PBS, pH 7.4, and applied to
reverse-phase Sep-Pak C-18 cartridges (Waters Corp.,
Milford, MA). The proteins were eluted with 3 ml of 80% isopropanol
containing 0.125 N HCl and lyophilized. Extracts were
reconstituted in 0.4 ml of RIA buffer (10 mM phosphate, 50
mM EDTA, 135 mM NaCl, 5 mM
NaHCO3, 0.05% Triton X-100, 0.1% Tween-20,
0.1% alkali-treated casein, 20 mg/l phenol red, pH 7.4), and spun to
remove any solid matter. After a 24-h preincubation of 0.1 ml of sample
with 0.1 ml of antihuman AM antibody (Phoenix Pharmaceuticals, Inc., Mountain View, CA) at 4 C, 0.1 ml of
125I-labeled AM (Phoenix Pharmaceuticals, Inc.) was added (10,000 cpm) and the mixture was incubated at 4
C overnight. Bound tracer was separated by
polyethylenglycol-facilitated precipitation with goat antirabbit IgG
and normal rabbit serum. After centrifugation, the supernatant was
discarded, and the radioactivity in the pellets was determined in a
-counter. Data were statistically evaluated by a two-tailed
Student’s t test using Prism 3.0 software. Differences were
regarded as significant at a value of P < 0.05.
Reporter Plasmid Constructs
A PCR product of 118 bp (-118, -1) containing the putative HRE
site closest to the transcription start site in the 5'-flanking region
of the AM gene (named HRE f in Fig. 8) was generated using human
genomic DNA as template and the following oligonucleotide primers:
sense, 5'-GCTGAGGAAAGAAAGGGAAG-3' and antisense,
5'-TGTCACCAAGAAACCACTGA-3'. Similarly, primers: sense,
5'-AGCCCCAAAGGAAGCAATGC-3' and antisense,
5'-TGTCACCAAGAAACC-ACTGA-3' were used to generate a PCR product of
166 bp (-166, -1) comprising the two potential HREs closest to the
transcription start site of the AM promoter (HREs named e and f in Fig. 8). Each of the resulting 118-bp and 166-bp DNA fragments were ligated
into the pCR2.1 vector (Invitrogen, Carlsbad, CA) to
generate pCR2.1–118 and pCR2.1–166. These plasmids were then digested
with HindIII and XhoI, and the resulting DNA
fragments were cloned into the same sites of a promoterless luciferase
reporter pGL2basic (Promega Corp., Madison, WI) to
generate pGL2b-1 and pGL2b-2, respectively.
The entire 5'-flanking region of the human AM gene was amplified by
standard PCR from human genomic DNA using primers: sense,
5'-GAATTCAGGTCCGCTCAGGTGACTCCTTCC-3' and antisense,
5'-GAGCTCGCTAGCCAGTGTCACCAAGAAACC-3' (the
antisense primer introduced a SacI site
[underlined] and an NheI site
[bolded]). The resulting 1755 bp (-1751, +4) product was
ligated into the pCR2.1 vector to generate pCR2.1–1755. An
NheI/NheI fragment from pCR2.1–1755 was
subcloned into the same site of pGL2basic generating pGL2b-4 (which
carries 4 putative HREs from the 5'-end of the AM promoter, namely c,
d, e, and f in Fig. 8). In the same way, a
SacI/SacI fragment from pCR2.1–1755 was also
subcloned into the SacI site of pGL2basic, generating
pGL2b-8, which encompasses the 8 putative HREs in the 5'-flanking
region of the AM gene (HREs a, b, c, d, e, f, q, and r in Fig. 8). The
fidelity of all PCR-derived sequences was verified by sequence
analysis. All positions are referred relative to the transcription
start site of the AM gene (+1; see Fig. 8).
The HIF-1 expression vector (pCMVß-HA-HIF-1) (34) was
generously provided by Dr. D. Livingstone (Dana Farber Cancer
Institute).
Transient Transfections and Luciferase Reporter Assay
Approximately 20 h before transfection 1.5 x
105 MCF7 cells were seeded onto 60-mm plates.
Each dish was then transfected for 4 h in the presence of
lipofectAMINE and Optimem medium I (Life Technologies, Inc.) with 1 µg of pSV-ß-galactosidase control vector
(Promega Corp.) and 3 µg of one of the following
plasmids: pGL2basic, pGL2b-1, pGL2b-2, pGL2b-4, or pGL2b-8. For the
HIF-1 transient overexpression assays, 5 µg of the
pCMVß-HA-HIF-1 vector were cotransfected with an equal amount of
one of the following plasmids: pGL2basic, pGL2b-4, or pGL2b-8, together
with 1 µg of pSV-ß-Galactosidase control vector (quantities
referred per dish). All transfections were carried out in duplicate
with aliquots of transfection mixture from a single pool. After
transfection, cells were incubated in RPMI 1640 medium supplemented
with 10% FBS and were either treated with DFX at 260 µM
for 24 h or left under normoxic conditions for the same time.
Cells were then collected in EBC lysis buffer with protease inhibitors
(40 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40,
1 mM AEBSF, 10 µg/ml aprotinin, 1 mM
NaVO4, 10 µg/ml leupeptin), and luciferase and
ß-galactosidase activities were determined according to the
manufacturer’s instructions using a TopCount NXT Packard luminometer
and a Bio-Rad Laboratories, Inc. 3550 Microplate Reader.
Luciferase readings were normalized by the ß-galactosidase values to
correct for differences in transfection efficiency and extract
preparation. For each construct transfectants, data were expressed as
fold increase of the luciferase value obtained with the DFX treatment
as compared with the luciferase value obtained in normoxic conditions,
which was arbitrarily defined as 1. Data were statistically evaluated
by a two-tailed one-sample Student’s t test using Prism 3.0
software. Differences were regarded as significant at a value of
P < 0.05.
|
ACKNOWLEDGMENTS |
|---|
antibody, and to Dr. R. H. Wenger
for critical reading of the manuscript (Institute of Physiology,
University of Zürich-Irchel). We gratefully acknowledge Dr. Y.
Ward (Department of Cell and Cancer Biology, National Cancer Institute)
for her expertise in confocal microscopy. Authors are also thankful to
Drs. E. Maltepe and C. Simon (Howard Hughes Medical Institute,
University of Chicago) for the HIF-1ß knockout mouse cell line, and
to Dr. D. Livingstone (Dana Farber Cancer Institute) for the
pCMVß-HA-HIF-1 vector. |
FOOTNOTES |
|---|
M. Garayoa was supported by a postdoctoral fellowship from the Dirección General de Enseñanza Superior, Ministerio de Educación y Cultura, Spain (PF96 0029138440). R. Pío was recipient of a fellowship from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (Grant 98/9172). This work was also supported in part by the Swiss National Science Foundation (3100-56743.99).
Received for publication November 23, 1999. Revision received February 1, 2000. Accepted for publication March 1, 2000.
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