From the Department of Pathology, University of Texas
Health Science Center, San Antonio, Texas 78229, the ¶ Institute
of Genetic Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21287, and the
Department of Medicine,
Massachusetts General Hospital, Harvard Medical School, Charleston,
Massachusetts 02129
Received for publication, December 28, 2000, and in revised form, February 21, 2001
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ABSTRACT |
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Hypoxia is a key determinant of tissue pathology
during tumor development and organ ischemia. However, little is known
regarding hypoxic regulation of genes that are directly involved in
cell death or death resistance. Here we report the striking induction by severe hypoxia of the anti-apoptotic protein IAP-2. Hypoxic cells
with IAP-2 up-regulation became resistant to apoptosis. IAP-2 was
induced by hypoxia per se rather than by the secondary effects of hypoxia, including ATP depletion and cell injury. The inductive response did not relate to alterations of cellular redox status or arrest of mitochondrial respiration. On the other hand, IAP-2
induction was attenuated by actinomycin D, suggesting a role for gene
transcription. In vitro nuclear run-on assays demonstrated specific increases in IAP-2 transcriptional activity after hypoxia exposure. HIF-1, the primary transcription factor that is responsible for multiple gene activation under hypoxia, does not have a role in
IAP-2 expression. HIF-1 and IAP-2 were induced by different degrees of
hypoxia; severe hypoxia or anoxia was required for IAP-2 induction.
Moreover, cobalt chloride and desferrioxamine activated HIF-1 but not
IAP-2. Finally, IAP-2 was induced by severe hypoxia in mouse embryonic
stem cells that were deficient of HIF-1. Thus, this study not only
provides the first demonstration of hypoxic regulation of an
anti-apoptotic gene but also suggests the participation of novel
hypoxia-responsive transcription mechanisms.
Lack of oxygen, i.e. hypoxia, plays a fundamental role
in many pathologic processes. In ischemic diseases, including stroke, myocardial infarction, and acute renal failure, hypoxia leads to cell
death and determines tissue pathology (1). In solid tumors, hypoxia
selects death-resistant cells, which confer poor prognosis and
contribute to cancer progression (2-4). Under hypoxia, some cells are
irreversibly injured and die whereas others can adapt to the stressful
environment and survive. The factors that determine the fate of
individual cells under hypoxia are poorly understood. However,
expression of specific genes within these cells appears to be a key
(5).
In response to hypoxia, mammalian cells express a variety of gene
products, including erythropoietin, vascular endothelial growth
factor, glucose transporter, and glycolytic enzymes (5, 6).
These proteins either increase oxygen delivery or enhance glycolysis to
facilitate metabolic adaptation. In 1992, the critical transcription
factor HIF-1,1 which is
responsible for hypoxic activation of multiple genes, was identified
(7). HIF-1 is a heterodimeric basic helix-loop-helix-per-aryl hydrocarbon receptor ARNT-sim (PAS) domain protein, consisting of Despite intense investigation of gene expression under hypoxia, little
has been learned about hypoxic regulation of genes that are directly
involved in cell death or death resistance (5). In the current study,
we show that the cell death inhibitory protein IAP-2 is strikingly
induced by severe hypoxia. IAP-2 is a member of the family of
inhibitors of apoptosis (IAPs), which were originally discovered in
baculoviruses and subsequently cloned from metazoans, including human
(11-18). In baculovirus, IAPs halt the death of host cells and thereby
preserve the microenvironment for virus proliferation (11, 12). In
Drosophila, loss of function of IAPs leads to inappropriate
cell deletion during development (19). In mammals, high levels of IAP
expression are shown in neoplasia (20, 21), and genetic defects of
neural IAPs are associated with the neurodegenerative disorder spinal
muscular atrophy (15). These studies suggest a pivotal role for IAPs in
maintenance of tissue homeostasis and protection against deleterious
insults. Our results show that hypoxic cells overexpress IAP-2 and
become resistant to apoptosis. IAP-2 up-regulation under hypoxia
does not result from ATP depletion, cellular redox status alteration, or respiration arrest; rather, the key to the inductive response is
lack of oxygen per se. IAP-2 induction involves gene
transcription; however, HIF-1, the primary hypoxia-responsive
transcription factor, is shown to be not involved. Thus, the results
not only demonstrate the striking induction of an anti-apoptotic gene
by hypoxia but also imply novel hypoxia-responsive transactivation mechanisms.
Cells--
Rat kidney proximal tubule cells (RPTC) were provided
by U. Hopfer (Case Western Reserve University, Cleveland, OH). RPTC overexpressing Bcl-2 were obtained by stable transfection of a vector
containing bcl-2 cDNA (22). The bcl-2 vector was a gift from J. Yuan (Massachusetts General Hospital, Boston, MA). Wild type and
HIF-1 Reagents--
The monoclonal antibody to Bax was a gift from R. Youle (NINDS, National Institutes of Health, Bethesda, MD). The
monoclonal antibody to HIF-1 Hypoxic Incubation--
An in vitro model of
hypoxia-reoxygenation has been described previously (22, 24) and was
adopted in this study. Briefly, cells were washed with
phosphate-buffered saline, transferred to an anaerobic chamber with
95% N2/5% CO2, and incubated in Krebs-Ringer bicarbonate buffer. This buffer was pre-equilibrated with 95% N2/5% CO2. EC Oxyrase, a biocatalytic
oxygen-reducing agent, was added at 1.2 unit/ml to the incubation
medium to consume residual O2 and maximize the degree of
hypoxia. For reoxygenation, cells after hypoxic incubation were
transferred back to full culture medium in 95% air/5%
CO2.
Immunoblot Analysis--
Cells were lysed with a buffer
containing 2% SDS, 100 mM DTT, and 62.5 mM
Tris-HCl (pH 6.8). Proteins (100 µg/lane) in whole-cell lysates were
subjected to reducing SDS-polyacrylamide (4-12%) gel electrophoresis.
The resolved proteins were then electroblotted onto a polyvinylidene
difluoride membrane. After 1 h of blocking in 2% bovine serum
albumin, the membrane was incubated overnight with primary antibodies
at 4 °C. By the next morning, the membrane was thoroughly washed and
incubated with horseradish peroxidase-conjugated secondary antibodies.
Antigens on the membranes were finally revealed by exposure to
chemiluminescent substrates (Pierce, Rockford, IL).
Northern Hybridization--
Total RNA was extracted from control
or experimental cells with TRI reagent (Molecular Research Center,
Cincinnati, OH). mRNA was subsequently purified with poly(A)Tract
(Promega, Madison, MI). Northern hybridization was carried out with a
NorthernMax kit purchased from Ambion (Austin, TX), following the
manufacturer's instruction. Briefly, mRNA (2 µg/lane) was
electrophoresed on formaldehyde-containing agarose gels. Resolved
mRNA was transferred to BrightStar-Plus positively charged nylon
membranes by downward capillary transfer and cross-linked to the
membranes using a UV cross-linker. Hybridization was carried out at
42 °C, using an iap-2-specific probe. The probe was generated
by reverse transcription-polymerase chain reaction, using primers
designed on the basis of the cDNA sequences of rat
IAP-2,2 and was
32P-labeled with the Ready-To-Go random labeling beads
(Amersham Pharmacia Biotech Inc., Piscataway, NJ). To assess
variation in loading and transfer of RNA, the blots were stripped and
reprobed with 32P-labeled glyceraldehyde-3-phosphate
dehydrogenase cDNA. Levels of gene-specific mRNA was revealed
by autoradiography.
In Vitro Nuclear Run-on Analysis of Transcriptional
Activity--
Nuclei from control and experimental cells were isolated
by a method modified from Spector et al. (25). Briefly,
cells were collected by scraping and centrifuging at 500 g/5 min and
lysed by 0.5% Nonidet P-40 in the homogenization buffer containing (in mM): 340 sucrose, 60 KCl, 15 NaCl, 2 EDTA, 0.5 EGTA, 1 DTT,
0.1 phenylmethylsulfonyl fluoride, 0.15 spermine, 0.5 spermidine, and
15 Tris-HCl, pH 7.5. Nuclei in cell lysates were purified by
centrifugation at 800 g/5 min through a 25% glycerol cushion. Purified
nuclei were resuspended in the nuclear storage buffer containing 40%
glycerol, and kept frozen in liquid nitrogen. During isolation, nuclear
morphology was monitored by phase contrast microscopy. For run-on
reaction, ~75 × 106 nuclei were incubated for 30 min at 30 °C in the reaction buffer containing (in mM):
150 KCl, 5 MgCl2, 2.5 DTT, 30 Tris-HCl, pH 8.0, 0.5 ATP,
0.5 CTP, 0.5 GTP, 100 µCi of [ Analysis of Apoptosis--
Apoptosis was assessed by cell
morphology and caspase activity (22, 24). Typical morphological
alterations used to characterize apoptosis included cellular shrinkage,
nuclear condensation and fragmentation, and formation of apoptotic
bodies (22). Five fields with ~200 cells/field in each 35-mm dish
were monitored. The experiments were repeated for five times with
duplicate dishes for each condition in every experiment. To
biochemically analyze apoptosis, caspase activity was measured using
the fluorogenic peptide substrate,
carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin
(DEVD.AFC) as described in our previous studies (24). Briefly, cells
were extracted with 1% Triton X-100. The lysates (25 µg of protein)
were added to the enzymatic reactions containing 50 µM
DEVD.AFC. After 60 min of reaction at 37 °C, fluorescence was
monitored at excitation 360 nm/emission 530 nm. For each measurement, a
standard curve was constructed using free AFC. Based on the standard
curve, the fluorescence reading from the enzymatic reactions was
translated into the molar amount of liberated AFC to indicate caspase
activity (24).
Chemical Analyses--
Protein was quantitated with the
bicinchoninic acid reagent purchased from Pierce Chemical Co.,
Rockford, IL. The concentration and purity of RNA were determined
spectrophotometrically by absorbance readings at 260 and 280 nm. To
measure ATP, cells were extracted with trichloroacetic acid. ATP in
cell extracts was determined by luminometry of the luciferin firefly
luciferase reaction using a kit purchased from Sigma Chemical Co.
(22).
IAP-2 Induction by Hypoxia--
We initially screened the
expression of apoptosis regulatory genes by immunoblotting. For this
purpose, RPTC cells were subjected to hypoxic incubation alone or
hypoxia followed by reoxygenation. Whole cell lysates were extracted
with a buffer containing 2% SDS and analyzed by immunoblotting. The
results are shown in Fig. 1. No
significant changes were detected in the expression of the anti-apoptotic protein Bcl-2 or its homologue, the pro-apoptotic protein Bax. Consistent with our previous observations, Bcl-2 was
barely detectable in control cells and remained low throughout hypoxia
and reoxygenation (Fig. 1A, lanes 1-7), whereas
abundant Bcl-2 was shown in cells stably transfected with a bcl-2
vector (Fig. 1A, lane 8). Cellular levels of Bax
were also stable (Fig. 1A), despite its translocation from
cytosol to mitochondria during hypoxia (22). In sharp contrast, IAP-2,
an apoptosis inhibitory protein was markedly induced by hypoxia. As
shown in Fig. 1A, basal IAP-2 expression in control cells
was quite limited (lane 1), whereas abundant IAP-2 was
detected after hypoxic incubation (lanes 2-4). IAP-2
induction reached a maximum during 1 h of hypoxia exposure and, of
interest, returned toward basal levels after reoxygenation (Fig.
1A, lane 5). The identity of IAP-2 was verified using three antibodies recognizing different epitopes (data not shown).
The antibodies were prepared separately by Santa Cruz Biotechnology
Inc. and by our laboratory.
The inductive response was rather specific for IAP-2, because no
induction was detected in IAP-1 (Fig. 1A) or XIAP (data not shown). Hypoxic induction of IAP-2 was not suppressed by glucose, a
glycolytic substrate that prevented declines of ATP in hypoxic cells,
negating the involvement of ATP depletion in the inductive response
(Fig. 1A, lane 6, Glu). Moreover,
neither the caspase inhibitor VAD
(carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone) nor Bcl-2 overexpression
affected IAP-2 induction (Fig. 1A, lanes 7,
8). The results indicate that IAP-2 expression was not a
result of cell injury/death, because VAD and Bcl-2 abolished apoptosis during hypoxia reoxygenation (22, 24).
Hypoxic expression of IAP-2 is not cell-type-specific, and was detected
in 3T3 fibroblasts, human kidney proximal tubular epithelial cells and
primary cultures of human umbilical vein endothelial cells (Fig.
1B, "C " for control; "H " for
hypoxia). Similar induction was also shown for malignant human renal
carcinoma cells (Fig. 1B, lanes 7, 8,
HRC).
Oxygen Deficiency, Not ATP Depletion, Leads to IAP-2
Expression--
Under hypoxia, cells are exposed to two kinds of
immediate stress, i.e. lack of oxygen and declines of
cellular ATP. Many pathologic processes are initiated during hypoxia by
ATP depletion rather than by oxygen deprivation per se (1).
Thus, to investigate the mechanisms responsible for IAP-2 induction, we
first determined whether it is a result of ATP depletion. In our
experimental model of hypoxia, progressive decreases of cellular ATP
were demonstrated. After 5 h of hypoxic incubation in the absence
of metabolic substrates, cell ATP declined to less than 1% of control.
Provision of 1 mg/ml glucose, the glycolytic substrate, maintained
cellular ATP to a substantial level, ~70% of control (22). The fact
that glucose given during hypoxia did not mitigate IAP-2 expression
(Fig. 1A, lane 6, Glu) suggests that
the induction was not caused by ATP depletion. To substantiate this
observation, we examined the effects of stepwise ATP declines. Cells
were incubated under normal oxygen atmosphere in a medium containing
the mitochondrial uncoupler CCCP, which abolishes ATP production by
oxidative phosphorylation (26). Glucose, the glycolytic substrate, was
provided at 0-0.5 mg/ml along with CCCP to achieve graded degrees of
ATP depletion (Fig. 2A). As a
positive control, one group of cells was subjected to 1 h of
hypoxic incubation and showed striking IAP-2 induction (Fig.
2B, lane 2). In sharp contrast, IAP-2 expression
remained minimal in cells incubated with CCCP in the presence of
various amounts of glucose (Fig. 2B, lanes
3-10). Thus, ATP depletion, either severe or mild, did not
activate this anti-apoptotic gene. The data imply that, independent of
ATP depletion, oxygen deficiency plays a critical role in hypoxic
induction of IAP-2.
Hypoxic Expression of IAP-2 Does Not Depend on Changes of Cellular
Redox Status--
Cellular redox status has been shown to affect gene
expression under conditions of oxygen deficiency (27-30). To test
whether it played an essential role in IAP-2 induction under hypoxia, we adjusted the redox status of the cells pharmacologically, using two
classes of reagents. The first class of chemicals tested were antioxidant, including the ROS scavenger N-acetyl-cysteine
(NAC), the cell permeant superoxide dismutase mimetic MnTBAP, and the glutathione peroxidase mimic Ebselen (31). The second class of
chemicals tested included H2O2 and
t-butylhydroperoxide (t-BHP), which by their
nature should augment cellular oxidative status. The results are shown
in Fig. 3. It is clear that, despite
manipulation of cellular redox status, neither the anti-oxidants nor
the pro-oxidants could induce IAP-2 expression (lanes 3-7)
under normal oxygen tensions. On the contrary, this anti-apoptotic gene
was activated by hypoxia in a remarkable manner (lane 2).
Data not shown have also demonstrated that the anti-oxidant NAC was not
able to ameliorate IAP-2 expression during hypoxia. Collectively, the
results suggest that alterations of cellular redox status do not have a
critical role in IAP-2 induction under hypoxia.
Mitochondrial Inhibition Does Not Lead to Apoptotic Gene
Expression--
We subsequently examined whether hypoxia up-regulated
IAP-2 through its actions on the mitochondrial function. Electron
transport in mitochondria is disrupted in cells under situations of
oxygen deficiency. This is due to the lack of the terminal electron
acceptor, molecular oxygen. Moreover, as depicted in Fig.
4A, hypoxia and its secondary
effects, including ATP depletion can also damage the machinery
responsible for respiration and oxidative phosphorylation (32). Key
components of the respiratory chain have been postulated as the
O2 sensors that signal gene expression in hypoxic cells (5,
33-35). To investigate the roles played by respiratory macromolecules in apoptotic gene expression, we examined the effects of respiration inhibitors. The rationale for this experiment was that, if hypoxic expression of IAP-2 was triggered by defects in electron transport, then a somewhat similar inductive response should be elicited by
poisons targeting the key components of the respiratory chain. The
inhibitors tested included the complex I inhibitor rotenone, complex
III inhibitors antimycin and myxothiazole, complex IV inhibitor sodium
azide, and potassium cyanide. Efficacy of the inhibitors to block
respiration in RPTC cells was indicated by their ability to prevent
mitochondrial ATP production (data not shown). In addition, we examined
the effects of oligomycin, an F0- and
F1-ATPase/synthase inhibitor, on IAP-2 expression. The results are shown in Fig. 4B. Clearly, none of the
mitochondrial poisons could mimic hypoxia, with respect to inducing
IAP-2. The data suggest that hypoxic expression of this anti-apoptotic
gene does not result from respiration defects.
Transcriptional Mechanisms Are Involved in IAP-2 Expression under
Hypoxia--
Our results presented so far have demonstrated hypoxic
induction of IAP-2 at the protein level by immunoblotting. Accumulation of a protein could be the result of increased transcription, mRNA stabilization, or decreased protein turnover. To examine the role of
transcription in IAP-2 expression, we initially tested the effects of
actinomycin D (Act.D), a DNA-primed RNA polymerase inhibitor that
blocks transcription. Cells were pretreated with Act.D for 1 h and
then shifted to hypoxic incubation. The results are shown in Fig.
5A. Consistent with our
previous observations, IAP-2 was barely detectable in control cells and
was markedly induced after hypoxia exposure (lanes 1,
2). Of significance, the inductive response was suppressed
by 10-100 µg/ml Act.D (lanes 5-8), suggesting a role for
transcription. This observation was further supported by Northern
analysis of mRNA, using 32P-labeled rat IAP-2-specific
probes. As shown in Fig. 5B, Northern hybridization detected
an IAP-2 transcript of ~3.5 kb. Sizes of the transcript agreed well
with the full-length cDNA of rat IAP-2 that we have
cloned.2 Moreover, expression of IAP-2 mRNA was
significantly increased during hypoxia (Fig. 5B, lanes
2-4); this increase was again suppressed by the transcription
inhibitor, Act.D (Fig. B, lane 5). Together, the
results suggest that gene transcription plays an important role in
IAP-2 induction under hypoxia.
Cellular Transcriptional Activity for IAP-2 Is Increased during
Hypoxia: in Vitro Nuclear Run-on Assays--
To further establish a
role for transcription in IAP-2 induction, we measured the cellular
transcriptional activity for this anti-apoptotic gene directly by
in vitro nuclear run-on assays. Nuclei were isolated from
control and hypoxic cells, and used for run-on reactions in the
presence of [ HIF-1 Does Not Have an Essential Role in IAP-2 Induction under
Hypoxia--
To pursue the transcriptional mechanisms underlying IAP-2
expression, we initially examined the role of HIF-1, a ubiquitous transcription factor that is responsible for hypoxic activation of
several classes of genes (5). HIF-1 is a heterodimer, consisting of Hypoxic Cells Overexpressing IAP-2 Are More Resistant to
Apoptosis--
Our results have demonstrated IAP-2 induction under
severe hypoxia by HIF-1-independent mechanisms. An intriguing question arose: what is the biological meaning of this inductive response? Given
the recognized role of IAP-2 as an apoptosis suppressor, we determined
whether cells with IAP-2 up-regulation became more resistant to
apoptotic insults. To this end, we compared apoptosis induced by
staurosporine in control, hypoxic, post-hypoxic, azide-treated, and
post-azide cells. Staurosporine (STA) was chosen as the apoptotic trigger, because this broad-spectrum protein kinase inhibitor potently
induces apoptosis in various kinds of cells and has been widely used in
apoptosis research (38-42). The results are shown in Fig.
8A. Control cells after 5 h of incubation in physiological buffer showed ~5% apoptosis
(condition "a"). Four hours of STA treatment following
1 h of control incubation led to apoptosis in ~40% cells
(condition "b"). STA-induced apoptosis was completely blocked by the general caspase inhibitor VAD (condition
"c"). Neither 1 h of azide preincubation nor
continuous presence of azide prevented STA-induced apoptosis
(conditions "e " and "d"). Of
significance, STA-induced apoptosis was inhibited to ~11%, when the
cells were incubated continuously under severe hypoxia (condition
"f"). On the other hand, if the cells were first exposed to 1 h of hypoxia and then returned to normal oxygen for 4 h
of STA treatment, apoptosis developed in 38% cells (condition
"g"). The results show that hypoxic cells and not
post-hypoxic cells were more resistant to apoptosis. To substantiate
the morphological observations, we measured caspase activity using the
fluorogenic peptide substrate DEVD.AFC (24). Again, significant
inhibition of STA-triggered caspase activation was shown for continuous
hypoxia (condition "f "), whereas azide and prior
hypoxia were without effect. We subsequently determined IAP-2
expression under these experimental conditions (Fig. 8B). It
is clear that only cells exposed to continuous hypoxia expressed high
levels of IAP-2 (lane 6, condition "f"). No
IAP-2 induction was shown for azide (lanes 4, 5;
conditions "d " and "e"). Consistent with
previous results (Fig. 1), reoxygenation led to decreases of IAP-2
expression in post-hypoxic cells to basal levels (lane 7;
condition "g"). Taken together, these results have
provided compelling evidence that hypoxic cells overexpressing IAP-2
are indeed more resistant to apoptosis.
In response to hypoxia, mammalian cells activate and express
multiple genes (5). Products of these genes can increase oxygen delivery in tissues via erythropoiesis (erythropoietin) and
angiogenesis (vascular endothelial growth factor), facilitate
metabolic adaptation via glycolysis (glycolytic enzymes and glucose
transporters), and enhance cellular ability of survival (insulin-like
growth factor 2) (6). Recent studies have documented hypoxic
accumulation of p53, a potent trigger of apoptotic cell death (36, 43). Despite these important findings, hypoxic regulation of genes that are
directly involved in cell death or death resistance remains largely
unknown (5, 44). The current study has shown for the first time the
hypoxic regulation of IAP-2, an anti-apoptotic gene. The regulation is
specific for IAP-2, because neither Bcl-2/Bax nor IAP-1/XIAP was
significantly induced in hypoxic cells. Moreover, the inductive
response is unique for severe hypoxia or anoxia; mild or moderate
decrease of oxygen tensions was not effective. On the other hand,
up-regulation of IAP-2 by severe hypoxia appears to be a universal
response, and has been detected in several cell types. IAP-2 induction
does not result from secondary effects of hypoxia such as declines of
cellular ATP, alterations of redox status, or mitochondrial inhibition.
The key to the induction appears to be oxygen deficiency per
se. Of significance, HIF-1, the primary transcription factor that
mediates hypoxic activation of multiple genes, is not responsible for
IAP-2 gene activation under severe hypoxia.
Strong evidence has been obtained to indicate that IAP-2 induction is
not a result of cell injury. First, maximal IAP-2 expression was
detected after 1 h of hypoxic incubation, a period during which
cells remained viable and injurious processes, including cytochrome
c translocation and caspase activation, had not yet occurred
(22, 24). Second, the general caspase inhibitor VAD blocked caspase
activation and progression of apoptosis but did not halt IAP-2
expression. Moreover, IAP-2 was induced by hypoxia in RPTC cells
overexpressing Bcl-2, a powerful anti-death molecule that prevents
injury and cell death in our experimental model of hypoxia
reoxygenation (22). Finally, when glucose was provided during hypoxic
incubation, cells generated ATP through anaerobic glycolysis. These
cells were not injured by 1- to 5-h hypoxia and were able to recover
fully after reoxygenation (data not shown). IAP-2 was induced in these
cells as well. Collectively, these results have provided clear-cut
evidence that IAP-2 up-regulation under hypoxia does not result from
cell injury.
Despite its recognized role in gene regulation under hypoxia, IAP-2
induction appears to be independent of cellular redox status. The
significance of cellular redox status in hypoxic gene expression has
been suggested by recent studies (27-30). Based on these studies, two
hypotheses have been formulated. One model proposes that the
availability of molecular O2 determines the levels of
reactive oxygen species (ROS) within cells. As such, less ROS is
generated during hypoxic incubation. This allows the full expression of
particular genes that are otherwise suppressed by ROS (30, 45). This
model predicts that, with respect to gene regulation, inhibition of ROS
production or scavenging of ROS would mimic the effects of hypoxia.
Opposite to this model, Schumacker and his colleagues (10) have
proposed that ROS generated by mitochondria signals gene expression
under hypoxia. Their results show that cellular ROS production is
increased under 1-5% O2 (46). Inhibition of ROS
generation during hypoxia through either depletion of mitochondrial DNA
or the use of scavengers is accompanied by suppression of hypoxic gene
expression (27). Moreover, their recent studies have further
demonstrated a role for ROS in stabilization of HIF-1 Our results have demonstrated a critical role for gene transcription in
IAP-2 expression under severe hypoxia. IAP-2 was induced at the
mRNA as well as the protein level. Both increases in mRNA and
protein were suppressed by actinomycin D, the transcription inhibitor.
In addition, in vitro nuclear run-on assays demonstrated marked increases in transcriptional activity for IAP-2 after hypoxia exposure. Thus, IAP-2 transcription is indeed activated by hypoxia. However, these results do not exclude potential IAP-2 regulation at
post-transcriptional levels. As a matter of fact, actinomycin D could
noticeably reduce but could not completely abolish IAP-2 expression
under hypoxia (Fig. 5). Furthermore, the nuclear transcriptional activity for IAP-2 reached a maximal level at 1 h of hypoxia and declined thereafter (Fig. 6), whereas expression of IAP-2 mRNA as
well as protein remained high during 3 or 5 h of hypoxic
incubation (Figs. 1 and 5). The results suggest that transcription is
responsible for the rapid phase of IAP-2 induction, whereas
stabilization of mRNA and probably of protein may contribute to
IAP-2 expression during prolonged hypoxia exposure.
Post-transcriptional regulation of IAP-2 is also suggested by protein
changes shown in our experiments. For example, incubation with
mitochondrial inhibitors resulted in decreases of IAP-2 expression
(Figs. 2 and 4). On the other hand, proteins cross-reactive with the
IAP-2 antibody were detected in cells exposed to chemicals that modify
cellular redox status (Fig. 3). The protein changes were apparently not
due to variations of electrophoresis and immunoblotting, because
similar amounts of actin were detected in each lane; rather,
modifications that include proteolysis, degradation, cross-linking,
phosphorylation, and dephosphorylation could be involved.
The critical factors responsible for IAP-2 up-regulation under hypoxia
remain to be identified. Nevertheless, we have obtained compelling
evidence against the involvement of HIF-1, a ubiquitous transcription
factor that has been shown to mediate hypoxic activation of multiple
genes. Our results show clearly that expression of IAP-2 and the
activity of HIF-1 can be dissociated. These experiments focused on
HIF-1 A classical biological function of IAPs is to suppress cell death (52,
53). The first IAP genes were identified in baculovirus through their
ability to substitute p35 in prevention of host cell apoptosis (11,
12). IAPs may antagonize cell death at multiple levels, and the
critical targets include caspases, a family of cysteine proteases that
mediate the disassembly of apoptotic cells (54-56). Thus, an apparent
role played by the induced IAP-2 under severe hypoxia could be to
protect cells from injury/death and facilitate cell survival in the
highly stressful environment. We have compared the apoptotic
sensitivity of cells incubated under normal oxygen and severe hypoxia.
The results show clearly that hypoxic cells expressed high levels of
IAP-2 and became more resistant to apoptosis (Fig. 8). Although
apoptosis was specifically induced by staurosporine in this experiment,
a general increase of apoptotic threshold would be expected for the
hypoxic cells, because up-regulated IAP-2 in these cells would
antagonize apoptosis at several levels, including caspases (54-56). In
this context, IAP-2 induction can be considered as an important
mechanism of cellular adaptation. Such adaptive response has been
demonstrated for the neuronal-specific IAP under in vivo
situations of severe hypoxia or ischemia (57). During forebrain
ischemia, neuronal-specific IAP is induced in specific populations of
cells that are injury/death resistant. In addition to cytoprotection,
recent studies have documented at least two distinct functions for
IAPs. Survivin, a unique baculovirus IAP repeat with a single
BIR and no RING domains, localizes to the mitotic spindle
microtubules and regulates cytokinesis (58). On the other hand,
ubiquitin protein ligase activity has been shown for several IAPs,
which requires the integrity of the RING domain and may determine
proteasome-mediated degradation of IAPs as well as other key molecules
involved in cell death or survival (59). Therefore, in addition to its
cytoprotective potential, IAP-2 induced by severe hypoxia or anoxia may
regulate other important biological processes as well.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
subunits (8). Although HIF-1
is constitutively expressed, HIF-1
is precisely regulated by cellular oxygen levels. Under hypoxia, HIF-1
is induced, dimerizes with
subunits,
translocates to the nucleus, and initiates gene transcription (6).
Originally identified by its regulation of erythropoietin, HIF-1 has
now been shown to play a central role in hypoxic expression of a
variety of genes and is considered to be a master transcription factor that governs adaptive gene expression under situations of oxygen deficiency (5, 9, 10).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
null mouse embryonic stem cells were generated as described
previously (23). Primary cultures of human umbilical vein endothelial
cells were kindly provided by N. Pinckard (University of Texas Health
Science Center, San Antonio, TX). Other cells used in this study were
purchased from ATCC. The cells were maintained following standard procedures.
was purchased from Novus Biologicals,
Inc. (Littleton, CO). Polyclonal antibodies to Bcl-2, IAP-1, IAP-2, and
XIAP were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
We prepared two polyclonal IAP-2 antibodies recognizing different
epitopes to verify the identity of IAP-2. VAD was from Enzyme Systems
Products (Dublin, CA). Ebselen, MnTBAP, rotenone, antimycin A,
myxothiazole, oligomycin, and carbonyl
cyanide-m-chlorophenyl hydrazone (CCCP) were purchased from
Calbiochem-Novabiochem Co. (San Diego, CA). Radioactive chemicals were
purchased from PerkinElmer Life Sciences (Boston, MA). Other reagents
were from Sigma Chemical Co. (St. Louis, MO).
-32P]UTP, and 20%
glycerol. RNase inhibitor at 20 units/reaction was included to prevent
RNA degradation. The reaction was terminated by adding DNase I and
proteinase K. RNA was extracted with TRI reagent LS (Molecular Research
Center, Cincinnati, OH). Nascent 32P-uridine-labeled RNA
species were hybridized to an excess of IAP-2 cDNA or
-actin
cDNA (2 µg) that had been immobilized on Zeta-Probe GT membranes
by a Bio-Dot SF Microfiltration apparatus (Bio-Rad, Hercules,
CA). After 36 h of hybridization at 65 °C, blots were washed
and subjected to autoradiography. Quantitation of the signal was
performed by densitometry.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IAP-2 induction by hypoxia.
A, expression of apoptotic genes in RPTC cells. RPTC
(Con: control) were subjected to 1, 3, or 5 h of
hypoxic incubation (1H, 3H, 5H), or
5 h of hypoxia followed by 2 h of reoxygenation
(5H/2R). 5H+Glu: 1 mg/ml glucose was provided
during 5 h of hypoxia. 5H+VAD: 100 µM VAD
was provided during 5 h of hypoxia. 5H+Bcl-2: 5 h
of hypoxia was performed on cells overexpressing Bcl-2. Proteins in
whole cell lysates (100 µg/lane) were analyzed for the expression of
Bcl-2, Bax, IAP-2, or IAP-1 by immunoblotting. IAP-2 was shown to be
markedly and specifically induced by hypoxia. Data not shown in this
figure indicate that IAP-2 expression in control cells with normal
O2 is not affected by glucose, VAD, or Bcl-2 transfection.
B, induction of IAP-2 by hypoxia in various types of cells.
3T3 fibroblast cells (3T3), human kidney proximal tubule
cells (HPT), human umbilical vein endothelial cells
(HUV), and human renal carcinoma cells (HRC) were
subjected to one hour of hypoxia. Proteins in whole cell lysates (100 µg/lane) were examined by immunoblot analysis. C, control;
H, hypoxia.
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Fig. 2.
IAP-2 is induced by hypoxia per se
and not by declines of cell ATP. A, stepwise ATP
depletion induced by CCCP ± glucose. RPTC were
incubated for 1 or 3 h under normal oxygen tension (21%
O2) with the mitochondrial inhibitor CCCP and 0-0.5 mg/ml
glucose. Cellular ATP was measured by luminometry of the
luciferin-firefly luciferase reaction. Results are means ± S.E.
(n = 4). B, neither severe nor mild ATP
depletion leads to IAP-2 expression. Cells (Con: control)
were exposed to 1 h of hypoxia (1H), or 1-3 h of 15 µM CCCP with 0-0.5 mg/ml glucose. Proteins in whole cell
lysates (100 µg/lane) were analyzed for IAP-2 by immunoblotting. To
control sample loading and transfer, the blot was stripped and reprobed
for -actin.
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Fig. 3.
Alterations of cellular redox status do not
result in IAP-2 expression. RPTC cells (Con: control)
were exposed for 3 h to hypoxia (hypoxia), or to
various chemicals that were able to modify cellular redox status.
Concentrations of the chemicals used in this experiment were 10 mM NAC, 100 µM MnTBAP, 100 µM
Ebselen, 5 mM H2O2, 1 mM and 5 mM t-BHP. Proteins in whole
cell lysates (100 µg/lane) were analyzed for IAP-2 by immunoblotting.
To control sample loading and transfer, the blot was stripped and
reprobed for -actin.
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Fig. 4.
Mitochondrial inhibition does not induce
IAP-2. A, schematic of the respiratory chain and the
sites targeted by hypoxia and mitochondrial inhibitors. B,
induction of IAP-2 by hypoxia and not by mitochondrial inhibition. RPTC
(Con: control) were exposed for 3 h to hypoxia, or to
mitochondrial inhibitors (10 µg/ml rotenone, 10 µM
antimycin, 10 µM myxothiazole, 1 mM azide, 5 mM cyanide, or 10 µM oligomycin) in the
presence of 1 mg/ml glucose. Proteins in whole cell lysates (100 µg/lane) were analyzed for IAP-2 by immunoblotting. To control sample
loading and transfer, the blot was stripped and reprobed for
-actin.
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Fig. 5.
Actinomycin D suppresses IAP-2 expression
under hypoxia. A, immunoblots. RPTC cells
(Con: control) were preincubated for 1 h with 0-100
µg/ml actinomycin D, and subsequently transferred to hypoxia for
3 h. Proteins in whole cell lysates were analyzed for IAP-2 and
-actin by immunoblotting. B, Northern blots. RPTC cells
(Con: control) were subjected to 1, 3, or 5 h of
hypoxia (1H, 3H, 5H), or 5 h of
hypoxia in the presence of 20 µg/ml actinomycin D (Act.D).
Total RNA was extracted, and mRNA was purified and analyzed (2 µg/lane) for IAP-2 transcripts by Northern hybridization as described
under "Materials and Methods." To control sample loading and
transfer, the blot was stripped and reprobed for
glyceraldehyde-3-phosphate dehydrogenase mRNA. The results show
that Act.D at 10-100 µg/ml suppressed IAP-2 expression under
hypoxia.
-32P]UTP. Nascent 32P-labeled
RNA species were extracted and hybridized to rat IAP-2 cDNA. Blots
of a typical nuclear run-on experiment are shown in Fig.
6A, and the densitometric
result is summarized in Fig. 6B. Exposure of cells to
hypoxia led to significant increases in nuclear transcriptional
activity for IAP-2. After 1 h of hypoxia, the IAP-2
transcriptional activity was 4.2-fold over control (Fig. 6B). Of interest, when hypoxia was prolonged, nuclear
transcriptional activity for the anti-apoptotic gene decreased toward
basal levels. Thus, at the end of 3 h of hypoxia, the IAP-2
transcriptional activity was ~3-fold over control (Fig.
6B). As an internal control, the nuclear transcription
activity for
-actin remained largely unchanged or slightly decreased
during 1-3 h of hypoxia exposure, indicating the specificity of IAP-2
gene transactivation. These results, along with the inhibitory effects
of actinomycin D (Fig. 5), demonstrate an important role for
transcription in IAP-2 gene activation under severe hypoxia.
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Fig. 6.
Increase of nuclear transcriptional activity
for IAP-2 after hypoxia exposure. RPTC cells (Con:
control) were subjected to 1 or 3 h of hypoxic incubation
(1H, 3H). Nuclei were isolated for in
vitro nuclear run-on transcription in the presence of
[ -32P]UTP. RNA was subsequently extracted and
hybridized to membranes containing pGEM-T-easy empty vector or
pGEM-T-easy-IAP-2 cDNA. To control variations in nuclear run-on
reactions and RNA extraction, the RNA samples were rehybridized to a
membrane containing
-actin cDNA. A, representative
blots from a nuclear run-on experiment. No specific binding was
detected for blots immobilized with pGEM-T-easy empty vector (not
shown). B, densitometric results summarized from three
separate experiments. Intensity of control signals was arbitrarily set
as 1, and the relative intensity of hypoxia samples was calculated. The
results demonstrate specific increases in nuclear transcriptional
activity for IAP-2 after hypoxia exposure.
and
subunits. Although HIF-1
is constitutively expressed, HIF-1
is induced by hypoxia (maximal induction at 0.5-2%
O2) and determines DNA binding and transcriptional activity
(6). Thus, we first examined HIF-1
expression under the experimental conditions that activate apoptotic genes: near zero levels of O2. As shown in Fig.
7A, after 1 or 5 h of
severe hypoxia, increases of HIF-1
were only modest (lanes
2, 3). On the contrary, IAP-2 was markedly induced. To
further investigate the role of HIF-1, we analyzed IAP-2 expression in
cells with up-regulated HIF-1. Cobalt chloride (CoCl2) and
desferrioxamine (DFO) are two pharmacological inducers of HIF-1.
Exposure of cells to CoCl2 or DFO has been shown to trigger
HIF-1
expression, increases of DNA binding activity, and
trans-activation of genes containing HIF-1 binding sites (36, 37).
Consistent with these observations, HIF-1
was induced in RPTC cells
by CoCl2 or DFO under normal oxygen tensions (Fig. 7A, lanes 4-9). Nevertheless, IAP-2 expression
in these cells remained minimal, negating a role for HIF-1 in
regulation of this anti-apoptotic gene. The results were further
supported by our observation that HIF-1
, but not IAP-2, was
dramatically induced by 2% O2 exposure (Fig.
7B, lanes 3-5). Thus, activation of HIF-1
and
IAP-2 are triggered by different degrees of hypoxia; severe hypoxia or
anoxia is required for IAP-2 induction. Finally, conclusive evidence to
exclude the role of HIF-1 was obtained using HIF-1
-deficient cells
(Fig. 7C). HIF-1
/
mouse embryonic stem
cells have been generated and characterized recently (23).
Expression of HIF-1
in wild type but not in mutant cells was
confirmed by immunoblot analysis (data not shown (23)). Both types of
cells were subjected to control or hypoxia incubation, and whole cell
lysates were analyzed for IAP-2. As shown in Fig. 7C, severe
hypoxia induced IAP-2, in HIF-1
+/+ as well as
HIF-1
/
cells, indicating that not HIF-1 but other
transcription factors are responsible for the inductive response.
Together, the results do not support a role for HIF-1 in hypoxic
expression of IAP-2; instead, novel transcriptional mechanisms that are
responsive to severe hypoxia or anoxia may be involved.
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Fig. 7.
Dissociation of IAP-2 induction under hypoxia
from HIF-1. A, induction of HIF-1 but not IAP-2 by
cobalt chloride (CoCl2) and desferrioxamine
(DFO). RPTC cells (Con: control) were subjected
to 1 or 5 h of hypoxia (1H, 5H), or
incubated under normal oxygen tensions with 50-200 µM
CoCl2 or 100-500 µM DFO. Proteins in whole
cell lysates were analyzed for HIF-1 and IAP-2 by immunoblotting.
Severe hypoxia induced strong expression of IAP-2 but not HIF-1; on the
other hand, CoCl2 and DFO induced HIF-1 but not IAP-2.
B, induction of HIF-1 and IAP-2 by different degrees of
hypoxia. RPTC cells (Con: control) were subjected to 1 h of severe hypoxia (near zero levels of oxygen) or 6-24 h of 2%
oxygen incubation. Proteins in whole cell lysates were analyzed for
HIF-1 and IAP-2 by immunoblotting. Although abundant HIF-1 was
expressed under 2% oxygen, induction of IAP-2 was only detected under
severe hypoxia/anoxia. C, IAP-2 induction by severe hypoxia
in HIF-1-deficient cells. Wild type (HIF-1 +/+)
and mutant (HIF-1
/
) mouse embryonic stem
cells were subjected to 1, 3, or 5 h of severe hypoxia. Expression
of IAP-2 was analyzed by immunoblotting. IAP-2 was induced by hypoxia
in HIF-1
+/+ cells as well as in
HIF-1
/
cells.
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Fig. 8.
Hypoxic cells with IAP-2 up-regulation are
more resistant to apoptosis. A, apoptosis and caspase
activity. B, immunoblots of IAP-2 and actin. Experimental
conditions were as follows. a, RPTC cells were incubated for
5 h in a physiological buffer. b, cells were first
incubated for 1 h in a physiological buffer, then 1 µM staurosporine (STA) was added to incubate
for another 4 h. c, cells were first incubated for
1 h in a physiological buffer, then 1 µM STA and 50 µM caspase inhibitor VAD were added to incubate for
another 4 h. d, cells were first incubated for 1 h
with 10 mM azide, then 1 µM STA was added to
incubate for another 4 h. e, cells were first incubated
for 1 h with 10 mM azide, then incubated with 1 µM STA in the absence of azide for another 4 h.
f, cells were first incubated for 1 h under severe
hypoxia, then 1 µM STA was added to incubate the cells
under continuous hypoxia for another 4 h. g, cells were
first incubated for 1 h under severe hypoxia, then returned to
normal oxygen tension and incubated with 1 µM STA for
another 4 h. Apoptotic cells with characteristic morphology were
counted to assess the percentage of apoptosis, as described under
"Materials and Methods." Caspase activity was measured using the
fluorogenic peptide substrate DEVD.AFC (24). The results in
A are expressed as means ± S.E. (n = 5). Lanes 1-7 in B correspond, respectively, to
conditions a-g of panel A. It is clear that
hypoxic cells overexpressing IAP-2 are more resistant to apoptosis
induced by staurosporine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, the primary
hypoxia-responsive transcription factor (47). These two interesting and
somewhat contradictory hypotheses prompted us to determine whether
hypoxic expression of IAP-2 was dependent on cellular redox status. Our
results show clearly that, despite manipulation of cellular redox
status, neither the anti-oxidants nor the pro-oxidants could induce
IAP-2 expression under normal oxygen tensions (Fig. 3). Moreover,
inclusion of the anti-oxidant N-acetyl-cysteine during
hypoxia was not able to ameliorate IAP-2 expression (data not shown).
In addition, both respiration inhibitors and mitochondrial uncouplers
failed to induce IAP-2 (Figs. 2 and 4), whereas these two classes of
chemicals were expected to alter NADH/NAD+ ratio and,
therefore, the redox status of the cells in the opposite directions.
Together, these results suggest that changes of cellular redox status
do not have an essential role in IAP-2 induction under hypoxia.
, because this subunit is the key component of HIF-1,
expression of which determines the cellular HIF-1 activity. We first
showed that, in our experimental model of severe hypoxia, IAP-2 was
markedly induced, whereas expression of HIF-1
was modest. Subsequently, we found that, in cells where HIF-1 was activated pharmacologically by cobalt or desferrioxamine, IAP-2 expression was
not enhanced. Moreover, our results demonstrated that HIF-1 and the
IAP-2 gene were activated by different degrees of hypoxia. Although
HIF-1 was strongly activated by 2% oxygen, activation of the IAP-2
gene required more severe hypoxia or anoxia. Finally, IAP-2 was induced
by severe hypoxia in mouse embryonic stem cells that were devoid of
HIF-1. Together, these results have provided conclusive evidence to
exclude a role for HIF-1 in hypoxic activation of the apoptosis
inhibitory gene IAP-2. In addition to HIF-1, several other related or
unrelated transcription factors have been shown to participate in
hypoxic gene activation (5, 6). For example, recent studies using
transgenic mice have demonstrated an important role for egr-1 in gene
regulation under situations of in vivo hypoxia or ischemia
(48). However, regulation by these transcription factors appears to be
gene- and cell-type-specific (49-51). Of note, an intriguing aspect of
IAP-2 gene activation is the requirement of complete absence of oxygen
or anoxia. Thus, many of the transcription factors that respond to mild
or moderate hypoxia may not qualify as the key elements mediating IAP-2
induction. Ongoing studies in our laboratory include the analysis of
IAP-2 gene promoter to identify the critical severe hypoxia/anoxia
responsive elements.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Junying Yuan (Massachusetts General Hospital, Boston, MA), Richard Youle (NINDS, National Institutes of Health, Bethesda, MD), Ulrich Hopfer (Case Western Reserve University, Cleveland, OH), and Neal Pinckard (University of Texas Health Science Center, San Antonio, TX) for providing plasmids, antibodies, and cells. The technical assistance of Qiu Zong in some of the experiments is highly appreciated.
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FOOTNOTES |
---|
* This work was supported by grants from the American Heart Association, the Texas Advanced Research Program, and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of Lyndon Baines Johnson Research Award from the American Heart Association. To whom correspondence should be addressed: Dept. of Pathology, University of Texas Health Science Center, 7703 Floyd Curl Dr., San Antonio, TX 78229. Tel.: 210-567-6703; Fax: 210-567-2367; E-mail: dong@uthscsa.edu.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M011774200
2 Z. Dong, J. Nishiyama, and M. A. Venkatachalam, unpublished data. The IAP-2 shown in this article is the rat homolog of human IAP-2, mouse IAP-2, and cellular IAP-1 (cIAP-1).
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ABBREVIATIONS |
---|
The abbreviations used are: HIF-1, hypoxia-inducible factor-1; Act.D, actinomycin D; CCCP, carbonyl cyanide-m-chlorophenyl hydrazone; DEVD.AFC, carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin; IAP, inhibitor of apoptosis protein; NAC, N-acetyl-cysteine; ROS, reactive oxygen species; RPTC, rat kidney proximal tubule cells; STA, staurosporine; t-BHP, t-butylhydroperoxide; VAD, carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone; DTT, dithiothreitol; kb, kilobase(s); DFO, desferrioxamine; MnTBAP, Mn(111) tetrakis (4-benzoic acid) porphyrin chloride; PAS, per-aryl hydrocarbon receptor ARNT-sim.
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REFERENCES |
---|
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---|
1. | Cotran, R. S., Kumar, V., and Collins, T. (1998) in Robbins Pathologic Basis of Disease (6th ed.), Cell Injury and Cellular Death (Cotran, R. S. , Kumar, V. , and Collins, T., eds) , pp. 1-29, W. B. Saunders Co., Philadelphia, PA |
2. |
Brown, J. M.
(1999)
Cancer Res.
59,
5863-5870 |
3. | Sutherland, R. M. (1998) Acta Oncol. 37, 567-574[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Semenza, G. L.
(2000)
Crit. Rev. Biochem. Mol. Biol.
35,
71-103 |
5. |
Bunn, H. F.,
and Poyton, R. O.
(1996)
Physiol. Rev.
76,
839-885 |
6. | Semenza, G. L. (1999) Annu. Rev. Cell Dev. Biol. 15, 551-578[CrossRef][Medline] [Order article via Infotrieve] |
7. | Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447-5454[Abstract] |
8. |
Wang, G. L.,
and Semenza, G. L.
(1995)
J. Biol. Chem.
270,
1230-1237 |
9. |
Semenza, G. L.
(2000)
J. Appl. Physiol.
88,
1474-1480 |
10. |
Chandel, N. S.,
and Schumacker, P. T.
(2000)
J. Appl. Physiol.
88,
1880-1889 |
11. | Crook, N. E., Clem, R. J., and Miller, L. K. (1993) J. Virol. 67, 2168-2174[Abstract] |
12. | Clem, R. J., and Miller, L. K. (1994) Mol. Cell. Biol. 14, 5212-5222[Abstract] |
13. | Hay, B. A., Wassarman, D. A., and Rubin, G. M. (1995) Cell 83, 1253-1262[Medline] [Order article via Infotrieve] |
14. | Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. (1995) Cell 83, 1243-1252[Medline] [Order article via Infotrieve] |
15. | Roy, N., Mahadevan, M. S., McLean, M., Shutler, G., Yaraghi, Z., Farahani, R., Baird, S., Besner-Johnston, A., Lefebvre, C., Kang, X., et al.. (1995) Cell 80, 167-178[Medline] [Order article via Infotrieve] |
16. | Duckett, C. S., Nava, V. E., Gedrich, R. W., Clem, R. J., Van Dongen, J. L., Gilfillan, M. C., Shiels, H., Hardwick, J. M., and Thompson, C. B. (1996) EMBO J. 15, 2685-2694[Abstract] |
17. | Liston, P., Roy, N., Tamai, K., Lefebvre, C., Baird, S., Cherton-Horvat, G., Farahani, R., McLean, M., Ikeda, J. E., MacKenzie, A., and Korneluk, R. G. (1996) Nature 379, 349-353[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Uren, A. G.,
Pakusch, M.,
Hawkins, C. J.,
Puls, K. L.,
and Vaux, D. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4974-4978 |
19. | Wang, S. L., Hawkins, C. J., Yoo, S. J., Muller, H. A., and Hay, B. A. (1999) Cell 98, 453-463[Medline] [Order article via Infotrieve] |
20. | Ambrosini, G., Adida, C., and Altieri, D. C. (1997) Nat. Med. 3, 917-921[Medline] [Order article via Infotrieve] |
21. | Vucic, D., Stennicke, H. R., Pisabarro, M. T., Salvesen, G. S., and Dixit, V. M. (2000) Curr. Biol. 10, 1359-1366[CrossRef][Medline] [Order article via Infotrieve] |
22. | Saikumar, P., Dong, Z., Patel, Y., Hall, K., Hopfer, U., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3401-3415[CrossRef][Medline] [Order article via Infotrieve] |
23. |
Iyer, N. V.,
Kotch, L. E.,
Agani, F.,
Leung, S. W.,
Laughner, E.,
Wenger, R. H.,
Gassmann, M.,
Gearhart, J. D.,
Lawler, A. M., Yu, A. Y.,
and Semenza, G. L.
(1998)
Genes Dev.
12,
149-162 |
24. | Dong, Z., Saikumar, P., Patel, Y., Weinberg, J. M., and Venkatachalam, M. A. (2000) Biochem. J. 347, 669-677[CrossRef][Medline] [Order article via Infotrieve] |
25. | Spector, D. L., Goldman, R. D., and Leinwand, L. A. (1998) Cells: A Laboratory Manual, Nuclear Run-on Analysis , pp. 25.4-25.15, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
26. | Dong, Z., Patel, Y., Saikumar, P., Weinberg, J. M., and Venkatachalam, M. A. (1998) Lab. Invest. 78, 657-668[Medline] [Order article via Infotrieve] |
27. |
Chandel, N. S.,
Maltepe, E.,
Goldwasser, E.,
Mathieu, C. E.,
Simon, M. C.,
and Schumacker, P. T.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11715-11720 |
28. | Ehleben, W., Bolling, B., Merten, E., Porwol, T., Strohmaier, A. R., and Acker, H. (1998) Respir. Physiol. 114, 25-36[CrossRef][Medline] [Order article via Infotrieve] |
29. | Morel, Y., and Barouki, R. (1999) Biochem. J. 342, 481-496[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Neumcke, I.,
Schneider, B.,
Fandrey, J.,
and Pagel, H.
(1999)
Endocrinology
140,
641-645 |
31. | Saikumar, P., Dong, Z., Weinberg, J. M., and Venkatachalam, M. A. (1998) Oncogene 17, 3341-3349[CrossRef][Medline] [Order article via Infotrieve] |
32. | Piper, H. M., Noll, T., and Siegmund, B. (1994) Cardiovasc. Res. 28, 1-15[Medline] [Order article via Infotrieve] |
33. | Acker, H. (1994) Ann. N. Y. Acad. Sci. 718, 3-10[Medline] [Order article via Infotrieve] |
34. |
Chandel, N. S.,
Budinger, G. R.,
Choe, S. H.,
and Schumacker, P. T.
(1997)
J. Biol. Chem.
272,
18808-18816 |
35. | Semenza, G. L. (1999) Cell 98, 281-284[Medline] [Order article via Infotrieve] |
36. | An, W. G., Kanekal, M., Simon, M. C., Maltepe, E., Blagosklonny, M. V., and Neckers, L. M. (1998) Nature 392, 405-408[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Jiang, B. H.,
Zheng, J. Z.,
Leung, S. W.,
Roe, R.,
and Semenza, G. L.
(1997)
J. Biol. Chem.
272,
19253-19260 |
38. |
Yang, J.,
Liu, X.,
Bhalla, K.,
Kim, C. N.,
Ibrado, A. M.,
Cai, J.,
Peng, T. I.,
Jones, D. P.,
and Wang, X.
(1997)
Science
275,
1129-1132 |
39. |
Bossy-Wetzel, E.,
Newmeyer, D. D.,
and Green, D. R.
(1998)
EMBO J.
17,
37-49 |
40. |
Samali, A.,
Cai, J.,
Zhivotovsky, B.,
Jones, D. P.,
and Orrenius, S.
(1999)
EMBO J.
18,
2040-2048 |
41. | Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103[CrossRef][Medline] [Order article via Infotrieve] |
42. | Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., and Reed, J. C. (2000) Nat. Cell Biol. 2, 318-325[CrossRef][Medline] [Order article via Infotrieve] |
43. | Graeber, T. G., Osmanian, C., Jacks, T., Housman, D. E., Koch, C. J., Lowe, S. W., and Giaccia, A. J. (1996) Nature 379, 88-91[CrossRef][Medline] [Order article via Infotrieve] |
44. | Khan, S., Cleveland, R. P., Koch, C. J., and Schelling, J. R. (1999) Lab. Invest. 79, 1089-1099[Medline] [Order article via Infotrieve] |
45. | Porwol, T., Ehleben, W., Zierold, K., Fandrey, J., and Acker, H. (1998) Eur. J. Biochem. 256, 16-23[Abstract] |
46. |
Duranteau, J.,
Chandel, N. S.,
Kulisz, A.,
Shao, Z.,
and Schumacker, P. T.
(1998)
J. Biol. Chem.
273,
11619-11624 |
47. |
Chandel, N. S.,
McClintock, D. S.,
Feliciano, C. E.,
Wood, T. M.,
Melendez, J. A.,
Rodriguez, A. M.,
and Schumacker, P. T.
(2000)
J. Biol. Chem.
275,
25130-25138 |
48. | Yan, S. F., Fujita, T., Lu, J., Okada, K., Shan Zou, Y., Mackman, N., Pinsky, D. J., and Stern, D. M. (2000) Nat. Med. 6, 1355-1361[CrossRef][Medline] [Order article via Infotrieve] |
49. |
Ema, M.,
Taya, S.,
Yokotani, N.,
Sogawa, K.,
Matsuda, Y.,
and Fujii-Kuriyama, Y.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4273-4278 |
50. | Tian, H., McKnight, S. L., and Russell, D. W. (1997) Genes Dev. 11, 72-82[Abstract] |
51. |
Yan, S. F.,
Zou, Y. S.,
Gao, Y.,
Zhai, C.,
Mackman, N.,
Lee, S. L.,
Milbrandt, J.,
Pinsky, D.,
Kisiel, W.,
and Stern, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
8298-8303 |
52. | LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. (1998) Oncogene 17, 3247-3259[CrossRef][Medline] [Order article via Infotrieve] |
53. |
Deveraux, Q. L.,
and Reed, J. C.
(1999)
Genes Dev.
13,
239-252 |
54. | Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. (1997) Nature 388, 300-304[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Deveraux, Q. L.,
Roy, N.,
Stennicke, H. R.,
Van Arsdale, T.,
Zhou, Q.,
Srinivasula, S. M.,
Alnemri, E. S.,
Salvesen, G. S.,
and Reed, J. C.
(1998)
EMBO J.
17,
2215-2223 |
56. |
Roy, N.,
Deveraux, Q. L.,
Takahashi, R.,
Salvesen, G. S.,
and Reed, J. C.
(1997)
EMBO J.
16,
6914-6925 |
57. | Xu, D. G., Crocker, S. J., Doucet, J. P., St-Jean, M., Tamai, K., Hakim, A. M., Ikeda, J. E., Liston, P., Thompson, C. S., Korneluk, R. G., MacKenzie, A., and Robertson, G. S. (1997) Nat. Med. 3, 997-1004[Medline] [Order article via Infotrieve] |
58. | Li, F., Ambrosini, G., Chu, E. Y., Plescia, J., Tognin, S., Marchisio, P. C., and Altieri, D. C. (1998) Nature 396, 580-584[CrossRef][Medline] [Order article via Infotrieve] |
59. |
Yang, Y.,
Fang, S.,
Jensen, J. P.,
Weissman, A. M.,
and Ashwell, J. D.
(2000)
Science
288,
874-877 |