THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 17, Issue of April 29, pp. 17084 –17092, 2005
Printed in U.S.A.
Small Interference RNA-mediated Gene Silencing of Human
Biliverdin Reductase, but Not That of Heme Oxygenase-1,
Attenuates Arsenite-mediated Induction of the Oxygenase and
Increases Apoptosis in 293A Kidney Cells*
Received for publication, November 19, 2004, and in revised form, February 28, 2005
Published, JBC Papers in Press, February 28, 2005, DOI 10.1074/jbc.M413121200
Tihomir Miralem, Zhenbo Hu, Michael D. Torno, Katherine M. Lelli, and Mahin D. Maines‡
From the Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14624
BVR reduces biliverdin, the HO-1 and HO-2 product,
to bilirubin. Human biliverdin (BVR) is a serine/threonine kinase activated by free radicals. It is a leucine
zipper (bZip) DNA-binding protein and a regulatory factor for 8/7-bp AP-1-regulated genes, including HO-1 and
ATF-2/CREB. Presently, small interference (si) RNA constructs were used to investigate the role of human BVR
in sodium arsenite (As)-mediated induction of HO-1 and
in cytoprotection against apoptosis. Activation of BVR
involved increased serine/threonine phosphorylation
but not its protein or transcript levels. The peak activity
at 1 h (4 –5-fold) after treatment of 293A cells with 5 ␮M
As preceded induction of HO-1 expression by 3 h. The
following suggests BVR involvement in regulating oxidative stress response of HO-1: siBVR attenuated Asmediated increase in HO-1 expression; siBVR, but not
siHO-1, inhibited As-dependent increased c-jun promoter activity; treatment of cells with As increased AP-1
binding of nuclear proteins; BVR was identified in the
DNA-protein complex; and AP-1 binding of the in vitro
translated BVR was phosphorylation-dependent and
was attenuated by biliverdin. Most unexpectedly, cells
transfected with siBVR, but not siHO-1, displayed a
4-fold increase in apoptotic cells when treated with 10
␮M As as detected by flow cytometry. The presence of
BVR small interference RNA augmented the effect of
As on levels of cytochrome c, TRAIL, and DR-5 mRNA
and cleavage of poly(ADP-ribose) polymerase. The findings describe the function of BVR in HO-1 oxidative
response and, demonstrate, for the first time, not only
that BVR advances the role of HO-1 in cytoprotection
but also affords cytoprotection independent of heme
degradation.
The final step in heme metabolism is carried out by the
soluble enzyme biliverdin IX␣ reductase (BVR)1 (1, 2). A second
* This work was supported by NIEHS Grants RO1/ES04066 and
RO1/ES12187 from the National Institutes of Health. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14624. Tel.: 585-275-5383; Fax: 585-2756007; E-mail: [email protected].
1
The abbreviations used are: BVR, biliverdin IX␣ reductase; As,
sodium arsenite; ATF-2, activating transcription factor-2; hBVR, human biliverdin IX␣ reductase; AP-1, activator protein-1; bZip, leucine
zipper; CRE, cAMP-response element; CREB, cAMP-response elementbinding protein; heme, Fe-protoporphyrin IX; DR-5, death receptor-5;
HO, heme oxygenase; siRNA, small interference RNA; ELISA, enzyme-
form, biliverdin IX␤ reductase, which is identical to NADPHflavin reductase, has also been described (3– 6); this form is
active only during fetal development and has little, if any,
similarity to BVR. BVR, an evolutionarily conserved enzyme
with unique dual pH/cofactor-dependent activity profile, is primarily found in the mammalian species and is responsible for
the production of bilirubin IX␣. In most other animal species,
biliverdin constitutes the end product of heme (Fe-protoporphyrin) macro-cycle cleavage. BVR catalyzes the reduction of
biliverdin by incorporating two hydrogen atoms derived from
pyridine nucleotides (NADH in acidic pH or NADPH in basic
pH range) into the open tetrapyrrole ring structure (1, 2).
Biliverdin IX␣ in turn is a product of the mixed function oxidase activity of two forms of heme oxygenase proteins, HO-1
and HO-2 (7); the HO enzymes are also known as the HSP32
family of stress/heat shock proteins (8 –10). HO-1 is the stressresponsive cognate and is induced by a wide array of stimuli
that have in common the ability to cause oxidative stress (reviewed in Refs. 7 and 11).
Whereas in the past BVR was solely considered in the context of disposal of biliverdin, and bilirubin was considered a
toxic waste product of the heme metabolic pathway, recent
findings have alluded to notable biological functions of heme
degradation products in the cell; bilirubin is an effective intracellular antioxidant and a modulator of signal transduction
pathways and immune effector functions (12–16). Bilirubin and
biliverdin inhibit phosphotransferase activity of protein kinase
C and cAMP-dependent protein kinase (17); bilirubin inhibits
BVR activity, and biliverdin inhibits HO activity (1, 18). Because of the noted activities of bile pigments, induction of HO-1
is now generally accepted to be a key component of anti-inflammatory/anti-apoptotic stress effector pathways. In addition, the
recently ascribed functions to CO (19 –21), which result from
oxidation of ␣-meso carbon bridge of heme when the tetrapyrrole is cleaved by HO enzymes, support cytoprotective functions of heme oxidation products.
BVR, however, has functions distinct from its reductase activity and separate from those of its substrate and product; the
protein is a serine/threonine kinase that is activated in response to hydroxyl radicals (22). BVR is a member of the
“leucine zipper” (bZip) family of mammalian and yeast transcription factors that include c-Fos, c-Jun, ATF-2/CREB, Bach
1, P45, NrF2 and -3, (bZip), sYAP, GCN4, and c-Myc (23–32);
the reductase in a homodimeric form binds to the 7- and 8-bp
AP-1 and cAMP target genes, among them being ATF-2/CREB
linked immunosorbent assay; EGFP, enhanced green fluorescent protein; oligo, oligonucleotide; PBS, phosphate-buffered saline; Pipes, 1,4piperazinediethanesulfonic acid; PARP, poly(ADP-ribose) polymerase;
wt, wild type.
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This paper is available on line at http://www.jbc.org
siRNA-mediated Gene Silencing of Biliverdin Reductase
and c-jun (33). Arsenite is a reported regulator of bZip factors
(44). BVR being a bZip transcription factor and activator of
HO-1 could be among the list genes that are modulated by
arsenite treatment. This is consistent with the observation that
the reductase translocates from cytosol into the nucleus in
response to HO-1 inducers, including free radicals of cyclic
hydrocarbons, cytokines, and cGMP (34). The induction of
HO-1 by the free radical generator, menadione, is attenuated in
the presence of antisense BVR (30).
Arsenite modulates the profile of HO-1 gene expression in
the cell by activating a number of bZip transcription factors
involved in stress signaling pathways including the mitogenactivated protein family of kinases, p70 (s6k), protein kinase C,
and NF-␬␤ as well as changes in cellular free Ca2⫹ and NO䡠
levels/production (35– 40). HO-1 and genes that are involved in
apoptosis are among the targets for arsenite (9, 41– 45). Arsenite activation and increased binding of a complex of proteins,
which includes the Jun protein, to the CRE/AP-1 site of the
HO-1 promoter are essential for stress response of the oxygenase to this stimulus as well as to other oxidants. Furthermore,
arsenic compounds are considered to be tumorigenic, and evidence points to their activity to disrupt normal control of apoptosis by inhibiting c-Jun N-terminal kinase phosphatase and
activating the NF-␬␤ signaling pathway (35, 39, 46). The effect
arsenite has on cell death, however, is concentration-dependent
and has been proposed to involve oxygen free radicals (47). The
features of BVR that communicate with other proteins known
to have gene regulatory function in the cell together with above
noted observations support the potential role of BVR in the
stress response of HO-1.
The present study was undertaken to define further the role
of BVR in the response of HO-1 to arsenite and to examine
whether BVR itself has input into the cellular anti-apoptotic
effector pathways. We show, using arsenite as the stimulus,
that BVR is a key component of HO-1 stress response and that
this involves activity of the reductase as a kinase, transcription
factor, and the inactivator of biliverdin. We further demonstrate for the first time that BVR offers cytoprotection against
cell death independent of HO-1 expression.
MATERIALS AND METHODS
Constructs—siBVR and siHO-1 motifs were selected according to the
amino acid Asn-19 rule by finding the pattern in human BVR and human
HO-1 cDNA sequences (48, 49). The target sequence for hBVR is located
96 bp downstream of the start codon (nucleotides 96 –116). For HO-1, the
targeting sequence is 106 –126 nucleotides downstream of the start codon.
A retroviral based vector pSuper-Retro for siRNA expression was purchased from OligoEngine Co. (Seattle, WA). Retrovirus/siRNA-expressing
vectors pSuper-Retro-siBVR and pSuper-Retro-siHO-1 were constructed
according to the manufacturer’s instructions. Briefly, oligos containing
the sequence of 21-mer small interference RNA were synthesized as
follows: BVR siRNA, 5⬘-GATCCCC (TCC TCA GCG TTC CTG AAC CTG)
TTCAAGAGA (CAG GTT CAG GAA CGC TGA GGA) TTTTTGGAAA and
3⬘-GGG (AGG AGT CGC AAG GAC TTG GAC) AAGTTCTCT (GTC CAA
GTC CTT GCG ACT CCT)AAAAACCTTTTCGA; HO-1 siRNA, 5⬘-GATCCCC (AAC TTT CAG AAG GGC CAG GTG) TTCAAGAGA (CAC CTG
GCC CTT CTG AAA GTT) TTTTTGGAAA and 3⬘-GGG (TTG AAA GTC
TTC CCG GTC CAC) AAGTTCTCT (GTG GAC CGG GAA GAC TTT
CAA)AAAAACCTTTTCGA.
Complementary oligos were gel-purified and annealed to form double-stranded DNA. To insert the oligos into the pSuper-Retro plasmid,
BglII and HindIII were used to digest the vector, and the digested
vector was gel-purified. The double-stranded oligos were phosphorylated by T4 kinase. Ligation of the vector and oligos was carried out by
incubation of 10 ␮l of reaction containing 2 ␮l of vector, 1 ␮l of oligo, 2
␮l of 5⫻ ligation buffer, and 1 ␮l (1 unit) of T4 ligation buffer for 1 h at
room temperature. 2 ␮l of the ligation reaction was then used to transform DH5␣ Escherichia coli. Clones containing the oligo inserts were
identified by restriction digest analysis. Selected positive clones
were further verified by DNA sequencing. The verified constructs were
named pSuper-Retro-siBVR and pSuper-Retro-siHO-1. The retroviral
17085
vectors generating BVR siRNA and HO-1 siRNA were transfected into
the 293A packaging cell line, and the supernatant containing the expressed siBVR or siHO-1 retrovirus was then titrated by using NIH3T3
cell line. The concentration of retrovirus expressing siRNA used to
infect cells in further experiments was at a multiplicity of infection of 4
plaque-forming units/cell.
Other constructs used in this study are as follows. The expression
vectors pEGFP-FLAG-hBVR and pEGFP-FLAG-hHO-1 were generated
by inserting full-length human BVR cDNA or full-length human HO-1
cDNA, respectively, into plasmid pEGFP (Clontech). The expression
vectors generate an EGFP-FLAG-hBVR fusion protein or an EGFPFLAG-hHO-1 fusion protein. The vectors used for luciferase reporter
assay are as follows: a pGL3 luciferase-containing vector ligated with a
c-jun promoter sequence (pGL3/c-jun) or with a mutant form of c-jun
promoter (pGL3/c-jun⫺). (Both are kindly provided by Drs. McCance
and Baglia, University of Rochester, Rochester, NY.) A pCMV ␤-galactosidase vector was used as internal control for transfection efficiency.
Arsenite Treatment of the Cells—Arsenite (Sigma) was freshly prepared for each experiment in PBS and added to the cell culture at a final
concentration of 5 or 10 ␮M. Cells were treated for the durations indicated in appropriate figure legends. Thereafter, cells were harvested,
washed with PBS twice, and then used for experiments.
Cell Culture and Transfection of 293A Cells with hBVR and hHO-1
Expression Vectors—293A cell line (a human embryonic kidney cell line)
was obtained from Invitrogen. Cells were grown in 10-cm plates with
Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum
and 1% penicillin-G/streptomycin for 24 h or until a 70% confluency was
reached. Cells were subsequently transfected with 4 ␮g of plasmid in
each 10-cm dish using Transfectin Reagent (Bio-Rad), according to the
manufacturer’s protocol. This protocol was found to be optimal for
overexpression of hBVR and hHO-1, resulting in ⬃85% transfection
efficiency as detected by fluorescent microscopy. 24 h after transfections, supernatant containing retrovirus expressing siRNA was added.
This time point was designated as the “0” point. 4 and 16 h after the
initial infection with retrovirus, additional virus was added at the same
concentration. Samples were obtained at time points indicated in appropriate figure legends. 3 ⫻ 106 cells were used for RNA analysis and
1 ⫻ 106 for protein analysis.
Isolation of Cytosolic Fraction—293 cells were washed with PBS and
then homogenized in the extraction buffer (220 mM mannitol, 68 mM
sucrose, 50 mM Pipes-KOH, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, and
protease inhibitors). The homogenate was centrifuged at 400 ⫻ g at 4 °C
for 10 min, and the supernatant fraction was further centrifuged at
10,000 ⫻ g for 10 min; the resultant supernatant was used as the
cytosolic fraction for Western blot analysis of cytochrome c levels. The
protein amount was determined with a Bradford protein assay.
Western and Northern Blot Analyses—These analyses were carried
out as before (33). Briefly, cells infected as above were collected, washed
with PBS, resuspended in 1 ml of lysis buffer (100 mM Tris, 150 mM
NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 ␮g/ml aprotinin, 1 ␮g/ml
pepstatin), and incubated at 4 °C for 1 h. Cell lysate was subjected to
electrophoresis on 14% SDS-polyacrylamide gels and transferred to
nitrocellulose membrane (Pall Corp., Ann Arbor, MI). hBVR and hHO-1
fusion proteins were detected using mouse anti-FLAG antibodies (1:
1000, v/v, Sigma) as the primary antibody and alkaline phosphataselabeled anti-mouse IgG (1:5000, v/v, Bio-Rad) as the secondary antibody. hBVR and hHO-1 proteins were visualized by nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate staining (Calbiochem) or ECL system (Amersham Biosciences). Anti-mouse monoclonal
anti-cytochrome c and rabbit polyclonal anti-TRAIL antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PARP
antibody (C-2-10) was purchased from BioMol (Plymouth Meeting, PA).
Total RNA was extracted with TRIzol Reagent (Invitrogen) from 293A
cells. 10 ␮g samples were subjected to denaturing formaldehyde gel
electrophoresis and subsequently transferred to Hybond membrane.
Membranes were probed with a full-length hBVR cDNA (48) or 0.8-kb
fragment of hHO-1 cDNA (49). A 1.1-kb fragment of human ␤-actin
cDNA was used as internal control for loading. pCDNA3 containing
DR-5 cDNA was cut by EcoRI/HindIII and used as probe for DR-5.
Probes were labeled using [␣-32P]dCTP with the random primers labeling system (Invitrogen). Pre-hybridization, hybridization, and autoradiography were performed as described previously (50).
ELISA—HO-1 protein was measured by using an ELISA kit developed by Stressgen Bioreagents (Victoria, British Columbia, Canada)
according to the manufacturer’s instructions.
Measurement of BVR Activity—Human 293A cells infected with
pSuper-Retro-siBVR were lysed in buffer containing 50 mM Tris-HCl
(pH 7.4), 75 mM NaCl, 20 mM MgCl2, 10 mM MnCl2, 1% Nonidet-P-40, 2
17086
siRNA-mediated Gene Silencing of Biliverdin Reductase
mM EDTA, 2 mM EGTA, 10% glycerol, protease inhibitor mixture (1
␮g/ml aprotinin, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin, and 0.1 mM
phenylmethylsulfonyl fluoride), and phosphatase inhibitors (10 mM
NaF, 1 mM NaVO4). Noninfected 293A cells were used as control. BVR
activity was measured at pH 6.7 as described previously (51). The rate
of conversion of biliverdin to bilirubin was determined as the increase in
absorbance at 450 nm at 25 °C. Specific activity is expressed as nanomoles of bilirubin/min/mg of protein.
Luciferase Assay—Transfection of 293A cells in 24-well plates was
performed by using Lipofectamine (Invitrogen) with 0.4 ␮g of plasmid
containing PGL31 c-jun promoter fragment (⫺225 to ⫹150) (52), 0.4 ␮g
of plasmid containing c-jun promoter mutant construct (pGL3/c-Jun⫺)
and containing mutated AP-1 site (53), 0.4 ␮g of either empty pcDNA3,
pCMV ␤-galactosidase plasmid, or pGL3 vector (without the inserts). To
test the role of BVR and HO-1 in arsenite-dependent c-jun induction,
293A cells were infected with retrovirus expression either siRNA for
BVR or HO-1. After 24 h of infection, cells were treated with arsenite for
an additional 8 h and then harvested, washed, and lysed. 5 ␮l of lysate
from each sample was used for luciferase assay using a Promega kit
(Madison, WI). ␤-Galactosidase activity was used to assess transfection
efficiency, and luciferase activity was normalized against ␤-galactosidase activity.
In Vitro BVR Protein Translation, Nuclear Extraction, ␭-Phosphatase
Treatment, and Gel Mobility Shift Assay—293A cells were treated or
untreated with arsenite. 107 cells were harvested at 8 or 24 h after
treatment; cells were washed with cold PBS and then lysed with hypotonic buffer. Nuclei were collected by centrifugation and used for extract
preparation. In vitro BVR protein translation was performed using a
TNT Quick Coupled Translation System (Promega, Madison, WI).
Briefly, full-length hBVR cDNA was cloned into a pcDNA3 expression
vector downstream from the T7 RNA polymerase promoter. 2.0 ␮g of
recombinant plasmid DNA obtained was used for protein translation
with TNT Quick Master Mix in a 50-␮l reaction volume for 90 min at
30 °C.
To determine whether dephosphorylation of BVR impairs its binding
to AP-1, the following experiment was carried out using ␭-phosphatase
(Sigma). 293A cells were transfected with pEGFP-FLAG-hBVR, using
Lipofectamine Reagent (Bio-Rad). 24 h later, cells were treated with 5
␮M arsenite for 8 h. Nuclear extract was isolated and was subsequently
treated with ␭-phosphatase at concentrations of 20 – 80 units/20-␮l concentrations for 30 min at 37 °C. Thereafter, the samples were used for
gel mobility shift assay for AP-1 binding.
The in vitro translated protein and nuclear extract were analyzed in
gel mobility shift assay for their DNA binding capability to an AP-1
consensus oligo (54). The sequences of oligonucleotides in double
strands used in the present study were 5⬘-CGC TTG ATG AGT CAG
CCG GAA CGC TTG ATG AGT CAG CCG GAA-3⬘. The oligos were
labeled with [␥-32P]ATP by T4 kinase (Invitrogen). For DNA binding
assay, 3 ␮l of in vitro translated protein or nuclear extract was preincubated with 2 ␮l of binding buffer (20% glycerol, 5 mM MgCl2, 2 mM
EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, and 50 mM Tris-HCl, 0.25
mg/ml poly(dI):poly(dC)) in an 8-␮l reaction volume for 10 min at room
temperature. Then 2 ␮l of labeled oligo was added, and samples were
incubated for an additional 20 min at room temperature. The DNAprotein binding complexes were subjected to electrophoresis on a 6%
nondenatured polyacrylamide gel and processed for autoradiography.
In order to identify BVR in the DNA-protein complex, supershift experiments were performed using rabbit polyclonal anti-human BVR antibodies (55). Each experiment was repeated at least three times to
ascertain the reproducibility of results.
Detection of Apoptotic Cells—To detect apoptotic cells after arsenite
treatment, 293A cells were treated with 10 ␮M arsenite for 12 h and
subsequently harvested. Cells were washed and fixed with 75% ethanol
and stained with 20 ␮M propidium iodide with 2 ␮g/ml of RNase A. The
cells were further analyzed by flow cytometry for apoptosis based on the
propidium iodide staining. 103 cells were analyzed for each sample.
RESULTS
Arsenite Activates BVR Prior to Induction of HO-1 Expression—Previous studies (30) had shown that in the presence
H2O2, BVR is activated and displays increased phosphorylation. To examine whether activation of BVR is relevant to HO-1
oxidative response to arsenite, 293A cells were treated with low
arsenite concentrations (5 ␮M), and in the following 8 h, BVR
activity and HO-1 protein levels were monitored. ELISA was
used for detection of HO-1 protein. As shown in Fig. 1, after the
FIG. 1. Arsenite-mediated activation of BVR precedes induction of HO-1. 293A cells were treated with 5 ␮M arsenite and harvested
at the indicated time points for detection of BVR activity and HO-1
protein levels. BVR activity was analyzed at pH 6.7 with NADH as the
cofactor. ELISA measured HO-1 protein levels. Data shown are mean ⫾
S.D. of three experiments. The experiments details are provided under
“Materials and Methods.”
treatment of the 293A cells with arsenite, a significant increase
(4 –5-fold) of BVR activity was observed at 1 h after treatment.
BVR activity gradually decreased thereafter and essentially
returned to basal level 8 h later. There was no change in HO-1
protein levels in the 1st h after addition of arsenite. A significant increase in HO-1 level was observed 4 h after the treatment that further increased at the 8-h time point.
Arsenite-mediated Increase in BVR Activity Is Not Accompanied by Induction in Its Expression but by Increase in Its Serine/
Threonine Phosphorylation—Fig. 2 shows that the increase in
BVR activity was not essentially due to activation of gene
expression, as indicated by the unchanged level of its 1.5-kb
transcript detected by Northern blot analysis. A significant
increase in HO-1 mRNA message, however, was observed 2 h
after the addition of arsenite followed by robust increases at 4and 8-h time points.
Arsenite is known to activate serine/threonine kinases (56).
We have shown recently (22) that an increase in the phosphorylation of BVR triggers an increase in its reductase activity.
To examine whether arsenite treatment also increases BVR
phosphorylation, 293A cells were treated with 5 ␮M arsenite for
30 or 60 min, lysed, and subjected to immunoprecipitation
using anti-hBVR antibodies. The immunoprecipitations were
used for Western blot analyses of phosphorylated BVR. For
detection, a mixture of anti-serine and anti-threonine phosphoantibodies in a 1:1 ratio was used. In order to test whether an
increase in BVR activity reflected an increase in BVR protein,
cells were similarly treated with arsenite and harvested at 1, 2,
4, and 8 h after treatment, and cell lysate was used for detection of BVR protein levels by Western blotting. Findings of the
study shown in Fig. 3 demonstrated that arsenite indeed induced a significant increase in BVR phosphorylation at 30 and
60 min after treatment (Fig. 3a), whereas total BVR protein
levels were not affected by arsenite up to 8 h after treatment
(Fig. 3b).
Retroviral Vector Based siRNA System Effectively Knocks
Down BVR mRNA in 293A Cells—The role of BVR in oxidative
stress response of cells to arsenite was examined using a retroviral vector-based siRNA system to create a BVR knock-down
model. 293A cells transfected with expression construct for
EGFP-FLAG-BVR fusion proteins were infected with pSuperRetro-siBVR constructs and were harvested at the 8- and 24-h
time points. Control cells were transfected with EGFP-FLAGBVR expression construct. The efficiency of the vector-based
siRNA-mediated Gene Silencing of Biliverdin Reductase
17087
FIG. 2. Arsenite treatment increases HO-1 mRNA but not that
of BVR. Total RNA was isolated from 293A cells treated with arsenite
(5 ␮M) for the indicated periods. Northern blotting analysis was performed as described in the text. Membrane was probed with an 800-bp
hHO-1 cDNA fragment, together with the full-length hBVR cDNA.
Each lane contained 10 ␮g of RNA. The membrane was subsequently
probed for ␤-actin, used as a control for loading.
FIG. 3. Serine/threonine phosphorylation of BVR is induced by
arsenite treatment. 293A cells were transfected with pCDNA3FLAG-BVR expression vector. 24 h after transfection, cells were treated
with 5 ␮M arsenite for the indicated periods. Total cell lysate in RIPA
buffer was subjected to immunoprecipitation with anti-FLAG antibodies. The precipitated proteins were separated on SDS-PAGE, transferred to polyvinylidene difluoride membrane, and probed with either
anti-phosphoserine/-threonine antibodies (a) or with anti-hBVR antibodies (b). The ECL system was used for detection of signals.
FIG. 4. Retroviral based siRNA system effectively knocked
down BVR mRNA and protein levels in 293A cells. A pSuper-Retro
viral based siRNA system was used to silence BVR mRNA in cells and
to create an siRNA knock down 293A cell models. Experimental details
are provided in the text. a, Northern blot analysis of BVR expression
24 h after infection with the inhibitory construct. The filter was reprobed for ␤-actin that was used as control for RNA integrity and equal
loading. b, Western blot analysis of BVR protein in 293A cells infected
with BVR siRNA. The blot was first probed with anti-FLAG antibodies
and re-probed with anti-␤-actin antibodies, which served as control for
loading. The bottom panel shows Coomassie staining of the same gel to
evaluate the effect of siRNA on total cellular protein. c, fluorescence
microscopy of the 293A cells transfected with EGFP-FLAG-hBVR or
with the expression vector plus BVR siRNA.
siRNA system to “knock down” BVR was assessed by Northern
and Western blot analyses and by fluorescence microscopy. As
shown in Fig. 4, BVR mRNA was nearly undetectable 24 h after
infection (Fig. 4a). Western blot analysis indicated that BVR
protein was also reduced to undetectable levels at this time point
(Fig. 4b). Fig. 4c shows that 8 h after infection with pSuperRetro-siBVR, the EGFP-FLAG-BVR fluorescent fusion protein
display was significantly attenuated when compared with control
FIG. 5. BVR siRNA knocks down arsenite-dependent increases
in BVR activity and HO-1 protein levels. Cells were seeded into 6-well
plates and pretreated with retroviral construct containing BVR siRNA.
24 h later, cultures were treated with 5 ␮M arsenite. Analysis of BVR
activity and HO-1 protein levels were carried after 1 or 8 h. BVR activity
is expressed as nanomoles of bilirubin/min/mg of total cell protein. ELISA
was used to determine HO-1 protein levels. The results are expressed as
average ⫾ S.D. of three experiments and normalized to total amount of
proteins in each sample. *, p ⱕ 0.05 when compared with 0 time control.
⫹, p ⱕ when compared with appropriate arsenite-treated control.
cells in the absence of infection with the inhibitory RNA. The
experimental data indicated that the pSuper-Retro-siBVR system effectively decreased the target mRNA and thus could be
used as an effective model for experiments described below to
evaluate the function of BVR in cytoprotection and induction of
HO-1 in 293A cells in response to arsenite treatment.
BVR Influences Arsenite-dependent HO-1 Expression—BVR
siRNA was used to test the role of BVR in HO-1 response to
arsenite treatment. Cells were infected with retroviral vector
expressing BVR siRNA for 24 h, followed by treatment with 5
␮M arsenite. BVR activity and HO-1 protein level were analyzed (Fig. 5a). As with the experimental results shown in Fig.
1, arsenite treatment significantly increased BVR at the 1-h
time point, thereafter returning to the normal levels at 8 h.
BVR siRNA treatment of the cells suppressed the arsenitemediated increase in BVR activity, as well as that of the control
cells. The effect of siBVR treatment on HO-1 response to arsenite was examined. A robust and time-dependent increase in
HO-1 protein was observed when measured at 8, 12, 16, and
24 h after arsenite treatment. This increase in HO-1 expression
was suppressed by about 60% at 16 and 24 h when cells were
pretreated with BVR siRNA. The finding that siBVR did not
completely block the induction of HO-1 by arsenite is consistent
with the reported involvement of a number of transcription
factors in the induction of HO-1 that are activated by arsenite
(44) and indicate that BVR is not the sole transcription factor
for the HO-1 response to arsenite. Furthermore, the finding
that BVR siRNA in the absence of arsenite exposure did not
significantly affect basal levels of HO-1 suggests that BVR
influences arsenite-driven HO-1 induction.
Arsenite Treatment of 293A Cells Enhances Binding Ability
of BVR Protein to AP-1 Regulatory Elements—To examine
whether involvement of BVR in arsenite-mediated induction of
17088
siRNA-mediated Gene Silencing of Biliverdin Reductase
FIG. 6. Arsenite treatment increases BVR-AP-1 binding that is
phosphorylation-dependent. a, arsenite treatment increases BVRAP-1 binding. 293A cell were treated with 5 ␮M arsenite for 8 or 24 h;
nuclear extract was obtained and used for gel mobility shift assay for
AP-1 binding in the presence and absence of anti-hBVR antibodies
(supershift) (55). b, dephosphorylation of BVR by ␭-phosphatase treatment abolishes BVR-AP-1 binding. Nuclear extract was isolated from
the 293A cells transfected with pEGFP-BVR and treated with 5 ␮M
arsenite for 8 h. The extract was treated with ␭-phosphatase for 30 min
at 37 °C and subsequently used for gel mobility shift assay for AP-1
binding. Experimental procedures are described in detail in the text.
FIG. 7. Biliverdin, but not bilirubin, impairs BVR-AP-1 complex
formation. In vitro translated BVR was obtained using the TNT Quick
Coupled Translation System (Promega, Madison, WI). The translated
BVR protein was used for gel mobility shift assay. Left panel represents
gel shift assay and shows that biliverdin inhibited BVR/AP-1 binding in
vitro in a dose-dependent manner. Right panel represents one of the
experiments to determine BVR/AP-1 binding in the presence of 60 ␮M
each of biliverdin and bilirubin. The DNA fragment (42 bp) was a synthesized oligo with two AP-1 sites (30). The experiment was repeated four
times. t indicates in vitro translated BVR.
HO-1 is accompanied by an increase in its AP-1 binding activity, gel mobility shift assay was performed at 8- and 24-h time
points. Nuclear extract was prepared from 293A cells treated
with 5 ␮M arsenite and used in the assay for detection of
protein binding to 32P-labeled AP-1 probe. Results are shown in
Fig. 6. As shown in Fig. 6a, the nuclear extract obtained from
the treated cells displayed an increased ability to form a protein-DNA complex. BVR was identified as a major component
of the complex in the supershift assay using antibodies to
hBVR. It is noteworthy that whereas activation of BVR, i.e.
increased phosphorylation, is detected 1 h after arsenite treatment (Fig. 3), AP-1 binding remains in effect as late as 24 h.
These observations are consistent with the likelihood of BVR
functioning as a kinase at an early phase of cellular response to
arsenite and as a transcription factor/DNA-binding protein at
the later phase.
To confirm further that increased BVR/AP-1 binding
caused by arsenite treatment is related to phosphorylation of
the protein, the effect of dephosphorylation of BVR on its
DNA binding was examined. Previous studies had shown
that treatment of hBVR with phosphatase effectively dephosphorylates the protein (22). Cells were transfected with
pEGFP-FLAG-hBVR expression construct, followed by treatment with 5 ␮M arsenite. 8 h later nuclear extract was obtained and treated with ␭-phosphatase at 20 – 80 units/20 ␮l
of concentration for 30 min at 37 °C. Thereafter, samples
were analyzed for AP-1 binding. As shown in Fig. 6b, dephosphorylation of BVR significantly reduced its AP-1 binding
capability, and the decrease in the formation of BVR-AP-1binding complex was concentration-dependent.
Biliverdin, but Not Bilirubin, Is an Effective Inhibitor of BVRAP-1 Binding in Vitro—Previous studies had shown that BVR is
inhibited by bilirubin, whereas biliverdin is an effective inhibitor
of HO-1 activity (1, 18). To examine whether this inhibition
involves interference of BVR substrate with its DNA binding,
TNT in vitro protein translation system was used to test BVRAP-1 complex formation in the presence or absence of biliverdin.
Bilirubin was used as the control. The newly translated BVR
protein was incubated with the 32P-labeled DNA fragment, containing 2 AP-1 sites, in the presence of either biliverdin or bilirubin. The results of gel mobility shift assay are shown in Fig. 7.
In the presence of biliverdin, there was a notable decrease in
signal for BVR-DNA complex formation. Bilirubin, however, did
not affect formation of the complex. The result defines biliverdin
as an effective inhibitor of BVR-AP-1 complex formation, an
effect that was not shown by its reduced form.
BVR Regulates Arsenite-mediated c-jun Promoter Activation—BVR-AP-1 complex formation using an adenovirus construct of hBVR to overexpress the protein was shown previously (33). BVR in a homodimeric form binds to DNA
containing two AP-1 sites but not to a fragment containing a
single AP-1 site (30). In order to establish involvement of BVR
in AP-1 activation by arsenite, a pGL3 vector containing the
c-jun promoter was used as a luciferase reporter system, where
the c-jun promoter with two AP-1 sites was placed downstream
from the luciferase reporter. Cells were co-transfected with
siBVR, whereas siHO-1-infected cells were used as a control. A
second control was cell-transfected with TRRE site-mutated
c-jun promoter to address the specificity of the c-jun promoter.
Data are shown in Fig. 8a. 293A cells transfected with the
pGL3 vector containing the c-jun promoter showed a base-line
luciferase activity. After treatment of the cells with 5 ␮M arsenite, the luciferase activity of the c-jun promoter was increased approximately 3-fold. The same treatment, however,
did not increase TRRE site-mutated c-jun promoter. When cells
transfected with pGL3/c-jun promoter were pretreated with
BVR siRNA and followed by arsenite treatment, luciferase
activity was essentially similar to that of the untreated controls
(PGL3/c-jun). Pretreatment of the cells with HO-1 siRNA followed by arsenite treatment did not significantly affect lucif-
siRNA-mediated Gene Silencing of Biliverdin Reductase
FIG. 8. Knock down of BVR but not that of HO-1 prevents
activation of c-jun promoter by arsenite. a, c-jun promoter reporter
assay. 293A cells seeded into 24-well plates were transfected with
either c-jun luciferase promoter reporter construct (pGL3/c-jun, 0.4 ␮g)
or with c-jun luciferase promoter reporter mutant construct (pGL3mt/
c-jun, 0.4 ␮g) cells were cotransfected with pCMV ␤-galactosidase (0.3
␮g) vector used as a control for normalization of transfection. 24 h after
transfection cells were synchronized overnight by replacing growth
medium with one containing 0.1% fetal bovine serum. Cells were
treated with either siRNA for BVR or siRNA for HO-1 as described
under “Materials and Methods.” After 24 h, cells were treated with 5 ␮M
arsenite. 8 h later, they were harvested, washed with PBS, lysed in
reporter lysis buffer, and assayed for luciferase and ␤-galactosidase
activities. Luciferase activity normalized for transfection efficiency
against ␤-galactosidase activity is presented as mean ⫾ S.D. for three
experiments with quadruplicate samples. b, Western blot analysis of
HO-1 protein in 293A cells transfected with EGFP-FLAG-hHO-1 expression construct and infected with HO-1 siRNA. Total protein stained
by Coomassie Blue was used as control for loading. c, 293A cells transfected with expression vector for EGFP-FLAG-HO-1 plus or minus
HO-1 siRNA. EGFP fluorescence was detected by a microscope.
erase activity. These observations were supportive of BVR involvement of HO-1 response to arsenite through its AP-1
binding capability.
To confirm that HO-1 siRNA indeed had effectively knocked
down HO-1 protein, cells were infected with the EGFP-HO-1
expression construct, and 24 h later they were infected with
siHO-1 for an additional 24 h. Cell lysate was prepared and
used for the detection of protein levels by Western blotting (Fig.
8b). Fluorescent microscopy was used to visualize HO-1 protein
in intact cells (Fig. 8c). As noted, the HO-1 siRNA infection was
highly effective in attenuating HO-1 levels, thus generating a
useful model for further experiments described below.
siRNA-mediated Knock Down of BVR Increases Sensitivity of
293A Cells to Arsenite-mediated Apoptotic Cell Death—To evaluate whether BVR itself, independent of HO-1, is an effective
cytoprotector, arsenite-mediated apoptosis by a high concentration of the compound was examined. 293A cells were infected
with BVR siRNA or HO-1 siRNA retrovirus for 24 h, followed
by treatment with 10 ␮M arsenite. 12 h later, cells were harvested, washed, fixed with 75% ethanol, and stained with 20 ␮M
propidium iodide. The cells were analyzed by flow cytometry for
apoptosis based on propidium iodide staining. As shown in Fig.
9, BVR siRNA-mediated knock down in 293A cells resulted in a
4-fold increase in the number of apoptotic cells when compared
with control. Most surprisingly, in the 293A cells infected with
HO-1 siRNA retrovirus, a significant effect was not observed
after arsenite treatment when compared with control cells. The
data reveal a previously unknown function of BVR as an effec-
17089
tive protector of cells against oxidative stress and suggest independence of this activity from that of reduction of biliverdin
and production of bilirubin.
To investigate the mechanism by which siBVR potentiates
arsenite-mediated apoptosis, 293 cells were depleted of hBVR
using siBVR prior to treatment, and the effects of modulation of
BVR on key factors involved in apoptosis were examined (Fig
10). An early event in apoptotic signaling cascade is the release
of cytochrome c from mitochondria, which precedes activation
of caspases, FasL-induced cleavage of poly(ADP-ribose) polymerase (PARP), followed by DNA fragmentation. Activation of
caspase-3 by TRAIL/Apo-2L, a member of tumor necrosis factor
superfamily, is triggered through its binding to death receptors
DR4 and DR5. To examine effect of BVR knock down on arsenite-mediated cytochrome c release, cells infected with siBVR
were treated with 10 ␮M arsenite, lysed 24 h after the treatment, and levels of cytochrome c in the cytosol were examined
by immunoblotting. Control cells were not infected with the
interfering RNA but received the same regimen of arsenite
treatment (Fig. 10a). We observed an increase in cytochrome c
levels in the cytosolic fraction of arsenite-treated cells when
compared with control cells. This increase was augmented
when cells were pre-exposed to BVR siRNA. When the total cell
lysate post-arsenite treatment was examined for the appearance of the 85-kDa cleavage product of a 116-kDa PARP was
detected by Western blot analysis in both control and siRNAtreated cells (Fig. 10a). The appearance of the cleaved product
was more pronounced in siRNA-treated cells. In cells transfected with wtBVR, the 85-kDa cleavage product was essentially undetectable.
To investigate further the contribution of BVR to anti-apoptotic events, the effect of transfection of cells with wtBVR or
siBVR on another pro-apoptotic factor TRAIL was assessed. As
noted in Fig. 10a, similar to PARP, the marked induction of
TRAIL by arsenite treatment was attenuated by overexpression of wtBVR and conversely increased by depletion of endogenous BVR (Fig. 10a).
Binding of TRAIL to death receptor DR5 is essential for the
induction of TRAIL signaling of apoptotic cell death. TRAIL
triggers apoptosis through death receptors DR4 and DR5; DR5
is a greater contributor to apoptotic signaling. Therefore,
whether modulation of cellular levels of BVR would affect
arsenite dependent DR-5 induction was examined. As shown in
Fig. 10b, a modest induction of DR-5 mRNA was observed at
12, 16, and 24 h after treatment of control cells with arsenite.
However, when cells were pre-treated with BVR siRNA, a
robust induction of DR-5 mRNA upon treatment with arsenite
was observed. The arsenite-dependent DR-5 induction was
again attenuated when cells were transfected with wtBVR.
Treatment of cells with empty virus did not have a significant
effect on DR-5 mRNA levels. The results indicate that the
presence of BVR is important in preventing induction of DR-5
mRNA in events that culminate in apoptosis. Together, the
findings of this series of experiments suggest that BVR
prevents cells from proceeding to apoptosis by interfering at
several points along the cell death pathway.
DISCUSSION
The findings of this study suggest that BVR has a prominent
and multidimensional role in the regulation of HO-1 stress
response. Furthermore, it identifies the reductase, independent of bile pigments, as an essential component of cytoprotection against oxidative challenge. In turn, these findings reflect
the collective functions of BVR as a reductase, with the ability
to produce bilirubin and inactivate biliverdin, and as a kinase
and component of cell signaling pathways, with the ability to
influence gene expression.
17090
siRNA-mediated Gene Silencing of Biliverdin Reductase
FIG. 9. BVR knock down, but not that of HO-1, decreases viability of 293A cells treated by arsenite. 293A cells were infected with BVR
siRNA or HO-1 siRNA vectors. 24 h later, cells were exposed to 10 ␮M arsenite, and after 12 h, cells were analyzed for apoptosis by flow cytometry
based on propidium iodide staining of DNA content. Upper panel, the set of histograms shows the effect of siBVR and siHO-1 on the number of
apoptotic cells. Lower panel, bar graph represents statistical analysis of apoptosis assay data of cells treated with arsenite. Data shown are mean ⫾
S.D. of five experiments.
Although the heme molecule can be degraded to pyrrolic
complexes in vivo and by chemical reactions in vitro, the HO/
BVR pathway is the only effective mechanism for production of
CO, biliverdin IX␣, and bilirubin IX␣ (11). Inference to the role
of the HO/BVR system in cell signaling and in cytoprotection is
based on the current understanding of biological activities of
the heme degradation products by this combination of enzymes. The reduction product of BVR activity is an effective
intracellular antioxidant (57) and together with CO modulates
immune effector functions (12–15, 19). The substrate for BVR,
biliverdin IX␣, is an effective modulator of signal transduction
pathways and inhibits protein phosphorylation and kinase activity (17, 58 – 60). Direct evidence for the role of biliverdin in
cell signaling pathways was offered by the identification of
biliverdin IX␣ as the determinant factor in the dorsal axis
development in Xenopus laevis embryos (61).
The observation that biliverdin attenuates BVR-DNA complex formation (Fig. 7) is a novel finding and further supports
a role for the tetrapyrrole in cell signaling and identifies biliverdin as one component of the multidimensional mechanisms
by which BVR regulates HO-1 expression. In this context, the
reductase activity of BVR would serve as the means to inactivate the inhibitor of its binding to AP-1 sites in the HO-1
promoter. Biliverdin-mediated impairment of AP-1-BVR complex formation is consistent with the earlier work that had
shown inhibition of HO activity in vivo by the tetrapyrrole (18).
In fact, with the exception of biliverdin, to date no other heme
pathway products or normal cell constituents have been found
to inhibit HO activity in vivo or in vitro.
The occurrence of a regulatory loop in the cell to control the
heme degradation process, which would involve a combination
of inhibitory effects of biliverdin on HO-1 expression and its
catalytic activity, and activation of BVR by oxidant inducers of
HO-1 can be considered. In this scenario, activation of BVR and
rapid conversion of biliverdin to bilirubin would allow for derepression of HO-1 expression and increased activity; product
(bilirubin) inhibition of BVR activity then would allow for
buildup of biliverdin levels; this in turn, would cause product
(biliverdin) inhibition of the oxygenase, hence permitting return to normal of the heme degradation process. The various
components of this potential loop have been demonstrated here
as well as in previous reports. For instance, inhibition of BVR
activity by bilirubin and the initial suppression of HO activity
in vivo by biliverdin followed by a rebound increase were reported long ago (1, 18). The inhibitory effect of biliverdin on
BVR/AP-1 was documented in the present study. The concentration of biliverdin used for in vivo experiments (18) was
comparable with that employed in the gel mobility shift assay
(Fig. 7). It is relevant to note that exceedingly high concentrations of biliverdin can accumulate in the cell as the consequence of exposure to compounds that disrupt cell signaling
pathways (58). A wide range of functions in the cell are subject
to a change in activity of HO-1; this includes those that are
controlled by NO radicals (16, 19, 20, 62– 65). Therefore, interplay between BVR, its substrate, and its product in the regulation of the stress response of HO-1 would have a major impact
on the cell.
However, the mechanism for cytoprotection offered by BVR
as indicated by the striking increase in the number of apoptotic
cells when cells infected with siBVR are exposed to arsenite
(Fig. 9) is likely not to involve inhibition of bilirubin formation
and exacerbation of damaging effects of oxygen free radicals
siRNA-mediated Gene Silencing of Biliverdin Reductase
FIG. 10. BVR attenuates arsenite-mediated apoptosis by interfering with cytochrome c and TRAIL-dependent apoptotic pathway. a, Western blot analysis effect of BVR knock down on cytochrome
c, TRAIL, and PARP. Cells were either transfected with wild type hBVR
expression construct (pcDNA3-BVR) or infected with construct containing siRNA for BVR. Transfected cells treated with 10 ␮M As for 24 h
were used for preparation of total cell lysate and the cytosolic fractionation. The cytosolic fraction was subjected to 15% SDS-PAGE, and the
blotted proteins were used for detection of cytochrome c using monoclonal antibodies to the protein followed by the ECL detection system.
Total cell lysate was used for immunoblot analysis of PARP and TRAIL
using appropriate antibodies and the ECL detection system. Intact and
cleaved PARP proteins are indicated as 116- and 85-kDa bands, respectively. Same blot was stripped and probed with anti-actin antibodies
used as a control for loading. b, Northern blot analysis of DR5 levels in
cells treated with arsenite in the presence of increased or knocked down
levels of BVR expression. Cells transfected with wtBVR or infected with
BVR siRNA were treated for the indicated periods with 10 ␮M arsenite.
Total RNA was isolated and used for Northern blot analysis. The
membrane was probed with cDNA for DR5. Equal loading is indicated
in the lower panel showing 28 S and 18 S RNA. The above series of
experiments were performed twice. Experimental details are provided
under “Materials and Methods.”
(66). Rather, this element of the BVR role in cytoprotection is
clearly independent of HO-1 expression and must involve the
kinase activity of BVR and its input into the signal transduction pathways.
Kinases control a wide variety of cellular functions, including
apoptosis. Previous work with gene array analysis had identified several members of the signal transduction pathways as
potential targets for regulation by BVR. This includes HSF1
(heat shock transcription factor1), heat shock proteins 90 and
27, Bcl-2, Cox-2, protein kinase C␣, and WISP3 (inducible
signaling pathway protein 3). Among the list were ATF-2
(CREB-2) and c-jun (33). The finding that the c-jun promoter
activation by arsenite can be blocked when BVR expression is
knocked down (Fig. 8) places the reductase in the position of
having a direct role in influencing not only HO-1 gene expression but rather influencing that of an extended list of stressresponsive genes. In the case of c-Jun, because it can form a
DNA complex as a homodimer or a heterodimer not only with
c-Fos but also with ATF-2/CREB (54, 67, 68), a change in the
expression and activation state of c-Jun predictably would alter
the profile of the cell signaling pathways. The fact that the
knock down of BVR augmented cell killing activity of arsenite
17091
is clearly consistent with the role of the reductase in the expression of genes that affect cell growth and proliferation.
Among those, the ATF-2/CREB and mitogen-activated protein
kinase pathways are the most notable because of their input in
the response of the cell to growth factors, mitogens and
cytokines.
The unexpected finding that attenuation of BVR expression
markedly increased the proportion of apoptotic cells in arsenite-treated preparations and increased the levels of factors
associated with the apoptotic mode of cell death, including
cytochrome c, TRAIL, death receptor-5 (DR-5) mRNA, and
PARP, further supports the suggestion that the role of BVR in
cytoprotection against oxidants extends beyond regulating bilirubin and CO production by HO-1. We were indeed surprised
by the observation that siHO-1 did not influence arseniteinduced apoptosis in 293A cells. This was particularly unexpected considering a recent report on the enhancement of ischemia/reperfusion-induced lung injury caused by introduction
of the lung-specific siHO-1 (69). The divergence of two sets of
findings is perplexing because in the present study we carefully
established that in fact the HO-1 siRNA construct was effective
at suppressing both the HO-1 protein and its RNA. Accordingly, the only explanation that can be offered at this time is
activation of different pathways of signaling by arsenite and
ischemia/reperfusion. Arsenite induces production of oxygen
radicals, whereas ischemia/reperfusion injury has the initial
hypoxic component. Thus, the profile of genes activated by
arsenite and ischemia/reperfusion could differentially affect
pathways of cell death and survival. The differences in the
model system and constructs are also among the possible
explanations.
Based on the data and reasoning offered in this report, it is not
unreasonable to suggest that experimental modulation of BVR in
the cell could be used to enhance or attenuate the response of the
cell to external stimuli. The observation that by silencing BVR
expression the cell killing activity of arsenite is enhanced is a
strong argument in support of this suggestion. Arsenite is an
established human carcinogen that causes chromosomal aberrations, sister chromatid exchange, and micronuclei formation (47).
Although the mechanism of its carcinogenicity is poorly understood, a substantial amount of evidence points to the role of
hydroxy radicals, the activator of BVR (22), in the genotoxicity
(70, 71). Because oxygen radicals are suspected to underlie the
etiology of a long list of pathophysiological conditions, it would be
reasonable to suggest that BVR with its multidimensional input
into the regulation of oxidative stress-responsive genes may find
a useful place in therapeutic settings.
Acknowledgments—We are grateful to W. El-Deiry (University of
Pennsylvania, Philadelphia) for the gift of DR-5 cDNA; J. Boyce for the
preparation of the manuscript, and J. Smith for illustrations.
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