From the Laboratory of Molecular Immunology, Guthrie Research Institute, Sayre, Pennsylvania 18840
Received for publication, August 7, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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To determine how hyaluronidase
increases certain cancer cell sensitivity to tumor necrosis factor
(TNF) cytotoxicity, we report here the isolation and characterization
of a hyaluronidase-induced murine WW domain-containing oxidoreductase
(WOX1). WOX1 is composed of two N-terminal WW domains, a nuclear
localization sequence, and a C-terminal alcohol dehydrogenase (ADH)
domain. WOX1 is mainly located in the mitochondria, and the
mitochondrial targeting sequence was mapped within the ADH domain.
Induction of mitochondrial permeability transition by TNF,
staurosporine, and atractyloside resulted in WOX1 release from
mitochondria and subsequent nuclear translocation. TNF-mediated WOX1
nuclear translocation occurred shortly after that of nuclear
factor- Most cancer cells are known to secrete the matrix-degrading enzyme
hyaluronidase. Elevation of hyaluronidase levels is associated with
progression, invasion, and metastasis of breast, ovarian, endometrial,
prostate, and other cancers (1-5). Also, expression of hyaluronidase
by tumor cells induces angiogenesis in vivo (6). The growth
of murine lung carcinoma and melanoma, for example, is influenced by
Hyal-1, a locus determining hyaluronidase levels and
polymorphism (7). How hyaluronidase modulates cell growth is not known.
Both in vitro and in vivo studies have shown that
exogenous hyaluronidase reverses the resistance to chemotherapeutic
drugs in cancer cells and solid tumors by increasing their exposure to
the drugs (8-10). We have determined that hyaluronidase enhances cancer cell susceptibility to tumor necrosis factor
(TNF)1-mediated cell death
(11-13). For example, pretreatment of murine L929 fibroblasts and
human prostate LNCaP cells with hyaluronidase for at least 12 h
significantly increases their sensitivity to TNF-mediated death
(100-700%) (11-13). The hyaluronidase-enhanced TNF sensitivity in
L929 cells is associated, in part, with up-regulation of pro-apoptotic
p53 (11-13).
To further explore the mechanism whereby hyaluronidase enhances TNF
cytotoxicity, we report here the isolation of a novel murine WW
domain-containing oxidoreductase (Wox1) cDNA.
Hyaluronidase increased Wox1 gene and protein expression.
Ectopic expression of WOX1 in L929 cells enhanced their sensitivity to
TNF cytotoxicity, whereas antisense Wox1 raised TNF
resistance. Thus, WOX1 is involved in the hyaluronidase-increased TNF
sensitivity in various cancer cells. We produced polyclonal antibodies
against WOX1, examined WOX1 cellular localization, and determined the
possibility of WOX1 nuclear translocation in response to apoptotic
stimuli and inducers of mitochondrial permeability transition.
Overexpression of WOX1 was shown to induce cell death. We then examined
whether the cell death was p53- or caspase-dependent. Since
hyaluronidase up-regulates p53 expression, the role of p53 and WOX1 in
mediating cell death was determined.
Molecular Cloning of Wox1--
Isolation of novel cDNAs by
differential display, library screening, and functional analysis has
been described previously (14, 15). L929 cells were treated with bovine
testicular hyaluronidase (200 units/ml; Sigma) for 4 h, followed
by isolating total cellular RNA and performing first strand cDNA
synthesis and differential display (14, 15). A hyaluronidase-induced
cDNA of 300 base pairs was isolated and used to screen a In Vitro Translation--
The full-length murine Wox1
cDNA was constructed in the pCR3.1 vector. An in vitro
translation kit (Novagen, Madison, WI) was used to transcribe the
full-length Wox1-pCR3.1 construct into mRNA via the T7
promoter and to translate into [35S]methionine-labeled
WOX1 protein.
Expression Constructs--
Constructs, which were made with the
pEGFP-C1 vector (CLONTECH), for expressing
N-terminal green fluorescent protein (GFP)-tagged proteins are shown in
Table I. These constructs were used to express the full-length coding
region (construct 2), the antisense Wox1 mRNA
(expressing antisense Wox1 mRNA and GFP protein;
construct 3), the N-terminal WW domain region (construct 4), a partial
ADH domain (amino acids 180-392; construct 5), and the potential
mitochondrial targeting regions in the ADH domain (amino acids 180-273
and 209-273; constructs 6 and 7, respectively). To examine whether the
large-size GFP protein (28 kDa) affects the WOX1 function, similar
constructs, which were made with a small C-terminal v5 tag (5 kDa) in the pcDNA3.1.TOPO vector (Invitrogen), were used
to express full-length WOX1 (construct 8), the first WW domain
(construct 9), and the first and second WW domains (construct 10).
p53 Expression Construct--
A full-length p53 cDNA clone
from normal human tissues was found in the expressed sequence tag data
base (GenBankTM/EBI accession numbers AI243172 and
AF307851) and obtained from Incyte Genomics (St. Louis, MO). The coding
region was cloned into the pcDNA3.1/CTGFP-TOPO vector (Invitrogen)
and tagged with a GFP sequence at the C terminus (construct 11) (see
Table I).
Site-directed Mutagenesis--
Site-directed mutagenesis was
performed to alter several indicated sites in the Wox1
cDNA sequence using the QuikChange site-directed mutagenesis Kit
(Stratagene, La Jolla, CA). The aspartic acid residues of a putative
caspase recognition site (DIND, amino acids 267-270) were mutated to
glycine, i.e. D267G and D270G (construct 17) (see Table I).
The GKRKRV sequence (amino acids 50-55) of the nuclear localization
sequence (NLS) was also mutated to GQGTGV (construct 18) (see Table
I).
Antibody Production--
A WOX1 peptide (RLAFTVDDNPTKPTTRQRY,
amino acids 89-107) was synthesized by Genemed Biotechnologies, Inc.
(San Francisco, CA) and conjugated with keyhole limpet hemocyanin for
antibody production in rabbits using the Pierce antibody production
kit. The selected WOX1 sequence is identical between human and mouse.
Transient Transfection--
In most cases, transfection studies
were performed by our standard CaPO4 precipitation method
(14, 15). Forty-eight h post-transfection, the extent of cell death was
measured by crystal violet staining (a measure of both necrosis and
apoptosis). Additionally, DNA fragmentation assays (11) were performed
to determine the extent of apoptosis. To exclude the possibility of
nonspecific killing of cells caused by transfection reagents or vectors
alone, the observed results were repeated using liposome-based cell
transfection reagents such as LipofectAMINE (Amersham Pharmacia
Biotech), GeneFECTOR (Venn Nova, Pompano Beach, FL), and FuGENE 6 (Roche Molecular Biochemicals) and electroporation (BTX ECM830,
Genetronics, San Diego, CA).
Stable Transfectants and TNF Cytotoxicity Assays--
Stable
transfectants of L929 cells for expressing the desired proteins were
established as described previously (14, 15). Where indicated, L929
cells were electroporated with the above indicated purified
Wox1 construct DNAs (in pEGFP-C1 or pCR3.1), followed by
selecting neomycin-resistant stable transfectants or cell
colonies using 300 µg/ml G418, an analog of neomycin (Life Technologies, Inc.). Functional analysis of cellular sensitivity to TNF
cytotoxicity was performed as described (11-15, 18). The established
stable transfectants were cultured on 96-well plates overnight,
followed by exposure to recombinant human TNF (Genzyme Corp. (Boston,
MA) and R&D Systems (Minneapolis, MN)) for 16-24 h.
Cell Lines--
The following cell lines were from American Type
Culture Collection (Manassas, VA): murine L929 and NIH/3T3 fibroblasts;
human ovarian ME180 and HeLa cells, monocytic U937 and THP-1 cells, transformed 293 fibroblasts, and Molt-4 T cells; neonatal rat H9c2
cardiomyocytes; and monkey kidney COS-7 fibroblasts. The lymphotoxin-producing L929R cells (18), the human neural SK-N-SH cells,
and the human breast MCF-7 cells were gifts of Dr. D. Beezhold, R. Aronstam, and J. Noti (Guthrie Research Institute), respectively.
Confocal Microscopy--
Where indicated, confocal microscopy
analysis was performed to determine the colocalization of WOX1 and
mitochondria. Mitochondria were stained by antibodies against
cytochrome c or by the membrane potential-sensitive
mitochondrial stain Mitotracker Red CMXRos (Molecular Probes, Inc.,
Eugene, OR).
Northern and Western Blotting--
To perform Northern
hybridization, L929 cells were cultured in 100-mm Petri dishes and
treated with hyaluronidase (200 units/ml) for 1-24 h. Total cellular
RNAs were isolated from these cells, and Northern hybridization was
carried out using 40 µg of RNA/lane (14, 15). Antibodies used in the
Western blotting were against I Yeast Two-hybrid Interactions--
The CytoTrap yeast two-hybrid
system was from Stratagene. Unlike the traditional system, which
depends upon protein-protein interaction in the nucleus (20), this
assay system is based on the binding of an Sos-tagged bait protein to a
cell membrane-anchored target protein (tagged with a
myristoylation signal) that results in activation of the Ras
signaling pathway, thereby permitting mutant yeast cdc25H to
grow at 37 °C using a selective agarose medium or plate containing
galactose. Two constructs of Wox1 as baits (in the pSos
vector) and three constructs of p53 as targets (in the pMyr vector)
were made (see Table I). Binding interactions using combination of
these vectors were performed. Vectors that are included in the system
for positive binding interactions are pSos-MafB and pMyr-MafB (21), and
those that are included for negative binding interactions are empty
pSos and empty pMyr, pMyr and lamin C, or other vectors.
Molecular Cloning of Murine Wox1--
To further explore the
mechanism whereby hyaluronidase enhances TNF cytotoxicity, we isolated
a murine Wox1 cDNA (2197 bases; GenBankTM/EBI accession number AF187014) by differential
display and cDNA library screening. The cDNA possesses an open
reading frame, a typical Kozak sequence at the initiation site (ATG),
and an upstream in-frame stop codon. The deduced murine WOX1 protein sequence (414 amino acids, 46 kDa) possesses two N-terminal WW domains
(first domain, amino acids 18-47; and second domain, amino acids
59-87), an NLS (GKRKRV, amino acids 50-55), and a C-terminal short-chain ADH domain (amino acids 121-330) (Fig.
1). WW domains are known to bind proteins
with a particular proline motif, (A/P)PP(A/P)Y (22, 23). Whether the WW
domains of WOX1 bind to this motif is not known.
The human gene coding for WOX cDNAs (or known as
WWOX or FOR) has been mapped to a fragile site on
chromosome 16 (24-26). Murine WOX1 is highly homologous to full-length
human WWOX (46 kDa) (24) and FOR II (46.7 kDa) (25). Three
alternatively spliced variants are FOR I (41.2 kDa), FOR III (21.5 kDa), and FOR IV (4.1 kDa), which possess distinct C-terminal ends
(Fig. 1) (25). Two alternatively spliced variants we have identified and sequenced are human prostate WOX3 (identical to FOR III;
GenBankTM/EBI accession numbers AI669330 and AF187015)
(Fig. 1) and FOR I-related human WOX5 (a partial clone;
GenBankTM/EBI accession number AI219858), whose ADH domain,
but not the N terminus, is identical to the sequence of FOR I.
Gene and Protein Expression--
Murine L929 fibroblasts
constitutively expressed a low level of Wox1 mRNA, as
determined by Northern blotting (Fig.
2A). Exposure of L929 cells to
hyaluronidase for 2-24 h resulted in increased Wox1 gene
expression (~2.3 kilobases), peaking at 8-24 h (~150% increase) (Fig. 2A). The induced Wox1 gene
expression correlates positively with the induction of TNF sensitivity
in L929 cells, which requires pretreatment with hyaluronidase for at
least 10 h (11-13).
As predicted, in vitro translation of the full-length murine
Wox1 cDNA produced a protein of ~46 kDa, as analyzed
by reducing SDS-polyacrylamide gel electrophoresis (Fig.
2B). A 30-kDa product was also observed (Fig.
2B). This protein is most likely a degradation product of
46-kDa WOX1 since Wox1 mRNA, which is derived from the
cloned full-length cDNA, is unlikely to undergo alternative splicing.
WOX1 is a single chain protein and does not exist as a dimer, as
determined by nonreducing SDS-polyacrylamide gel electrophoresis. A
putative caspase recognition site is DIND (267). However, this site does not appear to be the proteolytic degradation site. Alteration of the DIND sequence to a non-caspase recognition sequence (GING) by
site-directed mutagenesis failed to prevent WOX1 degradation (Fig.
2B). The caspase inhibitor peptide
acetyl-Asp-Glu-Val-Asp-CHO (aldehyde) at 100-200
µM failed to block WOX1 degradation during in
vitro translation. Furthermore, the serine protease inhibitors leupeptin and leuhistin at 100 µM could not inhibit WOX1
degradation during in vitro translation.
Stimulation of L929 cells with hyaluronidase induced WOX1 protein
expression (Fig. 2C). Our produced antibodies, which
interacted with both human and mouse WOX1, recognized both 46- and
30-kDa WOX1 (Fig. 2C). Whether this 30-kDa WOX1 is a
degraded protein from 46-kDa WOX1 remains to be determined. Our
produced antibodies are specific since the preimmune serum failed to
interact with both WOX1 proteins, the synthetic WOX1 peptide blocked
the binding of the antibodies to WOX1, and the antiserum also
interacted with the in vitro translated WOX1 protein (data
not shown).
WOX1 Is Mainly Located in the Mitochondria, and TNF Mediates WOX1
Nuclear Translocation--
Immunostaining of COS-7 fibroblasts with
anti-WOX1 and anti-cytochrome c antibodies showed the
presence of WOX1 in the mitochondria and nuclei, as determined by
confocal microscopy and colocalization analysis (Fig.
3A). Similar results were
observed using neonatal rat heart H9c2 cells (Fig. 3B).
These results were further confirmed using human ovarian ME180 and
HeLa, breast MCF-7, and neural SK-N-SH cells; isolated rat heart
cardiomyocytes; and murine NIH/3T3 and L929 cells. The presence of WOX1
in the mitochondria was further confirmed by Western blotting using
purified rat liver mitochondria (Fig. 3C). COX4 was examined
as a marker protein for mitochondria (Fig. 3C).
Tagging of full-length murine WOX1 with an N-terminal GFP sequence
(construct 2) (Table I) and
expression in COS-7 cells revealed the presence of GFP-WOX1 in
the mitochondria, as determined 24 h post-transfection (Fig.
3D). Mitochondria were stained by the membrane
potential-sensitive stain Mitotracker Red CMXRos. Less than 10% of the
transfected cells had nuclear localization of this protein.
By making successive deletion constructs (constructs 5-7) (Table I)
and expressing these constructs in COS-7 cells, the mitochondrial targeting sequence in WOX1 was mapped within the ADH domain (amino acids 209-273; construct 7) (Fig. 3D). The truncated
GFP-WOX1ww protein (amino acids 1-95; construct 4), which contains
only the N-terminal WW domains and NLS, was expressed in the nucleus
(Fig. 3E). Similarly, the human prostate GFP-WOX3 protein
(see Fig. 1), which possesses the WW domains, the NLS, and a partial
ADH domain, was expressed in the nucleus (data not shown).
A time course study showed TNF-mediated GFP-WOX1 nuclear translocation
(Fig. 3F). This was determined by examining isolated nuclei
by Western blotting using an established L929 cell line stably
expressing the GFP-WOX1 protein (Fig. 3F). This observation correlates with the time point of TNF-mediated cytochrome c
release from mitochondria to the cytosol in L929 cells (Fig.
3F). Similarly, time-dependent endogenous WOX1
protein nuclear translocation was also observed in COS-7 and H9c2 cells
upon stimulation with TNF for 20 min (data not shown).
TNF-mediated WOX1 nuclear translocation took at least 20-40 min,
which occurred shortly after TNF-mediated p65 NF- WOX1 Enhances TNF Cytotoxicity by Up-regulation of p53, but
Down-regulation of Bcl-2 and Bcl-xL--
To determine the
effect of WOX1 on TNF cytotoxic functions, we established several L929
cell lines that stably expressed the above indicated GFP-WOX1 proteins
(using constructs 2, 4, and 5). Expression of these proteins was
determined by Western blotting using specific antibodies against GFP
(Fig. 4A). As expected, the
protein sizes of the expressed full-length GFP-WOX1 (predicted, 72 kDa;
observed, 57 kDa) and GFP-WOX1adh (predicted, 50 kDa; observed, 34 kDa)
were reduced by ~15 kDa, probably due to C-terminal degradation.
Exposure of these GFP-WOX1 stable transfectants to TNF for 24 h
resulted in enhancement of TNF-mediated cell death as compared with
control cells transfected with GFP alone (Fig. 4B). Both the
WW and ADH domains enhanced TNF-mediated L929 cell death (Fig.
4B). Expressing untagged full-length WOX1 (construct 1) in
L929 cells also increased their TNF sensitivity (data not shown),
indicating that GFP does not affect the WOX1 protein function.
In contrast, constitutive expression of antisense Wox1
mRNA (using construct 3) resulted in cellular resistance to TNF
killing (resistance increase by 65-90%). These observations indicate
that WOX1 participates in the TNF cytotoxicity pathway.
Although NF-
To exclude the possibility that the WOX1-increased TNF susceptibility
in the established L929 transfectants is due to mutation of these
cells, transient transfection experiments were performed. Transient
expression of TRADD (34), the first adaptor protein recruited by the
TNF receptor, in COS-7 fibroblasts resulted in activation of the TNF
killing pathway and cell death (Fig. 4D). The TRADD-mediated
death was significantly enhanced (2-3-fold increase) by WOX1 (using a
non-cytotoxic concentration) in cotransfection studies (Fig.
4D). Similar results were observed with ovarian ME180 and
L929 cells (data not shown).
We next examined whether the WOX1-increased TNF killing is associated
with down-regulation or up-regulation of apoptosis regulatory proteins.
Western blot analysis showed that p53 expression was significantly
increased (~200%) in L929 cells stably expressing full-length
GFP-WOX1 or the GFP-WOX1adh as compared with cells expressing GFP alone
(Fig. 4E). Both GFP-WOX1 and GFP-WOX1adh proteins were
present in the mitochondria (Fig. 3). In contrast, cells stably
expressing nuclear GFP-WOX1ww failed to increase p53 expression (Fig.
4E). Notably, the ADH domain significantly suppressed the
expression of the apoptosis inhibitors Bcl-2 and Bcl-xL
(>85%), whereas the WW domains had no effect (Fig. 4E). I WW Domain-mediated Apoptosis Is Independent of Caspases and Serine
Proteases--
Indeed, transient overexpression of WOX1 in cells
mediated death over a 48-h culture period. For instance, the
TNF-resistant NIH/3T3 fibroblasts were transfected with the full-length
Wox1 cDNA (construct 1) by CaPO4, and cell
death was observed 48-h post-transfection (Fig.
5A). The apoptotic cells
revealed condensation of cytoplasm and nuclei. Similarly, transient
expression of full-length Wox1 mediated the death of ME180,
L929, U937, and other TNF-resistant cells such as 293, L929R, and
neonatal rat H9c2 cardiomyocytes (data not shown).
As summarized in Table II, both the ADH
(construct 5) and WW (construct 4) domains induced DNA
fragmentation when overexpressed in NIH/3T3 cells. As a positive
control, overexpression of p53 induced apoptosis. In contrast, both
antisense Wox1 (construct 3) and NLS-mutated WOX1 (NLSqgtg,
construct 18) failed to mediate DNA fragmentation. Failure of
NLS-mutated WOX1 in inducing apoptosis suggests that nuclear
translocation of WOX1 is necessary for inducing cell death. Also, the
presence of the WW domains in WOX1 may suppress the apoptosis-inducing
activity of the ADH domain.
The WW domain-induced cell death is independent of caspases and serine
proteases. Transient expression of the N-terminal WW domains (first and
second domains; construct 10) or the first WW domain (construct 9) in
NIH/3T3 cells also resulted in cell death 48 h post-transfection
(Fig. 5B). These proteins contain the NLS, thus expressing
in the nuclei. Exposure of the transfected cells to the caspase
inhibitors acetyl-Asp-Glu-Val-Asp CHO (aldehyde) and
benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and the serine
protease inhibitors leupeptin and leuhistin (100 µM)
failed to block cell death (Fig. 5B). These results indicate
that the WW domain-mediated cell death is independent of caspases and
serine proteases.
WOX1 and p53 Are Partners in Apoptosis--
Since WOX1 increased
p53 expression, we then investigated whether p53 is involved in the
WOX1-mediated cell death. Transient expression of the WW domains
mediated the death of NIH/3T3 cells in 48 h, and the killing
function was increased by cotransfection with p53 (Fig.
6, A, panels a and
b). Also, using non-killing concentrations of p53 and WOX1
in transfecting monocytic U937 cells (by electroporation), a
synergistic killing effect was observed when combining p53 and WOX1
(data not shown).
Notably, antisense expression of WOX1 in NIH/3T3 cells abolished
p53-mediated cell death (Fig. 6A, panel c).
Similarly, antisense expression of WOX1 also inhibited p53 apoptosis of
THP-1 cells in cotransfection experiments (Fig. 6B). These
results suggest that there is a partnership between p53 and WOX1 in apoptosis.
However, using p53-deficient NCI-H1299 cells, transient expression of
both the WW and ADH domains mediated cytochrome c release from mitochondria and cell death, indicating that WOX1-mediated cell
death is independent of p53 (Fig. 6C). Similar results were observed using full-length WOX1 (data not shown). Together, these data
suggest that WOX1-mediated apoptosis is independent of p53, but p53
apoptosis requires the participation of WOX1.
The WW Domains of WOX1 Bind to the Proline-rich Region of
p53--
Confocal microscopy and colocalization analysis revealed that
p53 and WOX1 colocalized in the cytosol and partly in the nuclei in
MCF-7 cells (Fig. 7A). Similar
results were obtained using other cells such as ME180 and COS-7.
Immunoprecipitation of L929 cytosolic lysates with anti-p53 antibodies
resulted in coprecipitation of both p53 and WOX1 (Fig. 7B),
indicating the binding interactions between endogenous p53 and WOX1.
The presence of p53 in the precipitates was confirmed using anti-p53
antibodies in Western blotting (data not shown). Stimulation of L929
cells with TNF for 2 h resulted in migration of both proteins to
the nuclei (determined by immunostaining) (data not shown) and the
disappearance of both proteins from the cytosolic lysates in
coprecipitation studies (Fig. 7B).
Yeast two-hybrid analysis showed that the proline-rich region of p53
(amino acids 66-110) physically interacts with the WW domains of WOX1
in vivo (Fig. 7C). In negative controls, no
binding interactions were observed using antisense WOX1ww and p53,
empty vector (pSos) and empty vector (pMyr), and MafB and lamin C (Fig. 7C). MafB self-binding interactions were tested as positive
binding controls (Fig. 7C).
In this study, we cloned and functionally characterized the murine
WOX1 protein by antibodies, GFP tagging and expression, and other
approaches. The gene encoding WOX1 is located on a fragile chromosomal
site (24-26). Homozygous deletion of this gene has been found in
various cancers (24-26). Whether WOX1 plays a role in cancer
development remains to be established. We determined that WOX1 is
located mainly in the mitochondria. Mitochondrial intermembrane space
is a reservoir for a variety of apoptogenic proteins such as cytochrome
c; procaspase-2, -3, and -9; and apoptosis-inducing factor
(AIF) (35). Whether WOX1 is present in this intermembrane space remains
to be established. Apoptotic stimuli such as TNF and staurosporine
induce WOX1 nuclear translocation. WOX1 enhances TNF cytotoxic function
via its nuclear targeting WW domains and the mitochondrial targeting
ADH domain, suggesting that WOX1 functions at both cytosolic and
nuclear levels. Functionally, WOX1 mediates apoptosis when
overexpressed. WOX1 binds p53 in the cytosol. WOX1-mediated apoptosis
is independent of p53, whereas p53-mediated cell death requires the
participation of WOX1. This observation suggests that WOX1 is an
essential partner of p53 in apoptosis.
In agreement with other studies (24, 25), Wox1 mRNA is
ubiquitously expressed in most tissues and organs in mouse, as determined by reverse-transcription-polymerase chain reaction (data not
shown). Based on the gene structure, four splice variants of WOX
proteins are predicted (25). However, we observed additional WOX
protein species at high molecular sizes (65 and 100 kDa) in Western
blotting using human organs and cell lines (data not shown). Also,
three mRNA transcripts probably encoding high molecular mass WOX
proteins have been found (25). Indeed, additional splice variants have
also been found in the updated expressed sequence tag data base.
Accordingly, the functional properties of these proteins remain to be established.
TNF-mediated WOX1 nuclear translocation is independent of the TNF
signaling pathway that leads to phosphorylation and degradation of
I Nonetheless, TNF-mediated WOX1 release from mitochondria could be
dependent upon activation of BID (39). TNF mediates cleavage of
BID to truncated BID, which translocates to the mitochondria. Truncated BID oligomerizes BAK to generate membrane
pores, thus allowing cytochrome c and probably WOX1 release.
Truncated BID-mediated cytochrome c release does not appear
to be involved in the opening of mitochondrial transition pores (40).
However, atractyloside, an inducer of mitochondrial permeability
transition, also induces WOX1 nuclear translocation. This observation
suggests that WOX1 release from mitochondria could be dependent upon
mitochondrial transition pores as well as BAK oligomerization.
The WW domains of WOX1 are more potent than full-length WOX1 in
sensitizing the TNF-resistant COS-7 cells to TNF killing. We found that
when COS-7 cells were transiently transfected with the N-terminal WW
domains of WOX1 and cultured for 16-24 h, followed by exposure to TNF,
these cells underwent nuclear fragmentation in 3 h and subsequent
rupture of the cytosolic components and nuclear condensation in 6-16
h. Nonetheless, a prolonged treatment (>12 h) of the full-length
WOX1-expressing COS-7 cells with TNF is required to induce cell death.
An intriguing finding in our study is that stable expression of the ADH
domain or full-length WOX1 in the mitochondria resulted in
down-regulation of Bcl-2 and Bcl-xL, but up-regulation of
p53 in L929 cells. In contrast, the WW domains, when expressed in the
nuclei, failed to modulate the expression of these proteins. These
results suggest that WOX1 indirectly regulates the expression of p53,
Bcl-2, and Bcl-xL.
The anti-apoptotic Bcl-2 and Bcl-xL proteins block
mitochondrial permeability transition and prevent cytochrome
c release from mitochondria (41). Bcl-xL blocks
cytochrome c release by binding to the anion channel
voltage-dependent anion channel on the outer membrane of
mitochondria (42). Binding of Bcl-xL to voltage-dependent
anion channel results in closure of the voltage-dependent anion
channel. We determined that WOX1 significantly reduces the expression
of Bcl-2 and Bcl-xL in mitochondria. This event may result
in opening of the mitochondrial permeability transition pores and
release of apoptogenic proteins from the intermembrane space. This
notion is supported by the observation that transient overexpression of
the ADH domain in cells caused cytochrome c release and death.
Another intriguing finding is that when overexpressed, the
mitochondrial targeting ADH domain alone (using three regions of the
ADH domains; constructs 5-7) was capable of inducing cell death.
However, the apoptosis-inducing activity was not found in NLS-mutated
full-length WOX1 (construct 18), which indicates that nuclear
translocation is needed for the WW domains to mediate cell death. Also,
the presence of the mutated WW domains appears to suppress cell death
by the ADH domain. Suppression of the ADH domain function is probably
related to protein folding when the WW domains are present in the WOX1
protein. Other types of dehydrogenase domain proteins such as AIF (35)
and the CC3 protein (43) have been shown to induce cell death when overexpressed.
Endogenous WOX1 colocalizes with p53 in the cytosol. Ectopic expression
of both GFP-WOX1 and red fluorescent protein-tagged p53 in COS-7 cells
also results in cytosolic colocalization (>50% in p53/WOX1-expressing
cells) (data not shown). Co-immunoprecipitation studies further support
binding of p53 with WOX1 in the cytosol. Yeast two-hybrid experiments
show the binding of the WW domains of WOX1 to the proline-rich region
(amino acids 66-110) in p53. Based on these observations, it is
reasonable to suggest that both p53 and WOX1 migrate together to the
nucleus in response to TNF.
Ectopic expression of p53 and WOX1 showed that both proteins mediate
apoptosis in a synergistic manner. Although WOX1 can mediate apoptosis
independently of p53, blocking of WOX1 expression by antisense mRNA
abolishes p53 apoptosis. The inhibition of p53 apoptosis is not due to
blocking of p53 protein synthesis by the antisense Wox1
mRNA (data not shown). These observations strongly indicate that
WOX1 is an essential partner of p53 in apoptosis. The proline-rich
region has been shown to be necessary for p53-mediated apoptosis (44).
Our data suggest that binding of WOX1 to this region in p53 appears to
be essential for p53 apoptosis-inducing activity.
A wide range of transcription factors including c-Jun, AP-2, NF-E2,
CAAT/enhancer-binding protein- Susin et al. (35) isolated mitochondrial AIF, a homolog of
bacterial oxidoreductase. Once released from mitochondria, AIF translocates to the nucleus. Although recombinant AIF induces apoptosis
of isolated nuclei (35), whether nuclear AIF induces chromatin
condensation and nuclear DNA fragmentation in vivo is unknown. In response to apoptogenic signals such as staurosporine, both
WOX1 and AIF migrate to the nucleus and mediate cell death in a
caspase-independent mechanism. TNF induces WOX1 nuclear translocation. Whether AIF migrates to the nucleus in response to TNF is unknown. WOX1
enhances TNF cytotoxicity by increasing the expression of p53 (and
probably other pro-apoptotic proteins) as well as suppressing the
expression of Bcl-2 and Bcl-xL. In contrast, AIF does not appear to be involved in the regulation of protein expression. Whether
AIF and WOX1 act synergistically in mediating cell death is not known.
Although the TNF signaling pathway that leads to caspase activation and
cell death has been well defined,
we2 and others (47) have
shown that TNF-mediated cell death cannot be blocked by inhibitors of
caspases. This raises the possibility that TNF induces a
caspase-independent killing pathway. Data from us and Susin et
al. (35) support that both WOX1 and AIF are the downstream
mediators of the caspase-independent TNF killing pathway.
Finally, a high abundance of WOX1 was observed in rat heart (data not
shown). This suggests that WOX1 plays a homeostatic role in this organ.
The heart is a TNF-producing organ, and TNF plays a key role in the
pathogenesis of congestive heart failure (48, 49). Patients with
chronic and severe congestive heart failure have increased levels of
TNF in the circulation and cardiac tissues. TNF exerts a negative
inotropic effect and triggers the apoptotic process in cardiomyocytes.
Whether WOX1 plays a major role in the TNF-mediated apoptosis of
cardiomyocytes remains to be established.
B nuclear translocation, whereas both were independent
events. WOX1 enhanced TNF cytotoxicity in L929 cells via its WW and ADH
domains as determined using stable cell transfectants. In parallel with
this observation, WOX1 also enhanced TRADD (TNF
receptor-associated death
domain protein)-mediated cell death in transient expression
experiments. Antisense expression of WOX1 raised TNF resistance in L929
cells. Enhancement of TNF cytotoxicity by WOX1 is due, in part, to its
significant down-regulation of the apoptosis inhibitors Bcl-2 and
Bcl-xL (>85%), but up-regulation of pro-apoptotic
p53 (~200%) by the ADH domain. When overexpressed, the ADH domain
mediated apoptosis, probably due to modulation of expression of these
proteins. The WW domains failed to modulate the expression of these
proteins, but sensitized COS-7 cells to TNF killing and mediated
apoptosis in various cancer cells independently of caspases. Transient
cotransfection of cells with both p53 and WOX1 induced apoptosis in a
synergistic manner. WOX1 colocalizes with p53 in the cytosol and binds
to the proline-rich region of p53 via its WW domains. Blocking of WOX1
expression by antisense mRNA abolished p53 apoptosis. Thus, WOX1 is
a mitochondrial apoptogenic protein and an essential partner
of p53 in cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phage
cDNA library from murine NIH/3T3 fibroblasts
(CLONTECH, Palo Alto, CA). The isolated full-length
cDNA insert was amplified by polymerase chain reaction (using
-phage primers) (see Table I) and subcloned into the TA
cloning site of eukaryotic expression vector pCR3.1 (Invitrogen, San
Diego, CA). Protein domain analysis was performed using the SMART
Simple Modular Architecture Research Tool (16, 17). As compared
with the existing domains in the universal data bases, each resulting
positive domain is determined according to a calculated E-value (16, 17).
B
, Bcl-2, Bcl-xL, p53,
a phospho-SAPK/JNK peptide, NF-
B, and
-tubulin (Transduction
Laboratories (Lexington, KY) and Santa Cruz Biotechnology (Santa Cruz,
CA)). Anti-GFP and anti-cytochrome c oxidase subunit 4 (COX4) antibodies were from CLONTECH. Where indicated, images were analyzed by the NIH Image program. Purification of rat liver mitochondrial proteins for Western blotting was performed as described (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Murine and human WW domain-containing
oxidoreductase proteins. The deduced full-length murine
WOX1 (GenBankTM/EBI accession number AF187014)
amino acid sequence (414 amino acids, 46.5 kDa) possesses two
N-terminal WW domains (amino acids 18-47 and 59-87;
underlined), an NLS sequence (amino acids 50-55;
underlined), and a C-terminal short-chain ADH domain (amino
acids 121-330; underlined). A putative caspase recognition
sequence (DIND, amino acids 267-270) and its cleavage site
(arrow) are shown. Murine WOX1 is homologous to the
following WOX proteins: human WWOX (GenBankTM/EBI accession
number AF211943) (24); human FOR I (41.2 kDa; accession number
AF227526), FOR II (46.7 kDa; accession number AF227527), FOR III (21.5 kDa; accession number AF227528), and FOR IV (4.1 kDa; accession number
AF227529) (25); and human WOX3 (accession numbers AI669330 and
AF187015). WOX3 is identical to FOR III.
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Fig. 2.
WOX gene and protein
expression. A, exposure of L929 fibroblasts to
hyaluronidase (100 units/ml) resulted in a time-dependent
increase in Wox1 gene expression (~150% increase in
8 h; 40 µg of total RNA/lane). B, in vitro
translation of the isolated full-length Wox1 cDNA (in
the pCR3.1 vector) produced a 46-kDa protein and a 30-kDa protein. The
30-kDa protein is probably a degraded protein from 46-kDa WOX1. A
putative caspase recognition site (DIND) was altered to a non-caspase
recognition sequence (GING), designated WOX1(GING). This
alteration failed to prevent the degradation during in vitro
translation. C, stimulation of L929 cells with hyaluronidase
(100-200 units/ml) for 24 h resulted in the increased expression
of 46- and 30-kDa WOX1 proteins, as determined by Western blotting
using antibodies against a synthetic peptide of WOX1 at the N terminus.
At high concentrations (>400 units/ml), hyaluronidase suppressed WOX1
expression. The 30-kDa protein is probably a degradation product of
46-kDa WOX1.
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Fig. 3.
WOX1 is mainly present in the mitochondria,
and TNF mediates WOX1 nuclear translocation. A, COS-7
cells were stained with both anti-WOX1 and anti-cytochrome c
antibodies, followed by staining with secondary antibodies, and
subjected to confocal microscopy. Colocalization analysis showed that
WOX1 is mainly located in the mitochondria and nuclei. B,
similar results were observed by staining neonatal rat H9c2
cardiomyocytes using both antibodies. C, Western blotting
showed the presence of WOX1 in the purified rat liver mitochondria.
Also, the presence of COX4, a protein on the inner mitochondrial
membrane, is regarded as a marker protein for mitochondria.
D, when expressed in COS-7 cells, the full-length murine
GFP-WOX1 protein (construct 2) (Table I) is mainly located in the
mitochondria, as analyzed by counterstaining the cells with the
membrane potential-sensitive mitochondrial stain MitoTracker Red
CMXRos. Protein expression in cells was examined by fluorescent
microscopy 24 h post-transfection. Also, by successive deletion
and expression analyses, the mitochondrial targeting region was mapped
to amino acids 209-273 of the ADH domain (GFP-WOX1adh, construct 7).
E, however, the truncated GFP-WOX1ww protein (construct 4),
which possesses the N-terminal WW domains and the NLS, is present in
the nucleus. F, stimulation of an established L929 cell
line, which constitutively expresses full-length GFP-WOX1 (construct
2), with TNF (20 ng/ml) resulted in the appearance of GFP-WOX1 in the
isolated nuclei at the 40-min time point (detected by anti-GFP
antibodies). This correlates with the time course of cytochrome
c release from mitochondria to the cytosol.
Polymerase chain reaction primers and expression constructs
B nuclear translocation (at ~10-15 min), as determined by immunostaining and
fluorescent microscopy. Alteration of the NLS sequence (GKRKRV) to a
less hydrophilic sequence (GQGTGV) by site-directed mutagenesis (construct 18) abolished the TNF-mediated nuclear translocation of this
mutant protein. No nuclear translocation was observed when treating the
ADH domain (GFP-WOX1adh, construct 5)-expressing cells with TNF.
In parallel with the TNF-mediated mitochondrial permeability
transition, treatment of COS-7 cells with atractyloside (2 mM) (27) for 40 min to increase the opening of
mitochondrial transition pores also resulted in cytochrome c
release to the cytosol and WOX1 nuclear translocation (data not shown).
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Fig. 4.
WOX1 enhances TNF killing of L929 cells.
A, four stable L929 transfectants were established to
continuously express full-length GFP-WOX1 (predicted, 72 kDa; observed,
57 kDa) (construct 2), truncated GFP-WOX1ww (predicted, 37 kDa;
observed, 33 kDa) (construct 4), truncated GFP-WOX1adh (predicted, 50 kDa; observed, 34 kDa) (construct 5), or GFP protein (26 kDa) alone. A
degradation of 15 kDa was observed in the expressed GFP-WOX1 and
GFP-WOX1adh proteins. Anti-GFP antibody was used in Western blotting.
B, exposure of these WOX1-expressing cells to TNF for
24 h resulted in enhancement of cell death as compared with the
control GFP-expressing cells (n = 8). C, a
representative time course study showed that TNF-mediated I B
degradation was similar in both the GFP-WOX1- and GFP-expressing cells,
indicating that WOX1 enhancement of TNF cytotoxicity is not due to
impaired I
B
degradation and NF-
B activation. D,
transfection of COS-7 cells (in 96-well plates) with a
TRADD cDNA construct (in a cytomegalovirus-based
pRK vector) by CaPO4 resulted in cell death in 24 h
(white bars), and the cell death was significantly enhanced
by cotransfection with the full-length murine
Wox1-pCR3.1 cDNA (0.2 µg/well; construct 1;
black bars). At this concentration, WOX1 could not mediate
cell death. In controls, the empty vector pCR3.1 (0.2 µg/well) failed
to increase TRADD-mediated cell death. E,
down-regulation of Bcl-2 and Bcl-xL expression and
up-regulation of p53 expression were observed in the cells expressing
GFP-WOX1 or GFP-WOX1adh, but not in the cells expressing GFP-WOX1ww or
GFP alone. Both I
B
and
-tubulin levels were not changed in
these cells.
B is believed to play an essential role in blocking cell
death by TNF, ionizing radiation, and anticancer drugs (28-33), the
WOX1-increased TNF cytotoxicity is not due to impaired NF-
B
activation or nuclear translocation. Time course studies showed that
the kinetics of TNF-mediated I
B
degradation were similar in both
GFP- and GFP-WOX1-expressing L929 cells (Fig. 4C). Also, TNF
induced NF-
B (p65) nuclear translocation in both cells, as
determined by immunostaining and fluorescent microscopy (data not
shown). Additionally, TNF rapidly induced SAPK/JNK activation (peaking
at 5-20 min) in both GFP-WOX1- and GFP-expressing cells, as determined
using anti-phospho SAPK/JNK peptide antibodies in Western blotting.
Thus, the involvement of NF-
B and SAPK/JNK in WOX1-increased TNF
killing is unlikely.
B
levels in these cells were not changed (Fig. 4E).
The housekeeping protein
-tubulin was examined as control for
protein loading (Fig. 4E). These data suggest that
enhancement of TNF killing by WOX1 is associated in part with its
increased p53 expression and reduced expression of Bcl-2 and
Bcl-xL.
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Fig. 5.
WW domain-mediated cell death is independent
of caspases and serine proteases. A, transient
expression of full-length Wox1-pCR3.1 (construct 1)
in NIH/3T3 cells resulted in cell death, as observed 48-h
post-transfection (by the CaPO4 method), whereas the empty
pCR3.1 vector had no effect. The cells were stained by crystal violet.
B, transient expression of the first and second WW domains
(WOX1-2ww-pcDNA3.1, construct 10) or the first WW domain
(WOX1-1ww-pcDNA3.1, construct 9) resulted in the death of NIH/3T3
cells. The caspase inhibitors acetyl-Asp-Glu-Val-Asp-cho
(Ac-DEVDcho) and benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone (z-VAD.fmk) (100 µM) and the serine
protease inhibitors leuhistin and leupeptin (100 µM)
failed to block the WW domain-mediated cell death. The extent of cell
death was quantified (bar graph). The empty
vector-transfected cells (without treatment) were regarded as
0%.
WOX1-mediated DNA fragmentation of NIH/3T3 cells
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Fig. 6.
p53-mediated apoptosis is blocked by
antisense Wox1. A, shown
in panel a is the p53-increased killing of NIH/3T3 cells by
WW domains in transient expression experiments (by CaPO4)
using constructs of both WW domains (construct 10), the first WW domain
(construct 9), and/or p53 (construct 11). The extent of cell death is
demonstrated in panel b as a bar graph. The empty
vector-transfected cells are regarded as 0% killing. As shown in
panel c, blocking of WOX1 expression by antisense mRNA
(construct 3) resulted in inhibition of p53-mediated cell death in
cotransfection experiments. B, as determined by DNA
fragmentation assays, inhibition of p53 apoptosis by antisense
Wox1 mRNA was also observed in monocytic THP-1 cells
(electroporation with construct 2, 3, or 11 and/or an empty vector).
The sham electroporation is regarded as background DNA fragmentation
(0%). C, nonetheless, in the p53-deficient NCI-H1299 cells,
transient expression of both the WW (construct 4) and ADH (constructs 6 and 7) domains mediated cytochrome c release and cell death.
Similar results were observed using the full-length GFP-WOX1 construct
(data not shown). The sham transfection is regarded as 0%
killing.
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Fig. 7.
p53 and WOX1 colocalization and binding
interactions. A, confocal microscopy and colocalization
analysis revealed that p53 and WOX1 are colocalized in the cytosol and
partly in the nucleus in MCF-7 cells (magnification × 400).
B, immunoprecipitation (IP) of endogenous WOX1
with anti-WOX1 antibodies from the cytosolic lysates of L929 cells,
followed by blotting with anti-WOX1 antibodies, revealed the presence
of 46-kDa WOX1 (third lane). Also, precipitation
with anti-p53 antibodies, followed by blotting with anti-WOX1
antibodies, also resulted in the appearance of 46-kDa WOX1
(second lane), indicating binding of endogenous p53 to WOX1.
In the control without antibodies added, no precipitated protein was
observed (first lane). Exposure of L929 cells to TNF (20 ng/ml) for 1 h resulted in the disappearance of WOX1 from the
cytosol, due to migration of both p53 and WOX1 to the nuclei
(fifth and sixth lanes). C, yeast
two-hybrid analysis was performed (see "Experimental Procedures").
Interactions between target and bait proteins allow Sos-mediated
activation of the Ras signaling pathway, which permits the
temperature-sensitive yeast cdc25H to grow at 37 °C.
Positive binding interactions between full-length p53 (construct 14)
and full-length WOX1 (construct 12) or WOX1ww (both WW domains;
construct 13) are demonstrated, as evidenced by the growth of yeast at
37 °C. Similar results were obtained with the N-terminal
proline-rich region in p53 (amino acids 1-100 and 66-100; constructs
15 and 16, respectively) and full-length WOX1. p53 failed to bind to
the antisense construct of WOX1ww (reverse orientation of
WOX1ww cDNA). MafB protein self-interaction is a positive
control for the assay system. In negative controls, the yeast failed to
grow at 37 °C when testing MafB-lamin C or empty vector (pSos)-empty
vector (pMyr) interactions. Two representative colonies (out of 15-30)
are shown from each binding experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
and activation of p65 NF-
B and SAPK/JNK (36-38). For
example, the GFP-WOX1-expressing COS-7 cells were pretreated with the
proteasome inhibitor benzyloxycarbonyl-Leu-Leu-Leu CHO
(aldehyde) (10 µM) to block I
B
degradation
or with the I
B
phosphorylation inhibitors Bay 11-7082 and Bay
11-7085 (30 µM) for 1 h, followed by exposure to
TNF. These treatments inhibited TNF-mediated p65 NF-
B activation or
nuclear translocation, but failed to abolish the GFP-WOX1 nuclear translocation, as determined by immunostaining and fluorescent microscopy (data not shown).
, and PEBP2/CBF, contain the WW
domain-binding motif (22, 23). Thus, most of the WW domain-containing
proteins act as gene transcription activators or coactivators (45, 46).
When overexpressed, the WW domains of WOX1 mediate apoptosis. Whether
this is related to the transcriptional activation of apoptotic genes by
the WW domains of WOX1 remains to be established. Nonetheless, our data
show that the WW domains fail to increase p53 protein expression and
suppress Bcl-2 and Bcl-xL expression.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Derek Strassheim, Samira Khera, and Henry Puhl (Guthrie Research Institute); Dr. Steven P. Tammariello (Binghamton University), Dr. J. E. V. Watson (Imperial Cancer Fund, Edinburgh, United Kingdom), and Dr. R. Richards (Women's and Children's Hospital, North Adelaide, Australia) for carefully reviewing the manuscript. We thank Jeffery Mattison for sequencing analysis and SK-N-SH cells, Dr. D. Goeddel (Tularik Inc.) for the TRADD cDNA construct, Drs. D. Beezhold and J. Noti for L929R and MCF-7 cells, Dr. S. Ikeda for rat organs, Dr. R. Richards for the sequences of FOR proteins, and Mrs. Terrie Zimmer for assistance with antibody production.
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FOOTNOTES |
---|
* This work was supported by the Guthrie Foundation for Education and Research, the Wendy Will Case Cancer Fund, the American Heart Association, and National Cancer Institute Grants R01CA61879 and R55CA64423 from the National Institutes of Health (to N.-S. C.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF187014 (murine Wox1) and AI669330 and AF187015 (human WOX3).
To whom correspondence should be addressed: Lab. of
Molecular Immunology, Guthrie Research Inst., 1 Guthrie Square, Sayre, PA 18840. Tel.: 570-882-4620; Fax: 570-882-4643; E-mail:
nschang@inet.guthrie.org.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M007140200
2 N.-S. Chang, N. Pratt, J. Heath, and L. Schultz, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: TNF, tumor necrosis factor; WOX1, WW domain-containing oxidoreductase-1; GFP, green fluorescent protein; ADH, alcohol dehydrogenase; NLS, nuclear localization sequence; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; NF, nuclear factor; COX4, cytochrome c oxidase subunit 4; TRADD, TNF receptor-associated death domain protein; AIF, apoptosis-inducing factor.
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