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Chang NSA potential role of p53 and WOX1 in

mitochondrial apoptosis (review). Int J Mol Med
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ARTICLE in INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE FEBRUARY 2002
Impact Factor: 2.09 DOI: 10.3892/ijmm.9.1.19 Source: PubMed

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INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 19-24, 2002

19

A potential role of p53 and WOX1 in

mitochondrial apoptosis (Review)
NAN-SHAN CHANG
Guthrie Research Institute, Laboratory of Molecular Immunology,
Guthrie Medical Center, Sayre, PA 18840, USA
Received September 8, 2001; Accepted September 28, 2001

Abstract. A large number of p53-transcribed proteins have

been shown to mediate growth arrest and/or apoptosis in vitro,
whereas their in vivo roles remain largely unclear. p53 is
capable of initiating apoptosis without transcription of
apoptosis inducer genes, although the underlying mechanism
is unknown. p53 is present in the mitochondria and appears
to contribute to the biogenesis, function and apoptosis of this
organelle. We have recently cloned a p53-binding mitochondrial WW domain-containing oxidoreductase (WOX1).
Suppression of WOX1 expression abolishes p53 apoptotic
function, indicating that WOX1 is a likely partner of p53 in cell
death. In this review article, the potential role of WOX1/p53
as a signaling complex in the mitochondrial apoptosis is
discussed.

Contents
1. p53 functional domains and stability
2. p53 in mitochondria and its effect on biogenesis
3. Proteins involved in p53-mediated growth arrest and
apoptosis
4. Potential role of mitochondrial p53 in apoptosis
5. Mitochondrial WOX1 is a likely partner of p53 in apoptosis
6. Conclusion and perspectives

1. p53 functional domains and stability

p53 is a nuclear phosphoprotein and a transcription factor. p53
consists of two amino-terminal transactivation domains, a
proline-rich (or growth regulatory) region, a central DNAbinding domain, and a nuclear localization and oligomerization
domain at the carboxyl-terminus (reviewed in ref. 1). Of
particular interest, the N-terminal proline-rich region (residues
64-91) and the C-terminal basic region (residues 364-393) are

_________________________________________
Correspondence to: Dr Nan-Shan Chang, Laboratory of
Molecular Immunology, Guthrie Research Institute, Sayre, PA
18840, USA
E-mail: nschang@[Link]

Key words: p53, WW domain-containing oxidoreductase, WOX1,

apoptosis, mitochondria

essential for p53-mediated growth suppression and apoptosis

(2,3). Both the N-terminal transactivation domain 2 and the
proline-rich region appear to be essential for inducing proapoptotic genes or blocking antiapoptotic genes (3). A minimal
p53 repression domain (residues 339-346) for gene transcription
has been recently identified (4). The N-terminus of p53 controls
its binding and dissociation from target DNA (5).
Mdm2 controls the stability, transcriptional function and
nucleocytoplasmic translocation of p53 in resting cells.
Mdm2 mediates p53 degradation via a proteasome-dependent
mechanism. Mdm2 binds p53 and mediates the nuclear export
of p53 to the cytosol for degradation. Mdm2 is a RING fingerdependent ubiquitin protein ligase for itself and p53 (6).
Interestingly, the proline-rich region appears to provide stability
of p53 in resisting Mdm2-mediated degradation (7). Hdmx, a
human Mdm2 homologue, inhibits the p53 transcriptional
activity (8). However, it may prevent p53/Mdm2 complex from
degradation by a direct binding interaction (8).
Other protein factors may affect p53 stability. Hdm2
appears to enhance p53 degradation in neuroblastoma (9).
The coactivator protein TAF(II)31 competes with Mdm2 in
binding to the N-terminus of p53, thereby increasing p53
stability and transcriptional activity (10). NADH quinone
oxidoreductase 1 (NQO1), a p53 transcriptional target, also
stabilizes p53 (11). Inhibition of this enzyme by dicoumarol
increases p53 degradation by the proteasome system.
In stressed cells, expression of ARF, a nuclear inhibitor of
Mdm2, is upregulated. ARF binds to the p53/Mdm2 complex
in the nucleus. This results in dissociation and activation of
p53 from the complex and translocation of ARF/Mdm2 to the
nucleoli (1). p53 becomes transcriptionally active. ARF also
interacts with MdmX and sequesters MdmX within the
nucleolus, which results in an increase in p53 transactivation
(12). However, Wang et al could not demonstrate the
interaction between ARF and MdmX (13). Difference in the
observations from these investigators could be due to different
types of cells used in these studies.
Phosphorylation of p53 also tightly controls its stability. For
example, in response to stress, c-Jun N-terminal kinase (JNK)
phosphorylates p53 on Thr-81, which stabilizes and increases
the transcriptional activities of p53 (14). Phosphorylation of
serine-15 in the N-terminal nuclear export signal (NES) is
necessary for blocking p53 nuclear export (15). Intracellular
modification of wild-type p53 has been shown in the neuroblastoma cells, which confers p53 resistance to Mdm2mediated degradation and its cytoplasmic sequestration (16).

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CHANG: p53 AND WOX1 IN THE MITOCHONDRIAL APOPTOSIS

2. p53 in mitochondria and effect on biogenesis

p53 is present predominantly in the cytoplasm of primary
cultured cells and various cancer cells. However, less effort
has been devoted to establishing the cytoplasmic localization
of p53 and related functional roles.
In resting cells, the levels of cytosolic and nuclear p53 are
low. The p53 levels, for example, can be increased in normal
lymphocytes and fibroblasts by stimulating with mitogen
(17). p53 binds to the actin cytoskeleton and mitochondrial
membrane during the initiation of G1 phase in these cells
(17). Also, p53 translocates to the nucleus to initiate gene
activation and DNA synthesis for cell proliferation (17).
The mitochondrial localization of p53 has also been
demonstrated in human HT1080 and mouse C3H10T1/2 cell
lines (18). Mutant p53 complexes with chaperone heat shock
proteins (HSP) and the complexes appear to be located on the
outer membrane of the mitochondria (18).
p53 is present in the murine liver mitochondria (19).
Donahue et al isolated the outer membrane-removed submitochondrial particles, named mitoplasts, and demonstrated
the presence of p53 by Western blotting using p53-reactive
antibodies (19). Whether the mitochondrial p53 originates
directly from the cytosolic protein synthesis machinery, or
translocates from the nucleus, is unknown. Also, by transiently
expressing a dominant negative p53 in NIH/3T3 fibroblasts,
these investigators showed a decrease in mitochondrial
membrane potential as well as a reduction in the mitochondrial
16S rRNA expression (19). These observations imply that
p53 may have a direct effect on mitochondrial biogenesis and
function (19).
3. Proteins involved in p53-mediated growth arrest and
apoptosis
A large number of p53 transcriptional targets are known to
mediate growth arrest and apoptosis. For example, p21/WAF/
Cip1 is a downstream effector of p53-mediated growth arrest
in the G1 phase of the cell cycle (20). In addition, GADD45
(21), Wip1 (22), 14-3-3 sigma (23), BTG2 (24), IGF-BP3 (25)
and Seven in absentia (26) cause growth arrest to the G1 or
G2/M phase. p53 regulates the expression of these genes. p53
also targets the expression of inducers and inhibitors of
apoptosis. For example, both Fas/Apo-l (27) and Killer/DR5
(28) are cell membrane-associated apoptosis-inducing
receptors, which are upregulated in p53-mediated cell death.
In contrast, the membrane decoy receptors TRID (TRAILR3) (29) and TRUNDD (DcR2, TRAIL-R4) (30) could not
transduce a death signal by the ligand TRAIL, thus blocking
apoptosis. These receptors are also p53-transcriptional targets.
p53 regulates the expression of several mitochondrial
proteins that are either apoptotic or antiapoptotic. p53 controls
the transcriptional activation of Bax, a potent inducer of
mitochondrial apoptosis (31). Noxa, a mitochondrial protein
belonging to the Bc1-2 family, is a likely downstream effector
of p53 apoptosis (32). Interestingly, phosphorylation of
serine-46 in p53 is necessary for its transcriptional activation
of the mitochondrial apoptosis-inducer p53AIP1 (33). In
contrast, p53 also controls the expression of the mitochondrial
antiapoptotic Bcl-xL, a member of the Bcl-2 family (34). Most

recently, p53 is shown to induce the expression of mitochondrial PUMA (p53 upregulated modulator of apoptosis)
(35,36). PUMA binds to Bcl-2 and Bcl-xL through its BH3
domain and mediates apoptosis through the cytochrome c/
Apaf-1-dependent pathway (35,36).
Although p53 upregulates a multitude of proteins that
mediate apoptosis in vitro, the efficacy of these proteins in
mediating cell death in vivo remains controversial (reviewed
in ref. 37). Furthermore, p53 can initiate apoptosis without
transcriptional activation of its target genes (38,39). Caspase 8,
an enzyme in the cell death pathway, appears to be a downstream effector in the transcription-independent apoptosis of
p53 (40).
4. Potential role of mitochondrial p53 in apoptosis
Two recent studies have suggested that p53 participates in
mitochondrial apoptosis (41,42). A portion of nuclear p53
migrates to the mitochondrial surface during exposure of
various cancer cells to anticancer drugs or under hypoxia
(41,42). Also, some of the relocated nuclear p53 binds to the
mitochondrial import motor hsp70 (41). Cytochrome c release
from mitochondria occurs shortly after p53 accumulation on
the mitochondrial membrane. Whether the relocated nuclear
p53 directly initiates the cascade of mitochondrial apoptosis
is unknown. However, direct targeting of p53 overexpression
in the mitochondria induces cell death, indicating that p53 is
able to launch the cell death pathway in this organelle (41).
In light of the finding that p53 may participate in mitochondrial biogenesis and function in resting and proliferating
cells (19), it is reasonable to postulate that the residential
mitochondrial p53 has dual physiologic functions. In resting
normal cells, the mitochondrial p53 is antiapoptotic. In
stressed cells, it may become apoptotic. Testing the proposed
p53 function in mitochondria would require cloning and
identification of p53-interacting proteins that exert the downstream antiapoptotic or apoptotic effect.
5. Mitochondrial WOX1 is a likely partner of p53 in
apoptosis
Our recent finding of mitochondrial WW domain-containing
oxidoreductase (WOX1; also known as FOR or WWOX)
provides a likely molecular pathway on how p53 initiates cell
death in the mitochondria (43). WOX1 is a p53-binding protein
and p53 apoptosis depends upon WOX1 (43).
We isolated the murine Wox1 cDNA and showed that
ectopic expression of this gene in cancer cells increases their
sensitivity to the cytotoxic effect of tumor necrosis factor
(TNF) (43). Human WOX gene has been mapped to a fragile
site on chromosome 16q23.2 (44-46; reviewed in ref. 47).
The WOX gene has 9 exons. Homozygous deletion of this
gene has been demonstrated in breast and other cancer cells
(47,48), suggesting that WOX1 is a tumor suppressor and
loss of WOX proteins may lead to cancer development.
The full length WOX1 (46 kDa) possesses two N-terminal
WW domains, a nuclear localization sequence (NLS) and a
C-terminal short-chain alcohol dehydrogenase (ADH) domain.
Three alternatively spliced human WOX protein variants have
been described (43,45). Additional human and mouse WOX

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 19-24, 2002

21

Figure 1. WOX1 and p53 colocalization in mitochondria. Transient expression of ECFP-tagged WOX1 (WW domain-containing oxidoreductase; in pECFP-C1
vector) in Cos7 cells results in mitochondrial localization (x40 magnification; top panel). WOX1 induces clustering of mitochondria and hyper polarization, as
evidenced by an intense stain with the membrane potential sensitive dye Mitotracker Red. Cotransfection of NCI-H1299 cells with ECFP-WOX1 (in pECFP-C1)
and EGFP-p53 (in pEGFP-C1) results in colocalization of the expressed proteins in mitochondria (x40 magnification; bottom panel). Mitochondria were
identified by antibodies against cytochrome c and mouse rodamine IgG.

protein species have been found according to the updated NR

(non-redundant) and EST (expressed sequence tags) databases
(Chang et al, unpublished data). Also, quite a few of the WOXrelated cDNAs in the database require further sequence
confirmation. This is expected to result in identification of
additional WOX splice variants. Most intriguingly, the ADH
domain region, which is encoded by exon 4-8, is frequently
deleted in the splice variants (47,48). In contrast, the WW
domain region is rarely deleted and its amino acid sequence
is highly conserved among human, mouse and rat.
We determined that WOX1 enhances TNF cytotoxicity in
L929 cells via its WW and ADH domains. The enhancement
of TNF cytotoxicity by WOX1 is due, in part, to its significant
downregulation of the apoptosis inhibitors Bc1-2 and Bc1-xL
but upregulation of the proapoptotic p53 (43). Overexpression
of the full-length or the WW domain of WOX1 sensitizes the
TNF-resistant Cos7 cells to TNF killing (43).
WOX1 protein is mainly located in the mitochondria and
nuclei, and the mitochondrial targeting sequence has been
mapped within the ADH domain (43). WOX1 is also present
in the Golgi complex (Palcy and Bergeron, personal
communication). WOX1 induces clustering of mitochondria.
For example, transient overexpression of ECFP (enhanced cyan
fluorescent protein)-tagged WOX1 in Cos7 fibroblasts induces
clustering of mitochondria (Fig. 1). These mitochondria are
hyper polarized, as evidenced by an intense staining with the
membrane potential sensitive dye Mitotracker Red. Also,
cotransfection of p53-deficient NCI-H1299 cells with WOX1
and p53 constructs results in colocalization of the expressed
proteins in the mitochondria (Fig. 1). Similar results were
observed by transfecting L929 fibroblasts and other types of
cells with both WOX1 and p53 expression constructs (data
not shown).
Both p53 and WOX1 induce apoptosis synergistically in a
variety of cancer cells (43). WOX1 mediates apoptosis
independently of p53, as determined in p53-deficient cells
(43). Like the mitochondrial apoptosis-inducing factor (AIF)
(49,50), WOX1 apoptosis is caspase-independent (43).

Blocking of WOX1 expression by antisense WOX1 mRNA

inhibits p53-mediated apoptosis, suggesting that WOX1 is a
likely partner of p53 in apoptosis (43). In parallel with the
WOX1 and p53 colocalization, co-immunoprecipitation
showed that endogenous WOX1 physically interacts with p53
(43). Yeast two-hybrid analysis showed that WOX1 binds to
the proline-rich region of p53 via its WW domains (43). We
have recently generated a dominant negative WOX1. This
protein has no apoptosis-inducing activity probably due to an
altered conformation. However, this protein binds and blocks
p53 apoptosis (Chang et al, unpublished data). The observation
further supports the essential partnership of WOX1 and p53
in mediating cell death.
Exogenous stress stimuli mediate nuclear p53 translocation
to the mitochondria in certain tested cancer cells (41,42).
Nonetheless, this phenomenon does not appear to be true in
several other types of cells. For example, staining of breast
MCF-7 cells with antibodies against p53 and WOX1 shows the
cytosolic colocalization of both proteins (Fig. 2). Interestingly,
the nuclear WOX1 is located in the nucleoli and does not
appear to colocalize with the nuclear p53 (Fig. 2). We have
shown that perturbation of the mitochondrial permeability
transition pores by TNF, staurosporine and atractyloside results
in WOX1 release from the mitochondria and subsequent
nuclear translocation (43). Exposure of MCF-7 cells to
staurosporine for 1 h resulted in increased expression of both
cytosolic and nuclear p53 and WOX1, and these proteins
colocalized in the cytosol, suggesting p53/WOX1 complex
formation (Fig. 2). The increased expression was also
confirmed by Western blotting (data not shown). After 2 h of
exposure, the cells had undergone apoptosis, as evidenced by
a reduced cell volume and condensed nuclei (Fig. 2). A portion
of the cytosolic p53/WOX1 migrated to the nuclei. Most
interestingly, the nuclear WOX1 had migrated out of the
nucleoli (Fig. 2).
However, in neonatal heart H9c2 cardiomyocytes, ectopic
WOX1 and p53 are found to colocalize and are distributed
evenly in the cytosol. TNF mediates a rapid translocation of

22

CHANG: p53 AND WOX1 IN THE MITOCHONDRIAL APOPTOSIS

Figure 2. WOX1 and p53 colocalization and nuclear translocation during apoptosis in MCF-7 cells. MCF-7 cells were treated with staurosporine (1 M) for 0,
1 and 2 h. Cellular WOX1 and p53 were stained by specific primary and secondary antibodies. In resting cells, majority of the cytosolic p53 and WOX1 are
colocalized (x100 magnification). In contrast, the nuclear WOX1 is present in the nucleoli and not colocalized with the nuclear p53. During 1-h exposure,
staurosporine upregulates the expression of both cytosolic and nuclear p53 and WOX1 proteins in MCF-7 cells, and both proteins are still colocalized. Upon
exposure for 2 h, the cell volume of MCF-7 is reduced, indicative of apoptosis. A portion of cytosolic p53/WOX1 migrates to the condensed nuclei. The
nucleolar WOX1 is relocated to the nuclear matrix.

Figure 3. A schematic model for p53/WOX1 nuclear translocation and

inducing apoptosis in response to stress stimuli. In resting cells, a low level
of p53/WOX1 complex is in the mitochondria. In response to exogenous
stress such as TNF, the synthesis of p53 and WOX1 in cells is increased and
the synthesized proteins translocate to the mitochondria to assemble the p53/
WOX1 complex. This protein complex mediates clustering of mitochondria.
TNF mediates the opening of the mitochondrial transition pores, thus resulting
in release of the p53/WOX1 complex to the cytosol and further migrating to
the nuclei. p53 dissociates from WOX1 in the nucleus and WOX1 mediates
nuclear apoptosis.

the cytosolic WOX1/p53 complex to the mitochondria and

further to the nuclei during a prolonged exposure (Chang et al,
unpublished data).
Based on the above observations, a schematic pathway of
how p53/WOX1 mediates apoptosis is proposed (Fig. 3). In

resting cells, it appears that there is a small amount of p53/

WOX1 complex in the mitochondria. This complex has been
demonstrated in the cytoplasm of L929 fibroblasts (43). In
response to apoptotic stress, the cells start to synthesize p53
and WOX1 and both proteins migrate to the mitochondria to
assemble the p53/WOX1 complex. Some p53 may be recruited
from the nucleus in certain cells (41,42). The p53/WOX1
protein complex mediates clustering of mitochondria. Once
the mitochondrial transition pores open, p53/WOX1, along
with cytochrome c, AIF and other apoptogenic proteins, is
released to the cytosol. The p53/WOX1 complex further
translocates to the nucleus. WOX1 dissociates from p53 and
then initiates nuclear apoptosis.
6. Conclusion and perspectives
The salient feature of WOX1 is its mitochondrial colocalization,
interaction and synergism with p53 in cell death, indicating
that p53/WOX1 may act as a signaling complex in the mitochondrial and nuclear apoptosis. Accordingly, proteins that
interact with the p53/WOX1 complex in the death signaling
are predicted.
Nuclear p53 does not appear to bind WOX1. How p53
associates and dissociates with WOX1 in the cytosol and the
nucleus remains to be established. An intriguing feature is
the localization of nuclear WOX1 in the nucleoli and the
functional implication of its translocation to the nuclear matrix
during apoptosis also remains to be established.

INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 9: 19-24, 2002

Acknowledgements
I appreciate the research support from the American Heart
Association and the Guthrie Foundation for Education and
Research. I also appreciate Ms. Sue Chadwick's outstanding
work in preparing the manuscript, and the excellent interactive
discussion of the article with Joan Doherty, a summer research
student from University of Rochester, New York.
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