Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2

Head and Neck Oncology Group, King's College London Dental Institute, Floor 28 Tower Wing, Guy's Hospital Campus, London, SE1 9RT, UK.
Apoptosis (Impact Factor: 3.69). 04/2012; 17(8):762-76. DOI: 10.1007/s10495-012-0720-7
Source: PubMed
ABSTRACT
Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.

Full-text

Available from: Berna S Sayan
ORIGINAL PAPER
Apoptin induces apoptosis by changing the equilibrium
between the stability of TAp73 and DNp73 isoforms
through ubiquitin ligase PIR2
P. Taebunpakul
B. S. Sayan
M. Flinterman
P. Klanrit
J. Ga
¨
ken
E. W. Odell
G. Melino
M. Tavassoli
Ó Springer Science+Business Media, LLC 2012
Abstract Apoptin, a protein derived from the chicken
anaemia virus, induces cell death in various cancer cells
but shows little or no cytotoxicity in normal cells. The
mechanism of apoptin-induced cell death is currently
unknown but it appears to induce apoptosis independent
of p53 status. Here we show that p73, a p53 family
member, is important in apoptin-induced apoptosis. In
p53 deficient and/or mutated cells, apoptin induced the
expression of TAp73 leading to the induction of apop-
tosis. Knockdown of p73 using siRNA resulted in a sig-
nificant reduction in apoptin-induced cytotoxicity. The
p53 and p73 pro-apoptotic target PUMA plays an
important role in apoptin-induced cell death as knock-
down of PUMA significantly reduced cell sensitivity to
apoptin. Importantly, apoptin expression resulted in a
marked increase in TAp73 protein stability. Investigation
into the mechanisms of TAp73 stability showed that
apoptin induced the expression of the ring finger domain
ubiquitin ligase PIR2 which is involved in the degrada-
tion of the anti-apoptotic DNp73 isoform. Collectively,
our results suggest a novel mechanism of apoptin-induced
apoptosis through increased TAp73 stability and induc-
tion of PIR2 resulting in the degradation of DNp73 and
activation of pro-apoptotic targets such as PUMA causing
cancer cell death.
Keywords Apoptin p73 Ubiquitin ligase E3 PIR2
RNF144B PUMA
Abbreviations
AP Apoptin
DAPI 4
0
,6-Diamidino-2-phenylindole
Ad-GFP Adenovirus expressing green
fluorescent protein
BH3 Bcl-2 homology3
CIS Cisplatin
Lenti-GFP Lentivirus expressing green
fluorescent protein
Lenti-GFP-apoptin Lentivirus expressing green
fluorescent tagged apoptin
PKC Protein kinase C enzyme
PUMA p53 upregulated modulator of
apoptosis
P. Taebunpakul M. Flinterman E. W. Odell
M. Tavassoli (&)
Head and Neck Oncology Group, King’s College London Dental
Institute, Floor 28 Tower Wing, Guy’s Hospital Campus,
London SE1 9RT, UK
e-mail: mahvash.tavassoli@kcl.ac.uk
P. Taebunpakul
Department of Oral Surgery and Oral Medicine, Faculty of
Dentistry, Srinakharinwirot University, Bangkok, Thailand
B. S. Sayan
Cancer Sciences Unit, Faculty of Medicine, University of
Southampton, Southampton, UK
M. Flinterman
Research Division, Weill Cornell Medical College in Qatar, P.O.
Box 24144, Doha, Qatar
P. Klanrit
Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen
University, Khon Kaen, Thailand
J. Ga
¨
ken
Department of Haematological Medicine, The Rayne Institute,
King’s College London, London, UK
G. Melino
Medical Research Council, Toxicology Unit, Leicester
University, Leicester, UK
123
Apoptosis
DOI 10.1007/s10495-012-0720-7
Page 1
Introduction
The chicken anaemia virus (CAV) protein apoptin is 121
amino acids, rich in prolines, serines, threonines and basic
amino acids [1]. It has the ability to induce cell death in cancer
cells derived from different tissues but has negligible toxicity
in normal cells including fibroblasts, keratinocytes, smooth
muscle cells, T cells, and endothelial cells [26]. Although
apoptin is not toxic to normal cells, those cells become sen-
sitive to apoptin following oncogenic transformation by SV40
LT antigen suggesting that even a transient exposure to
transformation events sensitises cells to apoptin [3].
Apoptin seems to induce apoptosis through the intrinsic
pathway inducing cytochrome c release from the mitochon-
dria and caspase-3 activation but not caspase-8 [7, 8]. How-
ever, the exact mechanisms by which apoptin induces
apoptosis and its mode of tumour selectivity remain unclear.
So far two clear characteristics of apoptin have been observed;
(1) in cancer cells apoptin is localised to the nucleus where it
seems to interact with DNA, whereas in normal cells it
remains in the cytoplasm and becomes destabilised, (2)
apoptin is highly phosphorylated in cancer cells but signifi-
cantly less in normal cells of various types [9]. The upstream
signalling pathways that lead to the activation of apoptin as
well as the kinases responsible for apoptin phosphorylation
are currently unknown. Several kinases such as PI3-K, AKT
and CDK2 have been shown to be able to phosphorylate and
functionalise apoptin [10]. Recently, we have shown that in
certain tumour cell types apoptin is phosphorylated by protein
kinase C enzyme (PKC) [11]. Additionally, apoptin expres-
sion results in the activation and cellular redistribution of
PKCb and consequently activation of caspase-9/3, cleavage of
PKCd catalytic domain and downregulation of the MERTK
and AKT kinases [11].
Apoptin-induced apoptosis appears to be p53 indepen-
dent in many cancer cell lines [5, 12, 13]. The p53-inde-
pendent apoptotic function of apoptin is an important
feature for its use as a potential anticancer therapeutic.
However, the roles of the other p53 family members
including p63 and p73 in apoptin-induced apoptosis have
not been fully explored.
p73, first identified in 1997, shares a high level of
homology in the DNA-binding domain with the p53 protein
family [14]. The p73 gene is located on chromosome 1p36.3
and expresses seven differentially spliced C-terminal iso-
forms, p73a-g as well as at least four alternatively spliced
N-terminal isoforms that contain different parts of the
transactivation domain (TA). The DTAp73 is the collective
name for four different p73 isoforms lacking TA including
DNp73, DN
0
p73, Dex2 p73 and Dex2/3 p73 [1416].
The p73 gene can be transcribed from two distinct
promoters, P1 and P2. The P1 promoter is upstream of
exon 1 and drives the expression of transactivating TAp73
isoforms while P2 is the alternative promoter located in
intron 3, generating the amino-terminal truncated DNp73
isoforms [15]. Similar to p53, TAp73 transactivates genes
involved in cell cycle arrest and apoptosis in response to
cellular stress signals [17, 18]. In contrast, DNp73 isoforms
have an anti-apoptotic potential and establish a negative
feedback loop that controls the levels of TAp73 and p53
[19, 20]. Mutation of the p73 gene in cancer cells is very
rare [21]. However, frequent loss of heterozygosity of the
p73 gene has been shown in a number of different cancers,
suggesting an important role in tumourigenesis [2224].
Additionally, DNp73 has been shown to be up-regulated in
human cancers [2528] conferring an independent prog-
nostic value for such tumours [26, 29].
Under normal physiological conditions, TAp73 protein
levels are kept low, but p73 expression and activity increases
in response to a subset of DNA damaging agents [30]. The
DNA damage-mediated induction and activation of p73 is
mainly regulated by post-translational modification such as
phosphorylation, acetylation and ubiquitination [16, 31, 32].
In response to DNA damage TAp73 levels increase whilst
the DNp73 levels diminish, releasing the inhibitory effect of
DNp73 on TAp73 and p53 [28]. Several proteins including
E3 ubiquitin ligases Itch, FBXO45 and PIR2 have been
shown to regulate p73 stability and function [3335]. The
change in the equilibrium between pro-apoptotic and anti-
apoptotic isoforms of p73 is believed to be important in
regulating the induction of cell death under stress conditions.
Additionally, phosphorylation of p73 by several kinases such
as c-Abl tyrosine kinase, PKCd and PKCb serine/threonine
kinases has been shown to result in increased p73 activity and
apoptosis [3638].
p53 upregulated modulator of apoptosis (PUMA), one of
the Bcl-2 homology3 (BH3)-only subgroup of the Bcl-2
family members, plays an important role in p53-dependent
apoptosis induced by genotoxic stress [39, 40]. Similar to
p53, p73 can transactivate PUMA by binding to the same
p53-responsive element in the PUMA promoter in response
to DNA damage [41, 42]. We have recently shown that
apoptin expression induces the endogenous protein levels of
TAp73 in p53-mutated head and neck squamous cell carci-
noma H357 suggesting an important role for p73 in the
regulation of apoptin-induced apoptosis [43]. However, the
mechanism by which apoptin activates TAp73 remained
unknown.
Here we provide direct evidence that p73 is required for
apoptin-induced apoptosis as knockdown of p73 using
siRNA in HCT116 colon cancer cell lines increased
resistance to apoptin-induced killing. Importantly, apoptin
expression stabilised TAp73 and its downstream target
PUMA causing tumour cell death. The stabilisation of
TAp73 by apoptin resulted in increased expression of the
ring finger domain ubiquitin ligase, PIR2, consequently
Apoptosis
123
Page 2
degrading the anti-apoptotic DNp73 isoform and activating
the p73 mediated apoptotic pathway. Collectively, our data
proposes a novel mechanism for apoptin-induced apopto-
sis, which involves the stabilisation of TAp73 isoform and
concurrent reduction in the level of DNp73 through PIR2.
The resultant change in the equilibrium between the
expression of anti- and pro-apoptotic p73 isoforms seems
to determine sensitivity of cancer cells to apoptin.
Results
Apoptin induces G2/M arrest and activates TAp73a
Several studies have used apoptin fused to GFP for the
convenience of imaging and functional studies. However,
there has been some concern that GFP-apoptin may have a
somewhat altered function as compared to apoptin
alone [44, 45]. Although, GFP-apoptin behaves similarly to
native apoptin in cancer and transformed cells, it appears
to partially lose tumour cell selectivity. Additionally, in
contrast to apoptin, which is mainly cytoplasmic in normal
cells, GFP-apoptin when expressed can translocate to the
nucleus of some normal cells [44, 46].
We constructed an adenoviral vector expressing
Apoptin (Ad-apoptin) using the Ad-easy system (Q-bio-
gene) and infected p53-deleted Saos-2 cells with an MOI
20. Expression of apoptin was detected by indirect
immunofluorescence using a phospho-specific antibody
against Threonine-108-phosphorylated apoptin (Apoptin-
P). Nuclei were counterstained with DAPI. The percentage
of apoptin positive cells was determined by scoring of the
FITC stained cells (Ad-apoptin) and GFP marker (Ad-GFP)
using fluorescent microscopy. The percentage of apoptotic
cells was determined by scoring the cells with clear con-
densed or fragmented nuclei. The results showed that
already at 24 h post-infection, 84 ± 5 % of cells were
expressing apoptin and 76.5 ± 8 % of cells were express-
ing GFP. Apoptin mainly localised to the nucleus and was
phosphorylated whilst Ad-GFP infected cells showed
the presence of GFP both in the nucleus and cytoplasm.
Concomitantly, we observed a significant amount of
apoptotic nuclei in Ad-apoptin infected cells when com-
pared to Ad-GFP infected cells (P \ 0.05) (indicated with
white arrows, Fig. 1a). Cell cycle analysis by flow cytom-
etry indicated that in contrast to Ad-GFP, Ad-apoptin sig-
nificantly induced G2/M accumulation at 24 h post
infection with a further increase in G2/M arrest after 48 h
(P \ 0.05) (Fig. 1b).
To examine the effect of apoptin on p73 expression,
Saos-2 cells were mock infected or infected with either Ad-
apoptin or Ad-GFP or treated with cisplatin. Western blot
analysis was performed using p73a (p73SAM) antibody. At
48 h post infection, expression of apoptin in Saos-2 cells
resulted in the upregulation of endogenous TAp73a pro-
tein. At this time point apoptosis occurred in apoptin
expressing cells as detected by cleaved PARP p85 frag-
ment when compared to the control and Ad-GFP infected
cells (Fig. 1c). These results further confirmed the ability
of Ad-apoptin to induce apoptosis and provided clear evi-
dence that apoptin regulates p73 to induce apoptosis in the
p53 deficient Saos-2 cells.
p73 regulates p53 independent apoptin-induced cell
death
The importance of p73 in apoptin-induced cell death was
further investigated using human colon cancer HCT116
cell lines containing either wild-type p53 or hetero and
homozygous p53 (p53
?/?
, p53
?/-
and p53
-/-
) knockout.
The cells were infected with Ad-apoptin and cell viability
was measured at 24 and 48 h post-infection by the MTT
assay. As shown in Fig. 2a, no significant difference in the
sensitivity to apoptin was observed between p53 wild-type
and deficient HCT116 cells (P [ 0.05). This was further
confirmed by phase contrast microscopy showing mor-
phological changes associated with cell death in all cultures
infected with Ad-apoptin (Fig. 2b). Apoptin expression
induced TAp73 protein which was detected at both 24 and
48 h post-infection, in all three HCT116 cell lines, irre-
spective of their p53 status. p53-independent cell death was
detected by cleaved PARP at 48 h post-infection (Fig. 2c).
To examine whether p73 has a direct role in apoptin-
induced cell death, p53
-/-
HCT116 cells were transduced
with siRNA molecules directed against TAp73a and then
infected with Ad-apoptin at 48 h post-transduction. At 24 h
post-infection apoptin induced expression of endogenous
TAp73a but no apoptosis was detected in either control
siRNA transduced or p73 knockdown cells (data not
shown). However, at 48 h post-infection, Western blot
analysis showed a clear reduction in PARP cleavage in p73
knockdown cells indicating increased resistance of these
cells to apoptin-induced killing (Fig. 2d).
PUMA is important for apoptin-induced cell death
The effect of apoptin on a number of pro-apoptotic targets
including Bax, Bak and Noxa has been previously reported
[47]. Additionally, we have recently shown that apoptin
induces the expression of the p53/p73 pro-apoptotic target
PUMA [43]. To further investigate the importance of
PUMA in apoptin-induced apoptosis, PUMA knockout
HCT116 cells were investigated. MTT assay and phase
contrast microscopy showed that PUMA inhibition signif-
icantly reduced sensitivity to apoptin-induced apoptosis
(P \ 0.05) (Fig. 3a, b) as compared to PUMA expressing
Apoptosis
123
Page 3
Saos-2
a
Apoptin-P
(Ad-Apoptin)
Control
GFP
(Ad-GFP)
0
5
10
15
20
25
Ad-GFP Ad-AP
% Apoptotic cells
24 h
*
Mean+STDEV
0 200 400 600 800 1000
0
500
1000
1500
24 h post-infection
48 h post-infection
0
200 400 600 800 1000
0
300
600
900
1200
0 200 400 600 800 1000
0
500
1000
1500
# Cells
0 200 400 600 800 1000
0
500
1000
1500
2000
2500
0 200 400 600 800 1000
0 200 400 600 800 1000
0
500
1000
1500
2000
b
G1
49.50%
S
16.40%
G2-M
33.00%
G1
52.60%
S
22.00%
G2-M
23.90%
G1
32.80%
S
24.60%
G2-M
41.30%
G1
55.40%
S
19.70%
G2-M
23.30%
G1
52.20%
S
17.00%
G2-M
29.80%
G1
29.00%
S
24.60%
G2-M
44.80%
Control
Ad-GFP
Control
Ad-GFP
Ad-apoptin Ad-apoptin
Mean+STDEV
0
10
20
30
40
50
24 h 48 h
%G2/M arrest
Ad-GFP
Ad-AP
*
*
c
Saos-2
Apoptin-P
-Actin
PARP
TAp73
α
AP
GFP
CIS
C
GFP
β
# Cells
# Cells
# Cells
# Cells
0
500
1000
1500
# Cells
Apoptosis
123
Page 4
HCT116 cells. Interestingly, Western blot analysis showed
that apoptin increased the levels of p73 as well as p53,
which are upstream of PUMA, in both PUMA wild-type
and knockout HCT116 cells. However, diminished apop-
tosis was only observed in the PUMA knockout cells
(Fig. 3c) suggesting that the activation of the p53/p73
pathway is not sufficient for the full induction of apoptosis
by apoptin and that PUMA is one of the crucial mediators
of apoptin cell death induction. As apoptosis was not
completely inhibited in PUMA knockdown in HCT116
cells, there are likely to be other pro-apoptotic targets
involved in cell killing by apoptin. Interestingly, the level
of apoptin phosphorylation was reduced in PUMA knock-
down cells, this effect was not due to an altered infection or
expression efficiency of PUMA knockdown cells as con-
firmed by comparable levels of GFP expression in these
cells (Fig. 3c).
Apoptin stabilises the pro-apoptotic TAp73a isoform
We have previously shown that apoptin has no effect on the
transactivation of p73 isoforms [43]. We therefore inves-
tigated the effect of apoptin on the protein stability of
TAp73a and DNp73a isoforms using p73 isoform specific
inducible Saos-2 cells. As shown in Fig. 4a, expression of
apoptin increased TAp73a stability for up to 12 h, the
maximum time point in this experiment, as compared to
cells expressing Ad-GFP and non-treated controls. Impor-
tantly, apoptin did not increase the stability of the onco-
genic DNp73a isoform. Immunoprecipitation studies
demonstrated that apoptin interacted with both TAp73 and
DNp73a isoforms with higher affinity to TAp73a than
DNp73a (data not shown).
The role of specific p73 isoforms in sensitivity to
apoptin-induced cell death was also examined using
doxycycline inducible Saos-2 cells expressing either
TAp73a or DNp73a. Cells were infected with either Lenti-
GFP, Lenti-GFP-apoptin or left untreated in the presence
(? doxy) or absence (- doxy) of doxycycline induction.
Cells were analysed by indirect immunofluorescence and
apoptotic cells were scored. Doxycycline induction of
TAp73a but not DNp73a resulted in some cell death in
Saos-2 cells (Fig. 4b, c). However, apoptin expression
induced a significantly higher percentage of apoptosis in
cells expressing TAp73a as compared to apoptin expres-
sion without TAp73a induction (Fig. 4d, P \ 0.05). Con-
versely, the percentage of apoptotic cells induced by
apoptin in cells expressing DNp73a was significantly
decreased as compared to apoptin expression without
DNp73a induction (P \ 0.05) (Fig. 4d). The above results
indicate that apoptin regulates the p73 activity by
increasing the stability of TAp73a but not DNp73a.
Additionally, the expression of TAp73a sensitises cells
while DNp73a expression inhibits apoptin-induced cell
death.
Ubiquitin ligase PIR2 acts as a switch in apoptin-
induced apoptosis by increasing the degradation
of DNp73
As discussed in the introduction, the level of TAp73 is
believed to be regulated at post-translational level by various
modifications [32]. In order to identify the mechanism of
apoptin-induced TAp73 stability the effect of apoptin on the
expression of known p73 modulators was investigated. We
found that apoptin had no significant effect on the level of
ubiquitin ligase proteins, Itch and FBXO45 (data not
shown). However, apoptin expression resulted in a clear
increase in the level of ubiquitin ligase PIR2 and induction
of apoptosis in TAp73a inducible Saos-2 cells, as detected
by cleaved PARP. This effect was specific to apoptin as the
induction of p73 alone or the expression of control GFP had
no effect on PIR2 expression suggesting that cooperation
with apoptin is necessary for increased PIR2 expression
(Fig. 5a). Interestingly, PIR2 expression alone resulted in
increased TAp73 expression and the induction of apoptosis
in Saos-2 cells. Co-expression of PIR2 together with apoptin
in Saos-2 cells led to increased TAp73 and apoptosis at a
significantly higher levels than those reached by expressing
either apoptin or PIR2 alone (Fig. 5b). We also investigated
the effect of apoptin on the level of c-Abl and c-Jun proteins,
both of which have been implicated in the stabilisation of
p73 [48, 49]. No significant changes in the levels of either of
Fig. 1 Apoptin induces G2/M arrest and activates TAp73a. a Saos-2
cells were seeded in 8-well chamber slides, infected with either
Ad-apoptin or Ad-GFP. Cells were fixed after 24 h. Apoptin was
detected by a phospho-specific antibody against Threonine-108–
phosphorylated apoptin and Goat-anti-rabbit IgG FITC conjugate
antibody. Cells were mounted in DAPI-containing mounting medium
(Vector Laboratories). Apoptin and GFP stained green and nuclei
stained blue. The percentage of apoptosis was measured by scoring
apoptin and GFP positive cells which contained condensed or
fragmented nuclei. At least one hundred infected cells were counted
in triplicate experiments. Error bars indicate standard deviation
(STDEV). Asterisk represents significant difference of P \ 0.05.
b Cell cycle analysis of Saos-2 cells by PI FACS staining. The
analysis was performed using cell cycle analysis (Dean–Jett–Fox
model), FlowJo version 9.4.11 (Tree Star, Inc., USA). The graphs
represent the percentage of Saos-2 cells arrested in G2/M after
infection with 20 MOI of Ad-apoptin or Ad-GFP after 24 and 48 h.
Error bars indicate STDEV. Asterisk represents significant difference
of P \ 0.05. c Apoptin induces endogenous TAp73a protein level.
Saos-2 cells were infected with either Ad-apoptin or Ad-GFP at a
MOI of 40 or left untreated. Saos-2 cells treated with 10 lg/ml
cisplatin for 48 h were used as positive control. The cells were
collected at 48 h post-infection and were analysed by Western blot
analysis using indicated antibodies
b
Apoptosis
123
Page 5
0
20
40
60
80
100
120
% cell viability
24 h
Control GFP AP CIS
a
Mean+STDEV
0
20
40
60
80
100
120
control GFP AP CIS
% cell viability
48 h
HCT116 p53+/+
HCT116 p53+/-
HCT116p53-/-
control
GFP
AP
CIS
control
GFP
AP
CIS
control GFP AP
CIS
HCT116 p53+/+
HCT116 p53+/-
HCT116 p53-/-
24 h
c
p53
+/+
p53
-/-
p53
+/-
48 h
p53
+/+
p53
-/-
p53
+/-
Apoptin-P
C GFP AP CIS C GFP AP CISC GFP AP CIS
Tubulin
p53
TAp73
PARP
GFP
C GFP AP CISC GFP AP CIS C GFP AP CIS
Apoptin-P
Tubulin
p53
TAp73
PARP
GFP
0
0.1
0.2
0.3
0.4
0.5
0.6
control GFP AP
The relative amount of cleaved
HCT116 p53 WT
HCT116 p53 -/-
HCT116 p53 +/-
PARP (normalised with tubulin)
b
α
α
Apoptosis
123
Page 6
these proteins were observed with or without apoptin
expression (Fig. 5a).
To investigate a direct role of PIR2 in apoptin-induced
cell death, PIR2 was silenced by siRNA in TAp73a Saos-2
inducible cells. As shown in Fig. 5a, apoptin expression
resulted in increased levels of PIR2 and apoptosis in
TAp73a inducible Saos-2 cells both at 24 and 48 h post-
infection. PIR2 knockdown showed reduced apoptin-
induced apoptosis measured by the level of PARP cleavage
as compared with apoptin expression in siRNA control
transduced cells (Fig. 5c, 24 and 48 h).
In contrast to TAp73 inducible cells, expression of
apoptin in DNp73a inducible Saos-2 cells failed to induce
PIR2 levels with or without induction of DNp73a.Fur-
thermore, apoptin induced apoptosis only in cells lacking
DNp73a expression as shown by PARP cleavage (Fig. 5d).
These results indicated that PIR2 is induced by apoptin
downstream of TAp73a but not DNp73a hence upregula-
tion of TAp73a by apoptin leads to PIR2 induction and
consequently apoptosis. In agreement with a recent study
[33], we found that overexpression of PIR2 resulted in the
degradation of the anti-apoptotic DNp73 isoform conse-
quently inducing apoptosis in DNp73a overexpressing
Saos-2 cells (Fig. 5e). Taken together, these data suggest
that apoptin modulates TAp73a isoform function through
protein stabilisation resulting in the induction of its
downstream target ubiquitin ligase PIR2. The destabilisa-
tion of DNp73a by PIR2 removes DNp73a inhibitory effect
on the proapoptotic TAp73 isoforms triggering the apop-
totic machinery.
Discussion
Many viruses are able to reprogram cellular processes to
allow viral replication and propagation. Interaction of
certain viral proteins with key cellular components such as
p53 and pRB causes deregulation of the cell cycle program
and consequently cellular transformation. Additionally,
there is clear evidence for the role of viruses, through the
specific function of viral proteins such as E1A of human
adenovirus, in the induction of tumour specific cell death
[43, 50, 51]. The potential use of viruses and viral derived
proteins in cancer therapy is of major interest at present.
Therefore, understanding the cellular mechanisms which
determine the pro- and anti-apoptotic cellular response to
viral proteins is crucial for exploring their therapeutic
potential. In this study we have investigated the cellular
mechanisms responsible for the cytotoxic effect of CAV
derived protein VP3/apoptin, which shows tumour selec-
tive cytotoxicity. Previously, it has been shown that
apoptin-induced cell death was not dependent on p53 and
involved the intrinsic apoptotic pathway. However, both
the upstream signalling mechanism and the downstream
apoptotic components which regulate apoptin function are
currently unknown.
d
48 h
AP
GFP
CIS
C
AP
CIS
C
Apoptin-P
Tubulin
TAp73
PARP
p53
GFP
Control
siRNA
sip73
GFP
0
0.5
1
1.5
2
control GFP AP
Control siRNA
sip73
The relative amount of cleaved
PARP (normalised with tubulin)
α
Fig. 2 continued
Fig. 2 p73 regulates p53 independent apoptin-induced cell death.
a Human colorectal cancer cells HCT116 wild-type and its p53
knockdown derivative were left untreated or infected with either
Ad-GFP or Ad-apoptin at a MOI 20 or treated with 10 lg/ml
cisplatin. Cell survival was measured by the MTT assay at 24 and
48 h post-infection. Results are shown as percentage of viable cells
with respect to viable non-treated cells. All experiments were
performed in triplicate, error bars indicate STDEV. b Microscopic
pictures of wild type, p53
?/-
and p53
-/-
HCT116 cell lines 24 h
post-infection with either Ad-apoptin or Ad-GFP at a MOI 20, mock
infected or treated with 10 lg/ml cisplatin. c HCT116 wild-type and
its p53 knockdown HCT116 cells were left untreated or infected with
indicated recombinant adenoviruses and lysed 24 and 48 h post-
infection and analysed by Western blot. The percentage of cleaved
PARP p85 fragment was quantified using ImageJ (NIH, USA).
d HCT116 p53
-/-
cells were transfected with either siRNA against
TAp73 or siRNA control 24 h after transfection, cells were induced
with doxycycline overnight after which the cells were mock infected
or infected with either Ad-GFP or Ad-apoptin at a MOI 20 or treated
with 10 lg/ml cisplatin. Cells were lysed at 48 h after infection and
analysed by Western blot using the indicated antibodies. The
percentage of cleaved PARP p85 fragment was quantified using
ImageJ (NIH, USA)
b
Apoptosis
123
Page 7
It is known that the function of p73 is dependent on the
p53 status of the cell and differs in the presence/absence of
wild-type or mutated p53 [52]. Therefore, to investigate the
possible role of p73 in apoptin induced cytotoxicity we used
either heterozygous or homozygous p53 knockout HCT116
cells. Apoptin expression resulted in the induction of TAp73
and consequently apoptosis regardless of the cellular p53
status (Fig. 2b). Whilst p53 knockout did not reduce the
cytotoxicity of apoptin, downregulation of p73 by siRNA
significantly reduced apoptin-induced cell death. One of the
key features of apoptin is its selectivity for cancer cells; the
basis for this specificity might therefore involve the regula-
tion of p73 pathway. Indeed, apoptin resulted in an increase
in the endogenous TAp73a in human SV40 large T-antigen
0
20
40
60
80
100
120
control GFP AP CIS
% cell survival
48 h
HCT116 WT
HCT116 PUMA KO
*
Mean+STDEV
HCT116 WT
Control
Ad-apoptin
HCT116 PUMA KO
Control
Ad-apoptin
ab
c
C
AP
HCT116 WT
HCT116 PUMA KO
PARP
TAp73
PUMA
Apoptin-P
Tubulin
p53
GFP
C
CISCIS
AP
GFP GFP
0
0.5
1
1.5
2
2.5
3
control GFP AP
HCT116 WT
HCt116 PUMA KO
The relative amount of cleaved
PARP (normalised with tubulin)
α
Fig. 3 PUMA is important for
apoptin-induced cell death.
a Phase contrast microscopic
pictures of HCT116 cells and its
PUMA
-/-
cell lines at 24 h
post-infection with either
Ad-apoptin at a MOI 10 or
untreated cells. b The cells were
left untreated or infected with
either Ad-GFP or Ad-apoptin at
a MOI 10 or treated with
10 lg/ml cisplatin. Cell survival
was measured by the MTT
assay at 48 h post-infection.
Results are shown as percentage
of viable cells with respect to
viable non-treated cells. All
experiments were performed in
triplicate, error bars indicate
STDEV. Asterisk indicates a
significant difference of
P \ 0.05 c HCT116 cells and its
PUMA
-/-
cell lines were
infected with Ad-GFP or
Ad-apoptin at a MOI 10 or left
untreated or treated with
10 lg/ml cisplatin. After 24 h
post-infection, cell lysates were
prepared and analysed by
Western blot using the indicated
antibodies. The percentage of
cleaved PARP p85 fragment
was quantified using imageJ
(NIH, USA)
Apoptosis
123
Page 8
transformed fibroblasts 1BR3N but not in the matched nor-
mal human fibroblasts 1BR3 cells (data not shown). Addi-
tionally, apoptin phosphorylation levels were lower in
normal cells. However, as the regulation of different p73
isoforms in normal, unstressed cells remains unclear, the
precise role of p73 in increased sensitivity of tumour cells to
apoptin and its involvement in apoptin phosphorylation
needs further investigation in different normal cell types.
Our previous studies using luciferase reporter assays
showed that apoptin expression has no effect on the
b
DAPI GFP
Merge
Texas red (TAp73 )
Lenti-GFP-AP
Lenti-GFP
Control
Lenti-GFP
Lenti-GFP-AP
Control
TAp73
TAp73
Np73
0128421
Tubulin
p73
Tubulin
p73
Tubulin
Apoptin-P
Control
Ad-GFP
Ad-apoptin
Control
0128421
TAp73
Tubulin
Ad-apoptin
p73
Tubulin
Apoptin-P
Ad-GFP
p73
Tubulin
After CHX
After CHX
0
20
40
60
80
100
120
0124812
% p73 remaining
Time after Cycloheximide (h)
TAp73-C
TAp73-GFP
TAp73-AP
a
0
20
40
60
80
100
120
0124812
% p73 remaining
Time after Cycloheximide (h)
Np73-C
Np73-GFP
Np73-AP
α
Fig. 4 Apoptin stabilises the
pro-apoptotic TAp73a isoform.
a TAp73a or DNp73a
inducible-Saos-2 cell lines were
used to study the half-life of
p73. Cells were treated with
20 lg/ml cycloheximide (CHX)
and left untreated or infected
with either Ad-GFP or Ad-
apoptin at a MOI 10. 24 h post-
infection, cells were collected at
the indicated time points (0, 1,
2, 4, 8, 12 h). Cell lysates were
analysed by Western blot. Saos-
2 cells inducible for TAp73a
(b) and DNp73a (c) were
infected with either Lenti-GFP
or Lenti-GFP-apoptin at a MOI
of 4 in the presence or absence
of doxycycline. Cells were fixed
at 24 h after infection and
stained for p73 isoforms by
immunofluorescent staining
using mouse anti-HA primary
antibody and Texas red-labeled
secondary anti-mouse antibody.
Cells were mounted in DAPI-
containing mounting medium.
p73 stained red and Lenti-GFP-
apoptin stained green. d The
percentages of apoptotic cells in
Saos-2 inducible TAp73a and
DNp73a cell lines treated with
apoptin in the presence or
absence of doxycycline.
Apoptosis was examined by
scoring the GFP positive cells
which contained condensed or
fragmented nuclei. At least one
hundred infected cells were
analysed and experiments were
performed in triplicate. Error
bars indicate STDEV. Asterisks
represent significant difference
of P \ 0.05
Apoptosis
123
Page 9
transactivation of p73 [43]. Here we clearly demonstrate
that apoptin expression resulted in substantially increased
stability of the pro-apoptotic TAp73 but had no
significant effect on the stability of the anti-apoptotic
DNp73 isoform indicating the true pro-apoptotic function
of apoptin.
Fig. 4 continued
Apoptosis
123
Page 10
Differential protein stability is believed to be the main
mechanism that regulates the pro- and anti-apoptotic
activity of p73 isoforms thereby determining cellular fate.
This led us to investigate the role of E3 ubiquitin ligases
that are known to be involved in p73 stability. The ring
finger domain ubiquitin ligase, PIR2 was shown to be
directly regulated by TAp73 upon cellular stress and an
increase in the level of PIR2 leads to degradation of the
DNp73 isoform [33]. We observed that the induction of
TAp73 by apoptin leads to an increase in the PIR2 level
and subsequent apoptosis. PIR2 appears to play a role in
apoptin-induced apoptosis because PIR2 knockdown
showed increased resistance of cells to apoptin cytotoxic-
ity. Importantly, exogenous expression of PIR2 in DNp73
overexpressing cells, which are resistant to apoptin-
induced cell death, by itself was toxic further proposing an
important role for PIR2 in regulating the TAp73 protein
stability and DNp73 degradation to achieve an apoptotic
response. Moreover, our data suggests that TAp73 alone is
not sufficient to activate PIR2 expression and its activation/
stabilisation by apoptin prompts p73-mediated induction
of PIR2. The mechanism by which apoptin modulates
a
b
α
α
α
Fig. 5 Ubiquitin ligase PIR2 acts as a switch in apoptin-induced
apoptosis by increasing the degradation of DNp73 levels. a Saos-2
inducible cells expressing TAp73a were left untreated or infected
with indicated recombinant adenoviruses at a MOI 10 in the presence
and absence of doxycycline and lysed at 24 and 48 h post-infection
and analysed by Western blot. b Saos-2 cells were transfected with
plasmid expressing PIR2 and 24 h post-transfection, the cells were
treated with indicated adenoviruses at a MOI 20 or treated with
10 lg/ml cisplatin. Cells were lysed after 24 h treatment and
subjected to Western blot analysis with the indicated antibodies.
c siRNA PIR2 knockdown cells show increased resistance to apoptin-
induced apoptosis. TAp73a Saos-2 inducible cells were transfected
with either control siRNA or siRNA against PIR2. At 48 h post-
transfection, cells were infected with either Ad-apoptin or Ad-GFP or
treated with cisplatin. Cells were collected at 24 and 48 h post-
infection for Western blot analysis. The percentage of cleaved PARP
p85 fragment was quantified using ImageJ (NIH, USA). d DNp73a
inducible-Saos-2 cells were left untreated or infected with either
Ad-GFP or Ad-apoptin at a MOI 10. Cells were lysed at 48 h post-
infection and subjected to Western blot analysis with indicated
antibodies. e DNp73a inducible-Saos-2 cells were induced with
doxycycline and after 24 h the cells were transfected with either
empty vector or plasmid expressing PIR2. Cells were lysed at 24 h
post-transfection and subjected to Western blot analysis with
indicated antibodies. Anti-Myc antibody was used to detect PIR2
expression
Apoptosis
123
Page 11
transcriptional regulation of PIR2 through TAp73 remains
currently unclear.
We have shown that the expression of apoptin increases
the levels of endogenous TAp73 and its pro-apoptotic
target PUMA leading to apoptosis in p53 mutant HNSCC
cells [43]. In this study we further investigated the
importance of PUMA using PUMA KO HCT116 cells. The
results confirmed that despite the activation of p53 and/or
p73 by apoptin, inhibition of PUMA significantly increased
the resistance of tumour cells to apoptin-induced killing
when compared to PUMA wild type cells suggesting the
importance of PUMA in mediating apoptin-induced apop-
tosis. Interestingly, we found that the level of apoptin
phosphorylation in PUMA knockout cells was significantly
lower than wild-type cells. The GFP control showed that
this was not due to differences in infection efficiency or
expression levels in the knockout cells (Fig. 3c) indicating
a link between PUMA inhibition and apoptin phosphory-
lation. Recently, PUMA has been shown to be modulated
post-translationally through phosphorylation [53]. Whether
there is a functional interaction between PUMA and
apoptin phosphorylation needs further investigation.
Currently the upstream signalling pathways which sta-
bilise p73 in response to apoptin are not understood. A
recent study has shown that apoptin is activated by DNA
damage response (DDR) involving ATM and DNA-PK
α
α
Fig. 5 continued
Apoptosis
123
Page 12
[54]. Also independent studies have shown that p73
stabilisation is involved in DDR through ATM [55, 56].
Another mechanism involved in p73 stabilisation is
through phosphorylation and several kinases have been
shown to phosphorylate and hence stabilise p73 including
Abl, JNK, PKCd and PKCb. Recently we have shown the
activation of PKCd and PKCb by apoptin. However,
whether any of these kinases causes p73 phosphorylation
and are responsible for apoptin-induced TAp73 increased
stabilisation will form part of our future studies.
In summary, our findings suggest a novel pathway for
the induction of cell death by apoptin and possibly other
apoptotic inducing agents. Figure 6 demonstrates the pro-
posed model summarising the components which may be
involved in p53 independent, p73-mediated induction of
apoptosis by viral protein apoptin. Further investigation of
this mechanism will be very important for the design
of effective anticancer therapeutics.
Materials and methods
Cell lines, plasmids and reagents
Saos-2 cells with inducible TAp73a and DNp73a, Human
colon cancer HCT116, its PUMA
-/-,
p53
-/-
, p53
?/-
knockout and Osteosarcoma Saos-2 cells were cultured as
described previously [43]. Inducible TAp73a and DNp73a
were under the control of the TET-on system and induced
by the addition of 2.5 lg/ml doxycycline. The following
constructs were used: pcDNA4/TO/myc-His-PIR2, Lenti-
virus-GFP-apoptin, Lentivirus-GFP, Adenovirus-apoptin
and Adenovirus-GFP. Adenovirus amplification and puri-
fication was essentially done as described previously [51].
Flow cytometry
Cells were plated in 6-well plates and infected with ade-
novirus at a MOI 20. The samples were collected at 24 and
48 h after infection. Cells were fixed in 70 % ethanol and
stained with PI FACS staining solution. The samples were
incubated at 37 °C for 30 min then analysed on a
FACSCalibur within 24 h.
Immunofluorescent microscopy
Saos-2 cells were seeded in 8-well culture slides (Becton–
Dickinson, Oxford, UK) and infected with Ad-GFP or
Ad-apoptin. After 24 h phospho-specific antibody against
Threonine-108-phosphorylated apoptin and Goat-anti-
rabbit IgG FITC conjugate secondary antibody (Sigma,
Gillingham, UK) were used to detect Ad-apoptin. For the
Ad-GFP control GFP expression was directly detected
by fluorescence microscopy. Saos-2 cells with inducible
TAp73a and DNp73a were seeded in Falcon 8-well culture
slides and infected with Lenti-GFP-apoptin or Lenti-GFP.
After 24 h, TAp73a and DNp73a expression was detected
by mouse anti-HA antibody (Sigma, Gillingham, UK) and
followed by goat anti-mouse IgG (whole antibody) Texas-
Red (Vector Laboratories, Peterborough, UK). Fixation
and preparation of slides were carried out as described
previously [51].
MTT assay
Cell survival was measured by the 3-(4,5-dimethylthiaz-
olyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay as
described previously [51].
siRNA knockdown
The following siRNA reagents were used: On-TARGET
plus
Ò
control siRNA GAPD: Human (Thermo Scientific
Dharmacon
Ò
), Ambion Silencer
Ò
Select Pre-designed
siRNA: RNF144B (Applied Biosystems), On-TARGET
plus SMARTpool TP73 (Thermo Scientific Dharmacon
Ò
).
siRNA transfection was performed using Amaxa
TM
Nu-
cleofector
TM
as recommended by the manufacturer (Lonza
Biologics, Cambridge, UK).
Western blot analysis
Western blot analysis was performed as described previ-
ously [51]. The antibodies used were: antibody against
Threonine-108-phosphorylated apoptin, raised in rabbit
Apoptin
TAp73
PUMA
PIR2
Np73
Apoptosis
Fig. 6 Proposed model for p53 independent, p73-mediated induction
of apoptosis by viral protein apoptin. Apoptin expression leads to
activation of pro-apoptotic TAp73 which results in upregulation of
the downstream target PUMA and induction of cell death. The model
proposes that apoptin induces apoptosis by increased TAp73 stability
via the ring finger domain ubiquitin ligase PIR2. Consequently PIR2
expression, degrades anti-apoptotic DNp73 isoform to release DNp73
on TAp73 isoform
Apoptosis
123
Page 13
against peptide H2NSLITTT(PO3H2)PSRPRTA-CONH2
derived from the apoptin amino acid sequence (Eurogen-
tec) [11], mouse anti-Myc (Santa cruz), Rabbit anti-c-jun
(Santa Cruz), rabbit anti-GFP (cell signalling), rabbit anti-
p73SAM [57], rabbit anti-PIR2 [33], mouse anti-p53 clone
DO7 (Novacastra Laboratories), mouse-anti-Abl (BD
Biosciences), mouse anti-tubulin (Sigma), mouse anti-b
actin (Sigma), rabbit anti-PARP p85 fragment clone
G734A (Promega, Southampton, UK), mouse anti-HA
(Sigma), secondary anti-mouse (Sigma) and anti-rabbit
antibodies linked to horseradish peroxidase (GE Healthcare
Life Sciences, Little Chalfont, Buckinghamshire, UK).
Determination of p73 half-life
Determination of p73 protein turnover was performed by
adding 20 lg/ml cycloheximide to TAp73a and DNp73a
inducible Saos-2 cell lines 24 h after induction with
2.5 lg/ml doxycycline and infection with either Ad-apop-
tin or Ad-GFP or mock infected. Protein levels were
determined by collecting cells at different time points and
performing Western blot analysis as described above.
Statistical analysis
For statistical analysis, the Student’s t test was carried out.
Statistically significant difference was defined as P \ 0.05.
Acknowledgments We would like to thank Drs Franco Conforti
and Jie Jiang for technical advice, Prof. Bert Vogelstein for kindly
providing Ad-GFP, HCT116 cell line and its knockout p53
-/-
,
p53
?/-
and PUMA derivatives. This work was supported by Cancer
Research UK (Grant Numbers C1116/A6649) and Rosetrees Trust.
Patrayu Taebunpakul was supported by a Royal Thai Government
Scholarship.
Conflict of interest The authors declare no conflict of interest.
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    [Show abstract] [Hide abstract] ABSTRACT: ΔNp63 is a transcription factor that is critical for the development of stratified epithelia and is overexpressed or amplified in >80% of squamous cell carcinomas (SCCs). We identified the RING finger E3 ubiquitin ligase PIR2/Rnf144b as a direct transcriptional target of ΔNp63α and showed that its expression parallels that of ΔNp63α in keratinocytes, SCC cell lines and SCCs. We used primary keratinocytes as a model system to investigate the function of PIR2/Rnf144b in stratified epithelia. Depletion of PIR2/Rnf144b severely impaired keratinocyte proliferation and differentiation, associated with accumulation of p21WAF1/CIP1; a known target of PIR2/Rnf144b. More importantly, we found that PIR2/Rnf144b binds and mediates proteasomal degradation of ΔNp63α, generating a hitherto unknown auto-regulatory feedback loop. These findings substantiate PIR2/Rnf144b as a potentially critical component of epithelial homeostasis, acting downstream of ΔNp63α to regulate cellular levels of p21WAF1/CIP1 and ΔNp63α.
    Full-text · Article · Nov 2012 · Oncogene
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    [Show abstract] [Hide abstract] ABSTRACT: p73, has two distinct promoters, which allow the formation of two protein isoforms: full-length transactivating (TA) p73 and an N-terminally truncated p73 species (referred to as DNp73) that lacks the N-terminal transactivating domain. Although the exact cellular function of DNp73 is unclear, the high expression levels of the genes have been observed in a variety of human cancers and cancer cell lines and have been connected to pro-tumor activities. Hence the aim of this review is to summarize DNp73 expression status in cancer in the current literature. Furthermore, we also focused on recent findings of DNp73 related to the biological functions from apoptosis, chemosensitivity, radiosensitibity, differentiation, development, etc. Thus this review highlights the significance of DNp73 as a marker for disease severity in patients and as target for cancer therapy.
    Full-text · Article · May 2013 · Cell cycle (Georgetown, Tex.)
  • [Show abstract] [Hide abstract] ABSTRACT: The use of anticancer therapeutic agents is limited largely by their severe toxicity to normal tissues. The development of novel agents with tumor-specific cell-killing and effective gene delivery properties is thus very desirable. We used human adenovirus serotype 5 (AdHu5) as a vehicle to deliver the apoptin gene to specifically target gastric cancer in a recombinant gene delivery approach. AdHu5-apoptin is a safe and efficacious agent for the treatment of gastric cancer (GC). Our results show that apoptin protein encoded by the apoptin gene delivered via AdHu5 significantly inhibited the proliferation of SGC-7901 GC cells. Apoptin reduced the clone number by more than 75 % and resulted in cell cycle arrest in the G2/M phase for 48 % of the GC cells. It also induced cleavage of caspase-3, caspase-7, and caspase-9 in the GC cells. Intratumoral and peritumoral in vivo injection of AdHu5-apoptin significantly suppressed tumor growth and induced apoptosis in xenogeneic tumors in mice. The apoptosis induced by AdHu5-apoptin was independent of anti-apoptotic Bcl-2 and Bcl-xL proteins and the p53 pathway. Taken together, our results show that AdHu5-apoptin has great potential as a therapeutic agent for effective treatment of gastric tumors.
    No preview · Article · Jun 2013 · Tumor Biology
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