Báo cáo khoa học: Apoptosis and autophagy: Regulation of apoptosis by DNA damage signalling – roles of p53, p73 and HIPK2 ppt

MINIREVIEW
Apoptosis and autophagy: Regulation of apoptosis
by DNA damage signalling – roles of p53, p73 and HIPK2
Nadja Bitomsky and Thomas G. Hofmann
German Cancer Research Center (DKFZ), Cellular Senescence Group, DKFZ-ZMBH Alliance, Heidelberg, Germany
Introduction
Protection of the genome and maintenance of genomic
integrity following genotoxic stress is a crucial step in
counteracting tumorigenesis. Eukaryotic organisms,
from yeasts to humans, have developed efficient molec-
ular mechanisms to sense different types of DNA
damage. These different qualities of DNA damage
include DNA double-strand breaks (potently induced
by ionizing radiation), base modifications (e.g. induced
by alkyllating agents such as N-methyl-N-nitrosourea),
DNA crosslinks (e.g. induced by cisplatin) and stalling
of replication forks in the S phase of the cell cycle (e.g.
elicited by topoisomerase inhibitors such as camptothe-
cin and etoposide) [1]. Recent studies strongly suggest
an important tumour-suppressive role of the DNA
damage response (DDR) in humans: molecular markers
indicative for an active DDR, including site-specifically
Keywords
apoptosis; ataxia-telangiectasia mutated
(ATM); ataxia-telangiectasia mutated
and Rad3-related (ATR); DNA damage;
homeodomain-interacting protein
kinase 2 (HIPK2); nuclear bodies; p53;
p73; promyelocytic leukaemia
(PML)
Correspondence

T. G. Hofmann, German Cancer
Research Center, Cellular Senescence
Group A210, DKFZ-ZMBH Alliance, Im
Neuenheimer Feld 242, 69120 Heidelberg,
Germany
Fax: +49 (0)6221 424902
Tel: +49 (0)6221 424631
E-mail:
(Received 13 March 2009, revised 14
August 2009, accepted 27 August
2009)
doi:10.1111/j.1742-4658.2009.07331.x
Genomic stability is constantly threatened by DNA damage, caused by
numerous environmental and intrinsic sources, including radiation, chemi-
cals and oncogene expression. Consequently, cells have evolved a sophisti-
cated signal transduction network to sense DNA damage and to mount an
appropriate DNA damage response. Dysregulation of the DNA damage
response leads to genomic instability and cancer. Dependent on the cellular
background and extent of DNA damage, the DNA damage response trig-
gers cell cycle arrest and DNA repair, or in the case of irreparable damage,
inactivation of the cells by senescence or apoptosis. In this minireview, we
concentrate on the apoptotic response to DNA damage and signalling
pathways linked to the cell nucleus and nuclear bodies, with a particular
focus on the molecular players p53 and p73 and on the DNA damage-acti-
vated kinase homeodomain-interacting protein kinase 2 (HIPK2).
Abbreviations
ATM, ataxia-telangiectasia mutated; ATR, ATM and Rad3-related; Bak, Bcl-2 homologous antagonist ⁄ killer; CBP, CREB binding protein; Bax,
breakpoint cluster-2-associated x protein; Bcl-2, B-cell lymphoma 2; CtBP, C-terminal binding protein; DDR, DNA damage response; HDM2/
MDM2, human double minute/murine double minute 2; HIPK2, homeodomain-interacting protein kinase 2; IR, ionizing radiation; JNK, c-Jun
N-terminal kinase; NB, nuclear body; PML, promyelocytic leukaemia; Puma, p53-upregulated modulator of apoptosis; Siah1, seven in

absentia homologue 1; TGF-b, transforming growth factor-b; YAP1, Yes-associated protein 1.
6074 FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS
phosphorylated ataxia-telangiectasia mutated (ATM),
p53 and histone H2AX, have been found in early neo-
plastic lesions, but not in full-blown cancerous lesions
where the DDR is typically compromised [2].
The first signal transduction wave in response to
DNA damage is governed by rapid activation of
checkpoint kinases belonging to the family of phospha-
tidylinositol-3-OH-kinase-like protein kinases. The
currently best studied phosphatidylinositol-3-OH-
kinase-like protein kinase members are ATM, and
ATM and Rad3-related (ATR) [3–5]. Following DNA
double-strand breakage, cells primarily activate ATM,
which becomes recruited as an inactive dimer to the
DNA lesion by a sensor complex comprising the pro-
teins Mre11, Rad50 and NBS1 (i.e. the MRN com-
plex). The MRN complex and ATM locate at the
damaged DNA foci marked by phosphorylated histone
H2AX (c-H2AX) where ATM becomes fully activated
by autophosphorylation and phosphorylation-depen-
dently regulates numerous downstream mediators to
coordinate the DDR [1]. By contrast, the ATR kinase
mainly senses stress during DNA replication in the S
phase. Here, single-stranded DNA becomes opsonized
by the replication protein A, which recruits ATR via
the ATR-interacting protein to the DNA lesions and
orchestrates DNA-topoisomerase II beta-binding pro-
tein (TopBP1)-dependent ATR activation [6]. Both
ATM and ATR phosphorylate, and thereby activate,

further checkpoint kinases, including Chk1 and Chk2,
to transmit the damage signal to effector molecules
such as the tumour suppressor protein p53. Interest-
ingly, recent studies identified coordinated crosstalk
mechanisms between ATM and ATR, indicating that
their downstream signalling routes are actually not
running separately, as supposed initially [7–9].
Depending on the cellular context and the extent of
DNA damage – which determines whether or not dam-
age is reparable – the activated DDR can trigger differ-
ent cellular responses. Mild DNA damage is usually
handled through induction of cell cycle arrest through
the upregulation of cyclin-dependent kinase inhibitors,
such as p21, and subsequent repair of the lesions. To
achieve faithful repair, cells can engage numerous
sophisticated DNA-repair mechanisms [10]. In response
to irreparable DNA damage, the cellular response
switches towards induction of the senescence or cell-
death programme. The molecular basis underlying the
decision making is currently subject of intense investi-
gation. Although the cellular background appears to
play a major role, as for instance fibroblasts prefer to
undergo senescence whereas thymocytes favour cell
death induction, the molecular switch remains largely
unclear. In this minireview we focus on the apoptotic
signalling routes of the DDR regulated mainly from
the cell nucleus and the key molecules p53, p73 and
homeodomain-interacting protein kinase 2 (HIPK2).
Multiple functions of p53 in DNA
damage-induced apoptosis

The tumour suppressor and transcription factor p53 is
a major regulator of the cellular defence against neo-
plastic transformation and cancer development. Up to
50% of all human tumours show mutations in the p53
gene, which result in the expression of functionally
inactive p53 or in complete loss of p53 expression. In
tumours expressing wild-type p53, its ability to repress
cancer development often becomes functionally inacti-
vated via the upregulation of critical negative regula-
tors of p53, including its ubiquitin ligase
HDM2 ⁄ MDM2 [11,12]. Polyubiquitination and subse-
quent proteasomal degradation are major efforts to
keep the p53 protein levels low in healthy cells [12]. In
general, p53 activity in response to DNA damage is
tightly controlled by its post-translational modification
status and that of its E3 ubiquitin ligases, in particular
through site-specific phosphorylation, acetylation and
ubiquitination [13,14]. Consistently, p53 is phosphory-
lated by numerous DNA damage-activated protein
kinases, including ATM, ATR, Chk1, Chk2 and
HIPK2 [14].
Nuclear p53: regulation by HIPK2 and
promyelocytic leukemia protein
nuclear bodies
Upon DNA damage, as triggered by UV light, ionizing
radiation (IR) and chemotherapeutic drug treatment,
p53 is stabilized and activated. DNA damage-induced
p53 stabilization and activation is mediated primarily
by inactivating the negative regulatory effect of the
p53 ubiquitin ligase HDM2 (MDM2 in mouse). In this

context, ATM- and ATR-mediated phosphorylation of
p53 at Ser15, and Chk1 ⁄ 2-mediated phosphorylation
at Ser20, as well as phosphorylation of MDM2 at
Ser395 by ATM, are critical events (Fig. 1) [13,14].
Depending on the extent of damage, p53 induces
transcription of different sets of target genes, leading
to cell cycle arrest, apoptosis or cellular senescence.
Interestingly, nuclear p53 has been demonstrated to be
recruited to promyelocytic leukemia protein (PML)
nuclear bodies (NBs) through interaction with the
tumour suppressor PML in response to IR-induced
apoptosis as well as oncogene-induced senescence after
expression of oncogenic variant of the small GTPase
Ras [15,16]. In this context, the PML isoform PML IV
N. Bitomsky & T. G. Hofmann p53, p73 and HIPK2 in apoptosis
FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS 6075
has been shown to act as a cofactor in p53-dependent
transcription. In addition, a further critical p53 regula-
tor, the acetyltransferase CREB binding protein (CBP),
is corecruited by PML to PML-NBs, which results in
increased p53 Lys382 acetylation and p53 activation
[15]. Consequently, PML-NBs are envisioned as
macromolecular multiprotein complexes with a critical
role in regulating cell death, senescence and differentia-
tion [17–19].
Presumably the most prominent mark in priming the
apoptotic activity of p53 is phosphorylation at Ser46
[20]. This phosphorylation mark is clearly associated
with severe DNA damage (elicited by UV light, IR,
adriamycin ⁄ doxorubicin or cisplatin) and has been

shown to drive the expression of apoptotic p53 target
genes, such as p53-upregulated modulator of apoptosis
(Puma), p53 regulated apoptosis-inducing protein 1
(p53AIP1) and breakpoint cluster-2-associated x pro-
tein (Bax) [11]. Subsequently, HIPK2, a conserved
Ser ⁄ Thr kinase predominantly localizing to NBs, has
been identified as the p53 Ser46 kinase [21,22]. HIPK2
phosphorylates p53 at Ser46 in response to UV light,
IR and treatment with adriamycin and cisplatin
[21–25]. Upon severe damage induced by UV light,
HIPK2 binds p53 and is recruited to PML-NBs in a
PML-dependent manner [21,22,26]. Consistently, PML
Fig. 1. Regulation of DNA damage-induced cell death by p53 and HIPK2. Genotoxic stress-induced DNA damage facilitates activation of the
DNA damage-activated protein kinases ATM and ATR. ATR and ATM in turn phosphorylation-dependently activate the downstream check-
point kinases Chk1 and Chk2, respectively, and the tumour suppressor p53. Furthermore, ATM and ATR mediate HIPK2 activation by facili-
tating its stabilization through phosphorylation of the HIPK2 ubiquitin ligase, Siah1, which facilitates disruption of the HIPK2–Siah1 complex.
Once stabilized and activated, HIPK2 can bind p53 and is recruited to PML-NBs via interacting with PML. HIPK2 phosphorylates p53 at
Ser46 and stimulates pro-apoptotic p53 target genes, including caspase-6 (Casp-6) and Pml. In an autoregulatory feedback mechanism, cas-
pase-6 potentiates HIPK2 activity by removing its C-terminal autoinhibitory domain. In addition, HIPK2, and also JNK, can induce cell death in
a p53-independent manner by phosphorylation-dependent degradation of the anti-apoptotic corepressor CtBP. Beyond its nuclear function,
p53 is shuttled into the cytoplasm in response to DNA damage where it targets the mitochondria by activation of Bax and Bak, resulting in
the release of pro-apoptotic factors and apoptotic cell death.
p53, p73 and HIPK2 in apoptosis N. Bitomsky & T. G. Hofmann
6076 FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS
is a critical cofactor for efficient HIPK2-driven p53
Ser46 phosphorylation upon treatment with adriamycin
[26,27]. In addition, HIPK2 also interacts with the
CBP acetyltransferase and colocalizes with CBP and
p53 at PML-NBs [21]. HIPK2-mediated p53 Ser46
phosphorylation enhances CBP-mediated p53 acetyla-

tion at Lys382, which leads to full transcriptional
activation of p53, thereby potentiating the expression
of pro-apoptotic target genes [21]. The apoptotic signal
can be additionally boosted through the p53-dependent
upregulation of PML expression. This positive-feed-
back loop leads to PML accumulation and potentiation
of the apoptotic signal [29].
HIPK2 regulation
Tumour suppressor p53 not only serves as a critical
HIPK2 substrate, but also potentiates HIPK2 activity
by transcriptional upregulation of caspase-6 in response
to adriamycin-induced apoptosis. Caspase-6 cuts off the
C-terminal negative-regulatory domain of HIPK2,
which results in a hyperactivated truncated HIPK2 iso-
form and increased p53 Ser46 phosphorylation and
apoptosis induction [25]. As p53 can also negatively reg-
ulate HIPK2 stability (see below) in response to damage
by sublethal concentrations of adriamycin [30], or
during recovery from reparable UV damage [31], p53
shows an apparent split personality in regard to HIPK2
regulation. The switch between these opposing p53 func-
tions appears to be regulated by the extent of DNA
damage, which in turn determines whether DNA lesions
can, or cannot, be repaired. As unrepaired DNA dam-
age is characterized by constant activity of the DNA
damage checkpoint kinase ATM and ⁄ or ATR, continu-
ous ATM ⁄ ATR activity may represent a key regulatory
switch in apoptosis induction through facilitating
prolonged HIPK2 stabilization and activation [31].
Similarly to p53, HIPK2 is an unstable protein in

unstressed cells because it is constantly degraded
through the ubiquitin–proteasome system. HIPK2 pro-
tein levels are kept low in unstressed cells by polyubiq-
uitination, which is carried out by the E3 ubiquitin
ligases seven in absentia homolog-1 (Siah1), seven in
absentia homolog-2 (Siah2) and WD-repeat and sup-
pressor of cytokine signalling (SOCS) box-containing-1
(WSB1) [31,32]. In response to treatment with UV
light and adriamycin, HIPK2 degradation by Siah1
and WSB-1 is released, resulting in the accumulation
of HIPK2. In this context, Siah1 becomes phosphory-
lated by ATM and ATR at Ser19, which leads to dis-
ruption of the HIPK2–Siah1 complex, thus allowing
HIPK2 stabilization and activation [31]. Remarkably,
Fig. 2. Regulation of DNA damage-induced
cell death by the p73 pathway. In response
to DNA damage, JNK phosphorylation-
dependently liberates the tyrosine kinase
c-Abl from its cyctoplasmic anchor protein
14-3-3f. Subsequently, c-Abl is translocated
into the nucleus where it becomes fully
activated through site-specific phosphoryla-
tion by ATM. c-Abl regulates p73-induced
pro-apoptotic target gene expression by
direct phosphorylation of p73 at Tyr99 and
through phosphorylating YAP1, the cofactor
of p73. YAP1, in addition, stabilizes p73 by
protecting it against ubiquitination by the E3
ubiquitin ligase Itch. Furthermore, YAP1
interacts physically with PML, which in turn

stabilizes YAP1 through SUMOylation. p73,
YAP1, PML and p300 form a potent plat-
form for transcriptional activation of critical
pro-apoptotic target genes. As PML is a p73
target gene, p73 activates a positive
feedback loop, further stimulating p73
activity.
N. Bitomsky & T. G. Hofmann p53, p73 and HIPK2 in apoptosis
FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS 6077
during cellular recovery from reparable UV damage,
accumulated HIPK2 is rapidly removed through
Siah1-mediated degradation, which inhibits cell death
[31]. Although HIPK2 is stabilized upon repairable
UV damage, p53 Ser46 phosphorylation remains
absent under these conditions [31]. The function and
the substrates of HIPK2, in response to repairable
damage, remain elusive; however, it is tempting to
speculate that HIPK2 is also implicated in nonapop-
totic pathways, such as coordination of DNA repair.
Furthermore, HIPK2 also appears to be degraded
through involvement of the SCF
Fbx3
E3 ubiquitin
ligase complex, and the degradation is inhibited by
PML, thereby resulting in increased p53 transcriptional
activity [33]. Remarkably, overexpression of the acute
promyelocytic leukemia (APL)-causing chromosomal
translocation-derived fusion protein PML–RARa dras-
tically destabilizes HIPK2 [33]. How these pathways
affect HIPK2 activity and p53 Ser46 phosphorylation

in response to DNA damage remains to be elucidated.
Another means to keep the proapoptotic activity of
HIPK2 in check is sequestration to the cytoplasm.
Overexpression of the high-mobility group protein A1
(HMGA1) oncoprotein relocalizes HIPK2 into the
cytoplasm and inhibits p53 Ser46 phosphorylation
upon UV light-induced damage in HCT116 cells [34].
Collectively, these findings indicate that the apoptotic
function of HIPK2 is vulnerable and can be dysregu-
lated at different levels.
Cytoplasmic p53: targeting the
mitochondria
It is well established that p53 acts as a transcription
factor primarily located to the nucleus. However, there
is emerging experimental evidence that p53 has
additional functions in apoptosis induction in the cyto-
plasm (see Fig. 1). In the mid-1990s it was discovered
that p53 is capable of inducing apoptosis upon expo-
sure to UV light, not only by transcription-dependent
mechanisms but also by transcription-independent
mechanisms [35]. Remarkably, it has been demon-
strated that transactivation activity-deficient p53 is still
capable of inducing programmed cell death through
the intrinsic pathway in response to ectopic p53
expression [36], and that recombinant p53 is capable
of triggering mitochondrial membrane permeabiliza-
tion in cell-free systems [37,38]. Later on, p53 has been
reported to translocate to the cytoplasm in response to
numerous stress signals, including DNA damage,
hypoxia and oncogene expression, where it drives

mitochondrial outer membrane permeabilization and
caspase activation [39,40].
Transcription-independent cytoplasmic apoptosis-
inducing functions of p53 are carried out by regulating
the activity of Bcl-2 family members in IR-treated and
camptothecin-treated cells. p53 interacts with both
pro-apoptotic and anti-apoptotic members of the Bcl-2
protein family. p53 is able to interact (via its core
DNA-binding domain) with the anti-apoptotic mole-
cules B-cell lymphoma-extra large (Bcl-x
L
) and Bcl-2
[40]. Remarkably, nuclear p53-dependent upregulation
of Puma results in increased cytoplasmic Puma levels,
which facilitate liberation of cytoplasmic p53 from
Bcl-x
L
, thus contributing directly to the mitochondrial
cell-death route in response to UV light-induced dam-
age [41]. Additionally, p53 also interacts with the pro-
apoptotic Bcl-2 homologous antagonist ⁄ killer (Bak)
protein. This interaction seems to liberate Bak from its
inhibitor protein, myeloid cell leukaemia 1 (Mcl-1), to
induce Bak oligomerization, pore formation and subse-
quent mitochondrial outer membrane permeabilization
after treatment with adriamycin [42]. In the case of the
pro-apoptotic Bax protein, no physical interaction of
p53 and Bax was observed, although p53 can induce
Bax oligmerization and cytochrome c release [41,43].
So, how does p53 receive its signal for cytoplasmic

and mitochondrial translocation in respect of lacking a
classical mitochondrial translocation motif? As p53
post-translational modification is the most prominent
event to regulate its function in response to DNA
damage, it seemed to be promising to search for altera-
tions between cytoplasmic p53 and nuclear p53. How-
ever, the first analyses of the phosphorylation and
acetylation patterns of active cytoplasmic p53 failed to
detect any major differences between nuclear and cyto-
plasmic p53 in IR-damaged cells [44]. Interestingly,
ubiquitin ligase HDM2 ⁄ MDM2 has been previously
demonstrated to regulate p53 also by mono-ubiquitina-
tion. Mono-ubiquitination is not sufficient to target
p53 for proteasomal degradation. Consistently, it has
been shown that p53 mono-ubiquitination within its
C-terminus indeed assists its nucleocytoplasmic trans-
location [45–47]. A recently discovered novel p53 E3
ubiquitin ligase, called MSL2, which, unlike MDM2,
does not regulate p53 turnover, mediates p53 mono-
ubiquitination at Lys351 and Lys357, and MDM2-
independent nucleo-cytoplasmic translocation upon
treatment with etoposide [48]. Whether this simply
leads to p53 inactivation by removing it from its target
gene promotors, or contributes to the apoptotic func-
tion of p53 in the cytoplasm, remains to be investi-
gated. In addition, it is also conceivable that MSL2
contributes to the tumour suppressor function of p53
by inhibition of autophagy (self-eating), a recently
uncovered novel function for cytoplasmic (but not
p53, p73 and HIPK2 in apoptosis N. Bitomsky & T. G. Hofmann

6078 FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS
nuclear) p53 [49]. Through promoting catabolic reac-
tions, autophagy facilitates the maintenance of high
ATP levels and survival in response to nutrient deple-
tion, hypoxia and the DNA damage-inducing drug
etoposide [50,51]. Depletion, inhibition or loss of p53
leads to the induction of autophagy and increases cell
survival in response to stress [49].
p73 function in DNA damage-induced
cell death
Another key molecule critically involved in DNA dam-
age-induced cell death signalling is the p53-related
tumour suppressor and transcription factor p73 (see
Fig. 2). Similarly to p53, p73 is an unstable molecule
and is expressed in various isoforms [52]. In unstressed
cells, p73 forms a complex with the E3 ubiquitin ligase
Itch, which marks it for degradation by the ubiquitin–
proteasome system. Upon DNA damage (by UV
irradiation or the DNA-damaging chemotherapeutics
adriamycin, etoposide and cisplatin), the levels of Itch
become reduced and allow the accumulation of p73
[53]. p73 displays functions in apoptosis induction, and
many of its pro-apoptotic target genes indeed overlap
with those of p53, for example Puma, caspase-6 or
CD95 [54]. Like p53, p73 is also recruited to PML-
NBs upon DNA damage, like other key players in
DNA damage-induced cell death signalling (see below).
Moreover, p73 also binds to HIPK2, and both factors
colocalize in NBs [55]. Although HIPK2 has been
shown to drive p73-dependent transcription of an arti-

ficial reporter construct, the physiological role of the
HIPK2-p73 interaction is currently unclear [55].
Whether there exists a similar activation loop between
p73 and HIPK2, as previously described for HIPK2
and p53, also remains to be clarified.
Post-translational modifications of p73 by acetyla-
tion through p300 and by phosphorylation by the
DNA damage-activated, nonreceptor tyrosine kinase
c-Abl were found to be crucial for transactivating its
pro-apoptotic target genes after treatment with adria-
mycin [56]. In undamaged cells c-Abl is sequestered to
the cytoplasm by its interaction with 14-3-3f, which
becomes phosphorylated by c-Jun N-terminal kinase
(JNK) upon damage caused by treatment with
adriamycin, thus triggering the release of 14-3-3f and
translocation of c-Abl to the nucleus [57].
Once translocated to the nucleus, c-Abl is phosphor-
ylated by ATM at Ser465 after IR [58,59]. Phosphory-
lation at Ser465 leads to subsequent activation of
c-Abl and facilitates p73 transcriptional activation
through c-Abl-mediated phosphorylation of p73 at
Tyr99 [60]. In addition, a key regulator of p73 activity,
Yes-associated protein 1 (YAP1), also becomes phos-
phorylated by c-Abl. This phosphorylation mark is
essential to drive p73-mediated apoptosis by focussing
the co-activator function of YAP1 on p73 in cells
exposed to IR or cisplatin [61]. YAP1 is also critical to
protect p73 from proteasomal degradation upon dam-
age caused by treatment with cisplatin by competing
with its E3 ubiquitin ligase Itch for p73 binding.

Accordingly, YAP1 downregulation by RNA interfer-
ence decreases induction of apoptosis in p53-deficient,
p73-proficient H1299 cells following treatment with
cisplatin [62].
Recently, it has been demonstrated that PML is also
a direct target gene of the p73–YAP1 complex in
response to treatment with cisplatin. Moreover, PML
also interacts physically with YAP1 and promotes
YAP1 stabilization through facilitating modification
of YAP1 with the small ubiquitin-like modifier 1
(SUMO-1) [63]. Thus, p73 – similarly to what has been
previously reported for p53 [29] – further enhances its
pro-apoptotic activity through an autoregulatory feed-
back loop.
Interestingly, p73 becomes processed by caspases,
and the truncated versions of the p73 protein localize
at mitochondria and augment apoptosis induction in
response to treatment with the death receptor ligand
TRAIL (tumour necrosis factor related apoptosis indu-
cing ligand) [64]. However, whether p73 exerts a simi-
lar function after DNA damage-induced cell death is
currently unclear. It will be interesting to see whether
or not p73 has cytoplasmic functions similar to those
of p53. In summary, p73 apparently shares numerous
– but probably not all – regulatory principles and
effector pathways with its famous brother p53.
HIPK2 in p53-independent apoptosis
routes
In addition to its fundamental role in p53-driven apop-
tosis, HIPK2 also facilitates DNA damage-induced cell

death in the absence of p53. In UV light-damaged
cells, HIPK2 phosphorylation-dependently targets the
anti-apoptotic transcriptional corepressor C-terminal
binding protein (CtBP) for proteasomal destruction
(see Fig. 1) [65]. CtBP plays a critical role in repressing
pro-apoptic target genes, such as Bax [65,66]. After
treatment with UV light and cisplatin, HIPK2, and
also the stress-activated protein kinase JNK1, phos-
phorylate CtBP at Ser422 and thereby mark it for
degradation [67]. In addition, HIPK2 was also shown
to activate the JNK signalling pathway in hepatoma
cells after treatment with transforming growth factor-b
(TGF-b), making it likely that HIPK2 also contributes
N. Bitomsky & T. G. Hofmann p53, p73 and HIPK2 in apoptosis
FEBS Journal 276 (2009) 6074–6083 ª 2009 The Authors Journal compilation ª 2009 FEBS 6079
to p53-independent cell death in response to DNA
damage, both directly and via the JNK signalling path-
way [68,69], which is also capable of stimulating cell
death via the mitochondrial pathway [70]. Interest-
ingly, a recent report indicates that TGF-b mediates
activation of ATM in 293 cells, which results in p53
Ser15 phosphorylation [71]. Therefore, it will be inter-
esting to study whether HIPK2 also plays a role in
such a cell death-inducing setting.
Even though PML is a direct pro-apoptotic p53 and
p73 target gene in response to apoptotic stimuli, an
additional regulatory principle of PML regulation was
recently demonstrated: HIPK2 is able to stabilize PML
in a p53-independent manner following treatment with
doxorubicin by phosphorylating Ser8 and Ser38 [72].

PML phosphorylation is accompanied by an increased
SUMOylation and stability of PML, suggesting an
additional role of HIPK2 in regulating DNA damage-
induced cell death.
Collectively, these findings indicate that HIPK2 is
involved in DNA damage-induced cell death signalling
by using different downstream signalling routes involv-
ing p53, CtBP, PML and JNK.
Concluding remarks
Cell death activation from the nucleus is an important
regulatory principle in regulating the apoptotic
response to DNA damage. In the past decade, numer-
ous pathways and molecular players responsible for
controlling DNA damage-induced apoptosis have been
identified, including sensors, mediators and execution-
ers. In particular, tumour suppressor PML and its
associated PML-NB turned out to be a critical signal-
ling hub in coordinating the apoptotic arm of the
DDR. PML-NBs functionally cooperate with pivotal
apoptotic molecules, including p53, p73 and HIPK2,
by regulating their localization and pro-apoptotic func-
tion. However, much needs to be learned about poten-
tial crosstalk between these signalling pathways and
the molecular mechanisms underlying their regulation.
An additional pressing question is whether these sig-
nalling pathways operate in parallel in a given cell or
whether they act in a cell type- or tissue-restricted
manner. Last, but not least, the tumour-suppressive
activities of these players make it an attractive
approach to systematically mine their pathways for

novel targets in anticancer drug discovery.
Acknowledgements
We want to apologize to all the authors who made
important contributions to the field that could not be
cited here because of space restrictions. Work in our
laboratory is funded by the Landesstiftung foundation
of the State of Baden-Wu
¨
rttemberg, the German
Research Foundation, the German Cancer Aid, the
DKFZ-ZMBH Alliance, the Network Ageing Research
in Heidelberg and the Helmholtz Association.
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