Biochimica et Biophysica Acta 1793 (2009) 13351342

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Biochimica et Biophysica Acta

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a m c r

Invited review

Apoptin, a tumor-selective killer

Marek Los a,b,,1, Soumya Panigrahi c,1, Iran Rashedi d, Sanat Mandal e, Joerg Stetefeld f,
Frank Essmann a, Klaus Schulze-Osthoff a
a
Interfaculty Institute for Biochemistry, University of Tbingen, D-72076 Tbingen, Germany
b
BioApplications Enterprises, Winnipeg, MB, Canada R2V 2N6
c
Department of Immunology, Lerner Research Institute/NE40, 9500 Euclid Avenue, Cleveland, OH 44195, USA
d
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada M5G 2M9
e
Faculty of Medicine, Memorial University of Newfoundland, St. John's, NL, Canada A1B 3V6
f
Department of Chemistry, University of Manitoba, Winnipeg, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specically kills
Received 20 February 2009 tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial
Received in revised form 5 April 2009 for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells
Accepted 7 April 2009
it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its
Available online 15 April 2009
phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt
Keywords:
and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin
Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-
Apoptosis promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced
CDK2 from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In
Nur77 this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing
Tumor insights for the development of novel tumor-selective anticancer drugs.
2009 Elsevier B.V. All rights reserved.

1. Introduction Sequences in the 5-nontranscribed region of the CAV genome

Apoptin is a small apoptosis-inducing protein derived from
chicken anemia virus (CAV), which belongs to the Gyrovirus genus
and Circoviridae family and contains a single, minus-strand circular
DNA. CAV infects young chickens with the highest infectivity and
mortality on the hatch day, whereas chickens become completely
resistant to CAV-related death after about two weeks of hatching [1].
Hematopoietic cells in the bone marrow and T-cell precursors in the
thymus are the major targets for CAV infection. The early infection of
hematopoietic precursors results in the reduction of erythroid
precursor cells and megakaryocytes. This eventually leads to anemia
and thrombocytopenia, causing intramuscular hemorrhage and death.
The infection can be propagated within the ock both vertically and
horizontally. Interestingly, in chickens carrying tumors induced by
Rous sarcoma virus the intra-tumoral expression of apoptin causes
tumor regression [2]. Thus, the coevolution of CAV and the host may
offer benets to latently infected chickens.
function as promoter/enhancers [3]. The CAV genome contains three
partially overlapping open reading frames which produce a poly-
cistronic unspliced RNA encoding three different proteins [1,46]. All
proteins are expressed in CAV-infected cells [5,6]. VP1 is a 51-kDa
capsid protein and VP2 a 24-kDa dual specicity phosphatase.
Importantly, the induction of apoptosis by CAV has been attributed
to VP3, a 14-kDa non-structural protein, which is therefore also
referred to as apoptin [5,7,8]. Apoptin alone can induce apoptosis in a
broad range of transformed and cancer cells but not in non-
transformed or primary cells [9,10]. Recent data link apoptin's
complex mode of action to various cellular proteins, which play
prominent roles in both cell proliferation and cell death. It was
reported that late in infection, three other CAV mRNAs are synthesized
due to splicing events [11]. One of them encodes a putative truncated
apoptin protein consisting of the rst 58 amino acids only. The
signicance of these additional putative gene products for cell death
and other CAV manifestations is still unclear.

2. Apoptin sequence and structure

Corresponding author. University of Tbingen, Interfaculty Institute for

Apoptin is composed of 121 amino acids, and does not show
Biochemistry, Hoppe-Seyler-Str. 4, D-72076 Tbingen, Germany. Tel.: +49 7071
2974159; fax: +49 7071 295565.
signicant sequence homology with known cellular proteins [1]. The
E-mail address: mjelos@gmail.com (M. Los). C-terminal end of apoptin contains a bipartite nuclear localization
1
These authors contributed equally. sequence (NLS): NLS1 spans amino acids 8288, and NLS2 residues

0167-4889/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2009.04.002
1336 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342
Fig. 1. The primary structure of apoptin. The key domains and important sequences are indicated. Lower panel shows the sequence of apoptin (UniProtKB/Swiss-Prot entry P54094).
Amino acids marked in blue or green represent the indicated domains in the upper panel. See text for details.
111121 [12,13]. In addition, apoptin contains a putative nuclear
export sequence (NES) at residues 97105 (Fig. 1). These recognition
sequences drive the shuttling of apoptin in and out of the nucleus.
Apoptin also harbors a short hydrophobic leucine-rich stretch (aa 33
46) that is required for self-association as well as binding of
promyelocytic leukemia protein and other interaction partners
[9,14]. It is interesting to note that the biologically active form of
recombinant apoptin can form noncovalently attached globular
multimers consisting of 3040 monomers [15]. The formation of
this multimeric complex appears to be facilitated through the
interaction of the proline-rich hydrophobic regions in the N-terminus
(aa 169) of each monomer [16]. The C-terminal tip of each
monomer, on the other hand, contains a NLS with an accessible
phosphorylation site (Thr-108), which allows for its interaction with
other proteins and for modication by kinases. Microinjection of
apoptin multimers into the cytoplasm of tumor cells reveals that
apoptin complexes translocate to the nucleus resulting in apoptotic
death [15]. This conrms that apoptin multimers retain biological
activity. Unfortunately, a crystal structure of apoptin or its multimeric
complex is not available.
3. Induction of apoptosis in tumor cells by apoptin
Apoptin has been reported to induce the selective death of cell
lines derived from diverse human tumors including melanoma,
hepatoma, lymphoma, cholangiocarcinoma, colon carcinoma, breast
and lung cancer and others [14,17]. Indeed, to date more than 70
human tumor cell lines were shown to be susceptible to apoptin,
whereas apoptin does not induce apoptosis in a variety of normal
cells, including human endothelial cells, hepatocytes and hemato-
poietic stem cells. Thus, the sensitivity to apoptin presumably
depends on alterations related to the transformed status of the cell
[14]. Apoptin induces apoptosis not only in tumor cells, but also in
SV40-transformed broblasts or UV-irradiated cells from indivi-
duals with hereditary cancer-prone syndromes [18,19]. There is
only one single report of toxicity towards some non-cancerous
cells [20]. Nonetheless, this study provided cell death data only for
a fetal cell type (i.e. secondary cultures of human lung broblasts)
and not for other non-transformed cell types. This raises the
possibility that apoptin selectively affects normal cells at embryo-
nic stage, similar to apoptin's toxicity in young but not adult
chickens. However, the safety of apoptin is underlined by the fact
that continuous expression of apoptin under the H2-Kb promoter
in transgenic mice does not interfere with lymphocyte develop-
ment and proliferation [21]. This is in contrast to the deleterious
effects on the immune system observed in young chickens upon
CAV infection.
In addition to reports that apoptin effectively induces tumor-
specic apoptosis in animal models, intra-tumoral injection of
recombinant apoptin into nude mice with xenografted human
hepatoma cells reduces tumor growth and improves tumor regression
within a week of administration [22]. There are additional reports that
apoptin can enhance tumor size reduction. In fact, when apoptin is
used in combination with other chemotherapeutics, tumor regression
is more dramatic compared to conditions when apoptin or che-
motherapeutic drugs are administered alone [7,24].
4. Mechanisms of apoptin-mediated cell death
The mechanisms of cell death induced by apoptin are not clearly
understood, although there is a consensus on the existence of certain
molecular events. One of the key properties of apoptin is its ability to
induce tumor-specic cell apoptosis independently of p53 [25]. Thus,
apoptin is similarly effective in killing tumor cells that are p53-
decient or either express wildtype or mutant p53.
Apoptosis is typically mediated by intracellular cysteine proteases,
called caspases, that function as initiators and executioners of the
apoptotic process [26,27]. The activation of caspases is accomplished
by two major signaling routes: the extrinsic death receptor and the
intrinsic mitochondrial pathway. In the extrinsic pathway, ligation of
death receptors by their cognate ligands triggers the recruitment of
the adaptor FADD and initiator caspase-8 into a death-inducing
signaling complex. The mitochondrial pathway is initiated by the
release of cytochrome c into the cytosol, a process that is tightly
regulated by pro- and anti-apoptotic proteins of the Bcl-2 family. Once
released, cytochrome c binds to the adapter protein Apaf-1, which
enables the activation of the initiator caspase-9 in a high-molecular-
weight complex, called the apoptosome. Both pathways then
converge at the activation of downstream effector caspases, leading
to cell death via the cleavage of several cellular substrates [2628].
It is now well established that apoptin expression results in the
activation of caspases [29]. Apoptin-mediated cell death is however
independent of death receptors, as cells decient in FADD or caspase-
8, the key regulators of the extrinsic apoptotic pathway [30], remain
sensitive to apoptin. Moreover, blocking of death receptor CD95 with
neutralizing antibodies does not affect apoptin-induced cell death. In
contrast to components of the death receptor pathway, apoptin-
mediated apoptosis is strongly inuenced by regulators of the
mitochondrial pathway, as for instance a deciency of Apaf-1 strongly
protects tumor cells. Furthermore, apoptin triggers cytochrome c
release and the activation of caspase-9 [31].
Interestingly, the role of anti-apoptotic Bcl-2 in apoptin-mediated
cell death is still a matter of debate. Some research groups reported
that apoptin-mediated cell death is independent of the Bcl-2 status,
 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342 1337

and that co-expression of apoptin and Bcl-2 even accelerates cell
death [3234]. In contrast, Burek et al. [31] showed that both Bcl-2 and
Bcl-xL inhibit apoptosis triggered by apoptin in several tumor cell
lines. Notably, the proapoptotic Bcl-2 member Bax sensitizes cells to
apoptin-induced apoptosis, whereas cells devoid of Bax are strongly
protected [31]. Thus, at least in several cell types, apoptin-induced cell
death is regulated by pro- and anti-apoptotic Bcl-2 proteins and
involves the release of cytochrome c and activation of effector
caspases. The apparent controversy as to the role of Bcl-2 proteins
may arise from the differential involvement of Nur77, a member of the
steroid receptor family, in apoptin-induced cell death. It has been
shown that Nur77 can bind to Bcl-2 and change its properties from an
anti-apoptotic to a proapoptotic molecule, leading to the activation of
mitochondrial death pathway [35]. Because Nur77 is expressed in
different cell types at very varying levels, this might explain the
opposite effects of Bcl-2 on apoptin-induced cell death.
Maddika et al. [30] indeed proposed a role of Nur77 in apoptin-
mediated apoptosis (Fig. 2). Phosphorylated Nur77 appears to
transmit apoptotic signal from the nucleus to mitochondria, as it
was shuttled from the nucleus to the cytoplasm upon transient
expression of apoptin. Moreover, RNAi-mediated downregulation of
Nur77 strongly protected against apoptin-triggered apoptosis [30]. It
is presently unclear which kinase phosphorylates Nur77, however, Akt
that is strongly activated upon apoptin treatment is a viable candidate.
Upon transfer to cytoplasm, Nur77 may directly or indirectly cause
cytochrome c release and activation of the apoptosome-dependent
death pathway.
5. Apoptin-interacting molecules
The cellular localization of apoptin plays crucial role for its
selective toxicity [12,13]. However, the targeted translocation of
apoptin into the nucleus is not sufcient to induce apoptosis in
primary cells [12]. This nding suggests that for its tumor-specic
toxicity apoptin presumably requires additional interaction partners
that activate specic signaling pathways in cancer cells. Indeed, a
number of molecules have been described to interact with apoptin
and appear to be important for the nuclear localization of apoptin or
its tumor-selective cytotoxicity (Table 1).
A recent report [36] provides evidence that apoptin interacts with
peptidyl-prolyl isomerase-like 3 (Ppil3). Ppil3 belongs to the
cyclophilin family and shares 46% identity to cyclophilin A. Members
of this family catalyze the cistrans isomerization of peptidyl-prolyl
imidic bonds and play a role in mitochondrial maintenance, cell cycle
progression and apoptosis. The study showed that siRNA-mediated
depletion of Pipl3 leads to the migration of apoptin from cytoplasm to

Fig. 2. Proposed mechanism of action of apoptin in tumor cells. Upon heterologous expression or administration of cell-permeable constructs, apoptin associates with the regulatory
subunit of PI3K resulting in its constitutive activation, which leads to the sustained activation and nuclear translocation of Akt. The nuclear Akt activates CDK2 by both direct
phosphorylation and indirect mechanisms including the proteasome-dependent degradation of p27Kip1. The activated CDK2 phosphorylates apoptin at Thr-108 and thereby enforces
its nuclear accumulation in cancer cells. In the nucleus, apoptin can associate with different interaction partners including DEDAF, Nmi, APC/C and PML. A still unknown apoptin-
triggered event results in the phosphorylation of Nur77 and its nuclear export. In the cytoplasm Nur77 is known to modulate Bcl-2 proteins, for instance by converting Bcl-2 from an
anti-apoptotic to a proapoptotic molecule. This and additional events might induce apoptin-triggered apoptosis by the mitochondrial pathway of caspase activation.
1338 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342

the nucleus. Interestingly, proline-109 in apoptin was essential for the
Ppil3-dependent cytoplasmic localization of apoptin [36]. These
ndings shed a new light on the regulation of apoptin's cellular
localization.
Hippi, a protein interacting with huntington-interacting protein 1
(Hip 1), is also a binding partner for apoptin [37]. The HippiHip1
complex can recruit caspase-8 and regulates apoptotic cell demise in
Huntington's disease. In non-cancerous cells, apoptin colocalizes with
Hippi in the cytoplasm, whereas in tumor cells apoptin migrates to the
nucleus while Hippi remains in the cytoplasm [37]. The C-terminus
region of apoptin is vital for the interaction with Hippi. As mentioned
above, however, apoptin-mediated cell death is not compromised by a
genetic deciency of caspase-8 [30]. Nevertheless, the results suggest
that association of apoptin with cytoplasmic Hippi or Ppil3 may play a
role in sequestering apoptin in cytoplasm and preventing apoptosis of
normal primary cells.
Employing a yeast two-hybrid approach with apoptin as bait, N-
Myc-interacting protein (Nmi) has been found to associate with
apoptin [38]. It is an interferon-inducible protein that can interact
with transcription factors of Myc family. Nmi-like proteins enhance
the activity of Myc proteins that are involved in cell proliferation,
differentiation as well as in apoptosis. The interaction of apoptin and
Nmi has been proposed to account for the tumor specicity of
apoptin's cytotoxicity, because Nmi is only expressed at low levels in
normal tissues but highly upregulated in leukemia cell lines. Apoptin
has been hypothesized to switch the Nmi/Myc-mediated transform-
ing activity to an apoptotic activity. However, no functional analyses
have been provided as yet, and the restricted expression pattern of
Nmi does not explain the broad antitumor activity of apoptin.
Teodoro et al. [39] have shown that apoptin associates with APC1, a
subunit of anaphase-promoting complex/cyclosome (APC/C) and
thereby inhibits the ubiquitin ligase function of APC/C. This interac-
tion is present in cancer cells but is absent in non-cancerous cells,
which suggests another possible mechanism for the tumor-selective
action of apoptin. Overexpression of apoptin or RNAi-mediated
depletion of APC1 subunit induces apoptosis in p53-null tumor cells,
suggesting that apoptin may interfere with the APC/C pathway
through binding to APC1. These experiments provide evidence that
APC/C may serve as a potential target for the development of novel
anticancer therapies.
Due to its basic structure apoptin binds to naked DNA, preferen-
tially to heterochromatin. Biophysical studies revealed that bacterially
expressed apoptin forms active protein multimers consisting of 3040
monomers [15]. These apoptin multimers in turn can bind to DNA and
cooperatively form distinct superstructures consisting of 20 apoptin
multimers and approximately 3 kb DNA [16]. Whether apoptin's
binding to DNA results in transcriptional events is unknown. The
nding that apoptin does neither act as a transcriptional repressor, at
least in simple reporter gene assays, nor requires de novo macro-
molecular synthesis for apoptosis induction would, however, argue
against this possibility [12].
Apoptin can also interact with a proapoptotic protein, called death
effector domain-associated factor (DEDAF). DEDAF associates with
death effector domain-containing proapoptotic proteins and plays a
role in transcriptional regulation [40]. Similar to apoptin, transient
overexpression of DEDAF can induce cell death in cancer cells, but not
in normal cells. The cellular localization of DEDAF is similar to that of
apoptin, as both colocalize into the nucleus of tumor cells. Coexpres-
sion of apoptin and DEDAF increases the apoptotic death rate as
compared to the effects of expression of either of them alone [40].
Nonetheless, whether the interaction of apoptin with DEADF is
responsible for apoptin's toxicity awaits further investigation.
Apoptin also interacts with promyelocytic leukemia protein (PML)
and localizes in PML nuclear bodies [41]. PML nuclear bodies are
ordered protein complexes that associate with the nuclear matrix and
play a role in apoptosis, DNA replication, repair, transcription, RNA
transport and viral replication [42]. Many proteins residing in PML
nuclear bodies, including apoptin, undergo a specic posttranslational
modication, called sumoylation. However, the nding that a
sumoylation-decient mutant of apoptin can still trigger apoptosis
in wildtype as well as in PML-decient cells suggest that neither
sumoylation nor the interaction with PML are crucial for apoptin's
proapoptotic activity. Nevertheless, although apoptin's interaction
with PML appears dispensable for apoptosis, it was speculated to be
involved in CAV replication [41]. It has also been suggested that
apoptin colocalizes with FADD (Fas-associated protein with death
domain) and Bcl10, a caspase recruitment domain-containing protein,
in cytoplasmic structures, called death effector laments [20]. Both
FADD and Bcl10 are involved in apoptosis signaling by death receptors,
though the signicance of this colocalization for apoptin-induced cell
death is unknown.
Our group recently reported that, through its proline-rich
sequence (aa 8186), apoptin interacts with the SH3 domain of the
p85 regulatory subunit of phosphoinositide 3-kinase (PI3K, Fig. 2).
Class I PI3 kinases play a central role in various regulatory processes,
such as cell growth, survival and differentiation. Surprisingly, when

Table 1
Apoptin-interacting molecules.

Molecule Biological effect References

DNA Due to its basic structure, apoptin can directly interact with DNA, preferentially with double stranded DNA and [16,59]
heterochromatin.
Akt and PI3K Apoptin interacts with the SH3-domain of the p85 regulatory subunit of PI3K and activates PI3K. This event leads to [43,44]
sustained Akt activation and presumably aids Akt in nuclear translocation.
CyclinACDK2 CyclinACDK2 complex directly phosphorylates apoptin at Thr-108 and contributes to the regulation of its subcellular [49]
localization.
Peptidyl-prolyl isomerase like 3 (Ppil3) Apoptin's interaction with Ppil3 may favor its cytoplasmic localization. [36]

Hip-interacting protein (Hippi)
N-Myc interacting protein (Nmi)
APC1 (subunit of anaphase-promoting
complex/cyclosome)
Death effector domain associated factor Coexpression of apoptin and DEDAF increases apoptosis as compared to the effects of either protein alone.
(DEDAF)
Promyelocytic leukemia protein (PML)
Fas-associated death domain protein
(FADD)
Bcll0
Importin-1
In non-cancerous cells, apoptin colocalizes with Hippi in the cytoplasm, whereas in tumor cells apoptin migrates to the [37]
nucleus while Hippi remains in the cytoplasm.
The functional signicance of apoptin and Nmi interaction is not well understood. [38]
Apoptin's interaction with APC1 may lead to mitotic cell cycle arrest and apoptosis. [39]
[40]
SUMOylated apoptin interacts with PML in PML nuclear bodies. However, the disruption of the interaction of PML and [41]
apoptin does not affect apoptin's cytotoxicity.
Upon overexpression apoptin colocalizes with FADD, a component of the death receptor pathway, in so-called death effector [20]
laments. The signicance of this process is unknown.
Apoptin can colocalize with Bcl10, a regulator of apoptosis and NF-B activation. [20]
Importin-1 aids apoptin's nuclear translocation through the nuclear pore complex. [10,13]
 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342
p85 is downregulated, apoptin is shuttled out of the nucleus, thereby
imposing a negative regulation of apoptin-induced cell death [43].
Apoptin derivatives devoid of the proline-rich region do not interact
with p85, are unable to activate PI3K and show impaired apoptosis
induction. Thus, the interaction of apoptin with p85 subunit of PI3K
appears to be essential for the cytotoxic activity of apoptin [43]. In
addition to p85 regulatory subunit of PI3K, its downstream effector
kinase Akt1 was also found as an apoptin-interacting molecule [44].
Although the role of PI3K/Akt pathway in cell survival is well
established, there are examples in which PI3K and Akt also promote
cell death [4548]. Interestingly, as recently reported, the direct
interaction of apoptin with Akt1 triggers nuclear trafcking of Akt and
initiates apoptosis instead of a survival response [43,49]. Agents such
as etoposide, cisplatin, and interferon- induce Akt1 phosphorylation
that precedes the onset of caspase activation and apoptosis [50,51].
Importantly, transient activation of Akt1 supports cell survival,
whereas its sustained activation leads to apoptosis [5254]. These
observations therefore support a dual role of PI3K/Akt pathway in cell
survival and apoptosis.
6. Nuclear transfer of apoptin vital for its apoptotic action
The selective toxicity of apoptin is mainly attributed to its
differential subcellular localization in tumor and normal cells (Fig. 3).
Apoptin predominantly localizes to the nucleus of cancer cells, whereas
its accumulation in the nucleus is severely impaired in normal cells
[12]. Experimental data indicate the nucleo-cytoplasmic shuttling of
apoptin is regulated by both the bipartite NLS and the N-terminal NES
[10,12,13]. The NLS of apoptin is potentially active in both normal and
cancer cells, as small amounts of apoptin can translocate to the nucleus
in normal cells, albeit with markedly lower efciency [55,56]. Notably, a
CRM1-regulated NES (aa 97105) has been identied in apoptin that
appears to be active in normal cells but not in tumor cells, and thereby
retains apoptin in the cytoplasm of normal cells [10]. Apoptin also
contains a leucine-rich sequence (aa 3346) that is important for its
interaction with PML and other proteins [9,14]. Mutations that
inactivate this sequence stretch not only reduce the ability of apoptin
to associate with PML bodies, but also inhibit nuclear accumulation of
apoptin in tumor cells.
Fig. 3. Differential subcellular localization of apoptin in primary and transformed cells.
(A) In normal cells, such as human primary mononuclear cells, apoptin is localized in
the cytoplasm, as shown by the green staining of GFP-tagged apoptin and blue
counterstaining using the nuclear dye DAPI. (B) In transformed cells, such as U2OS
osteosarcoma cells, apoptin resides exclusively in the cell nucleus. Costaining for
promyelocytic leukemia protein (PML, red) reveals that a large fraction of apoptin
localizes in PML nuclear bodies.
1339
7. Apoptin phosphorylation role in apoptosis induction?
In addition to nuclear targeting signals, the phosphorylation of
apoptin is crucial in transducing apoptotic signals in cancer cells.
Mapping studies provide evidence that apoptin has a phosphorylation
site at threonine-108 (Thr-108) adjacent to the NES [57]. Thr-108
phosphorylation can be detected in vitro and in vivo in cell lines and
tumors. Phosphorylation at Thr-108 appears to drive tumor-specic
nuclear accumulation of apoptin via inactivation of the NES. In
contrast, two studies reported that Thr-108 phosphorylation only
partially affects apoptin's proapoptotic activity, as an apoptin mutant
devoid of this phosphorylation site retains the ability to induce
apoptosis [43,58]. Some ndings suggest that both N- and C-terminal
regions of apoptin have the capacity to bind DNA and induce apoptosis
independently, and that Thr-108 phosphorylation is required only for
the C-terminus [58,59]. An apoptin mutant consisting of the rst 69
amino acids is mainly located in the cytoplasm of human tumor cells,
but harbors a distinct, albeit weaker, proapoptotic activity [12].
Furthermore, there is a cluster of three threonine residues (aa 106
108) within the apoptin sequence. Thus, even though this possibility
has not yet been tested experimentally, it is conceivable that mutation
of Thr-108 may still allow for the phosphorylation of apoptin at the
adjacent threonine residues.
Due to the importance of apoptin phosphorylation for its tumor-
specic action, the apoptin kinase has intensively been sought for a
long time. We could recently identify the mitogenic cyclin-dependent
kinase CDK2 as the principal kinase that directly phosphorylates
apoptin at Thr-108 and is crucially required for apoptin-induced cell
death [49]. As mentioned above, an early event of apoptin signaling is
the interference with the PI3K/Akt pathway, which does not only
result in a very sustained activation but also the nuclear translocation
of Akt. This sustained Akt activation is in contrast to the transient
stimulation of kinase activity that is normally observed following Akt
activation by growth-stimulatory signals. It is therefore likely that the
prolonged activation of Akt might result in the phosphorylation of
unusual substrates.
During our studies we found that apoptin-induced Akt stimulation
results in a strong activation of CDK2, which is normally a S-phase
kinase involved in cycle progression [49]. Interestingly, pharmacolo-
gical inhibitors as well as dominant-negative mutants of PI3K and Akt
not only inhibited CDK2 activation, but also protected cells from
apoptin-induced cell death. The Akt-mediated activation of CDK2 was
caused by two mechanisms, including the direct phosphorylation of
CDK2 and the phosphorylation-induced degradation of its inhibitor
p27Kip1 (Fig. 2). Furthermore, cyclin A- but not cyclin E-associated
CDK2 was responsible for apoptin phosphorylation at Thr-108 as well
as its tumor-specic localization. The crucial role of CDK2 for the
biological activity of apoptin was further substantiated by the nding
that CDK2 inhibitors as well as the genetic deciency of CDK2 strongly
protected cells against apoptin-induced apoptosis [49]. These unex-
pected ndings therefore suggest that apoptin can convert mitogenic
pathways into cell death responses, which likely account for its tumor-
specic activity.
Interestingly, a sustained activation of Akt in nucleus, unlike the
transient activation induced by growth factors at the cell membrane,
has been implicated in other cell death pathways. For instance,
prolonged nuclear Akt/CDK2 activation does not protect but
sensitizes tumor cells for apoptosis induced by anticancer agents
such as methotrexate and docetaxel [23]. Also, in primary breast
tumor cells an elevated basal CDK2 activity has been found to
correlate with the sensitivity to paclitaxel [60]. In addition, elevated
CDK2 activity has been implicated in apoptosis in some other
conditions including negative selection of thymocytes [61]. Aberrant
activation of CDK2 might therefore cause disturbance of cell cycle
progression and cell death. These observations not only decipher
mechanisms of apoptin-induced cell death, but also provide novel
1340 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342
insights for the selective killing of tumor cells. Diverse regulators of
the PI3K/Akt pathway are involved in tumorigenesis and are highly
active in various types of cancers. Hyperactivation of these pathways
is associated with a poor clinical prognosis and contributes to drug
resistance. Thus, targeting the PI3K/Akt/CDK2 pathway by apoptin
might be responsible for its unique tumor-specic effects. We
propose that apoptin hijacks cell survival pathways and redirects
them towards induction of cell death, thereby establishing a novel
link between cell survival and cell death.
8. Clinical perspectives of apoptin
The ability of apoptin to induce apoptosis independently of p53
and to switch survival pathways into proapoptotic signaling carries an
enormous pharmacological potential. These remarkable features
might be directly employed for the use of apoptin in cancer therapy.
In addition, targeting of apoptin's interaction partners might be an
approach to achieve tumor specicity and to design new antitumor
therapies. Several preclinical studies have shown that apoptin
improves also the efcacy of traditional therapy, while apoptin itself
shows no adverse side effects.
Recent studies demonstrate that downregulation of survivin
together with the expression of apoptin signicantly increases the
percentage of apoptotic cells compared to the effects seen upon
survivin loss or apoptin expression alone [62,63]. Survivin belongs to
the family of inhibitors of apoptosis proteins (IAP) and is highly
expressed in several tumors. These ndings suggest that a combined
strategy using apoptin and a downregulation of survivin could be
effective in controlling the growth of tumors that are otherwise
resistant to traditional chemotherapy. Another study has shown that
the combined treatment of tumors with recombinant apoptin and IL-
18 induced a strong Th1 response against Lewis lung carcinomas that
was associated with a signicant inhibition of tumor growth [64].
Similarly, a combined therapeutic approach with recombinant
adenovirus expressing apoptin and etoposide showed an additive
cytotoxicity on human osteosarcoma U2OS cells [24,33]. Also, when
paclitaxel treatment was combined with apoptin, the survival of p53-
positive, p53-negative and p53-mutant cancer cells was compromised
at a signicantly lower dose of cytotoxic chemotherapy. These
observations indicate that apoptin in combination with conventional
chemotherapeutic agents offers an effective strategy for cancer
treatment [24].
The tumor-selective cell death by apoptin has now been demon-
strated in a great number of tumor cells using various techniques
including adenoviral transfer, transient overexpression or intro-
duction of cell-permeable apoptin constructs. Furthermore, several
groups have studied the effect of apoptin in different mouse modes.
While all these studies conrmed a tumor-specic effect, the most
challenging issue is still the development of efcient delivery forms of
apoptin at the tumor lesion. Noteborn and colleagues developed a
strategy for the use of apoptin in cancer therapy based on adenoviral
vectors. A single injection of the apoptin-producing adenovirus in
xenografted hepatomas resulted in a considerably delay in tumor
growth [22]. Furthermore, in order to increase the transduction
efciency some studies have investigated other viruses. A recent
study, for instance, showed that infection with a replication-decient
fowlpox virus containing the apoptin gene resulted in a signicant
induction of apoptosis in hepatoma cells and, moreover, efciently
repressed the growth of subcutaneous tumors in mice [65].
Another gene-therapeutic approach to express particular genes of
interest into the liver is the use of ligand conjugates of the
asialoglycoprotein receptor that is specically expressed on hepato-
cytes. Systemic administration of the apoptin gene via the asialogly-
coprotein receptor into mice bearing hepatocarcinomas resulted in
signicant tumor regression, whereas normal hepatocytes were
clearly not affected by apoptin gene delivery [66]. In addition, apoptin
derivatives could be directly targeted to other tissues, such as brain. In
this respect, apoptin conjugates that are taken up by the precursor of
heparin-binding epidermal growth factor, also known as the
diphtheria toxin receptor, are currently being tested [14]. This receptor
is constitutively present at the bloodbrain barrier and is strongly
upregulated in many tumors, such as glioblastoma.
Several studies have used apoptin derivates coupled to protein
transduction domains, including HIV-Tat, which allows for the
efcient intracellular delivery of exogenously administered apoptin
[20,30,43]. However, although apoptin fused to HIV-Tat-derived
peptide has been proven to efciently kill various tumor cell types
in culture, its use in preclinical and clinical settings is certainly
restricted. Nevertheless, HIV-Tat apoptin could be applied for the
treatment of squamous cell carcinoma or precancerous skin lesions.
The efcacy of apoptin for the treatment of squamous cell carcinoma
has recently been demonstrated in a mouse model using its ectopic
expression in primary oral tumor cells [67]. It might be also envisioned
to utilize the selective accumulation of apoptin in the nucleus of
cancer cells for drug development. Thus, fragments of apoptin could
be coupled to radionuclides emitting low energy radioactivity in order
induce DNA strand breaks selectively in cancer cells.
9. Other tumor-selective killer proteins
Beside apoptin, the identication of other proapoptotic proteins
with cancer-selective properties raises promises for new drug
development. The molecular pathways by which these proteins
exert their tumor-selective function are intensively investigated. The
cytokine TRAIL, a member of the TNF superfamily, induces apoptotic
cell death in a wide variety of tumor cells. Preclinical studies in mice
and nonhuman primates demonstrated that TRAIL induces apoptosis
in human tumors, whereas no cytotoxicity to normal tissues was
found [68]. The susceptibility of tumor cells to TRAIL and the apparent
lack of activity of TRAIL in normal cells have led to the proposed use of
TRAIL in cancer therapy [69]. Indeed, several phase 1 and 2 studies
with TRAIL or TRAIL receptor agonists are in progress in cancer
patients.
Interestingly, like apoptin, also the viral protein E4orf4 (adenovirus
type 5 E4 open reading frame 4) induces p53-independent cell death
in a wide range of tumor but not normal cells upon ectopic expression
[70]. In analogy to apoptin, E4orf4 is mainly localized in the nucleus
and employs the survival kinase c-Src for its apoptosis signaling [71].
Another similarity between E4orf4 and apoptin is the nding that
both proteins obviously target the APC/C complex for their tumor-
specic induction of apoptosis. This makes the APC/C complex
certainly an attractive target for future anticancer therapeutics. In
addition, a proapoptotic protein with structural similarity to apoptin
has been identied in some isolates of Torque teno virus (TTV) [72,73].
This protein, named TTV apoptosis-inducing protein (TAIP), triggers
p53-independent apoptosis in hepatocellular carcinoma and other
tumor cells, albeit mostly with less potency than apoptin. Unlike
apoptin, TAIP is localized in the cytoplasm of transformed cells.
Furthermore, the high frequency of TTV in human cancer biopsies
suggests an indirect tumor growth-stimulatory role of TTV infection
rather than a signicant oncosuppressive function [74].
Another remarkable protein with tumor-specic cytotoxic activity
has been isolated from a specic fraction of human milk and is
therefore called HAMLET (human -lactalbumin made lethal to tumor
cells). The proapoptotic activity of HAMLET was shown to reside in a
molecular complex consisting of the milk protein -lactalbumin and
oleic acid, which results in a change of protein conformation [75].
Similar to apoptin, HAMLET is a partly unfolded globule-like complex.
In cancer cells, HAMLET translocates to nuclei and induces p53-
independent cell death of tumor cells, but not normal cells. Recently, it
was shown that treatment with topical HAMLET has a benecial effect
on the regression of skin papillomas [76].
 M. Los et al. / Biochimica et Biophysica Acta 1793 (2009) 13351342
Ribonucleases have also attracted considerable attention as specic
anticancer agents. Onconase, an amphibian ribonuclease from oocytes
of the frog Rana pipiens is of particular interest, because it is cytostatic
and cytotoxic to various tumor cells and enhances their sensitivity to a
number of cytotoxic agents [77]. Onconase has demonstrated
signicant efcacy in phase III clinical trials of patients with malignant
mesothelioma that failed prior chemotherapy. Onconase's specic
mechanism of action is not fully understood, but it seems to target gene
regulation by microRNAs [77]. In conclusion, further investigations of
apoptin and related proteins with a tumor-specic proapoptotic
activity will be important not only for understanding the process of
carcinogenesis, but might eventually result in new therapeutic options
for anticancer treatment.
Acknowledgments
We apologize to those authors whose data were not discussed due
to space restrictions. M.L., F.E. and K.S.O. acknowledge the support
from Deutsche Forschungsgemeinschaft (SFB 773, GRK 1302) and the
Deutsche Krebshilfe.
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