REVIEW ARTICLE
Regulation of DNA fragmentation: the role of caspases
and phosphorylation
Ikuko Kitazumi and Masayoshi Tsukahara
Bio Process Research and Development Laboratories, Kyowa Hakko Kirin Co. Ltd, Gunma, Japan
Introduction
Apoptosis is a crucial cellular mechanism that is
involved in inflammation, cell differentiation and cell
proliferation. As a form of cell death, it is character-
ized by distinctive morphological and biochemical
changes, including plasma membrane blebbing, phos-
phatidylserine exposure, nuclear condensation and
DNA fragmentation [1]. These cellular changes are
largely mediated by caspases, a family of cysteinyl
aspartate-specific proteases whose target proteins are
important indicators of apoptotic cell death [2].
Keywords
apoptosis; caspase; DNA fragmentation;
okadaic acid; phosphorylation
Correspondence
M. Tsukahara, Bio Process Research and
Development Laboratories, Kyowa Hakko
Kirin Co. Ltd, 100-1 Hagiwara, Takasaki,
Gunma 370-0013, Japan
Fax: 81 27 353 7400
Tel: 81 27 353 7382
E-mail: masayoshi.tsukahara@kyowa-
kirin.co.jp
(Received 10 September 2010, revised 18
November 2010, accepted 26 November
2010)

Caspases almost exist in an inactive form whose acti-
vation is widely affected by protein phosphorylation ⁄
dephosphorylation [3–5]. Kinase ⁄ phosphatase activa-
tion initiates apoptotic signal pathways; protein
phosphorylation plays important roles in the signaling
cascade that contributes to the control of cell death
and survival signal transduction [6–8]. In this review,
we will discuss the role of caspases and phosphoryla-
tion in apoptosis, with particular emphasis on the
induction of DNA fragmentation, which is one of the
most typical characteristics of apoptosis.
Regulators of DNA fragmentation
One of the terminal processes of apoptosis is DNA
degradation. During apoptosis, DNA breakage usually
occurs in at least two stages: the first is initial cleavage
at chromatin loop domains (50–300 kb) to generate
high relative molecular mass DNA fragments; the sec-
ond is cleavage of loose parts of internucleosomal
DNA (in approximate multiples of 180 bp, oligonucle-
osomal size) into low relative molecular mass DNA
fragments [9]. Nuclear morphological changes vary
according to cell type and related factors, some of
which have been prevented using gene knockouts and
treatment inhibitors [10–13].
Several nucleases have been implicated in the degra-
dation of DNA during apoptosis, two major ones
being endonuclease G (EndoG) and DNA fragmenta-
tion factor (DFF). Each nuclease has a distinct cellular
location, is regulated in different ways and causes
DNA fragmentation by a different pathway. Translo-

between nucleosomes; DNA degraded by CAD can be
detected by agarose gel electrophoresis as a character-
istic ‘DNA ladder’ [20].
ICAD is an indispensable factor in normal CAD
function. ICAD acts as a specific chaperone for
CAD during its synthesis and, after translation,
forms a heterodimer with CAD and inhibits its
DNase activity [21,22]. It has been shown that the
CAD ⁄ ICAD complex forms a heterotetramer
(CAD ⁄ ICAD with CAD ⁄ ICAD) in nonapoptotic
cells [23]. Such ICAD ⁄ CAD complexes are mainly
localized in the nucleus due to the presence of a
nuclear localization signal at the C-termini of both
ICAD and CAD [24]. During apoptosis, activation
of capsase-3 results in ICAD cleavage, which releases
CAD to form an active homodimer in the nucleus
[25]. ICAD mutant overexpression does not affect
the extent of cell death [26], suggesting that ICAD
could be involved in the induction of DNA fragmen-
tation, but is not involved in the execution phase of
DNA fragmentation.
ICAD exists as both a long (ICAD ⁄ DFF45) and a
short (ICAD-S ⁄ DFF35) form. ICAD-S is a splicing
variant of ICAD that ends at residue 268 and lacks
the C-terminal 63 residues of ICAD [27]. This short
form also dimerizes with CAD, and partially main-
tains the function of the inhibitor and chaperone
[28]. ICAD-S cannot translocate to the nucleus
because of a splice-out nuclear localization signal in
its C-terminal [24,29]. Because ICAD cleavage and

JNK
Bcl-2
Bax
Bax
Cytochrome c
14-3-3
167
P38 MAPK
185
183
Degradation
125
p53
Bax
Akt
473
Caspase-3
ERK1
70
87
69
Apaf-1
Apoptosome
Caspase-9
Cleaved
caspase-3
Endo GAIF
Dimerization
PARP
DNA fragmentation

Degradation
Facilitatory effect
P
P
P
P
14-3-3
Bax
184
Bax
P P
P
P
P
159
121
163
184
P
P
P
Mcl-1
Bax
P
121
163
184
P
P
Mcl-1

P
P
364
Activation
Cleavage
465397
PP
P
Caspase-8
150
Caspase-3
P
Cleavage
Activation
P
Caspase-3
Endo GAIF
46
Apoptosis
Transcription of
Bax , Bcl-2
P
15
37
P
P
p53
Bad
Bcl-xL
Bcl-2

I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 429
Despite their similar localization, they have different
functions in DNA fragmentation. Mitochondrial nucle-
ase EndoG first induces higher order chromatin cleav-
age into high relative molecular mass DNA fragments
(> 50 kb in length), followed by inter- and intra-
nucleosomal DNA cleavages, resulting in products
with many internal single-stranded nicks spaced at
nucleosomal ( 190 bp) and subnucleosomal ( 10 bp)
periodicities. Hence, DNA fragmentation generated
by EndoG is broad compared with other nucleases
[19]. Although EndoG is both a double- and a
single-stranded DNase ⁄ RNase, it preferentially attacks
single-stranded regions in the presence of additional
co-activators [33].
Unlike EndoG, AIF does not have DNase activity.
It is a mitochondrial flavoprotein that plays an essen-
tial role in oxidoreductase activity in nonapoptotic
cells [34]. AIF has been reported to trigger chromatin
condensation and induce cleavage of DNA into high
relative molecular mass fragments through other nuc-
leases, but not to cause oligonuclesomal DNA frag-
mentation [35,36]. However, other studies have shown
that inhibition of apoptotic AIF does not prevent the
appearance of high relative molecular mass DNA frag-
ments [26]; the nuclear actions of AIF therefore remain
poorly understood.
Relationship between DNA fragmenta-
tion and caspases

Caspases are initially synthesized as inactive zymo-
gens, and their dimerization is crucial for stabilizing
the conformation of the active site, which is cleaved
prior to activation [44,45]. Initiator caspases are mono-
meric zymogens, which are activated by dimerization
during apoptosis, whereas the executioner caspases
exist as the inactive dimers [46]. Initiator caspases form
signaling complexes that are platforms for caspase acti-
vation. Pro-caspase-9 forms a large complex as the
apoptosome, which consists of released cytochrome c
from mitochondria and oligomers of Apaf-1 [47].
Pro-caspase-8 is activated through recruitment of the
death receptor complexes [41]. Executioner caspase
dimers are activated by upstream proteolysis or auto-
proteolysis to cleave sequentially and generate active
large and small subunits that form active hetero-
tetramers [5,38].
Caspases have multiple cleavage sites at specific
aspartic acid residues; the exact cleavage location
affects caspase activity and function [37]. In the case
of caspase-9, it is activated by autolytic cleavage via
the mitochondrial pathway [47]. Caspase-9 is also
cleaved by caspase-3 at another cleavage site. How-
ever, this fragmentation does not have caspase activity.
It enhances the activation of other caspases by alleviat-
ing endogenous X-linked inhibitor of apoptosis (XIAP)
inhibition of caspases [48]. Although cleavage is a sig-
nificant change for caspase activation, the cleaved frag-
ment does not always have caspase activity. It was
previously shown that cleavage of caspase still occurs

of CAD is a caspase-3-dependent process that occurs
in the nucleus [21,24]. Caspase-3 also affects the
induction of other factors involved in DNA fragmen-
tation by cleaving substrates. Poly(ADP-ribose) poly-
merase (PARP) is a major nuclear target for caspases
that is involved in many cellular functions, including
DNA repair and maintenance of genomic stability
[54]. PARP is activated in response to DNA damage,
and its activity is shown to regulate DFF40 activity
in vitro. Caspases cleave PARP and inactivate its
DNA-repairing abilities during apoptosis; hence, inhi-
bition of caspases mostly prevents PARP cleavage
and DNA fragmentation [10].
Caspases often share common substrates. Cells have
multiple cleavage mechanisms, as shown by the cleav-
age induction of ICAD and PARP in caspase-3-
deficient cells [13,55]. However, they exhibit different
levels of activity against substrates. The close relation-
ship between capsase-3 and caspase-7 is well docu-
mented. Although caspase-7 is as efficient as caspase-3
(in some cases more effective) for several substrates in
a cell-free system, caspase-3 is a major executioner cas-
pase [56]. The different localizations and substrates of
caspases contribute to functional distinctions. For
example, pro-caspases are often present in the cytosol
fraction (caspase-2, -3, -6, -7, -8 and -9) of living cells
to separate silent precursor caspases in the cytosol
from pro-apoptotic cofactors in the mitochondria and
nucleus [57,58], although caspase localization depends
on cell lines. In the case of pro-caspase-3 and -7, they

serine protease activity and its ability to act as an
inhibitor of apoptosis antagonist, which enhances cas-
pase activation [63]. The release of HtrA2 ⁄ Omi from
mitochondria into the cytosol and pro-apoptotic activ-
ity via XIAP inhibition is closely related to caspase
activity; HtrA2 ⁄ Omi activity contributes to the pro-
gression of caspase-independent cell death in mito-
chondria [64]. Although activation of these proteins is
highly dependent on caspase activation, DNA frag-
mentation has been shown to occur during caspase
inhibition [15,16]. In the case of cell death stimulation
that does not activate caspases, alternative pathways
induce caspase activity, resulting in DNA fragmen-
tation.
DNA fragmentation resulting from
phosphorylation-induced apoptotic
pathways
Cell signal transduction is regulated by the biochemical
modification of proteins that alters their conformation,
stabilization, reaction to substrates and function.
Reversible protein phosphorylation and dephosphory-
lation at serine and threonine residues can modulate
cell survival through positively or negatively changing
protein stability, transcriptional activity and apoptotic
ability [7,8]. Caspases play central roles in apoptotic
pathways, which induce DNA fragmentation [2,21].
Phosphorylation regulates many caspase activity-
induced signal pathways; phosphorylation is also
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 431

JNK ⁄ p38 kinases during apoptosis [70]; this represents
an example of cross-talk or cross-signaling in which
one signaling pathway is regulated by another
[66,69,71].
Additionally, phosphatases, which have an effect
opposite to kinases, play important roles in the down-
regulation of MAPK activity. Especially, the ser-
ine ⁄ threonine protein phosphatase (PP) is a key
regulator of cellular protein dephosphorylation. PP
can be classified as type 1 (PP1) or type 2 (PP2), and
PP2A regulates both cell survival and apoptotic cellu-
lar reactions [72]. PP2A has been shown to dephos-
phorylate p38 MAPK, thereby impairing its activity,
and its inhibition results in the induction of apoptosis
via caspase activation, for example [16,71].
Phosphorylation of mitochondrial apoptotic
proteins
The antiapoptotic Bcl-2 family members are important
regulators of cell survival in their control of mitochon-
drial pathways. These proteins both prevent and
induce entry into the apoptotic cell death cascade, for
example by activating caspases [73]. The family is
divided into three subfamilies: antiapoptotic proteins
(Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A1), pro-apoptotic
proteins (Bax, Bak and Bok) and BH3-only proteins
(Bad, Bid, Bik, Blk, Hrk, BNIP3 and BimL). Bcl-2
family proteins mostly mediate the activity of other
proteins in the same family [74]. Antiapoptotic Bcl-2
family members bind to pro-apoptotic family mem-
bers, interrupting cell death signals [75], but with very

with the release of cytochrome c and the formation of
the apoptosome [38]. The major pro-apoptotic protein
Bax exists mainly in the cytosol or loosely attaches to
mitochondria in an inactive form. Inactivated Bax is
phosphorylated at Ser184 by the physiological Bax
kinase Akt, and heterodimerizes with antiapoptotic
Bcl-2 family members such as Bcl-xL [83]. Activation
of Bax by dephosphorylation results in translocation
from the cytosol to mitochondria, where it forms large
oligomers. This translocation is inhibited by ERK-1
[69,84]. Bax dimerization leads to the formation of a
pore or channel in the mitochondrial outer membrane,
Phosphorylation and caspases in DNA fragmentation I. Kitazumi and M. Tsukahara
432 FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS
enabling multiple mitochondrial proteins to be released
into the cytosol with cytotoxic activities [85].
The activated BH3-only protein Bad is also localized
mostly in the cytosol in normal cells, and is phosphor-
ylated at Ser112, Ser136 and Ser155 in an ERK-1-
dependent manner [69]. Dephosphorylation of Ser136,
which is regulated by dephosphorylation of Ser112, is
a key action in mediating apoptosis. After dephosphor-
ylation of both Ser112 and Ser136, Bad is dephospho-
rylated at Ser155, which allows translocation to
mitochondria and the binding of Bcl-xL [86], and
increases the release of cytochrome c from mitochon-
dria into the cytosol through inactivation with Bcl-xL
and Bcl-2 [69]. Inactivated Bax and Bad bind 14-3-3,
the phosphoserine ⁄ threonine binding proteins in the
cytosol. 14-3-3 prevents Bax and Bad dissociation from

caspase-8 activity and function [91]. Moreover, p38
MAPK can directly phosphorylate and inhibit the
activities of caspase-8 at Ser364 and caspase-3 at
Ser150 [4]. After phosphorylation of Tyr310, caspase-8
is dephosphorylated at both Tyr397 and Tyr465 by the
Src-homology domain 2-containing tyrosine phospha-
tase-1, which allows its cleavage and activation [90],
and caspase-3 at threonine residues by PP2A interac-
tion [51] initiates apoptosis. Conversely, kinases
involved in the phosphorylation of caspases are regu-
lated by cleaved caspases [8]. Caspases, kinases and
phosphatases are regulated by each other and control
cell survival.
Phosphorylation of intranuclear protein
Core nucleosomal histone H2AX is phosphorylated at
sites of DNA double-stranded breaks in DNA-injured
cells. H2AX is a member of the histone H2A family,
which differs from other species by containing a
Ser139 phosphorylation site in the C-terminal tail.
Phosphorylation of H2AX on Ser139 is a key event in
the repair of DNA damage and the induction of DNA
degradation leading to cell death; therefore, the phos-
phorylated form of H2AX (cH2AX) is a sensitive
marker for DNA double-stranded breaks [92,93].
It has been reported that the last residue at C-termi-
nal Tyr142 is phosphorylated under normal conditions,
preventing recruitment of DNA repair factors to phos-
phorylated Ser139 [94]. The phosphorylation site
Ser139 is directly phosphorylated by JNK and p38b
MAPK [11,95]. cH2AX associates not only with DNA

OA is a component of diarrhetic shellfish poisoning
toxin [102]. It is a potent inhibitor of PP1 and PP2A
that increases the tyrosine phosphorylation and inacti-
vation of PP2A [68] with 100-fold greater selectivity
for PP2A over PP1 [103]. OA induces various cellular
reactions that can either induce or prevent apoptosis
through phosphorylation modulating (Fig. 1). Inhibi-
tion of PP upsets the balance between serine ⁄ threonine
phosphorylation and dephosphorylation of various
proteins, leading to altered signal transduction and
gene expression. The following section focuses on the
effects of OA on apoptosis.
Apoptotic effect of OA
The inhibition of PP positively regulates apoptosis by
activating pro-apoptotic factors and inactivating antia-
poptotic factors. Many PP dephosphorylation signals
are involved in the induction of DNA fragmentation,
such as activation of the caspase cascade and MAPK
family. Treatment with OA has been shown to alter
mitochondrial membrane permeability due to the
release of cytochrome c and AIF, and to enhance
apoptosis in HeLa cells [16], primary cultures of nor-
mal human foreskin keratinocytes [100] and Jurkat
T leukemia cells [104]. OA affects antiapoptotic Bcl-2
family members that are involved in mitochondrial
apoptotic pathways. PP2A plays a role in the dephos-
phorylation of Bcl-xL at Ser62 in response to oxidative
stress, and treatment with OA has been shown to
enhance phosphorylated Bcl-xL, leading to diminished
Bcl-xL ⁄ Bax interaction in human retinal pigment epi-

myeloid leukemia K562 cells [113]. Thus, OA effects
range from upstream of apoptotic signal pathways to
downstream proteins.
Antiapoptotic effect of OA
Although treatment with OA induces apoptosis, OA
also protects cells against other apoptotic signals.
PP2A can activate Bad via two different routes, direct
dephosphorylation of Ser112 and negative regulation
of the ERK pathway via p38 MAPK, both of which
lead to impaired phosphorylation of Ser112 [70]. After
dephosphorylation of Ser112, Ser136 becomes suscepti-
ble to multiple phosphatases. PP2A dephosphorylates
Bad mainly on Ser112, as well as on Ser136 and
Ser155 [6]. Treatment with OA was shown to
phosphorylate Bad at Ser112 and Bcl-2 at Ser70, and
activate ERK, thereby preventing tumor necrosis
factora ⁄ cycloheximide-induced JNK activation, cyto-
chrome c release and caspase activation in rat epithelial
IEC-6 cells [68].
Apoptotic activation of Bad results from 14-3-3 dis-
sociation after dephosphorylation of Ser112 and
Ser136, and sequential dephosphorylation of Ser155 by
PP2A. Activated Bad binds to Bcl-XL to prevent
antiapoptotic activation in both the interleukin-
3-dependent murine prolymphocytic cell line FL5.12
and the mouse embryonic fibroblast cell line NIH 3T3
[86]. Dephosphorylated Bax is directly increased by
PP2A, and indirectly through inhibition of Akt phos-
phorylation on Ser473 by p38a MAPK-mediated PP2A.
OA increases phosphorylation of Bax, then inhibits

inhibits PP2A at 10 000–40 000 times lower concentra-
tion than that required for PP1 inhibition [103]. The
apoptotic effects of OA and fostriecin (PP1 < PP2A)
and CA (PP1 = PP2A) were observed; however, TM
(PP1 > PP2A) did not exhibit any pro-apoptotic
effects in the interleukin-3-dependent murine pro-B cell
line [6], the endothelium-derived permanent human cell
line EA.hy926 [70] or Jurkat cells [104]. Inhibition of
PP2A equivalent to PP1 (PP1 = PP2A) or better than
PP1 (PP1 < PP2A) (OA, fostriecin, CA) induces
apoptosis; on the other hand, inhibition of PP1 rather
than PP2A (PP1 > PP2A) (TM) fails to induce apop-
tosis. It is possible that apoptosis is induced when PP1
has greater activation than PP2. Additionally, inhibi-
tion of PP1 by CA or TM prevents Fas-mediated
apoptosis, whereas inhibition of PP2A by OA protects
Jurkat cells from anisomycin [118]. The effects of OA
on apoptosis therefore depend on the kind of inducer,
as well as inhibition of PP type and cell type.
The effects of OA on cellular signaling are also
affected by intrinsic regulation. PP2A is a downstream
target of p38 MAPK, whose activity regulates the sub-
cellular localization of PP2A [70,114]; meanwhile,
PP2A dephosphorylates p38d MAPK [100,119]. p38
MAPK acts to limit the phosphorylation of JNK
through increased activation of PP2A [71]; thus the
MAPK family regulates its members via PP2A. PP2A
affects not only upstream but also downstream pro-
teins for apoptotic signaling. OA-induced activity of
the MAPK family mediates the downregulation of var-

drial membrane permeabilization in intact NIH 3T3
cells [86] and human hepatoma HepG2 cells [121].
Phosphorylation of ERK1 ⁄ 2, upstream of Bad, is simi-
larly degraded by ST in rat primary hepatocytes [101].
Interestingly, phosphatase and kinase inhibitors act
on identical cell death pathways and eventually induc-
tion of DNA fragmentation [26,104]. Both OA and ST
induce phosphorylation and activation of JNK and
p38 MAPK, which are involved in the increase of
release of cytochrome c into the cytoplasm and caspase
activation [16,71,122]. ST rapidly increased p53 cyto-
plasmic accumulation, which activated Bax in the
mouse cerebellar neural stem cell line C17.2 [123].
Treatment with OA increased levels of phosphorylated
p53 at Ser15 (at least one phosphorylated site), which
binds to microtubules and cannot be efficiently translo-
cated into the nucleus; this resulted in inhibition of its
transcriptional activity [124].
However, these inhibitors are essentially different,
although they lead in part to induce similar reactions.
Both ST and OA phosphorylate the same substrate
but at different phosphorylation sites. Stimulation with
I. Kitazumi and M. Tsukahara Phosphorylation and caspases in DNA fragmentation
FEBS Journal 278 (2011) 427–441 ª 2010 The Authors Journal compilation ª 2010 FEBS 435
ST induces JNK- and p38 MAPK-mediated phosphor-
ylation of Bax at Thr167, leading to its activation in
HepG2 cells [122]. On the other hand, treatment with
OA increases Akt-mediated phosphorylation of Bax at
Ser184, which is important in the cytosolic retention of
Bax [83,84]. The opposite reactions regulate functional

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