Báo cáo Y học: Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in human leukemia HL-60 cells potx

Requirement of caspase and p38
MAPK
activation in zinc-induced
apoptosis in human leukemia HL-60 cells
Masuo Kondoh
1,2
, Emi Tasaki
2
, Saeko Araragi
2
, Masufumi Takiguchi
2
, Minoru Higashimoto
2
,
Yoshiteru Watanabe
1
and Masao Sato
2
1
Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Machida, Tokyo;
2
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan
Zinc (Zn), an endogenous regulator of apoptosis, and has
abilities both to induce apoptosis and inhibit the induction of
apoptosis via the modulation of caspase activity. Due to the
multifunctions of Zn, the intracellular Zn level is strictly
regulated by a complex system in physiological and patho-
logical conditions. The commitment of Zn to the regulation
of apoptosis is not fully understood. In the present study, we
investigated the role of intracellular Zn level in the induction

of apoptosis in human leukemia cells (HL-60 cells) using a
Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells
with Zn for 6 h in the presence of Py (1 l
M
) exhibited
cytotoxicity in a Zn dose-dependent manner (25–200 l
M
).
Necrotic cells, assayed by trypan blue permeability,
increased in number in a Zn dose-dependent fashion (50–
100 l
M
), but the appearance of apoptotic cells, assayed by
formation of a DNA ladder and terminal deoxynucleo-
tidyltransferase-mediated dUTP-biotin nick end-labeling
method, peaked at 25 l
M
, suggesting the dependence of
intracellular Zn level on the execution of apoptosis. In fact,
treatment with Py resulted in increases in intracellular Zn
levels, and N,N,N¢,N¢-tetrakis (2-pyridylmethyl)ethylenedi-
amine, a cell-permeable Zn chelator, inhibited DNA ladder
formation induced by Py/Zn treatment (1 l
M
Py and 25 l
M
Zn). Py/Zn treatment activated the caspases, as assessed by
the proteolysis of poly(ADP-ribose) polymerase (PARP),
which is a substrate of caspase, and activated p38 mitogen-
activated protein kinase (p38

MAPK
), which is a transducer of
apoptotic stimuli to the apparatus of the apoptosis execu-
tion. Z-Asp-CH
2
-DCB, a broad-spectrum inhibitor of
caspase, attenuated proteolysis of PARP and DNA ladder
formation by Py/Zn, indicating that apoptosis induced by
Py/Zn is mediated by caspase activation. The p38
MAPK
-
specific inhibitor SB203580 also inhibited induction of
apoptosis by Py/Zn. Although SB203580 suppressed the
proteolysis of PARP, Z-Asp-CH
2
-DCB did not inhibit the
phosphorylation of p38
MAPK
, raising the possibility that
apoptosis triggered by Py/Zn might be mediated by the
p38
MAPK
/caspase pathway.
Keywords: pyrithione; apoptosis; zinc; caspase; p38
MAPK
.
There are two major mechanisms of cellular death: necrosis
and apoptosis. Necrotic cell death is an unregulated process
resulting from severe damage to the cell and is characterized
by ATP depletion, cell swelling, lysis, and the release of

intracellular contents resulting in tissue inflammation [1–3].
In contrast, apoptosis is a highly regulated, energy-depend-
ent event that leads to the elimination of excess or damaged
cells from tissues. There are numerous pathological and
physiological stimuli of apoptotic cell death, including death
factors, reactive oxygen species, and genotoxic agents [4–7].
Despite differences in their morphology and most of their
biochemical features, apoptosis and necrosis frequently
coexist following insult [8–11].
Various cellular functions are influenced by essential
trace-elements such as the divalent cations zinc (Zn). The
physiological concentrations of these cations are strictly
regulated, and the maintenance of discrete subcellular
pools of Zn is critical for the functional and structural
integrity of cells and contributes to a number of important
biological processes, including not only gene expression,
DNA synthesis and enzymatic catalysis but also regulation
of apoptosis. For example, Zn is present in presynaptic
vesicles of central excitatory neurons and is released by
synaptic activity or membrane depolarization [12–17].
Exposure to excessive Zn is neurotoxic to cortical neurons,
and this toxicity has been shown to be mediated by Zn
influx through glutamate receptor- or voltage-gated Ca
2+
channels [18–20]. Zn also regulates apoptosis in thymo-
cytes. A high concentration of zinc (0.5–5 m
M
) inhibited
glucocorticoid-induced apoptosis in mouse thymocytes
[21], but Zn itself induced apoptosis at a low concentra-

tion (80–200 l
M
) in mouse thymocytes [22]. Schrantz et al.
[23]reportedthatZn(atconcentrationof10l
M
to 50 l
M
)
inhibited manganese-induced apoptosis, dependent on
the inhibition of caspase-3 activation, but that higher
concentrations of Zn (50–100 l
M
) did not prevent
Correspondence to M. Sato, Faculty of Pharmaceutical Sciences,
Tokushima Bunri University, Yamashiro-cho 180, Tokushima
770-8514, Japan. Fax: +81 88 6553051, Tel.: +81 88 6229611 ext.
5611, E-mail:
Abbreviations: ERK, extracellular signal-regulated kinase; ICP-MS,
inductively coupled plasma mass spectrometry; JNK, c-Jun NH
2
-
terminal kinases; MAPK, mitogen-activated protein kinases; MTT,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide;
PARP, poly(ADP-ribose) polymerase; Py, pyrithione; TPEN,
N,N,N¢,N¢-tetrakis-(2-pyridylmethyl)ethylenediamine; TUNEL,
terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick
end-labeling.
(Received 9 August 2002, revised 20 September 2002,
accepted 30 October 2002)
Eur. J. Biochem. 269, 6204–6211 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03339.x

manganese-mediated apoptosis but rather increased cell
death in human Burkitt lymphoma B cells.
Caspases, a conserved family of cysteine proteases, play
a central role in apoptosis [24]. Caspases themselves are
present as proenzymes that are readily cleaved and
activated during apoptosis, providing the cell with a
means to rapidly amplify its apoptotic response [25,26].
Recently, there has been growing attention to the mech-
anisms of transduction of apoptotic stimuli to the caspase.
One of the most relevant aspects in the regulation of
apoptosis is the involvement of mitogen-activated protein
kinases (MAPKs), a family of serine/threonine kinases
that mediate intracellular signal transduction in response
to various stimuli [27]. To date, three major MAPKs have
been identified: extracellular signal-regulated kinases
(ERK1/2), stress-activated protein kinases [c-Jun
NH
2
-terminal kinases (JNK)], and p38 mitogen-activated
protein kinases (p38
MAPK
). ERK1/2 are activated mainly
by growth factors and are critically involved in the
regulation of mitogenesis [28,29]. On the other hand, JNK
and p38
MAPK
are activated mainly by cytotoxic insult and
are often associated with apoptosis [30–36]. Moreover,
activation of p38
MAPK

was observed in Zn-treated cells
[37–39]. Intracellular Zn exists as fixed pools of Zn or as
more dynamic and labile Zn pools, which are thought to
be associated with the regulation of apoptosis by Zn [40].
However, the molecular mechanism of induction of
apoptosis by Zn remains to be unknown.
The present study was carried out to determine whether
the commitment of apoptosis is dependent on the
activation of caspase via activation of p38
MAPK
using a
Zn ionophore in human leukemia HL-60 cells. An acute
increase in the intracellular Zn level caused cytotoxicity in
an intracellular Zn level-dependent manner. At a low Zn
level, typical features of apoptosis such as DNA fragmen-
tation were observed. At a higher concentration, the
feature of cell death was necrosis. Moreover, the induction
of apoptosis was accompanied by activation of caspase
and p38
MAPK
. Both a broad-spectrum inhibitor of caspase
and an inhibitor of p38
MAPK
attenuated the induction of
apoptosis by Zn. Moreover, although the p38
MAPK
inhibitor also inhibited the caspase activation, the caspase
inhibitor did not attenuate the activation of p38
MAPK
.

Based on the results, it was concluded that induction of
apoptosis by intracellular labile Zn is mediated via the
p38
MAPK
/caspase pathway.
MATERIALS AND METHODS
Materials
All reagents were of analytical grade. Zinc sulfate, sodium
pyrithione (Py) and N,N,N¢,N¢-tetrakis-(2-pyridylmethyl)-
ethylenediamine (TPEN) were purchased from Sigma (St
Louis, MO, USA). Zinc sulfate and Py were dissolved in
sterile water at 10 m
M
and 100 l
M
, respectively, and
stored at )20 °C before use. TPEN was dissolved in
dimethyl sulfoxide. Z-Asp-CH
2
-DCB, a broad-spectrum
inhibitor of caspases, was obtained from Calbiochem-
Novabiochem Co. (San Diego, CA, USA) [41,42].
SB203580, an inhibitor of p38
MAPK
, was obtained from
Calbiochem-Novabiochem. Poly(vinylidene difluoride)
membranes were purchased from Millipore Co. (Bedford,
MA, USA). An antibody for poly(ADP-ribose) poly-
merase (PARP) was purchased from BD-PharMingen.
Anti-p38

MAPK
and a phosphorylated form of p38
MAPK
antibodies were purchased from New England BioLabs
(Beverly, MA, USA).
Cell culture
HL-60 cells, a human leukemia cell lines, were cultured in
RPMI 1640 containing 10% fetal bovine serum in a 5%
CO
2
atmosphere.
Cytotoxicity of Py/Zn in HL-60 cells
The cytotoxicity of Py/Zn in HL-60 cells was analyzed
by colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) assay with some modifica-
tion [43]. Briefly, after the addition of MTT (0.5 mgÆmL
)1
),
cells were incubated at 37 °C for 4 h. SDS (10% w/v) in
0.05
M
HCl was added to the wells and then incubated at
room temperature overnight under dark conditions. The
absorbance was measured at 595 nm.
Assessment of apoptosis and necrosis in Py/Zn-treated
cells
Apoptotic cells were assessed by the appearance of a DNA
ladder and by terminal deoxynucleotidyltransferase-medi-
ated dUTP end labeling (TUNEL) analysis. DNA ladder
formation was assayed as described previously [44]. Briefly,

HL-60 cells (5 · 10
5
cellsÆwell
)1
in a six-well plate) were
treated with Zn in the presence or absence of Py at the
indicated concentrations for various periods. The treated
HL-60 cells were harvested and incubated in lysis buffer
[10 m
M
Tris/HCl (pH 8.0), 10 m
M
EDTA, 0.5% w/v SDS,
and 0.1% w/v RNase A] for 60 min at 50 °C. Phenol/
chloroform-extracted DNA was subjected to a 1.8%
agarose electrophoresis and stained with ethidium bromide.
The TUNEL assay was performed using an apoptosis
detection system according to the manufacturer’s protocol
(Promega Co., Madison, WI, USA). The criteria used to
determine necrosis was the loss of membrane integrity,
which was determined by permeability to trypan blue in
nonpermeabilized cells [45].
Measurement of intracellular trace metals
The treated HL-60 cells were washed once with phosphate-
buffered saline (NaCl/P
i
) and twice with NaCl/P
i
containing
10 m

M
EDTA. The washed cells was incubated with HNO
3
for 16 h at room temperature and for an additional 2 h at
60 °C. Amounts of intracellular trace elements were then
measured using an inductively coupled plasma-mass spec-
trometer (ICP-MS) (HEWLETT PACKARD 4500) or an
atomic absorption spectrophotometer (HITACHI Z-8200),
and the amount of each elements expressed as the concen-
tration of Zn per mg of the cellular protein. Protein assay
was performed using a Bio-Rad protein assay kit.
Western blot analysis
Vehicle- or Py/Zn-treated cells were lysed in lysis buffer
consisting of 1% NonidetP-40, 20 m
M
Tris/HCl (pH 8.0),
Ó FEBS 2002 Zn and apoptosis (Eur. J. Biochem. 269) 6205
137 m
M
NaCl, 10% glycerol, 1 m
M
phenylmethylsulfonyl
fluoride and 1 m
M
EDTA by sonication. Equal amounts of
samples were subjected to 10% SDS/PAGE and then
transferred to poly(vinylidene difluoride) membranes. The
membranes were blocked with 10 m
M
Tris/HCl (pH 7.5),

100 m
M
NaCl, and 0.05% Tween-20 containing 5% (w/v)
non fat milk for overnight at 4 °C. Anti-PARP, p38
MAPK
or
a phosphorylated form of p38
MAPK
was used as a primary
antibody, and a horseradish peroxide-labeled antibody was
used as a secondary antibody. The antibody-reactive bands
were revealed by ECL-based detection (Amersham Phar-
macia Biotech).
Statistical analysis
Data were analyzed by analysis of variance, followed by
Bonferroni multiple comparison test, or where applicable,
by Student’s t-test. The acceptable level of significance was
set at P < 0.05.
RESULTS
Determination of features of cell death induced
by elevation of intracellular Zn levels
Although Zn is used as an inhibitor of cell death via caspase
activation, direct evidence of Zn-mediated cell death has
been obtained in a transient global ischemia [46]. There are
complex systems for the regulation of intracellular zinc level
such as the zinc transporters, Zn-1, -2, -3 or -4 [47–51].
Therefore, we used a zinc ionophore (Py) to investigate the
cytotoxicity induced by elevation of intracellular Zn level.
The addition of extracellular Zn (at concentrations of up to
200 l

M
) to HL-60 cells did not cause cytotoxicity. However,
cytotoxicity of Zn was observed in the presence of Py
(Fig. 1A). To determine the characteristics of cell death
induced by Py/Zn treatment, we counted the number of
necrotic and apoptotic cells by trypan blue staining and
TUNEL methods, respectively. As shown in Fig. 1B,
Fig. 1. Characterization of the features of cell death induced by treatment with Zn plus a Zn ionophore. (A) Cytotoxicity of Zn in the presence or
absence of a Zn ionophore, pyrithione (Py). HL-60 cells (5 · 10
5
cellsÆmL
)1
)weretreatedwithZnattheindicatedconcentrationsinthepresenceor
absence of Py (1 l
M
) for 6 h. Then the viability was assessed by MTT assay. Data are means ± SD (n ¼ 4). *Significantly different from the
Zn-treated cells without Py (P < 0.05). Data represent two independent experiments. (B) Induction of necrosis and apoptosis by Py/Zn treatment.
After 6 h of treatment of Zn at the indicated concentration with or without Py, the number of necrotic and apoptotic cells were determined by
trypan blue staining and TUNEL assay, respectively. Data are means ± SD (n ¼ 4). *Significantly different from the vehicle-treated cells
(P < 0.05). ND, not determined. The results are representative of two independent experiments. (C) DNA fragmentation assay. After 6 h of
treatment with Zn at the indicated concentration with or without Py, extracted DNA was subjected to electrophoresis on a 1.8% agarose and then
stained with ethidium bromide. (D) Attenuation of Py/Zn-induced apoptosis by Zn chelator. After 2 h of treatment with TPEN (2 l
M
), cells were
treated with 25 l
M
Zn plus 1 l
M
Py for 6 h, and then the appearance of DNA ladder was assayed. The results are representative of three
independent experiments.

6206 M. Kondoh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
apoptotic cells appeared at 25 l
M
Zninthepresenceof
1 l
M
Py, while necrotic cells appeared Zn concentrations of
more than 50 l
M
in the presence of 1 l
M
Py. DNA ladder
formation, a characteristic feature of apoptotic cells, was
also observed at 25 l
M
Zn in the presence of 1 l
M
Py
(Fig. 1C). Thus, the features of cell death changed
depending on the intracellular Zn levels.
Induction of apoptosis by the elevation of intracellular
Zn levels at a low concentration but not a high
concentration
Next, we investigated the involvement of intracellular Zn
levels in the induction of apoptosis by cotreatment of 1 l
M
Py and 25 l
M
Zn (Py/Zn treatment). Determination of trace
elements in Py/Zn-treated cells by ICP-MS was performed.

As described in Table 1, treatment of Py or Zn alone did not
change intracellular Zn levels, but Py/Zn elevated intracel-
lular Zn levels to a level about twofold greater than that of
the vehicle-treated control. The intracellular Cu level was
also increased by Py/Zn treatment. Although Py treatment
alone, but not Zn treatment, enhanced intracellular Cu
levels, Py did not induce apoptosis, indicating that the
enhanced level of Cu is not sufficient to induce apoptosis
(Fig. 1B,C). Therefore, elevation of the intracellular Zn
levels was probably an essential event in the induction of
apoptosis by Py/Zn. Indeed, TPEN, a specific Zn
2+
chelator [52], abolished DNA ladder induced by Py/Zn
treatment (Fig. 1D). To determine the relationship between
intracellular Zn levels and induction of apoptosis, we
investigate the appearance of a DNA ladder induced by Zn
at concentrations in a narrow range in the presence of 1 l
M
Py. Although intracellular Zn levels increased with Zn
concentration in a Zn dose-dependent manner, Zn-induced
DNA ladder formation at concentrations of 20–30 l
M
with
maximal induction at 25 l
M
(Fig. 2A,B). These findings
indicated that the intracellular Zn level is an important
factor in the determination of type of cell death, necrosis or
apoptosis.
Involvement of caspase in apoptosis induced by Zn

There are various components of cell death machinery that
induce apoptosis [53,54]. Caspases, a family of cysteine
proteases, play a pivotal role in the execution of apoptosis
[26]. We therefore examined the involvement of caspases in
the apoptosis induced by Py/Zn. PARP is a substrate of
caspases, and proteolysis of PARP is an index of activation
of caspases [55–58]. Therefore, we first examined changes in
the time courses of induction of apoptosis (Fig. 3A) and
proteolysis of PARP (Fig. 3B) by Py/Zn. Induction of
apoptosis and activation of caspases were observed at 6 h of
treatment. Moreover, Z-Asp-CH
2
-DCB, a broad-spectrum
inhibitor of caspases, attenuated the induction of apoptosis
[41,42] (Fig. 3C) and also inhibited the proteolysis of PARP
(Fig. 3D) induced by Py/Zn. These data suggest that
Py/Zn-induced apoptosis is dependent on the activation
of caspases.
Involvement of p38
MAPK
in apoptosis induced by Zn
Apoptotic signals are transferred to the apoptotic apparatus
via various signal transduction pathways [53,54]. Recent
studies have suggested that apoptotic stimuli are transferred
to caspases via a MAPK cascade such as p38
MAPK
and
JNK [59–62], and treatment with Zn has been shown to
activate p38
MAPK

[37–39]. Therefore, we investigated the
requirement of p38
MAPK
in the activation of caspases in Py/
Zn-induced apoptosis. Activation of p38
MAPK
was deter-
mined by Western blot analysis using an antibody for the
activated form of p38
MAPK
. As shown in Fig. 4A, activation
of p38
MAPK
, determined by the phosphorylation of
p38
MAPK
, occurred at 3 h prior to the caspase activation
(6 h) and induction of apoptosis (6 h) (Figs 3A,B and 4A).
Moreover, SB203580, a specific inhibitor of p38
MAPK
,
inhibited the activation of p38
MAPK
and the induction of
apoptosis (Fig. 4B,C). SB203580 also inhibited the activa-
tion of caspases (Fig. 4D). Z-Asp-CH
2
-DCB did not inhibit
the activation of p38
MAPK

(Fig. 4E). These data indicate
that activation of p38
MAPK
is required for the caspases
activation in Py/Zn-induced apoptosis.
DISCUSSION
Apoptosis, an endogenous suicide program, plays a central
role in the maintenance of homeostasis, and there is an
endogenous mechanism by which induction of apoptosis is
controlled [53,54]. Several trace elements such as Zn,
calcium and magnesium are known to be endogenous
regulators of the induction of apoptosis. Calcium and
magnesium are components of endonuclease, which plays a
role in fragmentation of chromosomes into nucleosome
fragments in apoptosis [63,64]. Interestingly, Zn functions as
a inhibitor of apoptosis dependent on the inhibition of
activation of caspases, and also as an inducer of apoptosis,
dependent on the activation of caspases [23,65]. However,
the mode of action of Zn in induction of apoptosis remains
to be unclear. In this study, we investigated the molecular
mechanism underlying the mode of action of the induc-
tion of apoptosis by elevation of intracellular Zn levels.
Table 1. Changes in levels of trace elements by Py/Zn treatment. After 2 h of treatment of cells, the intracellular levels of trace elements were
determined as described in Materials and methods. Data are means ± SD (n ¼ 4). Groups without a common superscript letter are significantly
different at P < 0.05.
Treatment (l
M
) Trace elements (ng mg
)1
protein)

Py Zn Mg Ca Mn Fe Cu Zn
0 0 1219.86 ± 30.66
a
42.86 ± 11.89
a
2.32 ± 0.03
a
20.59 ± 6.61
a
13.42 ± 0.28
a
99.46 ± 3.91
a
1 0 1194.45 ± 13.30
a
25.44 ± 1.70
b
1.81 ± 0.02
b
14.49 ± 1.92
a
26.68 ± 0.60
b
95.03 ± 1.15
a
0 25 1167.09 ± 13.16
a
23.42 ± 4.96
b
1.06 ± 0.04

c
11.27 ± 1.06
b
11.99 ± 0.42
a
90.48 ± 1.87
a
1 25 1376.34 ± 57.47
b
20.82 ± 6.60
b
1.02 ± 0.07
c
13.68 ± 3.09
a
31.66 ± 1.21
c
200.24 ± 8.51
b
Ó FEBS 2002 Zn and apoptosis (Eur. J. Biochem. 269) 6207
Organisms are equipped with systems for intracellular Zn
levels, and it has been shown that exogenous addition of Zn
at a physiological concentration did not elevate intracellular
Zn level [47–51]. Therefore, in this study, we used a Zn
ionophore for the purpose of specific elevation of intracel-
lular Zn levels. As the intracellular Zn level increased, the
cytotoxicity of Zn in HL-60 cells was enhanced. However,
the mode of cell death was dependent on the concentration
of intracellular Zn. DNA ladder formation, a typical feature
of apoptotic cells, was observed at 141 ng ZnÆmg protein

)1
(basal level, 119 ng ZnÆmg protein
)1
), and DNA ladder
formation decreased above 211 ng ZnÆmg protein
)1
,
indicating that the ability of Zn to induce apoptosis is
dependent on the intracellular Zn level. In fact, it has been
shown that treatment with Zn (80–200 l
M
) induced apop-
tosis in mouse thymocytes [22], and it was found in another
study that Zn (0.5–5 m
M
) inhibited apoptosis in glucocor-
ticoid-treated mouse thymocytes [21]. It is well known that
Zn is an inhibitor of apoptosis mediated by inhibition of
caspases at a millimolar concentration [65]. Indeed,
although treatment with 25 l
M
Zn caused proteolysis of
PARP in the presence of Py (1 l
M
), 50 l
M
Zn did not
induce cleavage of PARP, indicating that a low level of
intracellular Zn triggers activation of caspases (data not
shown). Schrantz et al. [23] reported that caspase activation

is required for Zn-induced apoptosis in human Burkitt
lymphoma B cells. An increase in labile Zn in the cytoplasm
may suppress apoptosis by inhibition of the action of
caspase, cytoplasmic apoptosis inducer, via direct associ-
ation of Zn with caspases [40]. Although we did not examine
the localization of Zn introduced by Py, Zalewski et al.[66]
reported that labile Zn introduced by Py was localized in the
cytosol. Taken together, the results suggest that labile Zn
released from Zn pools is a potent regulator of apoptosis via
regulation of the activities of caspases.
It has been reported that execution of caspase activation
is preceded by intracellular signal transduction such as the
activation of p38
MAPK
triggered by apoptotic stimuli
[45,62]. Influx of Zn into the cytosol had already occurred
at 2 h after the addition of Py/Zn in the present study (data
not shown). Therefore, we turned our attention to the
MAPK cascade as an apoptotic signal transduction
machinery, which is involved in the early events of induc-
tion of apoptosis [37,59–62,67]. Especially, p38
MAPK
was
activated by Zn [37–39]. In this study, we found that
Fig. 3. Involvement of activation of caspases in the induction of apop-
tosis by Py/Zn. (A,B) Time-course study. Cells were treated with 25 l
M
Zn plus 1 l
M
Py. DNA fragmentation (A) and proteolysis of PARP

(B) were investigated at the indicated periods. (C,D) Effects of Z-Asp-
CH
2
-DCB. Cells, after pretreatment with Z-Asp-CH
2
-DCB (100 l
M
)
for 1 h, were incubated with 25 l
M
Zn plus 1 l
M
Py for 6 h. DNA
fragmentation (C) and proteolysis of PARP (D) were investigated. The
results are representative of three independent experiments.
Fig. 2. Relationship between intracellular Zn levels and induction of
apoptosis. Cells were incubated with Zn at the indicated concentration
in the absence or presence of Py (1 l
M
). (A) Induction of apoptosis.
After 6 h of treatment, induction of apoptosis was assessed by DNA
ladder formation. The results are representative of three independent
experiments. (B) Intracellular Zn level. Intracellular Zn levels were
measured using an atomic absorption spectrophotometer after 2 h of
treatment. Data are means ± SD (n ¼ 4). Groups without a common
superscript letter are significantly different at P < 0.05. The results are
representative of two independent experiments.
6208 M. Kondoh et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Py/Zn-induced apoptosis was mediated by activation of
caspases followed by rapid activation of p38

MAPK
. Previous
studies have demonstrated induction of apoptosis by
dopamine or nitric oxide dependent on p38
MAPK
activation
followed by activation of caspase-3 [61,62]. These findings
support our results. The rapid phosphorylation of p38
MAPK
suggests that MAPK plays a role in the early stage of
induction of Zn-induced apoptosis. The precise mechanism
of activation of p38
MAPK
remains to be elucidated. One
possible explanation is the involvement of production of
reactive oxygen species induced by Zn. In fact, Kim et al.
[68] showed that Zn-induced cytotoxicity was caused by the
production of reactive oxygen species. p38
MAPK
was also
reported to be activated by the production of reactive
oxygen species in Cd-treated cells [45].
In summary, we demonstrated in this study that optimal
intracellular Zn levels can be an initiator of apoptosis.
Considering the ability of Zn to inhibit the activation of
caspase, Zn might play a major role as an endogenous
regulator of apoptosis in physiological conditions. More-
over, activation of p38
MAPK
followed by caspase activation

was found to be required for induction of apoptosis by Zn.
This is the first study to show the involvement of a
p38
MAPK
/caspase-dependent pathway in the induction of
apoptosis by a low level of intracellular labile Zn.
ACKNOWLEDGMENTS
This work was supported in part by a Grant-in-Aid for General
Scientific Research from the Ministry of Education, Sciences, Sports
and Culture of Japan.
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Fig. 4. Involvement of activation of p38
MAPK
in the induction of apop-
tosis by Py/Zn. (A) Time-course study of activation of p38
MAPK
. Cells
were treated with 25 l
M
Zn plus 1 l
M
Py for the indicated periods.
Activation of p38
MAPK
was assessed by the phosphorylation of
p38
MAPK
by Western blotting analysis using an antibody for the
activated p38
MAPK
. (B,C) Involvement of p38
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in the Py/Zn-
treated cells. Cells, after pretreatment with SB203580 (5 l
M
)for1h,
were incubated with 25 l
M

Zn plus 1 l
M
Py for 6 h. Activation of
p38
MAPK
(B) and formation of DNA fragmentation (C) were inves-
tigated. (D) Involvement of p38
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in the activation of caspases in
Py/Zn-treated cells. Cells were treated with SB203580 for 1 h and then
treated with 25 l
M
Zn plus 1 l
M
Py for 6 h. Activation of p38
MAPK
was then examined. (E) Involvement of caspases in the activation of
p38
MAPK
in Py/Zn-treated cells. Cells were treated with Z-Asp-CH
2
-
DCB for 1 h and then treated with 25 l
M
Zn plus 1 l
M
Py for 6 h.
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