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

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

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

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
.

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,

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),

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.

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

[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

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

)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

(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

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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