Requirement of caspase and p38MAPK activation in zinc-inducedapoptosis in human leukemia HL-60 cells Masuo Kondoh1,2, Emi Tasaki2, Saeko Araragi2, Masufumi Takiguchi2, Minoru Higashimoto
Trang 1Requirement of caspase and p38MAPK activation in zinc-induced
apoptosis in human leukemia HL-60 cells
Masuo Kondoh1,2, Emi Tasaki2, Saeko Araragi2, Masufumi Takiguchi2, Minoru Higashimoto2,
Yoshiteru Watanabe1and Masao Sato2
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 lM) exhibited
cytotoxicity in a Zn dose-dependent manner (25–200 lM)
Necrotic cells, assayed by trypan blue permeability,
increased in number in a Zn dose-dependent fashion (50–
100 lM), 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 lM, 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 lMPy and 25 lM
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 (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execu-tion Z-Asp-CH2-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 p38MAPK -specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn Although SB203580 suppressed the proteolysis of PARP, Z-Asp-CH2-DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway
Keywords: pyrithione; apoptosis; zinc; caspase; p38MAPK
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 evenergy-depend-ent 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 Ca2+ channels [18–20] Zn also regulates apoptosis in thymo-cytes A high concentration of zinc (0.5–5 mM) inhibited glucocorticoid-induced apoptosis in mouse thymocytes [21], but Zn itself induced apoptosis at a low concentra-tion (80–200 lM) in mouse thymocytes [22] Schrantz et al [23] reported that Zn (at concentration of 10 lMto 50 lM) inhibited manganese-induced apoptosis, dependent on the inhibition of caspase-3 activation, but that higher concentrations of Zn (50–100 l ) 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: msato@ph.bunri-u.ac.jp
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)
Trang 2manganese-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
NH2-terminal kinases (JNK)], and p38 mitogen-activated
protein kinases (p38MAPK) 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 p38MAPKare activated mainly by cytotoxic insult and
are often associated with apoptosis [30–36] Moreover,
activation of p38MAPK 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 p38MAPK 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 p38MAPK Both a broad-spectrum inhibitor of caspase
and an inhibitor of p38MAPKattenuated the induction of
apoptosis by Zn Moreover, although the p38MAPK
inhibitor also inhibited the caspase activation, the caspase
inhibitor did not attenuate the activation of p38MAPK
Based on the results, it was concluded that induction of
apoptosis by intracellular labile Zn is mediated via the
p38MAPK/caspase pathway
M A T E R I A L S A N D M E T H O D S
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 mM and 100 lM, respectively, and
stored at )20 C before use TPEN was dissolved in
dimethyl sulfoxide Z-Asp-CH2-DCB, a broad-spectrum
inhibitor of caspases, was obtained from
Calbiochem-Novabiochem Co (San Diego, CA, USA) [41,42]
SB203580, an inhibitor of p38MAPK, 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-p38MAPK and a phosphorylated form of p38MAPK antibodies were purchased from New England BioLabs (Beverly, MA, USA)
Cell culture HL-60 cells, a human leukemia cell lines, were cultured in RPMI1640 containing 10% fetal bovine serum in a 5%
CO2atmosphere
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 addimodifica-tion of MTT (0.5 mgÆmL)1), cells were incubated at 37C for 4 h SDS (10% w/v) in 0.05MHCl 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· 105 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 mMTris/HCl (pH 8.0), 10 mMEDTA, 0.5% w/v SDS, and 0.1% w/v RNase A] for 60 min at 50C 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/Pi) and twice with NaCl/Picontaining
10 mMEDTA The washed cells was incubated with HNO3 for 16 h at room temperature and for an additional 2 h at
60C 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 Tris/HCl (pH 8.0),
Trang 3137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl
fluoride and 1 mMEDTA 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 mMTris/HCl (pH 7.5),
100 mMNaCl, and 0.05% Tween-20 containing 5% (w/v)
non fat milk for overnight at 4C Anti-PARP, p38MAPKor
a phosphorylated form of p38MAPKwas 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
R E S U L T S
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 lM) 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 · 105cellsÆmL)1) were treated with Zn at the indicated concentrations in the presence or 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.
Trang 4apoptotic cells appeared at 25 lM Zn in the presence of
1 lMPy, while necrotic cells appeared Zn concentrations of
more than 50 lMin the presence of 1 lMPy DNA ladder
formation, a characteristic feature of apoptotic cells, was
also observed at 25 lM Zn in the presence of 1 lM 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 lM
Py and 25 lMZn (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 Zn2+
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 lM
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 lMwith
maximal induction at 25 lM (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-CH2-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 p38MAPKin 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 p38MAPK and JNK [59–62], and treatment with Zn has been shown to activate p38MAPK[37–39] Therefore, we investigated the requirement of p38MAPKin the activation of caspases in Py/ Zn-induced apoptosis Activation of p38MAPKwas deter-mined by Western blot analysis using an antibody for the activated form of p38MAPK As shown in Fig 4A, activation
of p38MAPK, determined by the phosphorylation of p38MAPK, 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 p38MAPK, inhibited the activation of p38MAPKand the induction of apoptosis (Fig 4B,C) SB203580 also inhibited the activa-tion of caspases (Fig 4D) Z-Asp-CH2-DCB did not inhibit the activation of p38MAPK (Fig 4E) These data indicate that activation of p38MAPK is required for the caspases activation in Py/Zn-induced apoptosis
D I S C U S S I O N 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 elevainduc-tion 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)1protein)
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 ± 13.16a 23.42 ± 4.96b 1.06 ± 0.04c 11.27 ± 1.06b 11.99 ± 0.42a 90.48 ± 1.87a
1 25 1376.34 ± 57.47b 20.82 ± 6.60b 1.02 ± 0.07c 13.68 ± 3.09a 31.66 ± 1.21c 200.24 ± 8.51b
Trang 5Organisms 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 intracelintracel-lular 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 lM) induced apop-tosis in mouse thymocytes [22], and it was found in another study that Zn (0.5–5 mM) 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 lM Zn caused proteolysis of PARP in the presence of Py (1 lM), 50 lM 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 p38MAPK 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, p38MAPKwas 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.
Trang 6Py/Zn-induced apoptosis was mediated by activation of
caspases followed by rapid activation of p38MAPK Previous
studies have demonstrated induction of apoptosis by
dopamine or nitric oxide dependent on p38MAPKactivation
followed by activation of caspase-3 [61,62] These findings
support our results The rapid phosphorylation of p38MAPK
suggests that MAPK plays a role in the early stage of
induction of Zn-induced apoptosis The precise mechanism
of activation of p38MAPK 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 p38MAPKwas 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 p38MAPKfollowed 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 p38MAPK/caspase-dependent pathway in the induction of apoptosis by a low level of intracellular labile Zn
A C K N O W L E D G M E N T S
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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apop-tosis by Py/Zn (A) Time-course study of activation of p38MAPK Cells
were treated with 25 l M Zn plus 1 l M Py for the indicated periods.
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