(Received for publication, March 24, 1997, and in revised form, May 19, 1997)
From the The prevention of apoptosis by
Zn2+ has generally been attributed to its inhibition
of an endonuclease acting in the late phase of apoptosis. In this study
we investigated the effect of Zn2+ on an earlier event in
the apoptotic process, the proteolysis of the "death substrate"
poly(ADP-ribose) polymerase (PARP). Pretreatment of intact Molt4
leukemia cells with micromolar concentrations of Zn2+
caused an inhibition of PARP proteolysis induced by the
chemotherapeutic agent etoposide. Using a cell-free system consisting
of purified bovine PARP as a substrate and an apoptotic extract or
recombinant caspase-3 as the PARP protease, Zn2+ inhibited
PARP proteolysis in the low micromolar range. To rule out an effect of
Zn2+ on PARP, a protein with two zinc finger domains, we
used recombinant caspase-3 and a chromogenic tetrapeptide substrate
containing the caspase-3 cleavage site. In this system,
Zn2+ inhibited caspase-3 with an IC50 of 0.1 µM. These results identify caspase-3 as a novel target of
Zn2+ inhibition in apoptosis and suggest a regulatory role
for Zn2+ in modulating the upstream apoptotic
machinery.
Apoptosis is a genetically programmed process of cell death
characterized by a series of distinct morphological changes (1). Normal
development and tissue remodeling of multicellular organisms are
dependent on apoptosis, whereas defects in this process have been
implicated in a number of pathological conditions. The agents which
induce cells to undergo apoptosis are diverse and include extracellular
agents such as TNF- The influence of Zn2+ on apoptosis is a well known
phenomenon (2). In both in vitro and in vivo
models, Zn2+ supplementation prevents apoptosis induced by
a variety of agents (3, 4). Moreover, cells grown under conditions of
Zn2+ deficiency will undergo spontaneous apoptosis
(5-7).
The protective effect of Zn2+ has been attributed to its
inhibition of a Ca2+- and
Mg2+-dependent endonuclease (8), thereby
causing inhibition of DNA fragmentation, a terminal step and hallmark
of apoptosis. However, a number of observations suggest the existence
of other and perhaps more relevant targets for Zn2+. First,
the concentrations of Zn2+ used to demonstrate
anti-apoptotic effects in either intact cells or isolated nuclei have
ranged from micromolar to millimolar levels. However, the
concentrations of Zn2+ used for inhibition of the purified
Ca2+-dependent endonuclease were in the
millimolar range (9). Second, it has recently been reported that in
L929 cells, Zn2+ inhibited both TNF- Recent studies have pointed to a role for a family of caspase proteases
(formerly the ICE/Ced-3 proteases) (12) in apoptosis that act upstream
of the endonuclease. Proteases in apoptosis came to the forefront with
studies on the proteolysis of poly(ADP-ribose) polymerase (PARP),
initially described in cells induced to undergo apoptosis by various
chemotherapeutic agents, including etoposide (13-15). This event was
later determined to be catalyzed by the protease resembling ICE (16).
The human homolog of this protease has been cloned and is now known as
caspase-3 (CPP32/yama/apopain).
To determine if Zn2+ inhibits an event upstream of
endonuclease activation, we investigated the effect of Zn2+
on etoposide-induced apoptosis using PARP proteolysis as an indicator of this process. We show that Zn2+ is a potent inhibitor of
PARP proteolysis in intact cells. We also demonstrate in a cell-free
system that Zn2+ potently inhibits PARP proteolysis induced
both by a caspase-3-containing apoptotic extract and by purified
recombinant caspase-3. These results identify caspase-3 as a novel and
proximal site of Zn2+ inhibition in the apoptotic pathway.
Moreover, in light of results demonstrating a labile pool of
Zn2+ in cells which is in rapid equilibrium with the
extracellular medium (17), these findings raise the possibility that
the cell may utilize Zn2+ as a regulator of the upstream
apoptotic machinery.
Molt4 cells from ATCC (Rockville, MD) were
maintained under subconfluent conditions in RPMI medium with 10% fetal
calf serum. For experiments, cells were diluted to 5 × 105/ml in RPMI with 2% fetal calf serum and treated with
either etoposide delivered in a solution of 50% ethanol, 50%
Me2SO or vehicle control.
The apoptotic extract was obtained by
harvesting the cells after treatment with 20 µM etoposide
for 6 h, resuspending once in phosphate-buffered saline, and then
resuspending in cell-free buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 3 mM NaCl, 4 mM
MgCl2) at a concentration of 108 cells/ml.
Cells were then lysed by N2 cavitation for 10 min at 450 p.s.i. The lysate was centrifuged at 100,000 × g for 30 min, and the supernatant was retained. PARP
protease activity was retained in this fraction for at least 6 months
when kept frozen at Reactions in the cell-free system
were initiated by the addition of 25 ng of purified bovine PARP to
either 10 µg of the apoptotic extract or to 40 ng of caspase-3 in a
total volume of 15 µl of cell-free buffer. After 20 min at 37 °C,
the reactions were stopped by the addition of Laemmli buffer. For
cation studies, the apoptotic extract or recombinant caspase-3 was
added to buffer containing the respective cations immediately prior to
the addition of the PARP substrate.
A full-length cDNA
encoding caspase-3 was cloned into pET23b from Invitrogen (San Diego,
CA) and expressed in Escherichia coli strain BL21(DE3)pLysS
as described previously (18). The caspase contained a C-terminal
His6-tag to facilitate purification by affinity
chromatography on Ni2+-NTA-agarose from Qiagen (Chatsworth,
CA) according to the manufacturer's instructions.
Samples from intact cells and the
cell-free system or the apoptotic extract from etoposide-treated cells
were boiled in Laemmli buffer and loaded onto 6 or 15% polyacrylamide
gels for determination of PARP or pro-caspase-3 proteolysis,
respectively. After transfer to nitrocellulose membrane, the membranes
were incubated with a rabbit polyclonal antibody (1:2000) to an epitope
in the automodification domain of PARP or with a mouse polyclonal
antibody from Transduction Laboratories (Lexington, KY) (1:1000) to a
peptide in the p17 domain of caspase-3. Detection was accomplished
using a horseradish peroxidase conjugate of a goat anti-rabbit (1:5000)
or goat anti-mouse antibody (1:5000) from Bio-Rad for PARP and
pro-caspase-3 proteolysis, respectively, and the ECL detection system
from Amersham.
N-Acetyl-Asp-Glu-Val-Asp-p-nitroanilide
(Ac-DEVD-pNA) from Biomol (Plymouth Meeting, PA) at a concentration of
200 µM was incubated with either 20 µg of the apoptotic
extract from cells treated with 20 µM etoposide for
6 h or with 280 ng of recombinant caspase-3 for 10 min at 37 °C
in the presence or absence of varying concentrations of
ZnCl2 in the cell-free buffer. Substrate cleavage was
quantified by measuring the absorbance at 405 nm in a Shimadzu spectrophotometer.
To determine the effect of Zn2+ on etoposide-induced
PARP proteolysis in intact cells, Molt4 culture medium was supplemented with varying doses of ZnCl2 prior to treatment of the cells
with etoposide. The data in Fig. 1 indicate that
Zn2+ caused a dose-dependent inhibition of PARP
proteolysis with complete inhibition occurring at 100 µM
ZnCl2. Zn2+ treatment alone had no effect on
PARP proteolysis (data not shown).
To ascertain if the inhibitory action of Zn2+ was directly
on PARP proteolysis, we used a cell-free system consisting of a soluble extract from etoposide-treated cells and bovine PARP as a substrate. We
have previously used this system to study PARP proteolysis (19). The
soluble extract contains processed caspase-3 as shown by the data in
Fig. 2. Caspase-3 is synthesized as an inactive 32-kDa
zymogen (20). During activation, the zymogen is cleaved to yield two
subunits of 17 and 12 kDa which dimerize to form an active enzyme (21).
Using an antibody which recognizes both the zymogen and the 17-kDa
subunit, the immunoblot in Fig. 2 demonstrates that processing of the
zymogen was initiated between 2 and 3 h of etoposide treatment as
assessed by the appearance of the 17-kDa subunit. This closely
correlates with the induction of PARP proteolysis by etoposide in this
cell line (19).
Using an apoptotic extract from cells that had been treated for 6 h with etoposide, the results of Fig. 3 show that
Zn2+ and Cu2+ caused complete inhibition of
PARP proteolysis at a concentration of 10 µM. The effect
was independent of the counter- ion as both ZnCl2 and
ZnSO4, as well as CuCl2 and CuSO4,
gave identical results. Furthermore, other cations including
Ba2+, Ca2+, Mn2+, and
Fe3+ had no effect on PARP proteolysis. These results
strongly suggest that the means by which Zn2+ inhibits PARP
proteolysis is by acting directly on the PARP proteolysis step, either
by inhibiting the protease(s) or by rendering PARP insensitive to
proteolysis.
To further investigate the mechanism of Zn2+ inhibition and
its target of action, we examined the effects of Zn2+ and
other cations on PARP proteolysis catalyzed by purified recombinant caspase-3. The results in Fig. 4 indicate that the
extent of inhibition of recombinant caspase-3 by Zn2+ is
similar to the extent of inhibition observed on PARP protease activity
using the apoptotic extract and that the cation profile is identical to
that obtained in the apoptotic extract (Fig.
3).2 Inhibition was also obtained with
Cd2+ and Hg2+ (data not shown). These results
demonstrate that Zn2+ directly inhibits PARP cleavage
induced by caspase-3.
PARP is a nuclear enzyme that contains two zinc finger domains near its
amino terminus. It has been demonstrated that Zn2+ is
required for PARP binding to damaged DNA (22); however, the influence
of Zn2+ on the ability of PARP to serve as a substrate for
caspase-3 has not been investigated. To eliminate the possibility that
Zn2+ binding to the zinc finger domains of PARP may render
it insensitive to proteolysis, we utilized a chromogenic tetrapeptide
substrate, Ac-DEVD-pNA, containing the caspase-3 cleavage site in PARP
and lacking the zinc finger domains. The results from Fig.
5 demonstrate that Zn2+ inhibited the
cleavage of this substrate by the apoptotic extract with an
IC50 value of nearly 1 µM (filled
circles). A more potent inhibition by Zn2+
(IC50
The mechanism by which Zn2+ inhibits caspase-3 awaits
further studies. However, investigations on the crystal structure of
ICE have suggested that His-237 and Cys-285 are involved in catalysis (23). These residues are conserved in all the caspase family members
identified to date (24). Given the affinity of these amino acids for
Zn2+, it is possible that Zn2+ inhibits
caspase-3 by coordinating with one or both of these conserved sites.
Moreover, Zn2+ inhibition of caspase-3 in the mid-nanomolar
to low micromolar range is indicative of Zn2+ binding to a
single (or perhaps two) amino acid residue, whereas the tetrahedral
coordination of Zn2+ found in many Zn2+ finger
domains is characterized by a much greater affinity
(kd It has previously been reported that Zn2+ does not inhibit
caspase-3 activity (11). The reason(s) for the discrepancy with our
results is unclear but a possible explanation is that we have used
purified recombinant caspase-3 in our study, whereas in the referred
study an E. coli lysate containing the recombinant enzyme was used. Therefore, it is possible that the lysate contained factors
which were responsible for mitigating the inhibitory effect of
Zn2+ on caspase-3.
In the family of caspase proteases, caspase-3 has the highest homology
to the Ced-3 protease which is required for developmental cell death in
Caenorhabditis elegans (20). It is also activated by a
variety of agents that induce apoptosis including TNF- The question arises as to whether the concentrations of
Zn2+ required to inhibit caspase-3 in this study are
attainable within the cell. It has been determined that serum
Zn2+ concentrations are 10-15 µM (29) and
that the free Zn2+ concentration in serum is approximately
1 µM (30). Furthermore, the intracellular
Zn2+ concentration has been estimated at 100 µM (17). Nearly 10% of this ( The results reported herein, in conjunction with a recent report
demonstrating that treatment of T cells with a Zn2+
chelator is able to induce caspase-3 activation (32), suggest that
physiologic modulation of intracellular Zn2+ may have
important consequences for the regulation of caspases and the induction
of apoptosis.
We thank Dr. Carol Fierke of Duke University,
Dr. Tariq Ghayur of BASF Bioresearch, and Dr. Thomas O'Halloran of
Northwestern University for helpful discussions and advice.
Department of Medicine,
Centre
Hospitalier de L'Universite Laval Research Center, Laval University,
Sainte-Foy, Quebec G1V 4G2, Canada
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,1 the Fas ligand,
and chemotherapeutic agents. Intracellular agents which regulate
apoptosis include proteases, phosphatases, and kinases, products of
lipid metabolism, and the cations Ca2+ and
Zn2+.
- and
etoposide-induced cytotoxicity, as assessed by methylene blue staining,
prior to effects on DNA fragmentation (10). Third, it is becoming more
evident that the endonuclease functions in the "execution" rather
than "regulation" phase of apoptosis, and therefore its inhibition
may not prevent cell death. Fourth, Zn2+ has been
demonstrated to inhibit the protease responsible for cleavage of lamins
in cell-free extracts (11).
Cell Culture
80 °C.
Fig. 1.
Effect of Zn2+ on
etoposide-induced PARP proteolysis. Molt4 cells of 5 × 105/ml were pretreated for 1 h with 0-300
µM ZnCl2 followed by a 6-h incubation with 20 µM etoposide. The cells were then harvested and analyzed
for PARP proteolysis by Western blot.
[View Larger Version of this Image (21K GIF file)]
Fig. 2.
Processing of pro-caspase-3 by
etoposide. Molt4 cells of 5 × 105/ml were
treated with 20 µM etoposide. At the indicated times, the
cells were harvested and lysed, and the 100,000 × g
supernatant was analyzed for caspase-3 processing by Western
blot.
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Cation effects on PARP proteolysis catalyzed
by an apoptotic extract. Ten µg of an apoptotic extract (active
cytosol) from cells treated with etoposide for 6 h were added to
cell-free buffer containing the respective cations at the indicated
concentrations. The reactions were then initiated by the addition of 25 ng of bovine PARP and incubated for 20 min at 37 °C. Reactions were stopped by the addition of Laemmli buffer and processed for PARP proteolysis by Western blot.
[View Larger Version of this Image (18K GIF file)]
Fig. 4.
Cation effects on PARP proteolysis catalyzed
by caspase-3. Forty ng of recombinant caspase-3 were added to
cell-free buffer containing the respective cations at the indicated
concentrations. The reactions were initiated by the addition of 25 ng
of bovine PARP and incubated for 20 min at 37 °C. Reactions were
stopped by the addition of Laemmli buffer and processed for PARP
proteolysis by Western blot.
[View Larger Version of this Image (17K GIF file)]
0.1 µM) was observed when
recombinant caspase-3 was used as the protease (open
circles). These results provide strong evidence that the
Zn2+ inhibition of PARP proteolysis is due to its
inhibition of caspase-3 and not to a modification of the PARP
substrate.
Fig. 5.
Zn2+ effects on Ac-DEVD-pNA
proteolysis catalyzed by an apoptotic extract or by recombinant
caspase-3. Twenty µg of an apoptotic extract from
etoposide-treated cells (filled circles) or 280 ng of
recombinant caspase-3 (open circles) were added to buffer
containing the indicated concentrations of ZnCl2. Reactions were then initiated by the addition of Ac-DEVD-pNA to a final concentration of 200 µM and incubated for 10 min at
37 °C. Product formation (protease activity) was monitored by
measuring the absorbance at 405 nm.
[View Larger Version of this Image (15K GIF file)]
10
9-10
12
M).
(25), Fas
(26), and chemotherapy agents such as etoposide (27). Moreover, in a
cell-free system comprised of apoptotic extracts and healthy nuclei,
the presence of caspase-3 in the apoptotic extract was necessary to
induce apoptotic changes in the nuclei (21). Also, it has recently been
demonstrated that knockout mice which are lacking caspase-3 incur
severe abnormalities in the development of the nervous system (28).
Thus, the relevance of these data in demonstrating a novel inhibitory
site for Zn2+ in apoptosis is underscored by the fact that
caspase-3 is both an important and common effector of the apoptotic
machinery.
10 µM)
exists in a labile pool which is in equilibrium with various
Zn2+-binding proteins. Therefore, the intracellular levels
of free Zn2+ may be in the range necessary to inhibit
caspase-3. Currently, little is known about changes in intracellular
Zn2+ or its movement between compartments, but new classes
of Zn2+-responsive fluorophores appear to be promising in
gaining new insight into this area (31).
*
This work was supported in part by United States Army Grant
DAMD AIBS-516 and NIH Grant GM-43825 to (Y. A. H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by a national research service award from NIH.
Supported by American Cancer Society Grant IRG 158L and Claude
D. Pepper Older Americans Independence Center Grant NIA 5 P60 AG11268.
§§
To whom correspondence should be addressed: Duke University
Medical Center, Dept. of Medicine, Box 3355, Durham, NC 27710. Tel.:
919-684-2449; Fax: 919-681-8253.
1
The abbreviations used are: TNF, tumor necrosis
factor; ICE, interleukin-1 converting enzyme; PARP, poly(ADP-ribose)
polymerase; Ac-DEVD-pNA,
N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide.
2
We have also observed that the thiols
dithiothreitol and -mercaptoethanol attenuate the inhibition
observed with Zn2+. This is presumably due to the ability
of these reagents to act as Zn2+ chelators (33).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.