THE JOURNAL OF BIOLOGICAL CHEMISTRY
31575–31583, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteome Analysis Associated with Cadmium Adaptation in
U937 Cells
IDENTIFICATION OF CALBINDIN-D28k AS A SECONDARY CADMIUM-RESPONSIVE PROTEIN THAT CONFERS
RESISTANCE TO CADMIUM-INDUCED APOPTOSIS*
Received for publication, January 26, 2004, and in revised form, April 19, 2004
Published, JBC Papers in Press, May 19, 2004, DOI 10.1074/jbc.M400823200
Hye-Kyung Jeon‡§, Hyung-Seung Jin‡§, Dong-Hee Lee‡§, Won-Seok Choi§, Chang-Kiu Moon¶,
Young J. Oh‡, and Tae H. Lee‡§储
From the ‡Department of Biology and §Protein Network Research Center, Yonsei University, Seoul 120-749 and ¶College
of Pharmacy, Seoul National University, Seoul 110-799, Korea
Cadmium is a well known environmental toxicant and
carcinogen. To identify proteins involved in cellular
adaptive responses to cadmium, we established cadmi-
um-adapted U937 cells that exhibit resistance to cadmi-
um-induced apoptosis, and we performed comparative
proteome analysis of these cells with parental cells that
were either untreated or treated with cadmium. Newly
identified proteins that were changed in expression
level in both adapted cells and cadmium-treated paren-
tal cells included proteins implicated in cell prolifera-
tion and malignant transformation. Most interesting, a
calcium-binding protein calbindin-D28k was increased
only in the adapted cells but not in cadmium-exposed
parental cells. The level of calbindin-D28k increased by
the degree of cadmium adaptation and was stably main-
tained without selective pressure of cadmium. Cadmi-
um-adapted U937 cells were resistant to the toxic effects
of cytosolic calcium rise by cadmium treatment and by
depletion of intracellular calcium stores, suggesting
that enhanced calcium buffering by up-regulated calbi-
ndin-D28k may be responsible for acquiring resistance to
cadmium-induced apoptosis. We demonstrated that
overexpression of calbindin-D28k in MN9D neuronal
cells resulted in reduced cadmium-induced apoptosis.
Our study documents for the first time that cells re-
spond to long term cadmium exposure by increasing
calbindin-D28k expression, thereby attenuating cadmi-
um-induced apoptosis.
Cadmium is a long-lived environmental hazard with potent
multiorgan toxicity that is well recognized as an industrial
pollutant and a food contaminant and as one of the major
components of cigarette smoke (1). Cadmium is taken up
through calcium ion channels of the plasma membrane and can
affect calcium homeostasis by activating or inhibiting several
calcium-related enzymes (2). Following cadmium uptake, dam-
* This work was supported by grants from the Korean Ministry of
Environment through Eco-technopia 21C Project, from the Ministry of
Science and Technology through 21C Frontier Project and the Brain
Research Center (to Y. J. O.), and from the Korea Science and Engi-
neering Foundation through Protein Network Research Center at Yon-
sei University. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
储 To whom correspondence should be addressed: Dept. of Biology,
College of Science, Yonsei University, 134 Shinchon-Dong, Seodae-
moon-Gu, Seoul 120-749, Korea. Tel.: 82-2-2123-4084; Fax: 82-2-312-
2242; E-mail: thlee@yonsei.ac.kr.
This paper is available on line at http://www.jbc.org
Vol. 279, No. 30, Issue of July 23, pp.
Printed in U.S.A.
age to cells leads to a complex series of events that can culmi-
nate in the death of the cells. To attenuate cadmium toxicity,
cells respond by inducing a variety of genes and by modulating
the expression levels or activities of proteins. These include
proteins that (i) sequester cadmium to decrease the availability
of this ion from interacting with cellular targets to elicit toxic-
ity, such as metallothioneins (MT)1; (ii) control GSH and re-
lated proteins to detoxify reactive oxygen species that are gen-
erated by cadmium; (iii) repair membrane and DNA damage;
and (iv) renature or degrade unfolded proteins (3). Cadmium
can induce apoptosis in vivo and in vitro, as shown in cells of
the respiratory system (4), the testis (5), the kidney (6), the
liver (7), and the immune system (8, 9). Although a high dose of
cadmium exposure of cells can induce apoptosis, low dose of
cadmium stimulates DNA synthesis, cell proliferation, and ma-
lignant transformation (2).
Upon repeated exposure of cells with increasing doses of
cadmium, cells exhibit adaptive survival responses that have
enabled many cells to become more resistant to subsequent
high doses of cadmium. The cellular adaptation process in-
volves the suppression of apoptosis induced by this metal. For
example, cadmium-adapted alveolar epithelial cells, which
have up-regulated expression of MT, GSH, and antioxidant
enzymes, showed better protection against oxidant-induced ap-
optosis compared with the nonadapted cells (10). In addition,
cadmium-transformed prostate epithelial cell line exhibited
down-regulated gene expression of caspases and pro-apoptotic
Bax and up-regulation of anti-apoptotic BCL-2 gene, when
compared with the nontumorigenic parental line (11), suggest-
ing that acquired resistance to apoptosis plays a role in cell
transformation and malignant progression.
In the present study, in order to gain further insights into the
mechanism for cellular long term survival response to cadmium,
we have searched proteins associated with cadmium adaptation
by the use of proteomics. Two cell lines of U937 cells were used
for proteome comparison, a parental cell line and a cadmium-
adapted cell line. The proteins were separated by two-dimen-
sional gel electrophoresis for proteome analysis. Protein spots
1
The abbreviations used are: MT, metallothionein; MALDI, matrix-
assisted laser desorption/ionization; TOF, time of flight; MTT, 3-(4,5-
dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide; annexin V/FITC,
fluorescence isothiocyanate-labeled annexin V; PI, propidium iodide;
IEF, isoelectric focusing; RhoGDI-␣, Rho GDP dissociation inhibitor-␣;
RanBP1, Ran-binding protein 1; MNEI, monocyte/neutrophil elastase
inhibitor; MTA-1s, metastatic tumor antigen-1 short form; PARP, poly-
(ADP-ribose) polymerase; Ab, antibody; DTT, dithiothreitol; PBS, phos-
phate-buffered saline; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,
N⬘-tetraacetic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-
1-propanesulfonic acid.
31575
31576 Proteome Analysis Associated with Cadmium Adaptation
altered in cadmium-adapted cells were identified by peptide
mass fingerprinting using matrix-assisted laser desorption/ioni-
zation-time of flight (MALDI-TOF)-mass spectrometry. Remark-
ably, calbindin-D28k was increased only in the adapted cells but
not in parental cells treated with cadmium, suggesting that this
protein is a secondary cadmium-responsive protein. Calbindin-
D28k has been known to possess an anti-apoptotic function by
chelating calcium (12–16). We demonstrated that the adapted
cells showed better calcium buffering capacity than nonadapted
cells, and its overexpression in neuronal cells protected cells from
apoptosis induced by cadmium. Thus, increased expression of
calbindinD28k represents an adaptive survival mechanism that
resists cadmium-induced apoptosis.
EXPERIMENTAL PROCEDURES
Reagents—All reagents used for cell culture were purchased from
Invitrogen. Electrophoresis reagents including acrylamide solution
(40%), Tris base, glycine, Immobiline DryStrip, IPG buffer, and IPG
cover fluid were purchased from Amersham Biosciences. Trypsin (se-
quencing grade) was purchased from Promega (Madison, WI). Thapsi-
gargin, Pluronic F-127, Fluo-2/AM, BTC-5N/AM and BAPTA-AM were
from Molecular Probes (Eugene, OR). CdCl2, ionomycin, CHAPS, ace-
tonitrile, trifluoroacetic acid, ␣-cyano-4-hydroxycinnamic acid, and
other reagents were purchased from Sigma.
Cell Culture and Establishment of Cadmium-adapted U937 Cells—
The human monocytic cell line U937 was obtained from the American
Type Culture Collection (Manassas, VA). Cells were grown at 37 °C
with 5% CO2 in RPMI 1640 medium supplemented with 2% sodium
bicarbonate, 10% fetal bovine serum, 100 units/ml penicillin G, and 50
␮g/ml streptomycin. Cadmium-adapted cells were developed by step-
wise exposure of parental U937 cells to cadmium with concentration
ranging from 5 to 50 ␮M. Cells surviving from the medium containing 5
␮M CdCl2 were maintained for 2 weeks until normal growth rate was
recovered. These cells were exposed to a subsequent higher concentra-
tion of cadmium that was increased by 5 ␮M at each step. MN9D
neuronal cells overexpressing calbindin-D28k (MN9D/cal-D28k) were cul-
tured as described (17).
MTT Assay—Cell cytotoxicity induced by cadmium and other re-
agents was quantified by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltet-
razolium bromide (MTT) reduction assay, as described previously (18).
Cells were seeded in 96-well plates (104 cells/well) 1 day before treat-
ment. The cells were then treated with CdCl2 for various times and
concentrations. After incubation with the reagent, the cells were
treated with 1 mg/ml MTT solution at a CO2 incubator for 2 h at 37 °C
and lysed in 20% SDS in 50% aqueous dimethylformamide for 24 h. The
absorbance of the dissolved formazan grains within the cells was meas-
ured at 540 nm in a microplate reader (Molecular Devices, Palo Alto,
CA). Values from each treatment were calculated as percent cytotoxic-
ity of the untreated control.
Flow Cytometric Assessment of Apoptosis—Phosphatidylserine exter-
nalization during cadmium-induced apoptosis in U937 cells was deter-
mined by two-color analysis of annexin V/FITC binding and propidium
iodide (PI) uptake using flow cytometry (FACSCalibur, BD Biosciences)
according to the instructions of the manufacturer. Annexin V-FITC
detection kit and PI staining solution were purchased from BD
Biosciences.
Two-dimensional Gel Electrophoresis and Image Analysis—Protein
samples for two-dimensional gel electrophoresis were extracted as fol-
lowed. Cells were lysed in extraction buffer (7 M urea, 2 M thiourea, 4%
CHAPS, 40 mM Tris base, 20 mM DTT, 1 mM phenylmethylsulfonyl
fluoride, and 1% IPG buffer) at 4 °C for 30 min. The lysate was then
centrifuged at 15,000 rpm for 30 min at 4 °C. The supernatants were
collected and treated with 250 units/ml endonuclease (Sigma) for 1 h at
room temperature.
The first-dimensional isoelectric focusing (IEF) was performed on an
IPGphor unit (Amersham Biosciences) using precasted 24-cm pH 4 –7 IPG
gel strips. 2 mg of total proteins was dissolved to 350 ␮l with rehydration
buffer (7 M urea, 2 M thiourea, 2% CHAPS, 40 mM Tris base, 10 mM DTT,
0.02% bromphenol blue, and 1% IPG buffer) and rehydrated at 20 °C for
more than 12 h at 30 V. IEF was performed in a stepwise voltage increase
procedure at 20 °C to achieve a total of 100,000 V-h. After IEF separation,
the gel strips were incubated with equilibration buffer (50 mM Tris, 6 M
urea, 20% v/v glycerol, 2% w/v SDS, and 1% DTT) for 15 min followed by
incubation in the same buffer with DTT replaced by 1.25% iodoacetamide
(w/v) for an additional 15 min. The second dimension separation was
performed on 1.0-mm-thick gradient polyacrylamide gels at 15 mA per gel
at 10 °C overnight using a DALT electrophoresis unit (Amersham Bio-
sciences). The gels were stained with colloidal Coomassie Blue. The
stained gels were scanned and analyzed using Melanie 3 software
(GeneBio, Geneva, Switzerland).
In-gel Digestion for Protein Identification—Each excised gel spot was
destained with 100 ␮l of 50% acetonitrile in 25 mM ammonium bicar-
bonate (NH4HCO3). The solution was replaced with a fresh aliquot, and
the gel spots were incubated with shaking for 10 min at 37 °C. The
above washing and dehydration steps were repeated three times. After
removal of the dehydration solution, gel spots were dried in a Speed-
Vac. The dried gel spots were incubated with 10 –15 ␮l of 20 ng/␮l
trypsin in 25 mM NH4HCO3 on ice for 45 min, and then the supernatant
was discarded and replaced with 25 mM NH4HCO3. The trypsin diges-
tion was performed at 37 °C for 14 –18 h. Peptides were first extracted
using 0.1% trifluoroacetic acid, 50% acetonitrile at 37 °C for 30 min, and
the above procedure was repeated twice and then the peptides were
extracted subsequently with 100% acetonitrile. The extracted solutions
were dried in a Speed-Vac.
MALDI-TOF-Mass Spectrometry and Data Base Searching—The ex-
tracted peptides were dissolved in 0.1% trifluoroacetic acid, mixed with
an equal volume of matrix solution (10 mg/ml ␣-cyano-4-hydroxycin-
namic acid in 0.1% trifluoroacetic acid, 50% acetonitrile), and 1 ␮l of the
mixture was spotted onto a MALDI sample plate. Peptide mass spectra
were obtained using an Applied Biosystem Voyager-DE STR MALDI-
TOF mass spectrometer in the reflector mode. External calibration was
carried out by using Sequazyme peptide mass standard kit (Perspective
Biosystems) and internal calibration by using the autolytic peaks of
trypsin. The protein identification was performed by searching the
NCBInr protein data base using MS-Fit (prospector.ucsf.edu/). The
criteria for searching were set at 50 ppm, at least four matching peptide
masses, and theoretical molecular mass and pI matching estimated
values from gels.
Western Blot Analysis—Cells were washed with PBS and lysed in
SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercap-
toethanol, 0.002% bromphenol blue, and 10% glycerol). The samples
were boiled for 5 min and centrifuged at 12,000 rpm for 3 min, and
equal amounts of proteins (20 –50 ␮g) were separated by SDS-PAGE
and transferred to nitrocellulose membranes. The membranes were
blocked with TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing
5% nonfat milk for 1 h at room temperature and then incubated with
various Abs for 1 h at room temperature. After they were washed with
TBST (TBS plus 0.025% Tween 20), the membranes were incubated
with the horseradish peroxidase-labeled secondary Ab for 1 h. After
washing, the blots were treated with ECL reagents (Amersham Bio-
sciences), followed by exposure to x-ray film. Abs used are anti-cal-
bindin-D28k Ab (Swant, Bellinzona, Switzerland), anti-caspase 3 Abs
(murine-specific, Cell Signaling, Beverly, MA; human-specific, Santa
Cruz Biotechnology, Santa Cruz, CA), anti-caspase-8 Ab (BD Bio-
sciences), and mouse monoclonal PARP Ab (Roche Applied Science).
Measurement of the Level of Cytosolic Calcium and Cadmium—Cells
for intracellular calcium measurements were preloaded with the cyto-
solic calcium indicator, 5 ␮M Fura-2/AM plus 0.1% Pluronic F-127, for
30 min at room temperature and washed with RPMI 1640 (serum- and
phenol red-free). Cells were resuspended in black opaque 96-well plates
(2 ⫻ 104/well) in RPMI 1640 and treated with 20 or 50 ␮M CdCl2 for the
times indicated at 37 °C. Fluorescence was monitored on a Bio-Tek
FL600 fluorescence absorbance spectrophotometer (Winooski, VT). The
wavelength for excitation was 340 nm, and emission was measured at
510 nm. For intracellular cadmium measurement, cells exposed to
cadmium for 1 h were processed as above using the heavy metal-specific
fluorescent probe, BTC-5N/AM (1 ␮M), instead of Fura-2/AM. The
BTC-5N fluorescence was measured at a wavelength of 415 nm (exci-
tation) and 500 nm (emission).
RESULTS
Cadmium-adapted U937 Cells Exhibit Resistance to Apo-
ptosis Induced by Cadmium—To study cellular adaptive re-
sponses to long term cadmium exposure, these metal-adapted
U937 cells were established by exposing the cells to stepwise
increased cadmium concentrations ranging from 5 to 50 ␮M.
Cadmium resistance of the adapted cell line was tested by MTT
assay (Fig. 1A). Upon treatment of cells with 20 and 50 ␮M
CdCl2, the nonadapted parental U937 cells showed 54 and 82%
cytotoxicity after 24 h of exposure, respectively. In contrast, the
cadmium-adapted cells showed only 5 and 14% cytotoxicity.
Resistance to cadmium was also observed with longer treat-
 Proteome Analysis Associated with Cadmium Adaptation 31577

FIG. 1. Cadmium-adapted U937 cells exhibit resistance to cadmium-induced apoptosis. A, parental and cadmium-adapted U973 cells
were treated with 20 or 50 ␮M CdCl2 for the indicated times. Cell injury to cadmium exposure was determined by MTT assay. The results represent
the percent cytotoxicity of cells treated with cadmium compared with untreated control. Three independent experiments in quadruplicate were
done, showing similar results. A representative result is shown as the mean ⫾ S.D. B, cells were treated with 50 ␮M CdCl2 for the indicated times,
and early apoptotic (annexin V-positive) and secondary necrotic cells (double-positive) were determined by flow cytometry with annexin V/FITC
and propidium iodide (PI) (left). Representative fluorescence profiles of cells that were left untreated and treated for 24 h were shown (right).
Numbers in the respective quadrant profiles indicate the percentage of the cells present in this area.

ment (48 h) in the adapted cells. Flow cytometric analysis using
annexin V/FITC and PI double staining revealed that after 6 h
of treatment with 50 ␮M CdCl2, early apoptotic parental U937
cells (annexin V-positive, PI-negative) increased to 24.8%; as
treatment time extended, the early apoptotic cells gradually
decreased, whereas the cells in later stages of apoptosis (an-
nexin V-positive and PI-positive) occupied the majority. Previ-
ous work by Li et al. (19) has indicated that these double-
positive U937 cells represent secondary necrotic cells whose
membranes were damaged by cadmium. In cadmium-adapted
cells, no significant time-dependent changes in apoptotic and
secondary necrotic cell populations were observed, indicating
that the adapted cells were resistant to cadmium-induced apo-
ptosis and subsequent to the plasma membrane damage (Fig.
1B, left panel). Flow cytometric profiles of the parental and
cadmium-adapted cells that were left untreated and treated
with 50 ␮M CdCl2 for 24 h are presented in the right panel of
Fig. 1B.
Caspase-3 and caspase-8 are suggested to be involved in
cadmium-induced apoptosis of U937 cells (19). We examined
whether both caspases can be cleaved by cadmium treatment.
Western blot analysis showed that in parental U937 cells, after
24 h of exposure to 50 ␮M CdCl2, a significant decrease in both
levels of procaspase-3 and -8 was observed. Although the
cleaved product of caspase-3 (17 kDa) was visible at 6 h in
parental cells, longer exposure of cadmium did not cause the
increase of the band intensity. Rather, it became undetectable
after 24 h of treatment, reflecting degradation of the cleaved
form due to secondary necrotic death. In contrast, cadmium-
adapted cells showed no apparent decrease in procaspase-3
and -8 levels after 24 h of exposure to cadmium, further con-
firming the apoptotic resistance of cadmium-adapted U937
cells (Fig. 1C).
Proteome Analysis Associated with Cellular Cadmium Adap-
tation—To identify proteins associated with cadmium adapta-
tion, protein expression patterns were compared in parental
and cadmium-adapted cells by using two-dimensional gel elec-
trophoresis. To ensure that proteins expressed differentially in
the adapted cells resulted from the response to cadmium, two-
dimensional protein profiles were also compared with that of
parental cells treated with 20 ␮M CdCl2 for 24 h. Fig. 2A shows
the Coomassie Blue-stained two-dimensional protein pattern
obtained after separation of 2-mg protein samples from the
parental U937 cells. About 700 protein spots ranging from 10 to
31578 Proteome Analysis Associated with Cadmium Adaptation

FIG. 2. Comparison of the two-dimensional protein expression pattern of parental, cadmium-adapted, and cadmium-treated U937
cells. A, two-dimensional gel image of parental U937 cells stained with Coomassie Blue. B, comparative close-up views of each area indicated in
the image shown in A with the respective area obtained from gel images of cadmium-adapted cells and cadmium-treated (20 ␮M CdCl2 for 24 h)
parental cells. The positions and numbers of the differentially expressed proteins listed in Table I are indicated.

100 kDa with pI values between 4 and 7 were detected. Fig. 2B
is a comparative close-up view of six regions shown in Fig. 2A,
where significant differences in protein expression level are
marked. These protein spots were excised and subjected to
trypsin digestion, MALDI-TOF mass spectra measurements,
and data base searching with MS-Fit. Table I summarizes the
identified proteins with quantitative alterations in cadmium-
adapted cells and cadmium-treated parental cells, compared
with untreated parental cells. The proteins commonly up-reg-
ulated in the adapted cells as well as cadmium-treated paren-
tal cells include retinaldehyde dehydrogenase 2, Rho GDP dis-
sociation inhibitor-␣ (RhoGDI-␣), Ran-binding protein 1
(RanBP1), glutathione S-transferase, heterogeneous nuclear
ribonucleoprotein C1/C2, c-Myc-binding protein MM1, and a
hypothetical protein. Monocyte/neutrophil elastase inhibitor
(MNEI) was identified as a down-regulated protein in both
cadmium-adapted and cadmium-treated parental cells. Most
interesting, protein levels of calbindin-D28k and metastatic tu-
mor antigen-1 short form (MTA-1s) were increased only in
cadmium-adapted cells and not in cadmium-treated parental
U937 cells.
Increased Expression of Calbindin-D28k Resulted from Long
Term Adaptation of Cells to Cadmium—In the protein profiling
data, the levels of calbindin-D28k and MTA-1s were increased
only in cells adapted to prolonged cadmium exposure, suggest-
ing that their up-regulation may be the secondary consequence
of cellular response to cadmium. We are particularly interested
in calbindin-D28k expression because it is a calcium-binding
protein belonging to the calmodulin superfamily with six EF
hands (20). In many cell types, including U937 cells, cadmium
exposure induced intracellular calcium increase, which is in
part responsible for mediating the cadmium-induced apoptotic
cell death (19). In addition, calbindin-D28k suppressed apo-
ptosis induced by a variety of agents, and the death inhibitory
action of calbindin-D28k has been attributed to its ability to
remove toxic calcium in cells (12–16, 21–25). Thus, we pre-
dicted that calbindin-D28k up-regulation of the adapted U937
cells is a part of the cellular responses to resist cadmium-
evoked apoptosis. As a confirmatory experiment, we performed
immunoblot analysis by using an anti-calbindin-D28k Ab. The
result showed that although the expression level of calbindin-
D28k was increased in cadmium-adapted cells, there was no
change in calbindin-D28k expression after 24 h of treatment of
parental cells with 20 ␮M CdCl2, which is consistent with the
result obtained from the proteome analysis (Fig. 3A). The ele-
vated calbindin-D28k expression was stably maintained even
after culturing the cells in cadmium-free medium for at least 15
days (Fig. 3B). The increased expression of calbindin-D28k was
also observed in independently established cadmium-adapted
U937 cell lines, showing that cells adapted to higher concen-
trations of cadmium expressed higher levels of calbindin-D28k
(Fig. 3C). Thus, during the cadmium adaptation process, cells
appear to counteract the toxic effects of this metal by increas-
ing the level of calbindin-D28k.
 Proteome Analysis Associated with Cadmium Adaptation 31579
TABLE I
List of identified proteins
Changes in spot
Spot no. Protein name NCBI accession no. Mass pI Coverage densitya
Adapted Treated

kDa %

1 hnRNP C1/C2 4758544 31.96 5.1 22 ⫹ ⫹

2 hnRNP C1/C2 13097279 32.33 4.9 16 ⫹ ⫹
3 Ran-binding protein 1 938026 23.23 5.2 31 ⫹ ⫹
4 Glutathione S-transferase 2204207 23.38 5.4 45 ⫹ ⫹
5 Rho-GDI-␣ 4757768 23.2 5.0 29 ⫹ ⫹
6 Calbindin-D28K 4826655 30.02 4.7 19 ⫹ ⫺
7 Transmembrane protein 4 7657176 20.65 4.8 57 ⫹ ⫹
8 Retinaldehyde dehydrogenase 2 2577726 53.06 5.8 31 ⫹ ⫹
9 Monocyte/neutrophil elastase inhibitor 13489087 42.74 5.9 32 ⫺ ⫺
10 Metastatic tumor antigen 1 (short form) 22550298 49.01 5.4 15 ⫹ ⫺
11 c-Myc-binding protein MM1 1731809 18.64 5.6 40 ⫹ ⫹
12 Unnamed protein product 10438010 24.16 6.8 32 ⫹ ⫹
a
Signs represent relative increase (⫹) and decrease (⫺) in the protein level of cadmium-adapted U937 cells (Adapted) and the parental cells
treated with 20 ␮M CdCl2 (Treated) as compared with the untreated parental U937 cells.

dependent changes of fluorescence of Fura-2 in cells treated

FIG. 3. Calbindin-D28k was increased as a consequence of long
term adaptation of U937 cells to cadmium but was not inducible
by short term exposure. A, nonadapted and adapted U937 cells were
either left untreated or treated with 20 ␮M CdCl2 for 24 h. Cell lysates
were prepared, and the level of calbindin-D28k was examined by West-
ern blot analysis with anti-calbindin-D28k Ab. B, the stable up-regula-
tion of calbindin-D28k in cadmium-adapted cells. Cadmium-adapted
cells were cultured in the absence or presence of 50 ␮M CdCl2. After the
indicated culture times, cell lysates were analyzed for calbindin-D28k
expression. C, the increased level of calbindin-D28k expression corre-
lated with the concentrations of cadmium that were used for selection of
the adapted cells. Parental U937 cells that had been independently
adapted to the indicated concentrations of cadmium were processed for
Western blot analysis to detect the level of calbindin-D28k expression
using the anti-calbindin-D28k Ab.
Cadmium-adapted U937 Cells Exhibit Better Calcium
Buffering Capacity and Resistance to Death Induced by In-
tracellular Calcium Overloading—In view of the calcium
buffering function of calbindin-D28k, it is likely that the ele-
vation of this protein in the adapted cells may play a role in
reducing the level of intracellular toxic calcium ion increased
by cadmium. Therefore, we compared the changes in intra-
cellular Ca2⫹ concentration of cadmium-adapted and paren-
tal U937cells by using the cytosolic calcium indicator Fura-
2/AM after exposure to cadmium. Fig. 4A shows the time-
with 20 and 50 ␮M CdCl2, revealing that the magnitude of
increase in the intracellular ion concentrations induced by
cadmium was significantly attenuated in cadmium-adapted
cells. We also examined the effect of BAPTA-AM, a commonly
used chelator of intracellular free Ca2⫹, on cadmium-induced
apoptosis. Fig. 4B illustrates that parental cells pretreated
with 20 ␮M BAPTA-AM and subsequently stimulated with 50
␮M CdCl2 for 24 h showed total inhibition of apoptosis, re-
ducing the percentage of apoptotic cells (annexin V-positive)
from 53.6 to 18.4%, which was almost equivalent to the
percentage value of cadmium-unexposed control cells. As ex-
pected, the cadmium-adapted cells were resistant to apo-
ptosis after treatment with 50 ␮M CdCl2, and pretreatment of
BAPTA-AM had no significant effect, suggesting that in the
adapted cells the availability of free calcium ions necessary
for cadmium-induced apoptosis is greatly reduced by the
binding of calcium to the overexpressed calbindin-D28k.
To acquire further evidence for the enhanced calcium buff-
ering property of the adapted U937 cells, we tested whether
they were better protected from toxicity triggered by intracel-
lular calcium overloading than nonadapted cells. Administra-
tion of thapsigargin (the endoplasmic reticulum Ca2⫹-ATPase
inhibitor) led to a massive increase in cytosolic Ca2⫹ by efflux
from vesicles in the endoplasmic reticulum (26). Calcium iono-
phore (ionomycin) also caused an intracellular Ca2⫹ rise that
was caused by the release of cytosolic Ca2⫹ stores and Ca2⫹
influx. These treatments ultimately resulted in cell death in
several cell types including U937 cells (27, 28). MTT assays
revealed that after 48 h of treatment with 10 –100 nM thapsi-
gargin, the nonadapted U937 cells resulted in 36 – 45% cytotox-
icity, whereas the cadmium-adapted cells showed 13–24% cy-
totoxicity. The difference in the susceptibilities of these two cell
lines was more conspicuous in response to ionomycin, in which
addition of this reagent up to 1000 ng/ml was not toxic to the
cadmium-adapted cells, rather promoting growth during the
48 h of exposure. However, the nonadapted parental cells ex-
hibited ⬃40% cytotoxicity with 500 –1000 ng/ml ionomycin
(Fig. 4C). Taken together, our results indicate that cadmium-
adapted cells are less sensitive to the disruption of intracellular
calcium levels than nonadapted cells due to better calcium
buffering capacity by up-regulated calbindin-D28k.
Ectopic Expression of Calbindin-D28k in a Neuronal Cell Line
Reduces Cadmium Toxicity Mediated by Intracellular Calcium
Increase—Apart from calbindin-D28k, other cadmium-response
proteins such as MT, glutathione S-transferase, and anti-oxi-
dant enzymes play roles in the protection of cadmium toxicity
31580 Proteome Analysis Associated with Cadmium Adaptation

FIG. 4. Cadmium-adapted U937 cells exhibit better calcium buffering capacity, resulting in the resistance to apoptosis induced by
depletion of intracellular calcium stores. A, the kinetics of intracellular Ca2⫹ concentration in parental and cadmium-adapted U937 cells in
response to cadmium exposure. Both cell lines of U937 cells were treated with 20 or 50 ␮M CdCl2 for up to 150 min. In each indicated time point,
cells were stained with Fura-2/AM, and the ratio of fluorescence value obtained from cadmium-treated cells to that of untreated control was plotted.
Calcium increase evoked by cadmium was significantly diminished in cadmium-adapted cells. Three separate experiments were done, and their
statistical results were expressed as the mean ⫾ S.D. (*, p ⬍ 0.05 compared with the nonadapted parental cells, analysis of variance t test.) B,
effects of pretreatment of intracellular calcium chelator BAPTA-AM on cadmium-induced apoptosis. Parental and cadmium-adapted cells were
either untreated or pretreated with 20 ␮M BAPTA-AM for 1 h, washed once with PBS, and then followed by 50 ␮M CdCl2 exposure for 24 h. Cells
were double-stained with annexin V/FITC and propidium iodide (PI) and analyzed by flow cytometry. Percentage values were obtained from a
representative experiment by measuring the fraction of annexin V-positive apoptotic cells. C, effects of thapsigargin- and ionomycin-evoked
intracellular Ca2⫹ increase in parental and cadmium-adapted U937 cells. Cells were treated with the respective reagents at the designated
concentrations for 48 h. Cytotoxicity induced by the disruption of intracellular calcium level was measured by MTT assay. The results represent
the percent cytotoxicity compared with the respective untreated control. Three independent experiments in quadruplicate showed similar results,
and representative data are shown as the mean ⫾ S.D.

by modulating the level of the reactive oxygen species (2, 10,
29 –31). Therefore, the direct contribution of calbindin-D28k in
the resistance to cadmium toxicity has to be determined with-
out the influence of these proteins. For this purpose, we have
tried to establish U937 cells stably expressing calbindin-D28k;
however, several attempts were unsuccessful because of high
lysosomal enzymatic activities in this particular cell type. In-
stead, we took advantage of using a dopaminergic neuronal
MN9D cell line that was permanently transfected with the
plasmid expressing calbindin-D28k (17), and we investigated
whether overexpression of calbindin-D28k per se can protect
against apoptosis induced by cadmium. The MN9D/cal-D28k
cells showed a high level of calbindin-D28k expression com-
pared with the control vector-transfected MN9D/neo cells (Fig.
5A). The MTT assay was done to compare the cytotoxic effect of
cadmium on these cells (Fig. 5B). After treatment of MN9D/neo
cells with 1 ␮M CdCl2, the percentage of cytotoxicity increased
by 28% at 24 h and by 67% at 48 h. In contrast, MN9D/cal-D28k
cells exhibited significantly less cytotoxicity than MN9D/neo
cells (12 and 28% cytotoxicity at each respective time period).
24 h of exposure to 10 ␮M CdCl2 resulted in 67% cytotoxicity in
MN9D/neo cells, whereas calbindin-D28k-expressing cells were
still more tolerant, exhibiting 43% cytotoxicity. Further incu-
bation of cells over 48 h resulted in complete cell death in both
cell lines. The results indicate that MN9D cells are highly
sensitive to cadmium, and calbindin-D28k has a protective func-
tion, although it cannot completely block cell damage caused by
this metal.
Experiments for examining the effect of BAPTA-AM in cad-
mium toxicity of MN9D cells revealed that a brief exposure of
this reagent in MN9D/neo cells decreased cadmium cytotoxicity
from 71 to 25%, indicating that cadmium-induced calcium in-
crease is responsible for cellular toxicity in this cell type. On
the other hand, in MN9D/cal-D28k cells, which exhibited less
cytotoxicity (47%) than MN9D/neo cells, pretreatment of
BAPTA-AM caused total inhibition of the cadmium toxicity,
 Proteome Analysis Associated with Cadmium Adaptation 31581

FIG. 5. Overexpression of calbindin-D28k in neuronal MN9D cells confers resistance to cadmium toxicity mediated by intracellular
calcium increase. A, detection of the ectopically expressed calbindin-D28k in MN9D cells stably transfected with the calbindin-D28k expression
plasmid (MN9D/cal-D28k) by Western blot analysis. B, MN9D/cal-D28k cells and the control vector-transfected cells (MN9D/neo) were treated with
1 and 10 ␮M CdCl2 for the indicated times, and cadmium toxicity was measured by MTT assay. The results represent the percent cytotoxicity of
cells treated with cadmium compared with untreated control. Three independent experiments in quadruplicate were done, showing similar results.
A representative result is shown as the mean ⫾ S.D. C, effects of pretreatment of BAPTA-AM on cadmium-induced apoptosis of MN9D/neo and
MN9D/cal-D28k cells. Cells were either untreated or pretreated with 20 ␮M BAPTA-AM for 1 h, washed with PBS, and then followed by 10 ␮M CdCl2
exposure for 24 h. Cadmium toxicity was measured by MTT assay. Three separate experiments in quadruplicate showed similar results. A
representative result is shown as the mean values ⫾ S.D. D, intracellular Ca2⫹ and Cd2⫹ concentration of MN9D/neo and MN9D/cal-D28k cells in
response to cadmium exposure. After addition of CdCl2 at indicated concentrations for 1 h, cells were stained with Fura-2/AM and also with
BTC-5N/AM, a specific dye to Cd2⫹. The ratio of fluorescence value obtained from cadmium-treated cells to that of untreated cells was calculated,
and their statistical results from four separate experiments were expressed as the mean ⫾ S.D. (*, p ⬍ 0.05 compared with untreated cells, analysis
of variance t test.)

suggesting an additive effect of BAPTA-AM and calbindin-D28k

on the removal of intracellular toxic calcium ion (Fig. 5C). The
enhanced calcium buffering capability of MN9D/cal-D28k cells
was evident from the results showing that after 24 h of expo-
sure to 10 ␮M CdCl2, the increase of Fura-2 fluorescence in
MN9D/cal-D28k cells was significantly diminished as compared
with that of MN9D/neo cells (1.9- versus 3.4-fold increase over
the respective untreated control) (Fig. 5D, left). When cadmium
influx into cells was measured by using a cell-permeable, cad-
mium-specific fluorescent dye (BTC-5N/AM) (32), no consider-
able difference in the increase of intracellular Cd2⫹ concentra-
tion was observed in MN9D/neo and MN9D/cal-D28k cells (Fig.
5D, right). These results suggest that both cell lines can take
up cadmium with similar efficiency and that the ectopically
expressed calbindin-D28k may not bind cadmium as efficiently
as calcium in vivo, although it has been shown that cadmium
was the potent inhibitor for calcium binding in in vitro exper-
iments using the purified calbindin-D28k (33).
Evidence That Calbindin-D28k Plays a Protective Role in
Cadmium-induced Apoptosis—To confirm further the tolerance
of MN9D/cal-D28k cells to cadmium-induced apoptosis, we com-
pared the cleavage kinetics of caspase-3 and its substrate
PARP (Fig. 6). Western blot analysis using the polyclonal an-
tibody of mouse caspase-3 showed that in MN9D/neo cells, the
cleaved caspase-3 product (19 kDa) was evident after 8 h of
exposure to 10 ␮M CdCl2, and the maximum amount of the
cleaved product was detected between 16 and 18 h of exposure.
In contrast, MN9D/cal-D28k cells showed a much delayed ap-
pearance of the cleaved product. Similar delayed cleavage ki-
FIG. 6. Cadmium-induced caspase-3 and PARP cleavage were
reduced and delayed in MN9D cells overexpressing calbindin-
D28k. MN9D/neo and MN9D/cal-D28k cells were treated with 10 ␮M
CdCl2 for the indicated time periods. Cell lysates were processed for
Western blot analysis with anti-caspase-3 Ab that specifically recog-
nizes the cleaved product of caspase-3, and also with anti-PARP Ab.
netics of PARP were observed in MN9D/cal-D28k cells. These
results clearly showed that overexpression of calbindin-D28k
per se can confer resistance to cadmium-induced apoptosis in
the dopaminergic neuronal cells. Based on the results obtained
from MN9D cells overexpressing calbindin-D28k, we concluded
that the elevated expression of calbindin-D28k in cadmium-
adapted U937 cells, at least in part, played a role in protection
from this metal-induced apoptosis.
DISCUSSION
We employed the proteomics approach to identify proteins
that are involved in the cellular adaptive response to cadmium
toxicity. To this end, we established the cadmium-adapted
31582 Proteome Analysis Associated with Cadmium Adaptation
U937 cells that exhibited tolerant phenotypes against cadmi-
um-induced apoptosis. Comparative analysis of proteome pro-
files between the parental and cadmium-adapted cells allowed
the identification of 12 proteins whose steady-state levels were
altered in the cadmium-adapted cells. Most of the identified
proteins were bona fide cadmium-responsive proteins because
altered expression was observed in both cadmium-adapted cells
and cadmium-treated parental cells. In addition, we found that
two proteins including calbindin-D28k were not up-regulated in
parental cells treated with cadmium, but their steady-state
level was increased in cadmium-adapted cells, representing
them as secondary cadmium-responsive proteins.
Some of the proteins identified in this study may be related
to the role of cadmium in cell proliferation and malignant
transformation. These include RanBP1, RhoGDI-␣, and c-Myc-
binding protein MM-1. It has been well established that low
doses (micromolar ranges) of cadmium can stimulate DNA
synthesis, cell proliferation, and malignant transformation (2,
34, 35). In particular, studies done by Misra et al. (34) demon-
strated that the Ras-dependent mitogen-activated protein ki-
nase signaling limb is of critical importance in the initiation of
cadmium-mediated mitogenesis in macrophages. RanBP1 and
RhoGDI-␣ are proteins that regulate the activity of Ras-like
small GTP-binding proteins, Ran and Rho, respectively.
RanBP1 binds directly to the GTP-bound Ran and enhances
the activity of Ran GTPase-activating protein 1. The regulated
RanBP1 activity is shown to be required for proper execution of
mitosis in somatic cells (36). RhoGDI-␣ is an inhibitor of Rho
that plays an important role in cellular processes such as cell
morphology, proliferation, malignant transformation and me-
tastasis, and apoptosis (37). Additionally, a putative cadmium-
responsive protein MM-1 has been proposed as a tumor sup-
pressor that controls the transcriptional activity of c-Myc (38).
Previous studies (39) have shown that cadmium treatment
alters the expression of c-Myc protein in a variety of cell types
and that cadmium-induced cell transformation and tumorigen-
esis may be associated with transcriptional activation of pro-
tooncogenes, including c-myc. More study is necessary to elu-
cidate possible involvement of RanBP1, RhoGDI-␣, and MM-1
in the cadmium-induced carcinogenesis.
MNEI was found to be down-regulated in cadmium-adapted
U937 cells and also in response to short term cadmium expo-
sure of the nonadapted cells. MNEI is a 42-kDa serpin (a serine
protease inhibitor) superfamily protein characterized initially
as a fast-acting inhibitor of neutrophil elastase. Cadmium,
which is present in cigarette smoke, is associated with the
development of emphysema and lung cancer. It has been sug-
gested that neutrophils, acting in part via the proteolytic ac-
tivities of neutrophil elastase, play a pivotal role in the devel-
opment of emphysema. For example, a synthetic neutrophil
elastase inhibitor (ZD0892) could prevent or reduce emphy-
sema in guinea pigs exposed to cigarette smoke acutely or daily
for 6 months (40). The neutrophil elastase-deficient mice were
significantly protected from the long term cigarette smoke-
induced emphysema compared with wild-type littermate con-
trols (41). Therefore, it is tempting to speculate that cadmium-
induced down-regulation of natural inhibitors of neutrophil
elastase MNEI may contribute in part to the inflammatory and
destructive effects of cigarette smoke leading to emphysema-
tous lung destruction.
Unlike the primary cadmium-responsive proteins described
above, MTA-1s and calbindin-D28k were up-regulated only in
cadmium-adapted resistant cells but not in cadmium-treated
parental cells. Therefore, these proteins can be classified as
secondarily acquired cadmium-responsive proteins. Concern-
ing the increased expression of calbindin-D28k in cadmium-
adapted U937 cells, we confirmed this by Western blot analysis
(Fig. 3A). Cells adapted to long term repeated exposure to
increasing doses of cadmium led to the concomitant elevation of
calbindin-D28k expression, as evident from the detection of
higher levels of calbindin-D28k in cells adapted to higher con-
centrations of cadmium (Fig. 3C). Its up-regulation was not the
result of direct transcriptional activation by cadmium. Re-
cently, cadmium has been reported to induce genes regulating
translation initiation, such as translational initiation factor 3
(TIF3) and translation elongation factor-1␦ (TEF-1␦) (42, 43).
Overexpression of these genes in mammalian cells resulted in
transformation and tumorigenesis, and their inhibition in the
cadmium-transformed BALB/c-3T3 cells by antisense mRNAs
caused a significant reversal of the transformed phenotype,
suggesting that the overall increase in the rate of protein
synthesis by these translation factors may in part be responsi-
ble for cadmium-induced cell transformation and tumorigene-
sis. Whether the observed overexpression of calbindin-D28k
resulted from the increase in the overall translation rate me-
diated by the overexpression of human TIF3 and TEF-1␦ in
cadmium-adapted cells requires additional studies.
As a member of calmodulin superfamily, calbindin-D28k can
modulate calcium homeostasis and can protect cells from cal-
cium-mediated apoptosis induced by a variety of apoptotic
stimuli (23–25, 44, 45). In particular, Bellido et al. (21) have
shown that the anti-apoptotic properties of calbindin-D28k may
result not only from calcium buffering but also from the ability
of the protein to interact with and to inhibit caspase-3, a
property that is independent of its calcium binding capability.
Several studies (11, 46 – 48) have indicated that cadmium ex-
posure results in a rapid and sustained intracellular calcium
elevation, followed by caspase-3 activation that leads to cell
death. In U937 cells, cadmium induces apoptosis via two inde-
pendent pathways, the calcium-calpain-dependent pathway
and the caspase-mitochondria-dependent pathway (19). There-
fore, this calcium buffering and/or direct caspase-3 inhibitory
effects of calbindin-D28k may account for the cadmium resist-
ance of the adapted U937 cells. In the present study, we have
provided strong evidence in support of this notion, based on the
following observations. (i) Cadmium-adapted U937 cells were
more resistant to apoptosis induced by intracellular calcium
overloading. (ii) Disruption of the intracellular calcium level by
BAPTA-AM had a significant blocking effect on cadmium-in-
duced apoptosis of the nonadapted cells but showed little effect
on the adapted cells because of the reduced level of available
free calcium ions. These results suggest that increased expres-
sion of calbindin-D28k in cadmium-adapted U937 cells can be
attributed to its calcium buffering effect during cadmium-in-
duced apoptosis. Finally, we demonstrated that neuronal cells
stably transfected with the plasmid expressing calbindin-D28k
exhibited less susceptibility to cadmium-induced, calcium-de-
pendent apoptosis and a much more delayed caspase-3 activa-
tion and PARP cleavage than cells transfected with the control
plasmid, providing direct evidence that calbindin-D28k contrib-
utes to the acquisition of resistance to apoptosis induced by
cadmium. It is worthwhile to note that in MN9D/neo cells the
cleaved product of caspase-3 was easily detectable, and its
amount was increased as cadmium exposure time was ex-
tended. On the other hand, in U937 cells the cleaved product
was not easily detectable, but the proform of caspase-3 was
degraded at a later stage of apoptosis, as it was in the degra-
dation of caspase-8 by cadmium (Fig. 1C). These results sug-
gest that for the initiation of apoptosis in U937 cells, cadmium
may use a more preferentially calcium-calpain-dependent
pathway than caspase-mitochondria-dependent pathway, and
at the late stage of apoptosis caspase-3 and caspase-8 may be
 Proteome Analysis Associated with Cadmium Adaptation
activated as a secondary consequence of apoptosis, which may
eventually result in necrotic cell death.
Calbindin-D28k has been shown to have a higher affinity for
binding cadmium than any other divalent cations (33). This di-
rect cadmium binding property of calbindin-D28k may also offer
protection from apoptosis. However, the level of intracellular free
cadmium was not much reduced in MN9D cells overexpressing
calbindin-D28k compared with MN9D/neo cells (Fig. 5D), suggest-
ing that the direct cadmium binding by calbindin-D28k may not
contribute much to protection from cadmium-induced apoptosis
in this neuronal cell type. It is likely that the anti-apoptotic
function of calbindin-D28k can be attributed to reducing the
intracellular toxic calcium level that is increased by cadmium
rather than its direct cadmium sequestering effect. The detailed
mechanisms by which calbindin-D28k protects cells against cad-
mium-induced apoptosis await future investigation.
In conclusion, our study focuses on the cellular adaptive
responses to long term cadmium exposure. An increase in the
expression of Rho GDI-␣, RanBP1, and c-Myc-binding protein
MM-1 in cadmium-adapted cells suggests the involvement of
these proteins in the metal-induced mitogenesis and cell pro-
liferation. Of interest is that acquisition of cadmium resistance
was partly caused by an increased expression of calbindin-D28k,
which enables cells to escape from cadmium-induced apoptosis.
Because calbindin-D28k is overexpressed in various cancer cells
(19, 24, 49), it seems reasonable to predict that the elevation of
this protein could promote neoplastic cell survival as well as
subsequent clonal expansion and tumor development. Unveil-
ing the precise mechanisms of this protein in rescuing cells
from cadmium-mediated apoptosis may shed light on the un-
derstanding of cadmium-mediated carcinogenesis.
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