J Biol Chem, Vol. 274, Issue 39, 27759-27767, September 24, 1999
Growth Stimulation Versus Induction of Cell
Quiescence by Hydrogen Peroxide in Prostate Tumor Spheroids Is Encoded
by the Duration of the Ca2+ Response*
Maria
Wartenberg,
Heike
Diedershagen,
Jürgen
Hescheler, and
Heinrich
Sauer
From the Department of Neurophysiology, University of Cologne,
D-50931 Cologne, Germany
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ABSTRACT |
With increasing size, multicellular prostate
tumor spheroids develop regions of quiescent, multidrug-resistant cells
expressing the cyclin-dependent kinase inhibitor
p27kip1. Treatment of small (diameter 60 ± 20 µm)
spheroids with 200 µM hydrogen peroxide
(H2O2) resulted in cell cycle arrest owing to
up-regulation of p27kip1 and down-regulation of the
transcription factor c-Fos. Incubation with 100 nM-1
µM H2O2 led to up-regulation of
c-Fos and enhanced tumor growth. Growth stimulation was inhibited by
bisindolylmaleimide I, indicating a role for protein kinase C in the
signaling cascade that involved the mitogen-activated protein kinase
members MEK1,2, ERK1, -2, and c-Jun N-terminal kinase. Changes in
Ca2+ influx underlined the differential effects of
H2O2. Incubation with 200 µM
H2O2 released [Ca2+]i
from intracellular stores followed by prolonged Ca2+
influx. Inhibition of influx by Ca2+-free media or
Ni2+, La3+, Mn2+ and SKF-96365
prevented the induction of quiescence and stimulated spheroid growth.
Consequently, treatment with 200 µM
H2O2 in Ca2+-free media
down-regulated p27kip1 and increased Fos protein. ATP exerted
effects comparably to those observed with H2O2.
Encoding growth stimulation by [Ca2+]i release
and induction of cell quiescence by prolonged Ca2+ influx
may provide a general mechanism for the control of tumor growth.
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INTRODUCTION |
The role of [Ca2+]i fluctuations in the
control of a variety of cell functions including contraction,
differentiation, motility, secretion, and proliferation is well
established (1). However, there is still uncertainty concerning the
mechanisms by which the one second messenger Ca2+ can exert
specific effects on discrete intracellular signal pathways that have to
be decoded by downstream effectors. Recently, some evidence has emerged
that the amplitude and duration of [Ca2+]i
signals mediates the differential activation of different transcription
factors, indicating that Ca2+ release from intracellular
stores and subsequent Ca2+ influx across the plasma
membrane activate distinct transcriptional pathways (2).
In the past years an increasing number of publications have
demonstrated that reactive oxygen species
(ROS)1 including
H2O2, O
2, and OH· play an
important role as second messengers (3-6) and may promote the
constitutive growth of neoplastic tissues (7). Recently, the production
of ROS has been found to be related with cytokins such as transforming
growth factor-
-1, interleukin-1, tumor necrosis factor-
(8-12)
as well as with hormones and peptide growth factors such as angiotensin
II, platelet-derived growth factor, and basic fibroblast growth factor
(13, 14). The action of ROS in signaling pathways involves
[Ca2+]i fluctuations in a variety of preparations
(15-18), indicating a fine-tuned interplay between ROS and
Ca2+ that may result in a distinct pattern of either gene
activation or down-regulation of transcriptional activity.
We have recently shown that 100 nM
H2O2 caused release of
[Ca2+]i from intracellular thapsigargin-sensitive
stores of Du-145 prostate cancer cells grown to the three-dimensional
tissue of multicellular tumor spheroids (19). Following
H2O2 treatment, a transient up-regulation of
c-Fos and a faster kinetics of tumor growth was observed. In the
present study treatment of multicellular spheroids with 200 µM H2O2 resulted in tumor growth
depression and up-regulation of the cyclin-dependent kinase
inhibitor p27kip1. p27kip1 acts as a negative regulator
of G1 progression (20) and has been suggested to be
associated with intrinsic multidrug resistance in three-dimensional
tumor tissues (21). Under these experimental conditions
Ca2+ release from intracellular stores was followed by a
prolonged period of Ca2+ influx. Since our previous studies
demonstrated that the effect of H2O2 on the
growth stimulation of tumor spheroids was
Ca2+-dependent we tested whether cell cycle
activation by 100 nM to 1 µM
H2O2 versus cell cycle arrest and
induction of cell quiescence by 200 µM
H2O2 was owing to the nature of the
H2O2-induced Ca2+ response. Our
data indicate that the [Ca2+]i release phase of
the [Ca2+]i response initiates cell cycle
activity. On the other hand prolonged Ca2+ influx is
mediating cell cycle arrest, which consequently results in the
depression of tumor growth.
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MATERIALS AND METHODS |
Culture Technique of Multicellular Spheroids--
The human
prostate cancer cell line DU-145 was kindly provided by Dr. J. Carlsson, Uppsala, Sweden. The cell line was grown routinely in 5%
CO2, humidified air at 37 °C with Ham's F-10 medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Roche Molecular Biochemicals), 2 mM glutamine, 0.1 mM -mercaptoethanol, 2 mM minimal essential
medium, 100 IU/ml penicillin, and 100 µg/ml streptomycin (ICN Flow,
Meckenheim, Germany). Spheroids were grown from single cells. Cell
monolayers were enzymatically dissociated with 0.2% trypsin, 0.05%
EDTA (ICN Flow) and seeded in siliconated 250-ml spinner flasks
(Integra Biosciences, Fernwald, Germany) with 250 ml of complete medium
and agitated at 20 rpm using a Cell-spin stirrer system (Integra
Biosciences, Fernwald). Cell culture medium was partially (100 ml)
changed every day.
Incubation of Spheroids with H2O2 and
ATP--
Small multicellular spheroids (diameter 60 ± 20 µm)
were washed in F-10 cell culture medium. They were placed in 8.5-cm
diameter plastic nonadhesive culture dishes (Greiner, Solingen,
Germany) and incubated for 1 h in E1 medium containing 135 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM Hepes (pH 7.4 at 37 °C)
and supplemented with different concentrations of H2O2 (Sigma) as indicated. In experiments with
nominally Ca2+-free solution, CaCl2 was omitted
from the incubation medium. The protein kinase C inhibitors
bisindolylmaleimide I (1 µM) and H-7 (10 µM) (both from Calbiochem-Novabiochem) were added to the spheroids 1 h before the addition of H2O2.
Spheroids were subsequently washed three times in F-10 medium and
cultivated in liquid overlay culture. Incubation with ATP (Sigma) was
performed in F-10 cell culture medium. ATP was present during an
incubation period of 24 h. Spheroid diameters in control and
treated samples were monitored every 24 h. Spheroid volumes were
calculated according to V = 4/3·
·r3. H2O2 did
not per se influence the volumes of tumor spheroids. The
lifetime of H2O2 in E1 buffer was determined by
a luminol-dependent chemiluminescence assay (22). In E1
buffer, no H2O2 degradation within 1 h of
incubation was observed (data not shown).
Ca2+ Imaging and Confocal Laser-scanning
Microscopy--
[Ca2+]i was monitored using the
fluorescent dye fluo-3, AM (Molecular Probes, Eugene, OR).
Multicellular spheroids were mounted to poly-L-lysine
(Sigma)-coated coverslips and were subsequently loaded for 60 min in
F-10 cell culture medium with 10 µM fluo-3, AM, dissolved
in dimethyl sulfoxide (final concentration 0.1%) and
pluronicTM F-127, which facilitates the solubilization of
fluo-3, AM (final concentration <0.025%). After loading, the
spheroids were rinsed three times in E1 buffer. Superfusion was
performed by gravity at a rate of 10 ml/min. A 90% volume exchange was
achieved within 10 s. The experiments were performed at 37 °C.
Fluorescence data were recorded using an inverted confocal
laser-scanning microscope (LSM 410; Zeiss, Jena, Germany) equipped with
a 25× objective, numerical aperture 0.80 (Plan-Neofluar, Zeiss).
Fluorescence was excited by the 488-nm line of an argon-ion laser.
Emission was recorded using a LP 515-nm filter set. Processing of
images (512 × 512 pixels, 8 bit) was carried out by the
Time-software facilities of the confocal setup. Full-frame images were
acquired and stored automatically at 4-s intervals to a 16-megabyte
video memory of the confocal setup. The minimum, maximum, mean,
standard deviation, and integrated sum of the pixel values in a region
of interest (selected using an overlay mask) were written to a data
file and routinely exported for further analysis to the commercially
available Sigma Plot (Jandel Scientific, Erkrath, Germany) graphic
software. Because fluo-3 does not permit the use of ratio measurements, data are presented in arbitrary units as the percentage of fluorescence variation (F/F0) with respect to the
resting level F0, which was set to 100%.
Immunohistochemical Techniques and Quantitative
Immunohistochemistry--
The c-Fos (AB-2) polyclonal antibody (5 µg/ml) was obtained from Calbiochem). The monoclonal antibody
anti-p27kip1 was obtained from Pharmingen (Hamburg, Germany)
and used in a concentration of 2.5 µg/ml. The anti-active MAPK
polyclonal antibody directed against ERK1, -2 (dilution 1:20), and the
anti-active JNK (dilution 1:20) polyclonal antibody were obtained from
Promega (Madison, WI). The polyclonal anti-active p38 MAPK (dilution
1:20) and MEK1, -2 (dilution 1:20) antibodies were obtained from
Calbiochem. Antibody staining was performed on whole-mount
multicellular spheroids. As secondary antibodies, a
Cy3TM-conjugated goat anti-rabbit IgG (H+L) (Jackson
ImmunoResearch, West Grove, PA), concentration 1.2 µg/ml, and a
Cy5 TM-conjugated F(ab')2 fragment goat
anti-mouse IgG (H+L) (Roche Molecular Biochemicals), concentration 3.25 µg/ml, were used. Excitation was performed using a 543-nm and a
633-nm helium-neon laser of the confocal setup. Emission was recorded
using LP570 and LP655 nm filter sets, respectively.
For quantitative immunohistochemistry, confocal images of whole mount
multicellular spheroids stained with only secondary antibodies
(background fluorescence image) and spheroids stained with primary and
secondary antibodies were recorded. The pinhole settings of the
confocal setup were adjusted to yield optical slices of 20-µm
thickness. After subtraction of background fluorescence, the
fluorescence signal (counts) was evaluated in 500-µm2
areas of interest by the image analysis software of the confocal setup
and was routinely exported for further analysis to the Sigma Plot
graphic software.
Immunoblotting--
Multicellular spheroids treated with
H2O2 and untreated controls were lysed in 125 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerine, 20 mM dithiothreitol, 1 mM EDTA, 0.01% bromphenol
blue. Equal amounts of proteins (15 µg/lane) were electrophoresed on
10 or 15% SDS-polyacrylamide gels. Immunoblots of c-Fos and
p27kip1 were prepared by electrophoretic transfer of proteins
from SDS-polyacrylamide gels to nitrocellulose by semi-dry Western
blotting. The nitrocellulose transfers were incubated for 1 h in
blocking buffer (5% lowfat milk powder in phosphate-buffered saline
containing 0.1% Tween 20) and then probed for 1 h with 1:300
dilution of polyclonal rabbit anti-c-Fos in blocking buffer or 1:200
dilution of monoclonal anti-p27kip1 in phosphate-buffered
saline, 0.1% Tween 20. As secondary antibodies, horseradish
peroxidase-conjugated rabbit (dilution 1:2 × 104) or
mouse (dilution 1:1 × 104) antibodies were used.
Statistical Analysis--
Data are given as mean values ± S.D., with n denoting the number of experiments that were
performed with at least three independent tumor spheroid cultures.
Student's t test for unpaired data was applied as
appropriate. A value of p < 0.05 was considered significant.
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RESULTS |
Expression of p27kip1 during the Growth of Multicellular
Tumor Spheroids--
The initial phases (day 1 to day 6) of tumor
growth follow an exponential growth kinetics with volume-doubling times
of approximately 24 h (19). With the development of quiescent cell
areas, which has been previously shown to occur at spheroid diameters
of approximately 180 µm (23), growth retardation occurs, and the
volume-doubling times increased to 96 ± 30 h
(n = 3) (Fig.
1A). By quantitative immunohistochemistry and immunoblotting, a correlation between the
growth kinetics of multicellular tumor spheroids and the expression of
p27kip1 became obvious (Fig. 1, B and C).
Our data indicate that p27kip1 protein levels continuously
increased during the first 6 days of spheroids culture. From day 7 on,
i.e the time where growth retardation of multicellular tumor spheroids
occurred, the increases of p27kip1 protein levels were
apparently more pronounced, indicating that with a critical diameter of
approximately 180 µm, quiescent cells develop in the tumor tissue of
multicellular spheroids.
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Fig. 1.
Growth kinetics (A) and
p27kip1 expression of multicellular tumor spheroids
(B and C). Note that
multicellular tumor spheroids grow with exponential growth kinetics
until they reach spheroid diameters of approximately 180 µm. The
decreased growth kinetics of larger spheroids is owed to the increased
expression of the cyclin-dependent kinase inhibitor
p27kip1 in larger spheroids. The inset in
B shows an immunoblot of p27kip1 protein in cell
extracts of 3-day and 18-day-old multicellular tumor spheroids. In
C, representative 3-day (left) and 18-day-old
(right) multicellular spheroids immunostained for
p27kip1 are shown. The bar represents 10 µm.
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Effects of H2O2 on the Growth Kinetics of
Multicellular Tumor Spheroids and the Activation of Mitogen-activated
Protein Kinase Pathways--
We have recently reported that nanomolar
concentrations of H2O2 induced an enhancement
of the growth kinetics of multicellular tumor spheroids (19). To
evaluate the differential activation of cell proliferation
versus cell quiescence by H2O2,
multicellular tumor spheroids with a mean size of 60 ± 20 µm
were incubated for 1 h with varying doses of
H2O2 ranging from 10 nM to 200 µM, and spheroid size was recorded 24 h thereafter.
Fig. 2A shows that 100 nM to 1 µM H2O2
significantly stimulated tumor growth, whereas
H2O2 concentrations exceeding 100 µM led to significant growth depression as compared with
the control in the absence of H2O2
(n = at least 7 for each experimental condition).
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Fig. 2.
Effects of different concentrations of
H2O2 on the growth of multicellular tumor
spheroids and mitogen-activated protein kinase signaling pathways.
A, growth stimulation of tumor spheroids was achieved with
100 nM and 1 µM H2O2.
Incubation with 100-200 µM H2O2
resulted in growth depression. Spheroids were incubated with
H2O2 for 1 h. B, growth
stimulation by 1 µM H2O2 is
inhibited in the presence of bisindolylmaleimide I (BIM).
Spheroid volumes were evaluated 24 h after treatment. The data are
presented as relative volume increase (%) in relation to the spheroid
volumes before treatment (set to 100%). C, activation of
JNK (10 min after the addition of H2O2), MEK1,
-2 (10 min after the addition of H2O2), and
ERK1, -2 (30 min after the addition of H2O2).
Tumor spheroids were probed with phospho-specific antibodies, and
immunofluorescence was evaluated by quantitative immunohistochemistry.
*p < 0.05, significantly different from control.
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Growth stimulation by H2O2 was apparently
mediated via activation of protein kinase C, since preincubation with 1 µM bisindolylmaleimide I (n = 3) (Fig.
2B) and 10 µM H7 (not shown) inhibited the
observed effects. Furthermore, activation of JNK, MEK1,2, ERK1, -2, but not p38 MAPK (not shown) was observed (Fig. 2C). MEK1, -2 and ERK1, -2 activation occurred after incubation with 1 µM and 200 µM H2O2
and was at its maximum 10 and 30 min, respectively, after the addition
of H2O2 to the incubation medium. Activation of
the ERK pathway was more pronounced with 200 µM as
compared with 1 µm H2O2. JNK activation was
observed 10 min after the addition of 1 µM
H2O2 to the incubation medium, whereas 200 µM H2O2 did not exert significant
effects (n = 3).
To determine possible toxic effects of H2O2,
lethal cell stainings were performed with the lethal cell stain
ethidium homodimer-1 after 4 days of spheroid culture. No cell
lethality was observed with H2O2 concentrations
below 0.5 mM. Incubation for 1 h with 0.5 mM and 1 mM H2O2
resulted in a cell lethality of 8 ± 5% and 43 ± 20%,
respectively (n = 3) (data not shown). To exclude that 200 µM H2O2 induced apoptosis
rather than cell dormancy, spheroids were screened for apoptosis
24 h after H2O2 exposure using terminal deoxynucleotidyltransferase-mediated dUTP-X nick end-labeling (TUNEL).
Under the applied experimental conditions, no apoptosis occurred (data
not shown).
Dose-dependence of the Duration and Amplitude of
H2O2-induced [Ca2+]i
Responses--
Cell cycle progression has been shown to be dependent
on changes in [Ca2+]i (24-27). To evaluate the
involvement of [Ca2+]i signals in the induction
of either cell proliferation or cell quiescence by
H2O2, single cell [Ca2+]i
changes were recorded in tumor spheroids incubated with different
concentrations of H2O2 ranging from 100 nM to 500 µM. Nonlethal concentrations of
H2O2 elicited a transient rise of
[Ca2+]i (Fig. 3,
A-C). The duration of the [Ca2+]i
response increased with the concentration of
H2O2 added to the incubation medium and
amounted to 60 ± 16 s, 85 ± 26 s, 246 ± 57 s, 370 ± 70 s, and 590 ± 150 s for 100 nM, 1 µM, 10 µM, 100 µM, and 200 µM
H2O2, respectively (Fig.
4A) (n = 4). Incubation of tumor spheroids with 500 µM
H2O2, which exerted cytotoxic effects in part
of the cells, resulted in a sustained rise of
[Ca2+]i (not shown). The amplitude of the
[Ca2+]i response was 37 ± 15%, 38 ± 20%, 45 ± 15%, 107 ± 28%, and 139 ± 34% that of
the resting [Ca2+]i for 100 nM, 1 µM, 10 µM, 100 µM, and 200 µM H2O2, respectively (Fig.
4B) (n = 4).
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Fig. 3.
Single cell [Ca2+]i
responses elicited with different concentrations of
H2O2 in multicellular tumor spheroids.
A, 100 nM H2O2;
B, 10 µM H2O2;
C, 200 µM H2O2.
H2O2 was present in the incubation medium
during the time indicated by the horizontal straight line.
Representative tracings are shown.
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Fig. 4.
Duration (A) and amplitude
(B) of the single cell [Ca2+]i
responses elicited with different concentrations of
H2O2 in multicellular tumor spheroids.
Note that both the duration and the amplitude are enlarged with
increasing concentrations of H2O2.
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Activation of Ca2+ Influx Pathways by
H2O2--
The data of the present study show
that the [Ca2+]i signals elicited by
H2O2 are prolonged with rising concentrations of H2O2. To evaluate whether the prolonged
[Ca2+]i responses were owing to the activation of
Ca2+ influx, spheroids were treated with 200 µM H2O2 under
Ca2+-free conditions and in the presence of either
Ni2+ (1 mM) and La3+ (50 µM), which have been previously shown to inhibit
Ca2+ influx across the plasma membrane (28), or SKF-96365
(10 µM), which is an inhibitor of nonselective
Ca2+ entry (29). These conditions led to a significant
shortening of the [Ca2+]i response, whereas its
amplitude remained unchanged (Fig.
5A and B). The mean
duration of the [Ca2+]i response under
Ca2+-free conditions and in the presence of
Ni2+, La3+, and SKF-96365 was 195 ± 65 s, 190 ± 30 s, 173 ± 35 s, and 194 ± 40 s, respectively (n = 4). A total inhibition
of the [Ca2+]i response was obtained by
preincubation with 2 µM carbonyl cyanide
m-chlorophenylhydrazone, which impairs the respiratory chain
and depletes mitochondrial Ca2+ stores (data not shown).
Hence, the [Ca2+]i signal elicited with 200 µM H2O2 is owing to mitochondrial Ca2+ release followed by prolonged Ca2+
influx.
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Fig. 5.
Effect of inhibition of Ca2+
influx pathways on the [Ca2+]i responses elicited
with 200 µM
H2O2. Inhibition of Ca2+
influx was achieved by Ca2+-free solution,
La3+, Ni2+, and SKF-96365. Effects on the
duration (A) and the amplitude (B) of the
[Ca2+]i responses elicited with 200 µM H2O2 are shown.
*p < 0.05, significantly different from the sample
treated with 200 µM H2O2 under
control Ca2+ conditions.
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Impact of Inhibition of Ca2+ Influx on the Growth
Kinetics of Multicellular Tumor Spheroids--
The working hypothesis
of the present study was the assumption that the switch between growth
stimulation and induction of cell quiescence by
H2O2 is encoded predominantly by the influx component of the [Ca2+]i response, which is,
according to the model of capacitative Ca2+ entry proposed
by Putney (30), composed of Ca2+ release from intracellular
stores followed by Ca2+ influx across the plasma membrane.
To validate this assumption, spheroids with a mean diameter of 60 ± 20 µm were treated with 200 µM
H2O2 under conditions where Ca2+
influx was inhibited, i.e. under Ca2+-free
conditions, in the presence of Mn2+ (50 µM),
La3+ (50 µM), and SKF-96365 (10 µM). Spheroid growth was evaluated after 24 h. Our
data demonstrate that these conditions led to a significant enhancement
of tumor growth as compared with both the untreated control and tumor
spheroids treated with 200 µM H2O2 in the presence of 1.8 mM
extracellular Ca2+ (n = 4) (Fig.
6). The spheroid volumes amounted to
5.17 × 105 ± 1.9 × 105
µm3, 5.75 × 105 ± 1.75 × 105 µm3, 6.46 × 105 ± 2.24 × 105 µm3, and 6.2 × 105 ± 2.16 × 105 µm3 for
Ca2+-free conditions, La3+, Mn2+,
and SKF-96365, respectively. This was in the same order of magnitude as
achieved after incubation with 1 µM
H2O2 (5.56 × 105 ± 2.02 × 105 µm3). The volumes of untreated control
spheroids and spheroids treated with 200 µM
H2O2 were significantly smaller and amounted to
2.57 × 105 ± 0.85 × 105
µm3 and 1.24 × 105 ± 0.87 × 105 µm3, respectively. Taken together, our
data suggest that under conditions where Ca2+ influx was
inhibited, 200 µM H2O2 stimulated
tumor growth to an extent not significantly different from spheroids
treated with growth-stimulating concentrations (100 nM to 1 µM) of H2O2, which indicates that
Ca2+ from intracellular stores resulted in cell cycle
activation, whereas prolonged Ca2+ influx induced cell
quiescence.
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Fig. 6.
Effect of inhibition of Ca2+
influx pathways by Ca2+-free solution, Mn2+,
La3+, and SKF-96365 on the growth of multicellular
spheroids treated with 200 µM
H2O2. Open bars show control
conditions. Multicellular spheroids remained either untreated or were
treated for 1 h with Ca2+-free solution,
La3+ (50 µM) Mn2+ (50 µM), or SKF-96365 (10 µM) in the absence of
H2O2. In the experiments indicated by
hatched bars either 1 µM or 200 µM H2O2 was present during the
time course of the experiment. Spheroid volumes were evaluated 24 h after incubation with H2O2. The mean spheroid
volume before treatment amounted to 0.7 × 105
µm3. The data show one of four experiments that yielded
comparable results. At least 15 tumor spheroids were evaluated for the
respective experimental condition in each experiment.
*p < 0.05, significantly different from control.
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Inhibition of Ca2+ Influx during Treatment of
Multicellular Tumor Spheroids with 200 µM
H2O2 Results in Down-regulation of
p27kip1 and c-Fos Up-regulation--
The data of the present
study demonstrate that treatment of multicellular tumor spheroids with
100 nM to 1 µM H2O2
resulted in cell cycle activation and enhanced tumor growth. Induction of cell quiescence and growth retardation of tumor spheroids was achieved after incubation with 200 µM
H2O2. Growth stimulation of multicellular tumor
spheroids should therefore be accompanied by a down-regulation of the
cyclin kinase inhibitor p27kip1 and up-regulation of the
transcription factor c-Fos, which has been demonstrated to be involved
in the induction of cell proliferation in multicellular Du-145 prostate
tumor spheroids (31). On the other hand, induction of cell quiescence
by 200 µM H2O2 should be mediated
by p27kip1 up-regulation and down-regulation of Fos protein.
Since inhibition of Ca2+ influx, which resulted in an
abridgement of the Ca2+ response elicited by 200 µM H2O2, was followed by an
enhancement of tumor growth, we expected an up-regulation of Fos
protein levels and down-regulation of p27kip1 under these
experimental conditions. To evaluate these issues, spheroids with a
diameter of 60 ± 20 µm, which express moderate levels of
p27kip1 (see Fig. 1), were incubated with either 100 nM or 200 µM H2O2 or
200 µM H2O2 in the absence of
extracellular Ca2+. Fos protein levels were evaluated by
immunohistochemistry and immunoblotting 1 h after incubation with
H2O2, and p27kip1 protein levels were
evaluated 24 h thereafter. Fig. 7,
A and B, demonstrates by quantitative
immunohistochemistry that p27kip1 protein levels were
down-regulated and Fos protein levels were up-regulated when spheroids
were treated with cell proliferation-inducing nanomolar concentrations
(100 nM) of H2O2 (n = 3). However, p27kip1 was up-regulated with tumor
growth-depressing micromolar concentrations (200 µM) of
H2O2, which consequently down-regulated Fos
protein. Under Ca2+-free conditions, i.e. under
conditions where Ca2+ influx was abolished, 200 µM H2O2 significantly
down-regulated p27kip1 protein levels as compared with the
untreated control sample and the sample treated with 200 µM H2O2 in the presence of 1.8 mM extracellular Ca2+. Under these conditions
p27kip1 expression was not significantly different from the
values achieved after stimulation of cell proliferation with 100 nM H2O2. Consequently, a
significant up-regulation of Fos was observed, which is indicative for
the induction of cell proliferation and tumor growth. The data obtained
with quantitative immunohistochemistry and confocal laser-scanning
microscopy could be confirmed by immunoblotting experiments (Fig.
8, A and B)
(n = 3). These findings clearly show that
[Ca2+]i release from intracellular stores by
H2O2 is encoding the induction of cell
proliferation, whereas prolonged Ca2+ influx is involved in
cell cycle arrest and the induction of cell quiescence.
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Fig. 7.
Effect of H2O2 on Fos
(A) and p27kip1 (B) protein
levels. Multicellular spheroids were treated either with growth
stimulating (100 nM) or with quiescence-inducing (200 µM) concentrations of H2O2. The
incubations with 200 µM H2O2 were
performed either in the absence or presence of extracellular
Ca2+ to evaluate the effect of inhibition of
Ca2+ influx pathways on the induction of cell
proliferation. The images show representative tumor spheroids. From the
upper left to the lower right: control; 100 nM
H2O2; 200 µM
H2O2; 200 µM
H2O2, Ca2+-free. The
bars represent 10 µm. The data show one of three
experiments, which yielded comparable results. At least 15 tumor
spheroids were evaluated for the respective experimental conditions in
each experiment. *p < 0.05, significantly different
from control.
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Fig. 8.
c-Fos (A) and
p27kip1 (B) immunoblots after treatment of
multicellular tumor spheroids with different concentrations of
H2O2. Blots were obtained from tumor
spheroids treated with 100 nM and 200 µM
H2O2. The incubations with 200 µM
H2O2 were performed either in the absence or
presence of extracellular Ca2+ to evaluate the effect of
inhibition of Ca2+ influx pathways on the induction of cell
proliferation. Note that incubation with proliferation-stimulating
concentrations (100 nM) of H2O2
resulted in up-regulation of c-Fos, whereas incubation with
quiescence-inducing concentrations of H2O2 (200 µM) resulted in up-regulation of p27kip1 and
down-regulation of c-Fos. Under Ca2+-free conditions, 200 µM H2O down-regulated p27kip1,
whereas c-Fos was increased. *p < 0.05, significantly
different from control.
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ATP-induced [Ca2+]i Signals and Growth
Stimulation of Multicellular Prostate Tumor Spheroids by ATP--
It
is possible that H2O2 recruits signals in
addition to the characteristic [Ca2+]i patterns
here described, which may be responsible for the observed effects on
proliferation. To investigate this issue, tumor spheroids were
incubated with ATP in concentrations ranging from 0.1 to 100 µM. It has been previously shown that in Du-145 prostate
cancer cells P2u purinergic receptors are present, which upon
activation lead to Ca2+ release from intracellular stores
by an inositol trisphosphate-mediated mechanism (32). Our data
demonstrate that incubation of tumor spheroids with 0.1 and 1 µM ATP significantly stimulated tumor growth, whereas a
concentration of 100 µM ATP resulted in growth depression
(n = 3) (Fig.
9A). As described previously
(32) ATP induced transient [Ca2+]i responses,
which partially exerted oscillatory behavior. The duration of the
[Ca2+]i responses increased with rising
concentrations of ATP added to the incubation medium and amounted to
36 ± 11 s, 90 ± 80 s, 270 ± 112 s, and
645 ± 260 s for 0.1, 1, 10, and 100 µM ATP,
respectively (n = 3) (Fig. 9B). The duration
of the [Ca2+]i responses elicited by 1 µM ATP, which exerted the most pronounced stimulatory
effect on tumor growth, was not significantly different from the
duration of the [Ca2+]i responses achieved with
growth-stimulating concentrations of H2O2. This
held likewise true for the duration of [Ca2+]i
responses elicited with growth-depressing concentrations of ATP, which
were not significantly different from the signals achieved with 200 µM H2O2.
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|
Fig. 9.
Growth effects and
[Ca2+]i responses elicited by ATP in
multicellular tumor spheroids. A, effect of different
concentrations of ATP on the growth of multicellular tumor spheroids.
Spheroid volumes were evaluated after a 24-h incubation with ATP. Data
are presented as relative volume increase (%) in relation to the
spheroid volume before the addition of ATP to the incubation medium.
B, increasing concentrations of ATP prolong the duration of
[Ca2+]i responses. Note that prolongation of the
ATP-induced [Ca2+]i responses with increasing
concentrations of ATP results in growth retardation of tumor
spheroids.
|
|
| |
DISCUSSION |
The major obstacle for the efficacy of cancer therapy is the
development of features of resistance toward anticancer agents, which
has been attributed to the development of a low proliferative cell
fraction in the depth of the tumor tissue (33-35). Recently, we have
shown that prostate cancer cells grown in multicellular tumor spheroid
culture develop with increasing size an intrinsic P-glycoprotein-mediated drug resistance that could be attributed to
quiescent cell areas (36). In corroboration with our previous results,
it has been demonstrated that compact multicellular spheroids that
express high levels of cell quiescence-related p27kip1 are less
susceptible toward anticancer agents, and p27 has therefore been suggested to be a survival gene (21). Since anticancer agents like
vinca alkaloids and antimetabolites are most active during a particular
phase of the cell cycle, recruitment of cells into the cell cycle may
chemosensitize for chemotherapy. Indeed it has been shown that
tumors with a high S-phase fraction respond better toward chemotherapy
than tumors with a low proliferating, quiescent cell fraction (33-35).
This implicates that a better knowledge of the molecular mechanisms
regulating cancer cell proliferation could potentially be exploited to
overcome problems encountered with conventional cell cycle recruitment strategies.
The data of the present study demonstrate that the
cyclin-dependent kinase inhibitor p27kip1 is
up-regulated in multicellular prostate spheroids with increasing spheroid growth, indicating the emergence of a low proliferating, quiescent cell population in the tissue. Our data furthermore indicate
that intracellular redox modulation by 100 nM to 1 µM H2O2 led to cell cycle
activation, up-regulation of the proliferation-associated transcription
factor c-Fos, down-regulation of p27kip1, and enhanced tumor
growth. Incubation of multicellular tumor spheroids with nonlethal 200 µM concentrations of H2O2
resulted in down-regulation of c-Fos, up-regulation of p27kip1,
and consequently in tumor growth retardation. Growth stimulation of
cells by nanomolar concentrations of H2O2 has
previously been observed in a number of different cell types,
e.g. cultured human and rat fibroblasts (37, 38), epidermal
cells (39), and smooth muscle cells (40, 41). Higher concentrations of
H2O2 in a range of 50-100 µM
have been shown to induce cell cycle arrest in the
G0/G1 phase of the cell cycle, whereas 300-400
µM H2O2 have been demonstrated to
result in apoptosis (42).
Although the differential effects of different concentrations of
H2O2 on cell cycle activation and the induction
of cell quiescence and apoptosis are now emerging, the signal
transduction mechanisms underlying these phenomena are not well
defined. [Ca2+]i changes after treatment of cells
with H2O2 have been observed in a variety of
preparations (15, 16, 43). However, the interrelation between
H2O2-induced signal transduction pathways and
[Ca2+]i changes lacks conclusive investigations.
Several redox-mediated signal transduction pathways seem to depend on
[Ca2+]i changes. It has been shown in a recent
study that big mitogen-activated protein kinase 1 (BMK1) or ERK5
activation in rat vascular smooth muscle cells by
H2O2 is Ca2+-dependent
(45). In Jurkat cells c-Jun expression following treatment
H2O2 was inhibited after chelation of
[Ca2+]i by BAPTA (18). The activation of the
redox-regulated transcription factor NF-
B seems to depend critically
on [Ca2+]i (47). Furthermore, in chicken B cells,
Ca2+ dependence of the nonreceptor tyrosine kinase Syk was
demonstrated. Syk acts upstream of JNK, which is activated upon
oxidative stress in this cell line (48). In a recent study of our
group, we have demonstrated that cell cycle activation following
treatment of multicellular tumor spheroids by nanomolar concentrations
of H2O2 is a
Ca2+-dependent process (19). The data of our
previous study demonstrated that the [Ca2+]i
transient induced by nanomolar concentrations of
H2O2 was predominantly mediated by
Ca2+ from intracellular stores, since a comparable
transient was observed in Ca2+-free solution. The present
study demonstrates that cell cycle activation by
H2O2 was mediated via an activation of protein
kinase C, since the protein kinase C inhibitor bisindolylmaleimide I inhibited the observed growth stimulation. Our data furthermore suggest
that the downstream signal transduction cascade is operating via
activation of MEK1, -2, ERK1, -2, and JNK but not p38. Interestingly, MEK1, -2 and ERK1, -2 activation was observed with both low (1 µM) and high (200 µM) concentrations of
H2O2. However, JNK activation was only observed
with cell cycle-activating concentrations of H2O2 (1 µM), whereas after
incubation with 200 µM H2O2,
which retarded tumor growth and increased p27kip1, JNK was not
significantly activated. This may indicate that a dynamic balance
between ERK and JNK pathway is important in determining whether cell
cycle activity is stimulated or cell quiescence is induced in
multicellular tumor spheroids. An activation of the ERK-signaling
cascade by oxidative stress has been demonstrated for several
preparations (49-52). Likewise, evidence for JNK activation by ROS has
been recently provided (9, 54). As is the case for ERK, JNK activation
has been shown to be involved in signal transduction pathways leading
to the activation of cell proliferation in various cell types
(55-60).
In the present study we showed that both the duration and the amplitude
of the [Ca2+]i response enlarged with increasing
doses of H2O2. Nonlethal concentrations (100 nM to 200 µM) of H2O2
induced transient [Ca2+]i responses, whereas
lethal (500 µM to 1 mM)
concentrations of H2O2 elicited sustained
[Ca2+]i responses. Because it has been recently
reported that JNK in B cells is selectively activated by short
transient [Ca2+]i responses (46), it sounds
reasonable that in the present study higher concentrations of
H2O2, which elicited prolonged [Ca2+]i signals, failed to induce a significant
activation of JNK. The increase in duration of the
[Ca2+]i transient observed with rising
concentrations of H2O2 was predominantly owing
to Ca2+ influx, since under Ca2+-free
conditions, after the addition of divalent and trivalent cations and in
the presence of SKF-96365, which has been demonstrated to inhibit
nonselective cation channels (29), the duration of the
[Ca2+]i response was significantly shortened.
Because high K+ solution did not raise
[Ca2+]i and the phenylalkylamine verapamil did
not impair the H2O2-induced
[Ca2+]i response, a participation of
voltage-dependent Ca2+ channels in the observed
Ca2+ influx was
excluded.2 To evaluate
whether the duration of the Ca2+ signal, i.e.
the Ca2+ influx phase, encoded the information for
induction of cell proliferation versus induction of cell
quiescence, multicellular tumor spheroids were incubated with
quiescence-inducing micromolar (200 µM) concentrations of
H2O2 under conditions where Ca2+
influx was inhibited, i.e. Ca2+-free solution,
presence of divalent and trivalent cations, presence of SKF- 96365. Subsequently c-Fos, p27kip1 and tumor spheroid growth was
monitored. Our data clearly show that abridgement of the
[Ca2+]i transient elicited with 200 µM H2O2 by inhibition of
Ca2+ influx resulted in up-regulation of Fos protein levels
and enhanced tumor growth, whereas p27kip1 was down-regulated,
and slow-cycling cells were obviously recruited for the cell cycle.
Under these experimental conditions the amplitude of the
[Ca2+]i response was not significantly different
from the amplitude observed with 200 µM
H2O2 in the presence of control extracellular
Ca2+. Hence, we concluded that the duration of the
[Ca2+]i response, which is mainly characterized
by prolonged Ca2+ influx, is the determinant for the switch
between H2O2-induced cell cycle activation and
induction of cell quiescence. This implies that
H2O2 does not act per se on
transcription factors and cyclins involved in the regulation of the
cell cycle but is critically dependent, at least as a cofactor, on
changes in [Ca2+]i.
The data of the present study demonstrate that
H2O2-induced [Ca2+]i
signals promote activation of the cell cycle when they are
short-termed, transient, and mediated predominantly by Ca2+
release from intracellular stores. They induce cell cycle arrest when
long-lasting, but transient Ca2+ influx is activated. In
the present study low concentrations of ATP stimulated tumor growth,
whereas higher concentrations led to tumor growth retardation. Since
increasing concentrations of ATP resulted in prolonged
[Ca2+]i responses, the duration of the
[Ca2+]i may provide a general means by which
Du-145 prostate cancer cells differentially regulate cell proliferation
versus cell quiescence. Furthermore this observation may
indicate that H2O2 may recruit additional
signal transduction pathways, i.e. by the activation of
hormone or growth factor receptors or their downstream targets. The
activation of fibroblast growth factor receptor type I (44) and
epidermal growth factor (53) by H2O2, which has
been recently reported, points to this direction. Further investigations on the interrelation between ROS and
[Ca2+]i signals are essential since constitutive
ROS production appears to be characteristic for tumor cells (7) and may
be one of the prerequisites for the loss of cell cycle control that occurs during neoplastic tissue growth.
| |
ACKNOWLEDGEMENTS |
Immunoblots and quantitative
immunohistochemistry of c-Fos are parts of the Ph.D. thesis of H. Diedershagen.
| |
FOOTNOTES |
*
This work has been supported by the Graduiertenkolleg
Molecular Basis of Pathophysiological Processes and by the Cologne
Fortune Program, University of Cologne (project 98).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.
To whom correspondence should be addressed: Dept. of
Neurophysiology, Robert-Koch-Str. 39, D-50931 Cologne, Germany. Tel.: 49-221-4786976; Fax: 49-221-4786965; E-mail:
hs@physiologie.uni-koeln.de.
2
H. Sauer, M. Wartenberg, and J. Hescheler,
unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
ROS, reactive oxygen
species;
ERK, extracellular signal-regulated kinase;
MEK1, -2,
mitogen-activated protein kinase kinase 1 and 2, respectively;
JNK, c-Jun N-terminal kinase.
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TOP
ABSTRACT
INTRODUCTION
