From the Hamilton Regional Cancer Centre and
¶ Department of Pathology and Molecular Medicine, Faculty of
Health Sciences, McMaster University, Hamilton, Ontario L8V 5C2,
Canada
Received for publication, May 29, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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High levels of reactive oxygen species (ROS) are
associated with cytotoxicity. Alternatively, nontoxic levels of ROS
like hydrogen peroxide (H2O2) can mediate
the transmission of many intracellular signals, including those
involved in growth and transformation. To identify pathways downstream
of endogenous cellular H2O2 production, the
response of Rat-1 fibroblasts exhibiting differential HER-2/Neu
receptor tyrosine kinase activity to removal of physiological
H2O2 concentrations was investigated. The
proliferation of all cells was abolished by addition of the
H2O2 scavenger catalase to the culture medium.
HER-2/Neu activity was not significantly affected by catalase
treatment, suggesting that the target(s) of the
H2O2 signal lie downstream of the receptor in
our model. ERK1/2 phosphorylation was blocked by catalase in
fibroblasts expressing wild type Neu, however such a response did not
occur in cells possessing activated mutant Neu. This indicates that the
ERK1/2 response contributes little to the growth inhibition observed.
By contrast, JNK1 activity increased following the addition of catalase
or H2O2, regardless of Neu activity or level of
cell transformation. Phosphorylation of p38 MAPK was induced by
H2O2 but not by catalase. These observations
suggest that scavenging of H2O2 from the
cellular environment blocks Rat-1 proliferation primarily through the
activation of stress pathways.
Oxidative stress is known to induce the cellular apoptotic program
by the activation of defined
ROS1-responsive signals: the
stress-activated protein kinase (SAPK) and nuclear factor kappa B
(NF- It follows that the deregulation of ROS levels may be a key factor in
neoplasia. Elevations in O In the present study the requirement of low levels of
H2O2 for Rat-1 fibroblast proliferation was
investigated. The effects on defined growth- and stress-associated
signaling pathways, produced by the scavenging of physiological
H2O2 levels from the extracellular environment,
were assessed. Rat-1 clones exhibiting differential growth and
transformation properties resulting from specific alterations of the
HER-2/Neu receptor were utilized as a model for the responses to
removal of the H2O2 stimulus (23, 24). Multiple
signals downstream of Neu are implicated in oncogenesis, and Neu
overexpression and/or activating mutation is observed in 25-30% of
human breast and ovarian cancers (for review see Ref. 25). The
activities of the Neu receptor, extracellular signal-regulated kinase 1 and 2 (ERK1/2) MAPK and JNK1 effector pathways were studied after the
addition of the H2O2 scavenger catalase. All
Rat-1 clones displayed comparable sensitivities to growth inhibition by
this treatment regardless of the level of Neu activity. It was found that scavenging of H2O2 caused stable
inhibition of the ERK1/2 signal, transient induction of the JNK1
pathway, and no effect on p38 MAPK. Constitutive Neu activation could
rescue the catalase-mediated block of ERK1/2 activity while only
slightly increasing resistance to growth inhibition. Therefore, these
results indicate that removal of extracellular
H2O2 can both down-regulate growth signals and activate stress-associated signals, however, the stress response appears to play a larger role in the anti-proliferative effects observed.
Cell Culture and Treatments--
All Rat-1 fibroblast clonal
cell lines were kindly provided by Dr. W. J. Muller and have been
previously characterized (23, 24). Rat-1NeuN (17) clone expresses wild
type Neu. The highly transformed Rat-1Neu8142(10) clone expresses Neu
having a 12-amino acid (aa 641-652) deletion in the extracellular
region of the receptor proximal to the transmembrane domain, resulting
in its constitutive dimerization. Rat-1NeuNT (3) is another highly transformed clone expressing Neu with an activating point mutation (V664E) in the transmembrane domain (26). Rat-1NeuNT-NYPD (6) is a
transformation-impaired clone expressing the NeuNT receptor with
C-terminal autophosphorylation sites (Tyr-1028, Tyr-1144, Tyr-1201,
Tyr-1226/7, Tyr-1253) replaced with phenylalanine residues. Cells were maintained at early passage number in 100-mm diameter tissue
culture plates (Falcon, Becton Dickinson) at 37 °C in a humidified
atmosphere of 5% CO2, 95% air. Rat-1 lines were grown in
Dulbecco's modified Eagle's medium with L-glutamine (Life
Technologies, Inc.), and all media were supplemented with 10%
heat-inactivated fetal calf serum (Life Technologies, Inc.), except in
serum-starved controls. Cells were passaged by trypsinization (1×
trypsin-EDTA; Life Technologies, Inc.).
Cultures were treated with various agents to manipulate extracellular
H2O2 levels and affect cellular functioning:
H2O2-scavenging bovine liver or fungal
(Aspergillus niger) catalase preparations (0-2000 units/ml,
Sigma), heat-inactivated (HI) catalase (boiled for 10 min),
H2O2-generating A. niger glucose
oxidase preparation (0-0.02 unit/ml, Sigma), exogenous
H2O2 (BDH inc.), and the MEK1 inhibitor PD98059
(20-30 µM, New England BioLabs).
Cell Proliferation and Active DNA Synthesis Assays--
The cell
growth rate was assessed by measurement of the total cell number after
4 days of culture. 1000 cells/well were seeded onto a 96-well plate
(Falcon, Becton Dickinson) and incubated until adherent. After
incubation for 4 days, cells were washed with H2O and total
cellular DNA was quantitated by the addition of 200 µl/well of 10 µg/ml Hoechst stain No. 33258 (bisbenzimide H 33258 fluorochrome,
Calbiochem-Novabiochem) in TNE buffer (5 mM Tris base, 0.5 mM EDTA, 1 M NaCl, pH 7.4). Fluorescence in wells (excitation
Quantitation of cellular DNA synthesis was performed on cells by the
addition of 10 µM 5-bromo-2'-deoxyuridine (BrdUrd, Sigma) for 30 min. Cells were then trypsinized and washed 2× with
phosphate-buffered saline. 100 µl of each cell suspension was added
to 2 ml of cold 70% ethanol with vortexing and left on ice for 30 min.
2 ml of 4 N HCl (BDH inc.) was then added, and
mixtures were left at room temperature for an additional 30 min. Tubes
were centrifuged, and cell pellets were washed in 1 ml of 0.1 M Borax (Sigma, pH 8.5), then stored in 70% ethanol at
Observation of ERK1/2 and p38 MAPK Phosphorylation
Status--
Cells were lysed for total protein in mTNE buffer
(50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1%
Nonidet P-40, 10mM NaH2PO4, 10 mM NaF, 2 mM EDTA, 1 mM
Na3 VO4, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). 50 µg of protein per sample was boiled in SDS gel sample
loading buffer (plus 5 mM EDTA) for 10 min and separated in
a 12% SDS-polyacrylamide gel. In Vitro Kinase Assay for the Measurement of JNK1 and p38 MAPK
Activity--
Cell cultures were washed 2× in ice-cold PBS and lysed
in mTNE buffer. 100 µg of total cellular protein was added to
60 µl of protein G-agarose beads and 0.5 µg of rabbit polyclonal
Low Level Generation of H2O2 by Rat-1
Fibroblasts Is Required for Efficient Growth in Culture--
Addition
of catalase preparations to the culture media has been shown by our
laboratory to block the proliferation of a variety of immortalized cell
types in a dose-dependent and reversible manner.2 Extracellular
catalase treatments have also been utilized to inhibit the effects of
exogenous H2O2 in cell culture (27) and forced
overexpression of catalase can inhibit DNA synthesis and cell growth
(28). To determine whether exogenous catalase activity could produce a
similar response in our Rat-1 clones, cells were treated with doses of
catalase or heat-inactivated preparation, and the total cell number
after 4 days culture was observed (Fig. 1A). The growth of all Rat-1
lines was blocked after catalase addition; removal of this treatment
during incubation or heat inactivation of the enzyme reversed this
effect. Catalase treatment caused growth arrest without significant
induction of the apoptotic program, as observed by cell morphology and
trypan blue exclusion (not shown). The cell cycle profile of
fibroblasts at increasing times of treatment (6-48 h) was assessed by
propidium iodide staining of total DNA and flow cytometry (not shown).
A discreet block of the cell cycle at the G1/S or
G2/M checkpoints was not observed, however, an
accumulation of cell populations in S and G2 phases suggests a prolonged S phase and inhibition of entry into mitosis. Coincubation of cells with an H2O2-generating
glucose oxidase preparation in addition to catalase also rescued the
proliferation block (Fig. 1B), providing evidence that
catalase-mediated effects are indeed a result of changes in
H2O2 levels. Treatment with glucose oxidase
alone at activities greater than 4 × 10
Consistent with the observations of lower cell number and a prolonged S
phase and mitosis, active DNA synthesis was slowed ~2- to 4-fold by
2000 units/ml catalase as observed by BrdUrd (Fig.
2A) or
[methyl-3H]thymidine (Fig. 2B)
incorporation into cellular DNA. Also, the addition of 200 µM H2O2 increased DNA synthesis
in all lines tested, in accordance with its well-documented
growth-promoting effects. Various mammalian cell types have been shown
to secrete H2O2 into the surrounding culture
medium (15, 16). This generation is thought to reflect cell growth
potential, because manipulation of cellular
H2O2 production results in changes to both the
amount of extracellular H2O2 and to levels of
proliferation and transformation (19, 20). The endogenous generation of
H2O2 by Rat-1 cells was examined (not shown),
with the amounts of H2O2 produced by each clone
(5-15 pmol/h/50,000 cells) corresponding to the rate of growth in
culture. Addition of A. niger catalase preparation to the
media efficiently lowered extracellular H2O2
levels (not shown). Rat-1Neu8142 cells consistently demonstrated a
2-fold or higher increase in H2O2 production
compared with the Rat-1NeuN line. Because of the differential in
HER-2/Neu activity, extracellular H2O2
generation, and level of transformation exhibited by these two clones,
they were compared directly in subsequent experiments focusing on
molecular responses to catalase treatment.
P44/42 MAPK Phosphorylation Is Blocked by Catalase Treatment, and
This Effect Is Rescued by Constitutively-activated Neu--
The
Ras-MAPK signaling cascade has also been shown to be activated by
H2O2 (11, 29). We wished to determine whether
the removal of H2O2 would cause ERK1/2 MAPK
down-regulation, as further evidence for the redox sensitivity of this
pathway. Thr-202/Tyr-204-phosphorylated ErK1 and 2 were detected using
phospho-MAPK-specific primary antibodies. Levels of phosphorylated ERKs
were compared with total ERK1/2 protein levels. Rat-1NeuN cells treated
with H2O2 displayed an increase in p44/42 MAPK
phosphorylation, whereas the addition of catalase for 24 h
(±10-min exposure to H2O2) abolished MAPK activity (Fig. 3A).
Heat-inactivated catalase did not produce such an effect. In contrast,
this decrease in ERK1/2 phosphorylation was not observed after the
identical treatment of Rat-1Neu8142 cells (Fig. 3B). The
MEK1 inhibitor PD98059 (30) was added to cells for 1 h as a
control for ERK1/2 down-regulation. These observations demonstrate that
the alteration of Neu receptor activity can modulate the response of
the MAPK pathway to a decrease in H2O2 levels. However, because the growth response of Rat-1Neu8142 cells to catalase
treatments differed little from that of Rat-1NeuN cells, this
proliferation block appears largely independent of the p44/42 MAPK
signal.
The response of Rat-1 clones to a range (0-500 units/ml) of catalase
activities was compared (not shown). Rat-1Neu8142 cells displayed a
slightly higher resistance to the effects of catalase than Rat-1NeuN:
This may result from differences in MAPK pathway activity or in the
amount of endogenous H2O2 production observed between the two clones. To test for the later possibility, SK-OV-3 cells (human ovarian adenocarcinoma) were also examined for resistance to catalase (not shown). These cells generate extracellular
H2O2 at a level ~6-fold higher than the
Rat-1Neu8142 line. This increased H2O2 did not
correlate to any increase in resistance; indeed the growth of SK-OV-3
cells was extremely sensitive to the scavenging treatment. Sensitivity
of cells to removal of H2O2 in the presence of
the MEK1 inhibitor PD98059 was also investigated (not shown). This
concurrent treatment blocked the growth of Rat-1NeuN cells to a greater
extent than catalase treatment alone, whereas this effect was less
marked in the Rat-1Neu8142 line, which exhibited increased p44/42 MAPK
phosphorylation. Such increased MAPK activity did not rescue cell
growth to a significant degree, however. Thus it appears that, although
the H2O2 signal can regulate ERK1/2 activities,
there are also other intracellular signals that are responsible for
blocking proliferation.
Extracellular Catalase Activates the JNK1 Pathway--
Because the
effects of H2O2 scavenging could not be fully
attributed to the down-regulation of mitogenic MAPK signals, we examined the response of a SAPK signal; the JNK1 pathway. The N-terminal phosphorylation of c-Jun by JNK1 (p46 JNK) is a
well-characterized cell stress response (1, 31). JNK1 activity was
examined by an in vitro kinase assay (Fig.
4). All treatments induced a transient
increase in JNK1 kinase activity as quantitated by level of substrate
(c-Jun peptide) phosphorylation. In Rat-1NeuN cells (Fig.
4A) a toxic level of H2O2 (2 mM) activated JNK1 up to 10-fold over basal levels, with
peak activity occurring 1 h after treatment. Nontoxic levels of
H2O2 could not induce JNK1 activity. 500 units/ml catalase (~IC75) induced the JNK1 signal
~5-fold with peak activity after 30 min. Heat-inactivated catalase
(500 units/ml) could also activate JNK1 to a lesser extent (~2-fold
over basal levels). Such stimulation did not result in any change in
cell viability and growth as discussed. The response of Rat-1Neu8142
cells (Fig. 4B) was similar, except that 2 mM
H2O2 increased phosphorylation of the c-Jun
substrate in an identical manner as catalase treatment (~5-fold
increase, peak at 30 min). These observations demonstrate that opposing
directions of H2O2 imbalance, namely, the
addition of high concentrations of H2O2 or
removal of endogenous H2O2 levels by catalase,
can stimulate the JNK1 pathway in a similar fashion. Also, the
activation of this SAPK by catalase is unaffected by differential Neu
receptor activity, suggesting that it may be a critical component of
the growth inhibition observed with all Rat-1 fibroblasts studied.
These data indicate that the up-regulation of JNK1 is a common response
to removal of extracellular H2O2 in our Rat-1
fibroblast model.
P38 MAPK Phosphorylation Is Induced by H2O2
but Not by Catalase Treatment--
In a similar manner to that
observed with JNK1 signal stimulation, the stress-induced p38 MAPK was
phosphorylated upon addition of exogenous H2O2
in all cells tested (Fig. 5). However,
this response to oxidative stress was sustained over time, and no p38 activation was observed after treatment of cells with catalase or
heat-inactivated catalase preparations. Levels of induction of p38
phosphorylation in both Rat-1NeuN and Rat-1Neu8142 lines by 2 mM H2O2 was comparable to that
observed under hyperosmotic conditions (0.2 M NaCl for 30 min). The phosphorylation of one p38 MAPK substrate, the transcription
factor ATF2, was also investigated under the same conditions using an
in vitro kinase assay (Fig. 5). No significant changes in
phosphorylation of GST-ATF2 peptide by p38 were observed after catalase
or H2O2 treatments. It is undetermined whether
this finding is a result of a functional impairment of p38 in the cells
tested or the inability of the substrate utilized to respond to the p38
signal. Regardless, the absence of any direct p38 activation by
catalase at levels observed to affect ERK1/2 and JNK1 suggests that the
block in Rat-1 fibroblast proliferation is independent of this
signal.
This is the first report describing the effects of the removal of
endogenously produced H2O2 upon specific
mitogenic and stress-responsive signaling pathways. To remove
H2O2 from the cellular environment, a catalase
preparation was directly added to the culture media. This strategy was
adopted so that the molecular signaling responses observed could be
attributed to changes in H2O2 status at the plasma membrane. One potential problem with this approach is enzyme purity, raising concerns that the effects observed could result from
contaminants. To address this issue, we compared the effects of
catalase to heat-inactivated catalase in all experiments. Incubation with the heat-inactivated solution did not affect cell viability and
growth, nor did it affect Neu receptor or ERK activities. JNK1 activity
did increase upon the addition of heat-inactivated catalase, but at a
2- to 3-fold lower level than that observed after treatment with active
catalase. A comparable A. niger catalase preparation
markedly reduced extracellular H2O2 levels,
whereas heat inactivation of this treatment blocked the effect. In
addition, catalase-induced growth inhibition was rescued upon
cotreatment of cells with H2O2-generating
glucose oxidase. The levels of glucose oxidase activity required
for this rescue were cytotoxic if added alone, indicating that the two
enzymes are inversely affecting H2O2 levels.
These results provide evidence that the responses to the catalase
preparation used specifically reflect its H2O2 scavenging activity. It is likely that changes in the concentration of
extracellular H2O2 will in turn affect
intracellular redox status, because H2O2 can
freely diffuse across cell membranes. Therefore, the effects of the
catalase treatment used here are best described as a response to the
alteration of the "pericellular" redox environment, which is both
outside and inside of the plasma membrane. Certainly the fact, that Neu
receptor activity was not greatly changed by the addition of catalase
to cell cultures whereas various MAPK signaling cascades were,
indicates that direct manipulation of intracellular signals by this
type of treatment occur.
One such affected signal was the ERK1/2 pathway. The inhibition of
ERK1/2 phosphorylation observed with catalase addition (and
up-regulation observed with H2O2) to Rat-1NeuN
cells agrees with the previous findings that demonstrate the
responsiveness of the MAPK cascade to H2O2 (11,
12). Constitutive activation of the Neu receptor in Rat-1 Neu8142 cells
induced basal ERK1/2 activity and protected it from catalase-induced
inhibition. Neu interacts with several signaling cascades, including
the Ras/Raf/MEK pathway, important in ERK1/2 regulation (25). The
rescue of ERK phosphorylation was accompanied by only a slight increase in resistance of fibroblasts to growth inhibition by catalase. This
result indicates that, although contributing to the cellular responses
observed, ERK1/2 signal down-regulation is not critical for them.
Unlike the ERK response, the stress-activated JNK1 signal was rapidly
and transiently induced by treatments that both increased and decreased
H2O2 in Rat-1NeuN and Rat-1Neu8142 lines. JNK1
is known to be responsive to oxidative stress (2, 32), but our findings
suggest an additional role for this signal in response to an
antioxidant. Rac1 and Cdc42, members of the Rho family of small GTPases
involved in actin cytoskeleton regulation and cell transformation, have
been shown to activate the JNKs through interaction with p21-activated
kinase (33, 34). In addition, Rac1 is a cytoplasmic component of the
plasma membrane-localized NAD(P)H oxidase complex that directs the
extracellular generation of O Because our data demonstrate MAPK inhibition and SAPK activation after
catalase treatment, it could be that loss of the
H2O2 stimulus blocks the Ras pathway while
inducing other JNK-dependent signals like phosphoinositide
3-OH kinase (PI3K) and Rac. Recent evidence suggests that PI3K
can play dual roles: the survival-promoting PI3K target Akt kinase
(protein kinase B) was shown to phosphorylate Rac1 at serine 71 and
abolish its GTPase activity (37). Treatment of our Rat-1 fibroblast
lines with a 5 µM concentration of the PI3K inhibitor
LY294002 (38) inhibited cell proliferation to a comparable extent
regardless of HER-2/Neu activity levels (not shown). Thus, changes to
the PI3K/Akt signal might contribute to the catalase-induced effects
upon JNK1 observed; however, this remains to be determined.
The role of JNK in cell cycle control is not known. c-Jun contributes
to a number of contrasting biological processes as part of the AP-1
transcriptional complex (3). The degree of SAPK activity balanced with
other AP-1-inducing signals such as the ERKs could decide which process
is followed. In our system, ERK inhibition and JNK activation are
observed with cell growth arrest. Studies by Wisdom et al.
(31) demonstrate that JNK-mediated phosphorylation of c-Jun is specific
for stress responses only, whereas c-Jun involvement in cell cycle
progression is triggered by other factors. Another report indicates
that the JNK signal can prolong the cell cycle at S and
G2/M phases in a p53-independent manner (39), however, the
mechanisms underlying this effect have yet to be elucidated. Indeed, we
observed an increase of the proportion of cells in S and G2
phases after incubation with catalase (results not shown). Because
cells remain trapped in the late stages of the cell cycle, apoptosis
may then be triggered by a "mitotic crisis."
In conclusion, this work demonstrates that H2O2
regulates components of various MAPK cascades to allow for the
efficient growth of cultured Rat-1 fibroblasts. ERK1/2 activities are
induced upon the addition of H2O2 and inhibited
by its removal. Expression of constitutively activated Neu receptor
reversed this inhibition but had little effect on the catalase-induced
block of proliferation. However, the stress-activated JNK1 pathway was
induced by both the addition and removal of the
H2O2 signal, regardless of Neu status. Another
stress signal, the p38 MAPK pathway, was activated by toxic levels of
H2O2 but not by its removal, indicating that the JNK1 response might be specific. A discreet range of JNK activation may act as a cell sensor of oxidative and reducing stresses, leading to
a response of growth arrest and death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B) pathways ((1), for review see Ref. 2). Activation of
c-Jun/AP-1 transcription factors by the c-Jun N-terminal kinase (JNK)
members of the SAPKs has been shown to induce apoptosis in several cell
types (3). This signal is in contrast to the other characterized roles
of c-Jun/AP-1 activity in survival and growth. For example, Jun
proteins associate with Nrf 1 and 2 transcription factors to
up-regulate the antioxidant/electrophile response
element-mediated gene expression as a defense against oxidative stress
(4). Similarly, NF-
B was originally identified as a mediator of
tumor necrosis factor
- and ROS-induced apoptosis but more recently
has also been characterized as part of a survival response by
stimulating antioxidant response element-regulated and
anti-apoptotic protein expression (5, 6). Indeed, ROS, like
H2O2, are associated with the regulation of
multiple cellular processes via interaction with proteins and lipid
species (7). These observations have led to the idea that ROS are
required "cofactors" in the regulation of many intracellular signal
transduction cascades. Addition of micromolar concentrations of
H2O2 to cells in vitro can activate
plasma membrane receptor tyrosine kinases, including the epidermal
growth factor receptor/ErbB-1 (8), platelet-derived growth factor
receptor (9), and insulin receptor (10). The p21 Ras-MAPK
(mitogen-activated protein kinase) pathway, a key growth signal linking
membrane receptors to the nucleus, is also stimulated by oxidative
stimuli such as H2O2 (11, 12). Rapid induction
of cellular H2O2 generation has been observed to follow the addition of a variety of peptide growth factors to cells,
suggesting an autocrine and/or paracrine role for ROS in growth
signaling (13-15). The specific molecular targets that are critical
for H2O2-mediated mitogenesis have yet to be
determined; the importance of a given pathway is also dependent on
factors such as cell type, environment, and level of differentiation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 350 nm; emission
, 460 nm) was measured using
a CytoFluor series 4000 multiwell plate reader (PerSeptive Biosystems).
The cell number was standardized to fluorescence for each cell type by
comparison with a standard curve generated from the seeding of known
cell numbers.
20 °C. Pellets were washed 3× in 0.5% Tween 20 (Sigma)/PBS,
suspended in 50 µl of Tween 20/PBS with 10 µl of fluorescein
isothiocyanate-conjugated
BrdUrd IgG1 antibody (Becton
Dickinson) for 30 min, rewashed 2×, and resuspended in 1 ml of PBS
with 5 µg/ml propidium iodide (Sigma) for 10 min. The percentage of
fluorescent cells in each sample was determined by flow cytometry
(Coulter Epics XL, FL1 versus FL3). DNA synthesis as
analyzed by [methyl-3H]thymidine incorporation
was performed by plating 50,000 cells in 60-mm plates, adding the
indicated cell treatments, then labeling with 1 µCi/ml isotope for
4 h. Cells were trypsinized and added to glass microfiber filters
(Whatman), and the filters were washed 3× with water, dried, and
immersed in scintillation mixture (Beckman) for assessment of
radioactivity using a Beckman LS 1801 scintillation counter.
-Protein phosphatase-negative
control was prepared as previously described. Proteins were
transferred onto nitrocellulose membranes, and the membranes were
blocked with 5% w/v skim milk and then incubated with
-phospho-p44/42 MAP kinase (Thr-202/Tyr-204) or
-phospho-p38 MAPK
(Thr-180/Tyr-182) antibody (New England BioLabs). After washing,
membranes were exposed to horseradish peroxidase-linked protein A
(ERK1/2 experiments) or goat anti-rabbit secondary antibody (p38
experiments, Bio-Rad), and chemiluminescence was detected. To compare
ERK1/2 and p38 phosphorylation levels with total protein levels,
membranes were stripped of antibodies and reprobed with
p44/42 MAP
kinase antibody (New England BioLabs) or
p38 MAPK antibody (C-20,
Santa Cruz Biotechnology).
JNK1 or
p38 antibody (C-17 or C-20; Santa Cruz Biotechnology),
and samples were incubated overnight at 4 °C for
immunoprecipitation. Beads were then washed 2× in cold mTNE, 2× in
cold "JNK" kinase buffer (2 mM HEPES, pH 7.2, 15 mM NaCl, 20 mM MgCl2, 10 mM
-glycerophosphate, 2 mg/ml p-nitrophenyl
phosphate, 100 µM sodium orthovanadate, 2 mM
dithiothreitol) and resuspended in a kinase reaction solution of 30 µl of JNK kinase buffer, 2 µg of JNK or p38 MAPK substrate: GST
fusion protein of c-Jun (1-79 aa) activation domain (Calbiochem) or
GST fusion protein of ATF-2 (19-96 aa) activation domain (Upstate Biotechnology) and 10 µCi of [
-32P]ATP (PerkinElmer
Life Sciences) at 30 °C for 20 min. Kinase reaction was stopped upon
addition of SDS gel loading buffer (plus 5 mM EDTA) and
heating of samples at 95 °C for 10 min. Protein from supernatants
was separated by SDS-polyacrylamide gel electrophoresis and transferred
to a polyvinylidene difluoride membrane, and phosphorescence of the
c-Jun substrate was quantitated. To compare c-Jun and ATF-2 N-terminal
phosphorylation levels with total JNK1 and p38 MAPK levels, the
membrane was incubated with
JNK1 or
p38 antibody, and membrane
chemiluminescence was detected.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 units/ml
was toxic to cells, suggesting that a defined window of extracellular
H2O2 concentration is required for cell
survival and growth.
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Fig. 1.
Growth inhibitory effects of catalase upon
Rat-1 fibroblast lines and rescue of this inhibition with the addition
of glucose oxidase. Cells plated at a density of 1000 cells/well,
96-well plate, were incubated until adherent and then treated.
Cat, catalase; HI cat, heat-inactivated catalase.
After 4 days, total DNA content in the wells was quantitated.
A, after adherence, cells were treated with 0-2000 units/ml
catalase or 2000 units/ml heat-inactivated preparation.
Dose-dependent growth inhibition by catalase was observed
at comparable levels in all Rat-1 lines. B, Rat-1NeuN and
Rat-1Neu8142 cells were plated as in part A and incubated with 0 or
1000 units/ml catalase alone, or coincubated with increasing
concentrations (2 × 10 3 to 2 × 10
2 units/ml) of an
H2O2-generating glucose oxidase preparation.
The addition of glucose oxidase rescued the catalase-induced
proliferation block in a dose-dependent manner.
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Fig. 2.
Inhibition of active DNA synthesis by
catalase in Rat-1 fibroblast lines exhibiting different growth rates
and levels of transformation. A, cells were dosed with
BrdUrd after the indicated treatments (200 µM
H2O2, 2000 units/ml catalase/heat-inactivated
(HI) catalase), labeled with fluorescein isothiocyanate-conjugated
anti-BrdUrd antibody and propidium iodide, and analyzed by flow
cytometry. Active DNA synthesis was stimulated after the addition of
H2O2 to the extracellular medium, whereas
scavenging of H2O2 decreased this synthesis in
all lines tested. B, measurement of
[3H]thymidine uptake after identical treatments.
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Fig. 3.
P44/42 MAPK (ERK1/2) activity is blocked by
catalase in wild type Neu-expressing cells but not in those with
dominant active Neu. A, Rat-1NeuN cells were treated
(2000 units/ml catalase/heat-inactivated (HI) catalase, 200 µM H2O2, PD = 30 µM PD98059) and protein lysates analyzed by Western
blotting. Membranes were labeled with phospho-specific anti-ERK
(Thr-202/Tyr-204) antibody to observe levels of activated p44/42 MAPKs,
then stripped and reprobed with anti-ERK antibody. B,
identical experiment using Rat-1Neu8142 cells.
PPase, incubation with
-protein phosphatase
(negative control). ERK1/2 phosphorylation was increased in response to
catalase treatment, the opposite effect to that observed in the wild
type Neu-expressing Rat-1NeuN line.
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Fig. 4.
Both cytotoxic levels of
H2O2 and growth inhibitory levels of
extracellular catalase cause a rapid and transient activation of
JNK1. JNK1 was immunoprecipitated from Rat-1NeuN (A)
andRat-1Neu8142 (B) protein lysates after the indicated
treatments (30 min 2 h).
Jun, no c-Jun (1-79
aa) substrate included; catalase/HI catalase = 500 units/ml.
Immunoprecipitates were then included in an in vitro kinase
reaction with JNK1 substrate (c-Jun (1-79 aa)) and label
([
-32P]ATP). 32P phosphorescence was
quantitated from polyvinylidene difluoride membrane. To compare c-Jun
phosphorylation with total JNK1 protein level in each sample, membranes
were then incubated with anti-JNK1 antibody.
View larger version (53K):
[in a new window]
Fig. 5.
p38 MAPK phosphorylation is stably induced by
H2O2 stress but unaffected by treatment with
catalase. Rat-1NeuN (A) and Rat-1Neu8142 (B)
cells were treated as shown (30 min 2 h). Catalase/HI
catalase = 500 units/ml. Protein lysates were analyzed by Western
blotting. Membranes were labeled with phospho-specific anti-p38
(Thr-180/Tyr-182) antibody to observe levels of activated p38 MAPK,
then stripped and reprobed with anti-p38 antibody to compare total
protein level in each sample. In both cell lines, p38 phosphorylation
was induced by 2 mM H2O2, but not
by catalase treatments. P38 MAPK was also immunoprecipitated from
lysates for use in a kinase reaction with p38 substrate (ATF2 (19-96
aa)) and label ([
-32P]ATP). No phosphorylation of this
substrate was observed. C-6 extract, untreated C-6 glioma
cell protein extract (negative control; NEB); C-6 + Anisomycin, anisomycin-treated C-6 extract (positive control;
NEB);
ATF2, no ATF2 substrate included in
reaction; Untr., no treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. A. Dorward, P. Siegel, N. Singh, J. Lee, M. Rozakis-Adcock, R. Austin, and W. Duivenvoorden for their helpful insight and comments regarding experimental procedures and manuscript preparation.
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FOOTNOTES |
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* This work was funded in part by the Medical Research Council of Canada (now the Canadian Institutes of Health Research) by Operating Grant MA14163 (to G. S.).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.
§ Sponsored by a Medical Research Council of Canada studentship.
To whom correspondence should be addressed: Hamilton Regional
Cancer Centre, 699 Concession St., Hamilton, Ontario L8V 5C2, Canada.
Tel.: 905-387-9711 (ext. 7007); Fax: 905-575-6330; E-mail: gurmit.singh@hrcc.on.ca.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M004617200
2 T. J. Preston, W. J. Muller, and G. Singh, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
ROS, reactive oxygen
species;
SAPK, stress-activated protein kinase;
NF-B, nuclear factor
kappa B;
JNK, c-Jun N-terminal kinase;
MAPK, mitogen-activated protein
kinase;
ERK1/2, extracellular signal-regulated kinase 1 and 2;
aa, amino acid(s);
HI, heat-inactivated;
MEK1, MAPK/ERK kinase 1;
BrdUrd, 5-bromo-2'-deoxyuridine;
PBS, phosphate-buffered saline;
GST, glutathione S-transferase;
PI3K, phosphoinositide 3-OH
kinase.
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