From the Division of Gastroenterology, ¶ Cancer
Center,
Department of Genetics, and § Abramson Family
Cancer Research Institute, University of Pennsylvania,
Philadelphia, Pennsylvania 19104
Received for publication, December 12, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Paclitaxel is a widely used chemotherapeutic
agent and is known to induce programmed cell death (apoptosis) in a
variety of cell types, but the precise underlying mechanisms are poorly
understood. To elucidate these mechanisms, we challenged human
esophageal squamous cancer cell lines with paclitaxel and investigated
its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nM) induced
apoptosis. All three mitogen-activated protein kinase (MAPK) family
members, c-Jun N-terminal kinase (JNK), p38 MAPK, and
extracellular signal-regulated kinase (ERK) were activated upon
paclitaxel treatment. Interestingly, JNK activation and p38 MAPK
activation were delayed and peaked at 48 h, whereas ERK activity
was sustained over 72 h. In addition, Ras activation and MAPK/ERK
kinase (MEK) phosphorylation were observed in concordance with ERK
activation. While ERK activation was completely ablated by MEK
inhibitors, immunoprecipitation and Western blot analysis revealed that
neither MEK-1 nor MEK-2 was involved, but instead another member of the
MEK family may potentially participate. Although pretreatment with a
general caspase inhibitor,
benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the
cell death, it did not prevent Ras or ERK activation. Furthermore,
inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the
cell death induced by paclitaxel. These results in aggregate suggest
that the delayed activation of JNK, p38 MAPK, and ERK was not
linked to activation of the cell death machinery.
Paclitaxel (Taxol) is a chemotherapeutic agent utilized for
the treatment of breast, ovarian, lung, and esophageal cancers (1, 2).
Paclitaxel exerts its cytotoxic effect by binding and stabilizing
microtubules (3) and induces apoptosis in a variety of cell types
(4-6) independently of p53 status (7). The mechanisms underlying
paclitaxel-induced apoptosis are unclear. Paclitaxel was recently
demonstrated to release cytochrome c by direct action on the
mitochondria (8), activate small GTP-binding proteins (9, 10), and
activate the NF- Akt (protein kinase B), a serine/threonine protein kinase, has a
pivotal role in exerting an antiapoptotic effect against various
stimuli (12). Overexpression of Akt was reported to confer resistance
to paclitaxel (13). However, it was recently reported that paclitaxel
induced apoptosis in human ovarian carcinoma cells independently of Akt
(14). Thus, it is unclear whether paclitaxel causes apoptosis through
modulation of the Akt survival pathway.
The mitogen-activated protein kinase
(MAPK)1 family comprises
c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular
signal-regulated kinase (ERK). The ERK pathway is known to be activated
by various stimuli including growth factors (15), lipopolysaccharide
(16), and chemotherapeutic agents (5). ERK activation may exert either an antiapoptotic (17-19) or a proapoptotic (5, 20) influence depending
upon the cellular context and by as yet unclarified regulatory
mechanisms. The JNK and p38 MAPK signaling pathways are also activated
by various and overlapping stimuli such as heat or osmotic shock,
radiation, and growth factors (15, 21). Although JNK and p38 MAPK have
been reported to be activated by paclitaxel (10, 22), the precise roles
of these activated kinases in this context remain obscure.
It has been reported that paclitaxel enhances ERK activity in various
cell lines (10, 23). By contrast, paclitaxel attenuated ERK activity in
some reports (24, 25). Since abrogation of the MEK/ERK pathway delayed
paclitaxel-induced apoptosis (23) or failed to prevent it (26), ERK
activation by paclitaxel and its relationship to apoptosis requires
elucidation. Investigation of the relationship of paclitaxel-induced
apoptosis and activation of the MAP kinases serves as the basis of our
studies and provides a venue to understand the interrelationship or
independence of MAPKs and apoptosis.
By utilizing human esophageal squamous cancer cells challenged with
physiological concentrations of paclitaxel, we demonstrate herein that
paclitaxel induces apoptosis, which is accompanied by delayed JNK and
p38 MAPK activation and prolonged ERK activation lasting over 72 h. Our studies, employing pharmacological inhibitors for MEK or
caspases, revealed that prolonged ERK activity was not mediated via the
same pathway as the apoptosis machinery and that MEK/ERK blockade did
not modify the cytotoxicity of paclitaxel, indicating that the
prolonged ERK activation may be secondary to cell death. The prolonged
ERK activation appeared to be mediated through a Ras- and
MEK-dependent pathway, in which neither the MEK-1 nor the
MEK-2 isoform was involved. This suggests that a different MEK family
member might be responsible for the prolonged ERK activity observed
with paclitaxel.
Materials--
Paclitaxel was obtained from Sigma. U0126,
PD98059, and SB202190 were purchased from New England Biolabs (Beverly,
MA) or Upstate Biotechnology, Inc. (Lake Placid, NY). Caspase
inhibitors, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
(Z-VAD-FMK), and Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), were obtained
from Biomol (Plymouth Meeting, PA). These compounds were dissolved in
Me2SO with final concentration never exceeding 0.1%.
Recombinant mouse epidermal growth factor (EGF) was purchased from
Roche Molecular Biochemicals. Antibodies against
p21waf1/cip1 (C-19), cyclin D1 (HD11), Elk-1 (I-20), and
MEK-1 (H-8) were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Anti-poly(ADP-ribose) polymerase (PARP), anti-MEK-2 (clone
96), and anti-caspase-3 antibodies were obtained from Pharmingen (San Diego, CA). An anti-Bcl-2 antibody (4-21) was obtained from
Calbiochem-Novabiochem Corp. (San Diego, CA). Polyclonal anti-rabbit
Akt-1 and Akt-2 antibodies were generated as described previously (27).
Anti-cleaved form-specific caspase-3, caspase-7, and caspase-9
antibodies, anti-Akt (Ser473), ERK1/2
(Thr202/Tyr204), p38 MAPK
(Thr180/Tyr182), JNK
(Thr183/ Tyr185), Elk-1 (Ser383),
and MEK1/2 (Ser217/Ser221) antibodies detecting
only the phosphorylated residues as indicated and
phosphorylation-independent antibodies for ERK, p38 MAPK, and JNK were
purchased from New England Biolabs. Secondary anti-mouse and
anti-rabbit horseradish peroxidase antibodies were obtained from
Amersham Pharmacia Biotech.
Cell Lines--
TE-5 and TE-8 cells are human esophageal
squamous cancer cell lines and were cultured in Dulbecco's modified
Eagle's medium, supplemented with 10% fetal bovine serum (Sigma), 100 µg/ml streptomycin, 100 units/ml penicillin (Life Technologies,
Inc.), and L-glutamine (Life Technologies) at
37 °C in a 5% CO2 incubator.
Cell Viability--
Cell viability was assessed by a modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay (WST-1 assay; Roche Molecular Biochemicals) following the
supplier's protocol. Briefly, cells were grown in 96-well plates
overnight at 37 °C in a 5% CO2 incubator and treated
with appropriate concentrations and durations of compounds as noted in
the figure legends. After 10 µl of WST-1 reagent was added,
A450 was measured by a microplate reader
(Molecular Devices, Inc., Sunnyvale, CA). The experiments were
repeated at least six times, and data were expressed as the mean ± S.E.
Nuclear Staining with Hoechst 33258--
Cells were grown in
chamber slides (Nunc Lab-Tek Chamber Slide System, Nunc Inc.
Naperville, IL) and treated with appropriate concentrations and time
courses of paclitaxel. Cells were fixed with ethanol/acetic acid
solution for 15 min at room temperature, washed with phosphate-buffered
saline (PBS), incubated with 25 µg/ml Hoechst 33258 (Sigma) for 10 min at room temperature, and washed again with PBS. The signals were
then visualized under a fluorescent microscope.
Flow Cytometry--
After treatment of cells with paclitaxel,
attached and floating cells were pooled in one tube. The cell pellets
were washed with PBS containing 1% fetal bovine serum, fixed in 70%
ethanol, stained with 0.5 mg/ml propidium iodide (Sigma) containing
3 Kunitz RNase (Roche Molecular Biochemicals) and 1% fetal
bovine serum in PBS, and then analysis was performed with a Coulter
Epics Elite ESP flow cytometer (Beckman-Coulter, Miami, FL).
Sample Preparation and Western Blotting--
After treatment of
cells with paclitaxel, floating and attached cells were combined,
washed with ice-cold PBS, and lysed in radioimmune precipitation buffer
(1% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and a protease inhibitor mixture tablet
(Roche Molecular Biochemicals) in PBS) for 15 min on ice. The total
cell lysate (10 µg/sample) was utilized for Western blotting as
described previously with minor modifications (27). When necessary, the membrane was stripped and reprobed with appropriate antibodies.
Immunoprecipitation--
Cell lysates harvested in
radioimmune precipitation buffer (250 µg) were immunoprecipitated
with 2 µg of anti-mouse MEK-1 or MEK-2 antibody for 1 h at
4 °C followed by incubation with 20 µl of Protein A/G Plus-agarose
beads (Santa Cruz Biotechnology) for an additional 1 h at 4 °C.
Pellets were washed four times with radioimmune precipitation buffer,
suspended in 3× SDS sample buffer (187.5 mM Tris, pH 6.8, 6% SDS, 30% glycerol, 150 mM dithiothreitol, and 0.03%
bromphenol blue), and heated at 95 °C for 3 min. The products were
dissolved on a 15% SDS-polyacrylamide gel, and Western blotting was
performed with the phospho-MEK1/2 antibody. The membrane was stripped
and reprobed with the anti-MEK-1 or anti-MEK-2 antibody.
Akt, JNK, p38 MAPK, and ERK Kinase Assays--
The Akt kinase
assay was performed as described previously (27). JNK, p38 MAPK, and
ERK kinase assays were performed with assay kits for the respective
kinases (New England Biolabs) following the company's instructions.
Briefly, treated cells were lysed in buffer (20 mM Tris, pH
7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM
MEK Kinase Assay--
Cells were immunoprecipitated as described
under "Immunoprecipitation" and "Akt, JNK, p38 MAPK, and ERK
Kinase Assays" except that 400 µg of total cell lysates was
utilized. The kinase reaction was conducted with 40 µl of kinase
buffer containing 50 µM unlabeled ATP, 5 µCi of
[ Ras Activity Assay--
Ras activity was assessed by employing
the Ras activation assay kit (Upstate Biotechnology). Briefly, treated
cells were lysed in Mg2+ lysis buffer (MLB buffer)
containing 25 mM HEPES, pH 7.5, 150 mM NaCl,
1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 2 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a
protease inhibitor mixture tablet. The cell lysates (650 µg) were
incubated with 10 µl of Raf-1 Ras binding domain-agarose conjugate
for 30 min at 4 °C. The beads were washed with MLB buffer and
resuspended in 2× SDS sample buffer. Western blotting was performed
with the anti-Ras antibody (Ras10; Upstate Biotechnology). The assays
were repeated three times, and the signals were quantified using the
NIH Image 1.62 program.
Paclitaxel Causes Cell Death in Human Esophageal Cancer
Cells--
We determined that physiologically relevant doses of
paclitaxel could induce cell death in human esophageal squamous cell cancer cells. These cells were employed, since esophageal cancer is one
of the targets of paclitaxel as a chemotherapeutic agent. We treated
TE-5 and TE-8 cells with 100 nM paclitaxel for 24, 48, and
72 h for the time course experiment and with 1, 10, 100, and 1,000 nM paclitaxel for 24 h for the dose-response
experiment, respectively, and then cell viability was assessed by the
WST-1 assay. Control cells were treated with 0.1% Me2SO
alone. Paclitaxel was able to induce cell death in a time- and
dose-dependent fashion in TE-5 (Fig.
1, A and B) and
TE-8 cells (data not shown).
Morphological features observed in cells dosed with paclitaxel
included rounding up and detachment from the plate (Fig. 1C, top panel). Hoechst 33258 staining demonstrated nuclear
condensation and fragmentation upon paclitaxel treatment (Fig.
1C, bottom panel). Flow cytometric analysis
revealed that in TE-5 cells paclitaxel treatment increased the
G2/M population consistent with previous reports (28, 29)
and also yielded a sub-G1 population (Fig. 1D).
Similar results were obtained in TE-8 cells (data not shown). Thus,
these data established the validity of using these cell lines for
further studies aimed at understanding the mechanistic basis underlying
paclitaxel's effects.
Paclitaxel Induces PARP Cleavage--
The proteolytic cleavage of
PARP, which synthesizes poly(ADP-ribose) from Caspase-7, Not Caspase-3, Is Responsible for the PARP Cleavage by
Paclitaxel--
Caspase-3 and caspase-7 have a central role in PARP
cleavage (reviewed in Ref. 31), and therefore we determined the
activation of these two caspases by Western blot analysis using
antibodies capable of detecting activated (cleaved) caspase-3 or
caspase-7. The expression level of caspase-3 did not significantly
change after paclitaxel treatment in TE-5 cells and TE-8 cells (data not shown), and the cleaved form of caspase-3 was not detectable at any
of the time points (data not shown). Cleavage of caspase-9, which is a
downstream target of caspase-3 in some cases (31), was also not
activated (data not shown). By contrast, cleaved caspase-7 fragments
were detected after 48 h of 100 nM paclitaxel treatment in TE-5 cells (Fig.
3A, lanes 1-4),
tracking a similar time course as PARP cleavage, and the caspase-7
cleavage was almost completely inhibited by preincubation with 40 µM Z-VAD-FMK (Fig. 3A, lane 6).
These results suggest that caspase-7, not caspase-3 or caspase-9, is
the caspase family member involved in paclitaxel-induced apoptosis in
TE-5 and TE-8 cells.
Furthermore, pretreatment with a general caspase inhibitor, Z-VAD-FMK,
but not a caspase-3-specific inhibitor, DEVD-CHO, partially rescued paclitaxel-induced cell death in TE-5 cells (Fig.
3B) and TE-8 cells (data not shown), underscoring the notion
that paclitaxel initiated the caspase machinery without involvement of
caspase-3.
Paclitaxel Induces Delayed JNK and p38 MAPK Activation and
Prolonged MEK/ERK Activation--
Although Akt (protein kinase B)
plays a pivotal role in the inhibition of apoptosis (12), we found that
paclitaxel exerted apoptotic effects independent of Akt. Neither the
expression of Akt-1 and Akt-2 nor Akt kinase activity was significantly
altered in TE-8 cells treated with paclitaxel (data not shown). This
suggests, in consonance with a recent report (14), that paclitaxel did not cause cell death via an inhibitory effect upon Akt, a finding also
in contrast to the notion that Akt is involved (13).
We observed next that ERK was activated upon paclitaxel treatment. In
TE-8 cells, phosphorylated ERK started to appear after 24 h of 100 nM paclitaxel treatment and was still detected at 72 h. ERK phosphorylation was detected even at 10 nM
paclitaxel at the 48-h time point (Fig.
4A, top panel).
Total ERK expression did not change throughout the time course (Fig.
4A, bottom panel). In TE-5 cells, ERK was weakly
phosphorylated at the 24-h time point and increasing thereafter through
72 h (Fig. 6A, top panel). This result was
corroborated by ERK kinase assay results, demonstrating 6-, 43-, and
51-fold activation after 24, 48, and 72 h of paclitaxel treatment,
respectively (Fig. 4B, lanes 1-4).
ERK activation induced by paclitaxel was dependent upon MEK as
supported by the use of two well established pharmacological MEK
inhibitors, PD98059 (32) and U0126 (33). The ERK kinase activity
observed at 48 h after dosing with paclitaxel was almost completely ablated by pretreatment with U0126 and partially by PD98059
(Fig. 4B, lanes 5-8), demonstrating that the ERK
activation is in part dependent upon MEK. We speculate that the
differential effects of these two MEK inhibitors might stem from the
different potencies of these two inhibitors or from differential
effects on the MEK-1 and MEK-2 isoforms (33).
Next, we examined whether JNK and p38 MAPK were activated by
paclitaxel. Both JNK and p38 MAPK phosphorylation were delayed upon
paclitaxel challenge in TE-5 cells, in which the phosphorylation started to appear after 24 h of paclitaxel treatment, peaked at 48 h, and then decayed (Fig. 4C). These results were
confirmed by JNK and p38 MAPK kinase assays (Fig. 4D,
lanes 1-4). Collectively, paclitaxel induces in a
reproducible fashion the delayed or prolonged activation of all three
MAPKs in human esophageal cancer cells.
Paclitaxel Induces Activation of ERK, JNK, and p38 MAPK Downstream
or Independently of the Programmed Cell Death Machinery--
Although
MAPKs have been reported to have a pivotal role in regulating apoptosis
(reviewed in Ref. 34), we found that the cell death induced by
paclitaxel was not modified by inhibiting ERK, JNK, or p38 MAPK
activation in TE-5 and TE-8 cells. MEK inhibitors alone did not
significantly affect the cell viability, although PD98059 tended to
increase it in both TE-5 cells (Fig.
5A) and TE-8 cells (data not
shown). Surprisingly, pretreatment of TE-5 cells (Fig. 5A)
and TE-8 cells (data not shown) with either 50 µM PD98059
or 20 µM U0126, which efficiently inhibited the ERK activation (Fig. 4B), did not affect the cell death induced
by paclitaxel.
A pyridinyl imidazole inhibitor, SB202190, which was originally
identified as a p38 MAPK-specific inhibitor (35), has been reported to
inhibit JNK at high concentration (36) and as efficiently as it
inhibits p38 MAPK (37). Therefore, we tested the effects of various
concentrations of SB202190 upon JNK and p38 MAPK activity in our model
systems. p38 MAPK activity was suppressed in a
dose-dependent fashion by pretreatment with SB202190
(Fig. 4D, top panel). Surprisingly, JNK activity
was abolished by SB202190 more efficiently than p38 MAPK even at 1 µM (Fig. 4D, bottom panel). Then we
pretreated cells with 50 µM SB202190, which abolished the
JNK and p38 MAPK activity, and cell viability was evaluated. As such,
simultaneous inhibition of the JNK and p38 MAPK pathway did not modify
the cell death caused by paclitaxel (Fig. 5B).
When cells were pretreated with MEK inhibitors followed by
paclitaxel treatment, the cleavage of neither PARP nor caspase-7 was
ablated (Fig. 2B, lanes 5 and 6, and
Fig. 3A, lanes 7-10). These results suggest that
the activation of ERK, JNK, and p38 MAPK induced by paclitaxel occurred
either downstream or independently of the programmed cell death machinery.
Ras and MEK Are Activated by Paclitaxel, and Prolonged MEK/ERK
Activation Is Independent of the Caspase Cascade--
We postulated
that upstream kinases of ERK could be different from the classical
Ras/MEK/ERK machinery utilized by growth factor stimulation because
paclitaxel caused a sustained ERK activation instead of the classic
transient one.
First, we observed that MEK was activated by paclitaxel treatment. We
employed Western blot analysis with an anti-MEK antibody specific for
phosphorylation at the Ser217/Ser221 sites and
capable of detecting both MEK-1 and MEK-2 isoforms. TE-5 and TE-8 cells
exhibited different time courses of MEK phosphorylation. The MEK
phosphorylation peaked at 24 h, returned to the basal level at
48 h, and further decreased below the basal level at 72 h in
TE-5 cells, whereas in TE-8 cells, the MEK phosphorylation peaked at
48 h and returned to the basal level at 72 h (Fig.
6, A and B). Of
note, the migration on electrophoresis gel of phosphorylated MEK
derived from paclitaxel-treated cells was faster than that in
EGF-treated cells in both TE-5 and TE-8 cells.
Next, since Ras is located upstream of the MEK/ERK pathway in the
growth factor-stimulated module, and Ras-independent ERK activation has
been reported (38), we noted that Ras activity was also enhanced upon
paclitaxel challenge. We determined Ras activity, which detects
active GTP-bound Ras. Ras activity was also induced by paclitaxel,
peaking at 48 h in TE-5 cells (Fig. 6C). This implies
that the MEK/ERK signaling pathway was activated by paclitaxel at or
upstream of Ras.
In order to explore whether the Ras/MEK/ERK pathway lies downstream of
the caspase cascade, we pretreated TE-5 cells with 40 µM
Z-VAD-FMK followed by paclitaxel treatment, after which ERK kinase and
Ras activity assays were performed. Neither ERK kinase activity nor Ras
activity induced by paclitaxel treatment was significantly altered by
Z-VAD-FMK (Fig. 4B, lane 9, and Fig. 6C, lane 8). We conclude that the Ras/MEK/ERK
signaling pathway was activated independently of the caspase cascade.
Differential Activation of MEK-1 and MEK-2 upon EGF and Paclitaxel
Treatment--
Since we observed disparate migration of phosphorylated
MEK1/2 upon paclitaxel treatment from that upon EGF stimulation (Fig. 6, A and B), we sought to identify the MEK
isoform(s) involved in ERK activation. TE-5 cells treated with EGF or
paclitaxel were immunoprecipitated with the anti-MEK-1 or MEK-2
antibody and immunoblotted with the phospho-MEK1/2 antibody. Without
immunoprecipitation (Fig. 7A,
lanes 1-6), we observed the same result as above, showing that EGF caused phosphorylation of the slow migrating band (Fig. 7A, lane 2) and paclitaxel induced
phosphorylation of the fast migrating band at the 24-h time point (Fig.
7A, lane 4). When immunoprecipitation was
conducted with the MEK-1 antibody (Fig. 7A, lanes
7-10) or the MEK-2 antibody (Fig. 7A, lanes
11-14), MEK phosphorylation was enhanced in EGF-treated cells
(Fig. 7A, lanes 8 and 12) but not in
paclitaxel-treated cells (Fig. 7A, lanes 10 and
14). Equal immunoprecipitation efficacy was confirmed by
reprobing the membranes with the MEK-1 (Fig. 7B, lanes
7-10) or the MEK-2 antibody (Fig. 7B, lanes
11-14).
The specificity of the antibodies was evaluated by reprobing the
membranes with the opposite antibody as used for immunoprecipitation. Faint bands were detected when cells were immunoprecipitated with the
MEK-1 antibody and immunoblotted with the MEK-2 antibody (Fig. 7C, lanes 7-10), and when immunoprecipitated
with the MEK-2 antibody and immunoblotted with the MEK-1 antibody (Fig.
7C, lanes 11-14). These results suggest that
both antibodies may cross-react in their recognition of MEK-1 and
MEK-2, although they are putatively isoform-specific. Therefore, we
cannot exclude the possibility that the MEK phosphorylation observed in
Fig. 7A (lanes 7-14) may not represent the phosphorylation
of the specific MEK isoform. Our finding was further substantiated by
MEK kinase assays. MEK-1 and MEK-2 kinase activity was enhanced by EGF
stimulation but not by paclitaxel treatment.
We observed that physiologically relevant concentrations of
paclitaxel induced cell death with features characteristic of apoptosis. Although pretreatment with a caspase inhibitor, Z-VAD-FMK, inhibited PARP cleavage induced by paclitaxel, cell death was rescued
partially by Z-VAD-FMK, implying that paclitaxel caused cell death via
both caspase-dependent and -independent pathways. The
caspase-independent pathway may be analogous with so-called "slow
cell death," which is often observed by chemotherapeutic drugs
(39).
Since caspase-3 is considered to be central to the apoptosis machinery
(Ref. 40; reviewed in Ref. 31), it was surprising that we observed
caspase-7, not caspase-3, activation upon paclitaxel treatment. Our
novel finding is nevertheless consistent with a report demonstrating
that upon VP-16 treatment, PARP was cleaved by caspase-7 but not
caspase-3 (30). Since caspase-3 knockout mice have normal apoptotic
response (41), and deficiency of a caspase member can be compensated by
activation of other caspases (42), there may be regulatory mechanisms
that preferentially utilize different caspase members in response to
apoptotic stimuli, and caspase-3 activation may be dispensable in some
cellular contexts. It is tempting to speculate that caspase-7 may be
preferentially activated by certain chemotherapeutic agents.
Paclitaxel has been reported to activate various signal transduction
pathways including JNK (22), p38 MAPK (10, 22), and ERK (10, 23), but
the primary target for the paclitaxel action is not defined. Although
microtubules are the well documented cellular targets of paclitaxel
(3), CD18 (43), Ki-Ras (44), and Bcl-2 (45), all have the potential to
bind to paclitaxel. In addition, Taxol mediates serine phosphorylation
of shc (46). Our data indicate that paclitaxel activated the ERK
pathway at the level of Ras or, not inconceivably, upstream of Ras.
However, since Ras and ERK can co-localize with microtubules (47, 48), it is conceivable that paclitaxel, when bound to microtubules, induces
ERK activation directly or via a Ras-dependent signaling pathway.
It has been reported that PARP cleavage may be suppressed by a MEK
inhibitor, demonstrating that the caspase cascade is downstream of the
MEK/ERK pathway (49). By contrast, in our model systems, when cells
were pretreated with MEK inhibitors or SB202190, which inhibited both
JNK and p38 MAPK at 50 µM, cell death induced by paclitaxel was unaltered. When these compounds were added
simultaneously prior to paclitaxel treatment, cell viability was also
unaffected (data not shown). Kinase assays revealed that these
compounds efficiently suppressed ERK activity. JNK and p38 MAPK
kinase activities were partially suppressed. Although we cannot exclude
the possibility that the residual kinase activity contributed to cell
death, we conclude that the delayed or prolonged activation of the JNK, p38 MAPK, and MEK/ERK kinases occurred independently of cell
death, which is underscored by several independent experiments. In
concert with this notion, anisomycin has been demonstrated to induce
JNK, p38 MAPK, and ERK activation independently of apoptosis (50).
Since we observed that MEK phosphorylation upon paclitaxel treatment
showed a distinct migration on Western blotting with the phospho-MEK1/2
antibody from that upon EGF stimulation, we sought to identify the MEK
isoform(s) involved. Immunoprecipitation/Western blotting and kinase
assays using anti-MEK-1 and MEK-2 antibodies revealed that neither
MEK-1 nor MEK-2 appeared to be responsible for ERK activation by
paclitaxel. Although among the MEK family members identified thus far,
MEK-5 is the member most closely structurally related to MEK-1 and
MEK-2, it is not known to activate ERK-1 or ERK-2 (51). Thus, it is
possible that another member of the MEK family, perhaps a novel one,
modulates ERK activation that is induced by paclitaxel. The
identification of a novel MEK member is currently under investigation.
Sustained activation of the MEK/ERK pathway has been reported upon
stimulation with growth factors such as nerve growth factor (52),
insulin (53), EGF (15), and hepatocyte growth factor (15) as well as
during recovery from cardiac ischemia (54). Postulated roles for
sustained MEK/ERK pathway activation include mediation of neuronal cell
differentiation (52) and matrix metalloproteinase-9 induction (38).
However, the duration of ERK activation in their reports is less than
24 h. We observed that ERK was activated in a
MEK-dependent fashion, and to our surprise, it was
sustained even after 72 h of paclitaxel treatment. To our
knowledge, this is the first report demonstrating such a prolonged ERK
activation with some form of extracellular stimulation. Although the
time course of MEK activation preceded ERK activation, there was a time
lag between MEK and ERK activation, and ERK activation was still
observed even after MEK activity decayed (Fig. 6, A and B). The significance of the time lag between MEK and ERK
activation and sustained versus transient ERK activation,
however, are not clear. Ultimately, the effects of delayed or prolonged
activation of different MAP kinases in the face of cell death
may be a means by which the host cell is attempting to maintain
cellular homeostasis (Fig. 8).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B/I
B-
signaling pathway (11). Although these
actions were implicated as the mechanisms through which paclitaxel
induces apoptosis, these studies were impaired by the employment of
high concentrations of paclitaxel.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin) for 15 min on ice. The cell lysates (250 µg)
were incubated with immobilized c-Jun, phospho-p38 MAPK, and phospho-ERK1/2 antibodies for JNK, p38 MAPK, and ERK kinase assays, respectively, for 4 h to overnight at 4 °C. The
immunoprecipitated products were washed twice with the cell lysis
buffer and twice with kinase buffer with 25 mM Tris, pH
7.5, 5 mM
-glycerolphosphate, 2 mM
dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl2. The pellets were suspended in the
kinase buffer containing 100 µM ATP for the JNK assay,
200 µM ATP and 2 µg of ATF-2 for the p38 MAPK assay,
and 200 µM ATP and 2 µg of Elk-1 fusion protein for the
ERK kinase assay. Each reaction was then incubated for 30 min at
30 °C. Western blotting was performed with the phospho-c-Jun, phospho-ATF-2, and phospho-Elk-1 antibodies, respectively, for the JNK,
p38 MAPK, and ERK kinase assays. The kinase assays were repeated three
times for each kinase, and the signals were quantified using the NIH
Image 1.62 program.
-32P]ATP (PerkinElmer Life Sciences), and 2 µg of
kinase-inactive ERK-2 fusion protein (Upstate Biotechnology) for 30 min
at 30 °C. The kinase reaction was terminated by the addition of 3×
SDS sample buffer, and the products were separated on a 10%
SDS-polyacrylamide gel. The gel was dried and subjected to autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Induction of cell death by paclitaxel
treatment in TE-5 and TE-8 cells. A, TE-5 and TE-8
cells were challenged with 100 nM paclitaxel for sequential
durations (24, 48, and 72 h), and cell viability was assessed by
the WST-1 assay, a modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
assay. When cells were untreated, vehicle alone (0.1%
Me2SO) was added. Cell viability
(A450) was measured by a microplate reader after
the addition of WST-1 solution in 96-well plates. The experiments were
repeated at least six times, and data are expressed as the mean ± S.E. B, TE-5 and TE-8 cells were untreated (0.1%
Me2SO) or treated with escalating doses of paclitaxel (1, 10, 100, and 1,000 nM) for 24 h, and cell viability
was assessed by the WST-1 assay, demonstrating a dose response against
paclitaxel. The experiments were repeated at least six times, and data
are expressed as the mean ± S.E. C, bright field views
of TE-5 cells untreated (0.1% Me2SO) (left,
top panel) or treated with 100 nM paclitaxel for
48 h (right, top panel) are demonstrated
(magnification, × 100). Nuclear staining was performed with Hoechst
33258 in chamber slides and visualized under a fluorescent microscope,
demonstrating nuclear condensation and fragmentation induced by
paclitaxel treatment (right, bottom panel) (magnification, × 100). D, TE-5 cells
were untreated (0.1% Me2SO) or treated with increasing
doses (10, 100, and 1,000 nM) of paclitaxel for 24 h
or with 100 nM of paclitaxel for 48 and 72 h, and
propidium iodide-labeled cells (~20,000 events) were analyzed by a
flow cytometer, demonstrating a sub-G1 cell population
after paclitaxel treatment. A representative result of triplicate
experiments is shown.
-nicotinamide
adenine dinucleotide (NAD) in response to DNA strand breaks, is an
early biochemical event during apoptosis (30). Since PARP cleavage is a
hallmark of caspase activation, we demonstrated that the apoptosis
machinery is activated upon paclitaxel challenge in the TE-5 and TE-8
cells. We assayed for PARP cleavage after paclitaxel treatment, using
an anti-PARP antibody that detects both intact and cleaved products of
PARP. As shown in Fig. 2A,
intact PARP (116 kDa) was converted into 85-kDa fragments in a
time-dependent fashion with 100 nM paclitaxel
treatment in TE-8 cells. Similar results were obtained when TE-5 cells
were treated with 100 nM paclitaxel (Fig. 2B,
lanes 1-4).
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Fig. 2.
PARP cleavage is induced by paclitaxel
treatment in TE-5 and TE-8 cells. A, PARP cleavage was
assessed by Western blotting with an anti-PARP antibody, which detects
intact (116-kDa) and cleaved (85-kDa) products, after TE-8 cells were
untreated (0.1% Me2SO) or treated with escalating doses
(1, 10, 100, and 1,000 nM) of paclitaxel for 24, 48, and
72 h. Intact PARP (116 kDa) and cleaved fragments (85 kDa) are
indicated as arrows. B, PARP cleavage was
assessed by Western blotting with the anti-PARP antibody after TE-5
cells were treated with 100 nM paclitaxel alone
(lanes 2-4) or in combination with MEK inhibitors, U0126
and PD98059 (lanes 5 and 6), and a general
caspase inhibitor, Z-VAD-FMK (lanes 7-9). The inhibitors
were added 30 min prior to paclitaxel treatment.
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Fig. 3.
Caspase-7 is cleaved by paclitaxel and
pretreatment with Z-VAD-FMK partially prevents paclitaxel-induced cell
death. A, caspase-7 activation was assessed by Western
blotting with a cleaved caspase-7-specific antibody after TE-5 cells
were treated with 100 nM paclitaxel alone (lanes
2-4) or in combination with the MEK inhibitors (lanes
7-10) and Z-VAD-FMK (lanes 5 and 6). The
inhibitors were added 30 min prior to paclitaxel treatment.
B, effects of Z-VAD-FMK and a caspase-3 inhibitor, DEVD-CHO,
upon cell viability were assessed by the WST-1 assay in TE-5 cells. The
inhibitors were added 30 min prior to paclitaxel treatment. Experiments
were repeated six times, and data are expressed as the mean ± S.E.
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Fig. 4.
Paclitaxel induces prolonged activation of
the MAPK signaling pathways in TE-5 and TE-8 cells. A,
ERK activity was assessed by Western blotting with an
anti-phosphorylation-specific ERK1/2 antibody after TE-8 cells were
untreated (0.1% Me2SO) or treated with escalating doses
(1, 10, 100, and 1,000 nM) of paclitaxel for 24, 48, and
72 h (upper panel). Equal protein loading was confirmed
by reprobing the membrane with an ERK1/2 antibody detecting both
unphosphorylated and phosphorylated forms (lower panel).
B, ERK kinase activity was assessed by nonradioactive ERK
kinase assay. TE-5 cells were treated with 100 nM
paclitaxel alone (lanes 2-4) or in combination with the MEK
inhibitors (lanes 5-8) or Z-VAD-FMK (lane 9)
(upper panel). The inhibitors were added 30 min prior to
paclitaxel treatment. Equal amounts of Elk-1 were confirmed by
reprobing the membrane with an anti-Elk-1 antibody detecting both
unphosphorylated and phosphorylated forms (lower panel). The
experiments were repeated three times, and a representative result is
demonstrated. The signals were quantified by a densitometer, and the
-fold activation compared with the control lane is demonstrated
below. C, JNK and p38 MAPK kinase activities were
assessed by Western blotting with anti-phosphorylation-specific JNK and
p38 MAPK antibodies after TE-5 cells were treated with 100 nM paclitaxel for 24, 48, and 72 h (top and
third panels). Equal protein loading was confirmed by
reprobing the membranes with a JNK or p38 MAPK antibody detecting both
unphosphorylated and phosphorylated forms (second and
bottom panels). D, nonradioactive p38 MAPK
(top panel) and JNK (bottom panel) kinase assays
were performed after TE-5 cells were treated with 100 nM
paclitaxel alone (lanes 2-4) or in combination with a p38
MAPK inhibitor, SB202190 (lanes 5-7), which turned out to
inhibit JNK as well. The inhibitor was added 30 min prior to paclitaxel
treatment.
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Fig. 5.
Effects of MAPK inhibitors upon
paclitaxel-induced cell death. A, cell viability was
assessed by the WST-1 assay after TE-5 cells were untreated (0.1%
Me2SO) or treated with 100 nM paclitaxel in
combination with MEK inhibitors, PD98059 and U0126, which were added 30 min prior to paclitaxel treatment. The experiments were repeated six
times, and data are expressed as the mean ± S.E. B,
cell viability was assessed by the WST-1 assay after TE-5 cells were
treated with 100 nM paclitaxel in combination with
SB202190, which was added 30 min prior to paclitaxel. The experiments
were repeated six times, and data are expressed as the mean ± S.E.
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Fig. 6.
Ras and MEK are also activated by paclitaxel
treatment in TE-5 and TE-8 cells. A and B,
Western blotting was performed after TE-5 cells (A) and TE-8
cells (B) were treated with EGF (10 ng/ml for 30 min) or 100 nM paclitaxel for 24, 48, and 72 h. Upper
and lower panels demonstrate the time courses of
paclitaxel-induced phosphorylation of ERK and MEK1/2, respectively. Of
note, the migration on the electrophoresis gel of phosphorylated MEK
derived from paclitaxel-treated cells was faster than that in
EGF-treated cells in both TE-5 and TE-8 cells. C and
D, Ras activity was assessed by precipitating GTP-bound
active Ras with Raf-1 Ras binding domain-agarose conjugate (top
panel). TE-5 cells were untreated (0.1% Me2SO) or
treated with 100 nM paclitaxel for 24, 48, and 72 h
(lanes 1-4). For EGF stimulation, cells were serum-starved
overnight, and 10 ng/ml EGF was added for 2 or 30 min (lanes
5-7). In lane 8, Z-VAD-FMK was added 30 min
prior to paclitaxel. The lower panel demonstrates
that equal amounts of Ras were present. The experiments were repeated
three times, and data are expressed as the mean ± S.E. The
signals were quantified by a densitometer, and the -fold activation
compared with the control lane is demonstrated below.
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Fig. 7.
Differential activation of MEK-1 and MEK-2
upon EGF and paclitaxel treatment. A, B, and
C, TE-5 cells treated with EGF (10 ng/ml for 30 min) or
paclitaxel (100 nM for 24 h) were immunoprecipitated
with an anti-MEK-1 or MEK-2 antibody, and Western blotting was
performed with the phospho-MEK1/2 antibody (A) and
anti-MEK-1 and anti-MEK-2 antibodies (B and C).
Lanes 1-6 show Western blotting without
immunoprecipitation, recapitulating the result of Fig. 6A.
The experiments were repeated three times, and a representative result
is shown. Ab, antibody, HC, heavy chains;
IP, immunoprecipitation; WB, Western blot.
A, when immunoprecipitation was conducted with the MEK-1
antibody (lanes 7-10) or the MEK-2 antibody (lanes
11-14), MEK phosphorylation was enhanced in EGF-treated cells
(lanes 8-12), but not in paclitaxel-treated cells
(lanes 10 and 14). B, the membranes
were reprobed with the anti-MEK-1 (lanes 7-10) or
anti-MEK-2 antibody (lanes 11-14), demonstrating equal
immunoprecipitation efficiency. C, the membranes were
reprobed with the anti-MEK-2 (lanes 7-10) or the anti-MEK-1
antibody (lanes 11-14) to evaluate the specificity of both
antibodies. D, TE-5 cells treated with EGF (10 ng/ml for 30 min) or paclitaxel (100 nM for 24 and 48 h) were
immunoprecipitated with the anti-MEK-1 or anti-MEK-2 antibody, and each
kinase assay was performed using a kinase-inactive ERK-2 fusion protein
as a substrate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Schematic of paclitaxel-induced effects upon
signal transduction pathways. Paclitaxel induced cell death or
apoptosis by activation of caspase-7 but not caspase-3. In addition,
there was parallel, but not apparent interdependent, activation of JNK,
p38 MAPK, and ERK. ERK activation was dependent upon Ras and an unknown
MEK family member.
Impaired CL100/MKP-1 activation upon cisplatin treatment was recently
demonstrated to be responsible for sustained JNK activation (55), and
negative feedback inhibition of the ERK pathway has been implicated to
be the most important factor for sustained ERK activation (56).
Therefore, we speculate that regulators of MAPKs, such as
JNK/SAPK-associated protein 1 (JSAP1) (57) and CL100/MKP-1 (55), might
be deregulated by paclitaxel and thus contribute to the sustained MAPK
activations observed in our study. Since a small GTP-binding protein,
Rap1, has been shown to mediate sustained ERK activation induced by
nerve growth factor (58), the involvement of a specific molecule in
sustained MAPK activations needs to be taken into account as well. The
clarification of such modulators may open new avenues to enhance the
efficacy of chemotherapeutic agents.
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ACKNOWLEDGEMENTS |
---|
We are indebted to members of the Rustgi laboratory for discussion and support.
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FOOTNOTES |
---|
* This work was supported by the Abramson Family Cancer Research Institute, National Institutes of Health (NIH) Grant R01 DK57735, and the Molecular Biology and Morphology Core Facilities of NIH Grant P30 DK 50306.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: 600A CRB, Division of Gastroenterology, University of Pennsylvania, 415 Curie Blvd., Philadelphia, PA 19104-6144. Tel.: 215-898-0154; Fax: 215-572-5412; E-mail: anil2@mail.med.upenn.edu.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M011164200
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ABBREVIATIONS |
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The abbreviations used are: MAPK, mitogen-activated protein kinase; DEVD-CHO, Asp-Glu-Val-Asp-aldehyde; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MEK, MAPK/ERK kinase; PARP, poly(ADP-ribose) polymerase; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; EGF, epidermal growth factor; PBS, phosphate-buffered saline.
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