Microtubule Dysfunction Induced by Paclitaxel Initiates
Apoptosis through Both c-Jun N-terminal Kinase
(JNK)-dependent and -Independent Pathways in Ovarian Cancer
Cells*
Tzu-Hao
Wang
,
Diana M.
Popp§,
Hsin-Shih
Wang¶,
Masao
Saitoh
,
Jane G.
Mural**,
Donald C.
Henley
,
Hidenori
Ichijo
, and
Jay
Wimalasena

From the
Department of Obstetrics and Gynecology,
Graduate School of Medicine, University of Tennessee Medical Center,
Knoxville, Tennessee 37920, the § Life Sciences Division and
the ** Environmental Sciences Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee 37831, the ¶ Department of Obstetrics and
Gynecology, Chang-Gung Medical School, Chang-Gung Memorial Hospital,
Taipei, Taiwan, and the
Department of Biomaterials Science,
Faculty of Dentistry, Tokyo Medical and Dental University,
Tokyo 113-8549, Japan
 |
ABSTRACT |
The antineoplastic agent paclitaxel
(TaxolTM), a microtubule stabilizing agent, is known
to arrest cells at the G2/M phase of the cell cycle and
induce apoptosis. We and others have recently demonstrated that
paclitaxel also activates the c-Jun N-terminal kinase/stress-activated
protein kinase (JNK/SAPK) signal transduction pathway in various human
cell types, however, no clear role has been established for JNK/SAPK in
paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK
signaling cascades in apoptosis resulting from microtubular dysfunction
induced by paclitaxel, we have coexpressed dominant negative (dn)
mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac,
JNKK, and JNK) in human ovarian cancer cells with a selectable marker
to analyze the apoptotic characteristics of cells expressing dn vectors
following exposure to paclitaxel. Expression of these dn signaling
proteins had no effect on Bcl-2 phosphorylation, yet inhibited
apoptotic changes induced by paclitaxel up to 16 h after
treatment. Coexpression of these dn signaling proteins had no
protective effect after 48 h of paclitaxel treatment. Our data
indicate that: (i) activated JNK/SAPK acts upstream of membrane changes
and caspase-3 activation in paclitaxel-initiated apoptotic pathways,
independently of cell cycle stage, (ii) activated JNK/SAPK is not
responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii)
apoptosis resulting from microtubule damage may comprise multiple
mechanisms, including a JNK/SAPK-dependent early phase and
a JNK/SAPK-independent late phase.
 |
INTRODUCTION |
Paclitaxel (TaxolTM) is an antineoplastic agent
specifically targeting microtubules (1, 2) and extensive studies
indicate that paclitaxel arrests cells at the G2/M phase of
the cell cycle (3). While mitotic arrest of paclitaxel-treated cells
has been observed to initiate apoptosis (4-6), the biochemical events downstream of kinetic stabilization of microtubule dynamics which lead
to apoptosis remain largely unclear (3). Furthermore, substantial
evidence indicates that the G2/M arrest of the cell cycle
may not be the only mechanism to induce apoptosis (7-10); additional phosphoregulatory pathways may be involved in inducing apoptosis (11-13).
We and others have recently demonstrated that paclitaxel activates the
c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK)1 pathways in a
variety of human cells through microtubular interactions (14, 15). The
JNK/SAPK signaling pathways respond to various stress-related stimuli
and are involved in initiation of apoptosis in many cell types
(16-20). Whether JNK/SAPK activation is required for
paclitaxel-induced apoptosis has remained unclear, however, it is known
that paclitaxel induces phosphorylation of Bcl-2 (11, 12) and that
Bcl-2 can be phosphorylated by activated JNK/SAPK (21).
The purpose of this study was to examine whether inhibition of the
JNK/SAPK signaling pathway protects cells from paclitaxel-induced apoptosis and/or abrogates paclitaxel-induced phosphorylation of Bcl-2.
Our results demonstrate that expression of dn-ASK1 (apoptosis signal-regulating kinase 1), dn-Rac, dn-JNKK, or dn-JNK, while exerting
no effects on phosphorylation of Bcl-2, inhibits apoptosis induced by
paclitaxel treatment up to 16 h. The present study clearly
indicates that activation of the JNK/SAPK signaling cascade promotes
early phases of paclitaxel-induced apoptosis, independently of cell
cycle stage or Bcl-2 phosphorylation.
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EXPERIMENTAL PROCEDURES |
Cell Culture, Transfection, and Paclitaxel
Treatment--
Ovarian carcinoma cells BR (22, 23) were cultured in
Dulbecco's modified Eagle's medium/F-12 (Sigma) supplemented with 10% newborn bovine serum, penicillin, and streptomycin and incubated at 37 °C in 5% CO2. Control vectors pCMV-lacZ and
pSR
empty vector as well as expression vectors for wild type
(wt)-JNK1 (pSR
-JNK), dominant-negative (dn)-Ras (pSR
-dn Ras), and
dn-Rac (pSR
-dn-Rac) (24) were from Z.-G. Liu and M. Karin
(University of California at San Diego). A Myc-epitope tagged
expression vector for wt-Rac, pEXV3-Rac, was from A. Hall (University
College, London, United Kingdom)(25). Dominant-negative expression
vectors for JNK/SAPK (pSR
-APF) and JNKK/SEK1 (pSR
-K116R) were
from G. L. Johnson (National Jewish Center for Immunology and
Respiratory Medicine, Denver, CO) (26). Expression vectors for wt- and
dn-ASK1 (pcDNA3-ASK1-HA, pcDNA3-dn ASK1-HA) were described
previously (27-30). In addition to pSR
and pcDNA3 (Invitrogen,
Carlsbad, CA) empty vectors, expression vectors used for control
purposes include: pCMV-CD20 that was from E. Harlow (Massachusetts
General Hospital, Boston, MA)(31) and pcDNA3-kinase dead
p70S6kinase (K100R) that was generated by polymerase chain
reaction mutagenesis from the p70S6kinase cDNA, which
was originally from J. Avruch (Massachusetts General Hospital).
Liposome-mediated transfections of BR cells using Tfx-50 (Promega,
Madison, WI) were performed as described previously (14). A CMV
promoter-driven enhanced green fluorescent protein construct, pEGFP
(CLONTECH, Palo Alto, CA), was co-transfected as a
selectable marker for transfected cells. Stock solutions of paclitaxel,
actinomycin-D, and cisplatin (all from Sigma) were prepared with
Me2SO at concentrations of 10, 1, and 50 mM,
respectively. In this study, 1 µM paclitaxel was used to
treat cultured cells.
Annexin-V Binding, Flow Cytometric Analyses, and
Sorting--
Twenty-four h after co-transfection of BR cells with
pEGFP and wt or dn expression vectors, 105 trypsinized
cells were incubated at room temperature for 15 min with 5 µl of
phycoerythrin (PE)-conjugated annexin-V (Pharmingen, San Diego, CA) and
0.125 µg/ml of 7-amino actinomycin D (7-AAD) (Sigma) in binding
buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and analyzed by flow cytometry with a
FACStarPlus (Becton Dickinson, San Jose, CA). Flurochromes
such as green fluorescent protein expressed by pEGFP, PE-annexin V, and
7-AAD were excited by laser tuned to 488 nm and emissions were detected at 507, 575, and 650 nm, respectively. Data of 10,000 cells from each
sample were analyzed with the CellQuest3 software (Becton Dickinson).
To compare other apoptotic characteristics in cells with or without
expression of dn vectors, the green fluorescent protein positive and
negative cells were sorted using a FACStarPlus flow
cytometer and analyzed separately.
Western Blot Analyses--
Aliquots of cell lysate containing
equal protein mass were resolved on SDS-polyacrylamide gel
electrophoresis, transferred to nitrocellulose membranes, and probed
with anti-HA epitope (Boehringer-Mannheim), anti-Myc (Calbiochem, San
Diego, CA), anti-JNK (Santa Cruz Biotechnology, Santa Cruz, CA),
anti-PARP (Upstate Biotechnology, Lake Placid, NY), or anti-Bcl-2
(DAKO, Carpinteria, CA) antibodies followed by relevant second
antibodies conjugated with horseradish peroxidase (Santa Cruz
Biotechnology). After washing, proteins were detected by
chemiluminescence (ECL, Amersham) as described previously (14).
Immunocomplex JNK Assay--
Activity of the JNK/SAPK was
measured as described previously (14). Endogenous JNKs were
immunoprecipitated from cell lysate using an anti-JNK antibody
conjugated to agarose beads (Santa Cruz Biotechnology) and the JNK
activity was assayed by levels of incorporated
[
-32P]ATP into its substrate GST-c-Jun (amino acids
1-79) (Santa Cruz).
Cell Cycle Analyses--
Green cells expressing control vector
or dn expression vectors were isolated by flow cytometric sorting,
fixed with 70% ethanol, treated with 0.1 mg/ml RNase (Sigma), stained
with 20 µg/ml propidium iodide, and analyzed with flow cytometry.
Propidium iodide-stained DNA content of each cell was used as the
parameter of cell cycle profile.
Caspase-3 Assay--
We used the colorimetric substrate
Ac-DEVD-p-nitroaniline (Calbiochem) for caspase-3 assays in
a procedure modified from the manufacturer's protocol. Briefly,
aliquots of sonicated cell lysate were prepared in lysis buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM dithiothreitol, 0.1 mM EDTA), incubated with 200 µM Ac-DEVD-p-nitroaniline in assay buffer
(50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS,
10 mM dithiothreitol, 0.1 mM EDTA, 10%
glycerol) in 96-well plates at 37 °C for 24 h. Absorbance of
the cleaved product was measured at 405 nm in a BioKinetic EL340
microplate Reader (Bio-Tek Instruments, Winooski, VT).
DNA Fragmentation Assays--
Identification of the ladder
pattern of DNA fragmentation in 1.6% agarose gel was previously
reported (32). To quantify fragmented DNA in apoptotic cells,
transfected cells isolated by flow cytometry were treated with
paclitaxel or vehicle (Me2SO) and levels of fragmented DNA
resulting from apoptosis were measured with a Cell Death ELISA kit
according to the manufacturer's protocol (Boehringer-Mannheim).
Statistics--
Analysis of variance (ANOVA) and Scheffe test
for post hoc comparisons were used for statistical analyses
with the STATISTICA software (Statsoft Inc., Tulsa, OK). The
p values equal to or greater than 0.05 are considered to be
not significant.
 |
RESULTS |
Expression of dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK Transiently
Alleviates Cytotoxicity Induced by Paclitaxel Treatment for 16 h--
To determine whether inhibition of the JNK/SAPK signaling
cascade abolishes apoptosis in cells treated with paclitaxel, we analyzed paclitaxel-induced apoptosis among ovarian cancer BR cells
transfected with dn-Ras, dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK, along
with pEGFP to allow selection of transfected cells. The efficacies of
these dn expression vectors for inhibition of the JNK/SAPK have been
demonstrated previously (14). Following paclitaxel treatment for the
indicated times, transfected cells were stained with both PE-conjugated
annexin-V (binding to both apoptotic cells and dead cells) and the
viability dye 7-AAD (staining dead cells but not early apoptotic
cells) to differentiate early apoptotic cells (annexin-V
positive/7-AAD negative) from dead cells (7-AAD positive) (Fig.
1).

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Fig. 1.
Three-color analyses for apoptosis and cell
death among transfected cells by flow cytometry. Ovarian cancer BR
cells were co-transfected with pEGFP and an expression vector for
dn-Ras for 24 h, treated with 1 µM paclitaxel for
16 h, stained with PE-conjugated annexin-V and 7-AAD, and analyzed
by flow cytometry. A, early apoptotic cells (annexin-V
positive/7-AAD negative) in LR region were differentiated from dead
cells (7-AAD positive) in UR region. The UL and LL regions contain dead
cells (7-AAD positive) and live cells (annexin-V negative/7-AAD
negative), respectively. B and C, to
analyze early apoptotic cells among transfected cells, we first
excluded dead cells (7-AAD positive) from the whole population shown in
B and only analyzed live cells by profiles of EGFP and
PE-conjugated annexin-V. Percent of early apoptotic cells among
transfected (green) cells were calculated by: 100% × UR/(UR + LR)
shown in C.
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Percentages of early apoptotic cells among transfected cells treated
with paclitaxel for 16 or 48 h are summarized in Table I. Data presented as the (percent in
paclitaxel-treated cells/percent in Me2SO-treated cells)
ratios among cells expressing different dn signaling proteins are
compared in Fig. 2. In transfected cells without paclitaxel treatment, expression of dn vectors decreased the
percentage of early apoptotic cells. These decreases in basal levels of
apoptosis were specific for expression of these dn expression vectors
because expression of irrelevant genes in pCMV-lacZ, pcDNA3-kinase dead-p70S6kinase, or pCMV-CD20 did not decrease basal
apoptosis (data not shown). Treatment with paclitaxel for 16 h
significantly increased apoptosis in both control
vector-transfected and dn-Ras-transfected cells (p < 0.05), but the induction of apoptosis by paclitaxel was decreased to
statistically insignificant levels (NS) in cells expressing dn-ASK1,
dn-Rac, dn-JNKK, or dn-JNK (Table I and Fig. 2A). In contrast to 16 h treatment with paclitaxel, 48 h of treatment significantly (p < 0.05) increased early apoptotic
cells in all transfected cells irrespective of expression of dn vector
types (Table I and Fig. 2B).
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Table I
Apoptotic effects of paclitaxel treatment on transfected cells
Human ovarian cancer BR cells were cotransfected with pEGFP and empty
(control) or dn expression vectors for 24 h, treated with 1 µM paclitaxel for 16 or 48 h, and stained with
phycoerythrin-conjugated annexin-V and 7-AAD followed by flow
cytometric analyses. Percentages of early apoptotic cells (annexin-V
positive/7-AAD negative) among green transfected populations, which
were set as 100%, are shown as the mean ± S.E.
(n = 6) of duplicates from three independent
experiments. Statistical data in the right column are comparisons
between dimethyl sulfoxide (Me2SO) and paclitaxel treatment
(horizontal comparisons). The symbols (* and ) indicate significant
differences (p < 0.05) between cells expressing dn
vectors and cells expressing control vector (italicized values) within
each treatment group (vertical comparisons).
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Fig. 2.
Expression of dn-ASK1, -Rac, -JNKK, or -JNK
significantly inhibits apoptosis induced by paclitaxel treatment for
16 h. Apoptosis among transfected BR cells were analyzed by
staining profiles of PE-conjugated annexin-V and 7-AAD.
A, comparisons of paclitaxel (PTX)-induced
apoptosis among cells expressing control vector or dn vectors after
paclitaxel treatment for 16 h. B, comparisons of
apoptosis among cells expressing different vectors after 48 h
treatment with paclitaxel. Bars in figures are calculated
from the data in Table I using the formula: 100% × (% in
paclitaxel-treated cells/% in Me2SO
(DMSO)-treated cells).
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To verify that expression of dn signaling proteins efficiently
inhibited endogenous JNK/SAPK activity, we co-transfected BR cells with
pEGFP and dn expression vectors, isolated transfected cells by flow
cytometry using expression of pEGFP (green fluorescence) as a
selectable marker, treated with paclitaxel, and analyzed by
immunocomplex JNK assay. We isolated only live (7-AAD negative) green
cells expressing transfected vectors (Fig.
3A) and verified expression of
dn signaling proteins by Western blot analyses (Fig. 3, B
and C).

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Fig. 3.
Isolation of transfected cells coexpressing
green fluorescent protein and verification of expression of dn
signaling proteins in these cells during the period of treatment up to
48 h. A, 24 h after co-transfection with
pEGFP and dn expression vectors, transfected cells were isolated by
flow cytometric sorting using expression of green fluorescent protein
as a selectable marker. Examples of isolating cells transfected with
dn-JNK and dn-ASK1 are shown. Three regions, Gr (green),
NG (non-green), and red (7-AAD positive) were
selected to represent live transfected cells (coexpressing pEGFP), live
non-transfected cells (EGFP negative), and dead cells, respectively.
B and C, Western blot analyses and immunocomplex
kinase assays were used to measure transfected protein levels and
endogenous JNK activity, respectively. Expression of HA-dn-JNK or
dn-ASK1-HA was not detected in non-green cells (NG) but was
substantial in green transfected cells (Gr) and those
protein levels remained largely intact up to 48 h of treatment.
Efficient suppression of endogenous JNK activity by expression of
dn-JNK or dn-ASK1 were demonstrated by the kinase assay using GST-c-Jun
as substrate for JNK. Data shown are from a representative experiment,
which was repeated twice with comparable results. Nonspecific bands
also recognized by the 12CA5 antibody are labeled with
asterisks (*). DMSO, Me2SO.
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The inability of dn vectors to protect cells from apoptosis following a
48-h treatment with paclitaxel did not result from decreased expression
of dn signaling proteins. We have confirmed substantial expression of
EGFP by fluorescent microscopy (data not shown) and expression of dn
signaling proteins by Western blot analyses up to 48 h of
paclitaxel treatment (Fig. 3, B and C). At
48 h of treatment, protein levels of dn-JNK or dn-ASK1 in
Me2SO-treated cells remained as high as those at earlier
time points; whereas those in paclitaxel-treated cells slightly
decreased, perhaps resulting from a general protein degradation during
apoptosis (Fig. 3, B and C). Despite its slight
decrease in later time points of paclitaxel treatment, expression of
these dn signaling proteins efficiently suppressed paclitaxel-induced
JNK activation through all the time points (Fig. 3, B and
C).
Collectively, these results suggest that expression of dn-ASK1, dn-Rac,
dn-JNKK, or dn-JNK transiently protects ovarian cancer cells from
paclitaxel-induced apoptosis up to 16 h. When treated with
paclitaxel for a longer time, such as 48 h, cells may undergo apoptosis through additional, JNK/SAPK-independent mechanisms.
Expression of dn Signaling Proteins of the JNK/SAPK Pathway Does
Not Alter Cell Cycle Profiles of Paclitaxel-treated Cells--
To
investigate whether these dn signaling proteins inhibited
paclitaxel-induced apoptosis through regulation on cell cycle progression, we analyzed cell cycle profiles of cells expressing dn
signaling proteins at 16, 24, and 48 h of paclitaxel treatment. Compared with cells expressing control vector, expression of dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK did not change cell cycle profiles, nor
interfere with mitotic arrest of cells after paclitaxel treatment (Fig.
4).

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Fig. 4.
Expression of dn signaling proteins does not
alter the cell cycle profiles nor prevent the G2/M arrest
of paclitaxel-treated cells. Twenty-four h after transfection,
cells expressing control or dn signaling proteins were isolated by flow
cytometric sorting, treated with paclitaxel for 16, 24, or 48 h,
and analyzed by flow cytometry using propidium iodide-stained DNA
content of each cell as parameter for the cell cycle profiles.
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Apoptotic Characteristics in Paclitaxel-treated Cells Are Abrogated
by Blockage of the JNK/SAPK Pathways--
To examine the time course
of inhibition of apoptosis by dn signaling proteins, we treated
isolated cells expressing control vector or dn signaling proteins with
paclitaxel and quantified DNA fragmentation with ELISA. Expression of
dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK significantly inhibited
paclitaxel-induced DNA fragmentation up to 16 h, whereas the
inhibition declined at 24 h and completely disappeared at 36 h of treatment (Fig. 5A). In
transfected cells treated with paclitaxel for 16 h, expression of
dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK significantly inhibited caspase-3
activation by paclitaxel (Fig. 5B) and suppressed
paclitaxel-induced PARP cleavage (Fig. 5C). These results
are consistent with data given above for annexin-V binding (Table I and
Fig. 2), again indicating that inhibition of the JNK/SAPK signaling
pathways transiently protects ovarian cancer cells from
paclitaxel-induced apoptosis.

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Fig. 5.
Expression of dn-ASK1, -Rac, -JNKK, or
-JNK transiently inhibits paclitaxel-induced DNA fragmentation,
caspase-3 activation, and cleavage of PARP. Twenty-four h after
transfection, sorted cells expressing different vectors were treated
with paclitaxel and apoptotic features were analyzed.
A, time course of paclitaxel-induced DNA fragmentation
measured by the Cell Death Detection ELISA. Data shown are mean ± S.E. (n = 6) of triplicates from two independent
experiments. B, after treated with paclitaxel for 16 h,
caspase-3 activity in cells expressing control vector or dn vectors was
measured using Ac-DEVD-p-nitroaniline as a colorimetric
substrate. Levels of caspase-3 activity are calculated by subtracting
the mean of OD405 readings of Me2SO-treated,
control-transfected cells from each reading of paclitaxel
(PTX)-treated cells, then the resultant levels of
paclitaxel-treated, control-transfected cells are set as 100 arbitrary
units. Data shown are mean ± S.E. (n = 6) of
triplicates from two independent experiments. C, after
treated with paclitaxel for 16 h, cleavage of PARP among
transfected cells was analyzed by Western blot. The ratios between
112-kDa (intact) and 86-kDa (cleaved) species of PARP were compared in
the bar graph. Data shown are from a representative
experiment, which was repeated three times with comparable results. A
nonspecific band also recognized by this anti-PARP antibody is labeled
by an asterisk (*).
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Overexpression of wt-ASK1, wt-Rac, or wt-JNK Promotes
Apoptosis--
Our data would predict that overexpression of wt
signaling proteins of the JNK/SAPK pathways may promote
paclitaxel-induced apoptosis. Flow cytometric analyses on
annexin-V binding confirmed that overexpression of wt-ASK1, wt-Rac, of
wt-JNK significantly (p < 0.05) promoted apoptosis in
both Me2SO-treated and 16 h paclitaxel-treated BR
cells (Fig. 6A). Consistent
with flow cytometric data, overexpression of wt-ASK1, wt-Rac, or wt-JNK
also significantly (p < 0.05) increased basal and
paclitaxel-induced DNA fragmentation (Fig. 6B) and the increases in apoptosis measured by two independent methods are very
similar. Western blot analyses with an anti-HA epitope antibody (12CA5)
and an anti-Myc epitope antibody (9E10) confirmed expression of
HA-wt-ASK1, HA-wt-JNK, and Myc-wt-Rac (Fig. 6C, upper panel) and immunocomplex kinase assays verified the augmentation of
paclitaxel-induced JNK activation by overexpressed signaling proteins
(Fig. 6C, lower panel). The increases in the apoptosis
induced by paclitaxel above those due to overexpression of wt signaling
proteins were the same as that in paclitaxel-treated,
control-transfected cells (Fig. 6, A and B).
Therefore, the effects of paclitaxel and overexpression of wt signaling
proteins appear to be additive. However, among cells overexpressing
these wt signaling proteins, the 8-12-fold JNK activation induced by
paclitaxel (Fig. 6C) was not proportional to the
2.5-3.5-fold increase of apoptosis (Fig. 6, A and
B), suggesting that activation of JNK is not linearly
related to apoptosis.

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Fig. 6.
Overexpression of wt-ASK1, wt-Rac, or wt-JNK1
promotes apoptosis. A, BR cells were co-transfected
with pEGFP and expression vectors for control, wt-ASK1, wt-Rac, or
wt-JNK1 for 24 h, treated with Me2SO (DMSO)
(solid bars) or 1 µM paclitaxel (hatched
bars) for 16 h, stained with PE-conjugated annexin-V and
7-AAD, and analyzed by flow cytometry as described in the legend to
Fig. 1. B, BR cells overexpressing wt-ASK1, wt-Rac, or
wt-JNK1 were isolated by flow cytometric sorting, treated with
Me2SO (solid bars) or 1 µM
paclitaxel (hatched bars) for 16 h, and DNA
fragmentation was quantified by ELISA. OD405 readings of
control-transfected cells treated with Me2SO are set as 100 arbitrary units. Data shown are mean ± S.E. (n = eight or four) from several independent experiments.
C, isolated BR cells expressing control vector,
HA-wt-ASK1, HA-wt-JNK1, or Myc-wt-Rac were treated with
Me2SO or 1 µM paclitaxel (PTX) for
3 h and expression of theses epitope-tagged, wt signaling proteins
were confirmed by Western blot analyses with an anti-HA (12CA5)
antibody or an anti-Myc (9E10) antibody. Endogenous JNK activities in
these lysates were measured by immunocomplex kinase assay using
an anti-JNK antibody for immunoprecipitation and GST-c-Jun as
substrate. Nonspecific bands also recognized by the 12CA5 antibody
are labeled by asterisks (*).
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Suppression of JNK/SAPK Activation Specifically Inhibits Apoptosis
Induced by 16-h Paclitaxel Treatment But Not That Induced by
Actinomycin D or Cisplatin--
To examine whether the JNK/SAPK
signaling cascade plays a specific role in apoptosis resulting from
microtubular dysfunction, we compared JNK activations and
cytotoxicities in ovarian cancer cells treated with paclitaxel or
treated with two DNA targeting agents, actinomycin D and cisplatin.
Treatment with 5 µM actinomycin D or 50 µM
cisplatin for 16 h induced a comparable ladder pattern of DNA
fragmentation with that induced by paclitaxel treatment (Fig.
7A), whereas only paclitaxel
significantly activated JNK (Fig. 7B). Moreover, expression
of dn-ASK1 or dn-JNK only inhibited DNA fragmentation induced by 16-h
paclitaxel treatment but not that induced by actinomycin D or cisplatin
(Fig. 7C).

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Fig. 7.
Expression of dn-ASK1 or dn-JNK
specifically inhibits DNA fragmentation induced by paclitaxel but not
that induced by actinomycin-D or cisplatin. A,
treatment with 1 µM paclitaxel (PTX), 5 µM actinomycin D (ACTD), or 50 µM cisplatin (CDDP) for 16 h induced the
ladder pattern of DNA fragmentation resolved in 1.6% agarose gel.
B, BR cells were treated with paclitaxel, actinomycin
D, or cisplatin for 8 or 16 h, and JNK activities and JNK protein
levels were measured by immunocomplex kinase assay and Western blot
analysis, respectively. Relative intensities of bands are shown as fold
(shown in parentheses) of Me2SO-treated control.
C, 24 h after transfection, sorted cells
expressing control vector, dn-ASK1, or dn-JNK were treated with
paclitaxel (solid bars), actinomycin D (hatched
bars), or cisplatin (checked bars) for 16 h. DNA
fragmentation were quantified by ELISA. Data shown are mean ± S.E. (n = 3) from a representative
experiment.
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Suppression of the JNK/SAPK Signaling Cascade Does Not Inhibit
Phosphorylation of Bcl-2 Induced by Paclitaxel--
To determine
whether paclitaxel-activated JNK/SAPK is required for phosphorylation
of Bcl-2 in paclitaxel-treated cells, we co-transfected BR cells with
pEGFP and dn expression vectors and treated cells with 1 µM paclitaxel for 16 h. Following treatment, green
fluorescent positive (transfected) and negative (non-transfected) cells
were analyzed separately for phosphorylation of Bcl-2. Phosphorylated Bcl-2 bands exhibiting slower mobility on Western blots were identified from both transfected and non-transfected cells, irrespective of which
dn vectors were overexpressed (Fig.
8A). To confirm these results
by an alternative approach, we co-transfected cells with pEGFP and dn
vectors, isolated transfected cells by flow cytometric sorting, then
treated control or dn vector-expressing cells with paclitaxel or
Me2SO for 16 h and analyzed the status of Bcl-2 phosphorylation. Consistent with previous experiments, treatment with
paclitaxel induced phosphorylation of Bcl-2 in all transfected cells,
including those expressing control vector or vectors for dn-ASK1,
dn-Rac, dn-JNKK, or dn-JNK (Fig. 8B). Collectively, these results indicated that inhibition of the JNK/SAPK signaling cascade did
not influence paclitaxel-induced phosphorylation of Bcl-2.

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Fig. 8.
Blockage of the JNK/SAPK signaling cascade
does not interfere with paclitaxel-induced phosphorylation of
Bcl-2. A, BR cells were co-transfected with pEGFP and
dn expression vectors for 24 h, treated with paclitaxel for
16 h, then transfected (green, Gr) and non-transfected
(non-green, NG) populations were separately isolated by flow
cytometric sorting. By Western blot analyses, phosphorylated Bcl-2
(p-Bcl-2) was identified as an additional band exhibiting
slower mobility on SDS-polyacrylamide gel electrophoresis when it was
compared with Bcl-2 in Me2SO-treated control.
B, Co-transfected BR cells with pEGFP and different dn
expression vectors were isolated by flow cytometric sorting, treated
with either paclitaxel (PTX) or Me2SO for
16 h, and analyzed for the status of Bcl-2 phosphorylation by
Western blots. Data shown are from a representative experiment, which
was repeated twice with comparable results.
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DISCUSSION |
Activation of the JNK/SAPK signaling pathways has been
mechanistically implicated in regulation of apoptosis (16-20),
however, the roles of JNK/SAPK in promoting (33-35) or preventing
apoptosis (36, 37) differ, depending on both cell type and
apoptosis-triggering stimuli (17, 19). Furthermore, in addition to
apoptosis, JNK/SAPK activation may be involved in proliferation (38,
39) and oncogenic transformation (40). On the other hand, apoptosis
itself can be considered as a form of stress, hence, JNK/SAPK
activation may be a stress response secondary to apoptosis, rather than
a primary mediator in apoptotic pathways (19). We have previously demonstrated that in BR ovarian cancer cells treated with paclitaxel, activation of JNK/SAPK reaches a peak at 2 h when apoptosis is still minimal (14), suggesting that JNK/SAPK activation is not a
secondary response to paclitaxel-induced apoptosis.
One of the early changes in apoptotic cell membranes is externalization
of phosphatidylserine, which exerts high affinity to annexin-V.
Therefore, increased binding of annexin-V is a sensitive indicator for
apoptosis (41). Apoptotic cells are also characterized by increased
activities of caspases, cleavage of 112-kDa poly(ADP-ribose) polymerase
(PARP) into a 86-kDa species, and DNA fragmentation (42, 43). We have
previously demonstrated that paclitaxel induces characteristic
apoptotic morphology (23, 44) and DNA fragmentation assayed by both gel
electrophoresis and the terminal deoxynucleotidyltransferase-mediated
dUTP nick end labeling (TUNEL) method in ovarian cancer cells (44). By
analyzing these biochemical apoptotic characteristics in
paclitaxel-treated cells overexpressing wt or dn signaling proteins of
the JNK/SAPK pathways, our results herein demonstrate an
apoptosis-promoting role for JNK/SAPK in ovarian cancer cells treated
with paclitaxel (Figs. 2, 5, and 6). Since suppression of the JNK/SAPK
signaling cascade only protects cells from paclitaxel-induced apoptosis
but not from apoptosis induced by DNA targeting agents, actinomycin-D
or cisplatin (Fig. 7), the present study further suggests an
apoptosis-promoting role of the JNK/SAPK cascade specifically in
apoptotic process resulting from microtubular dysfunction.
JNK/SAPK has been shown to be involved in activation of caspases that
are required for execution of the apoptotic process (34, 45-48). The
role of JNK/SAPK in activation of caspases is, however, not
straightforward. JNK/SAPK could be either upstream (34, 45) or
downstream (46-48) of caspase activation, depending on cell type and
apoptosis-initiating agents. Since paclitaxel has been shown to
activate caspases (49, 50), we analyzed the role of JNK/SAPK in
caspase-3 activation of paclitaxel-treated cells. Our data show that
inhibition of the JNK/SAPK cascade prevented paclitaxel-induced
caspase-3 activation, PARP cleavage, and DNA fragmentation (Fig. 5),
indicating that JNK/SAPK is upstream of caspase-3 activation in
paclitaxel-initiated apoptosis. These and other studies, where
anticancer agents were used to induce apoptosis (34, 45), suggest that
JNK/SAPK acts upstream of caspase activation in chemotherapy-initiated apoptosis.
Intriguingly, while expression of dn-Ras decreased basal levels of
apoptosis (Table I), it did not significantly abrogate paclitaxel-induced apoptosis (Table I and Fig. 2). Although we previously reported that both Ras and ASK1 are required for optimal activation of JNK/SAPK by paclitaxel (14), results of the present study
failed to clarify a pro- or anti-apoptosis role for Ras in the
apoptotic process initiated by paclitaxel. In addition to activation of
mitogen-activated protein kinase and JNK/SAPK pathways, Ras may also
provide a survival signal that is mediated by the phosphoinositide
3'-OH kinase-dependent activation of the protein kinase
B/Akt (51). Furthermore, inhibition of Ras activity induces apoptosis
(52) and exerts synergistic inhibition of cell growth with paclitaxel
(53). These studies further collaborate the complex and multifunctional
roles of Ras in both cell growth and apoptosis.
In contrast to Ras, it is clear that the related small G-protein, Rac,
has a critical role in paclitaxel-induced apoptosis at 16 h,
probably via activation of JNK/SAPK. Previous studies have demonstrated
that Rac activate the JNK/SAPK pathway (54-58) and our studies clearly
indicate that microtubule damage activates Rac (Fig. 6C) and
that Rac is at least partially responsible for JNK activation and the
resulted apoptosis (Figs. 2, 5, and 6). It is interesting that among
the upstream signaling proteins employed in this study, the wt-ASK1
appeared to have the highest apoptosis-promoting effect (Fig. 6).
Possible explanations for these observations include that ASK1, when
overexpressed, may also activate other apoptosis-related kinase such as
p38 (27-30).
Since expression of dn signaling proteins remained largely intact at
48 h after Me2SO treatment (Fig. 3), the disappearance of protection from basal apoptosis cannot be explained by a decrease of
dn signaling proteins. Forty-eight hours of treatment with Me2SO (vehicle control) at a 0.1% final concentration did
not activate JNK/SAPK (14) or initiate apoptosis. However,
liposome-mediated transfection with control DNA into BR cells was
observed to exert mild cytotoxicity and caspase-3
activation,2 hence it is
possible that transfection by itself may initiate an apoptotic process
that eventually overrides the transient protection by dn signaling
proteins of the JNK/SAPK pathway. Nevertheless, treatment with
paclitaxel for 48 h caused a further significant increase in
apoptosis above the relatively high basal levels (Fig. 2B
and 5A, Table I), suggesting coexistence of multiple
apoptotic pathways. This suggestion is supported by the ability of dn
signaling proteins to inhibit apoptosis in both control and
paclitaxel-treated cells at 16 h but not 48 h treatment.
Therefore, the pathways leading to apoptosis after the 16-h
versus 48-h treatment appear to be fundamentally different.
Multiple mechanisms have been suggested to be involved in
paclitaxel-induced apoptosis, such as: abortive mitosis after
paclitaxel-induced G2/M block (4-6), activation of
p34cdc2 (50, 59, 60) and other Cdks (61, 62), activation
and local release of an apoptosis-inducing cytokine (9), and induction of transcription regulators and enzymes that modulate apoptosis (10).
Whereas overexpression of wt-ASK1, wt-Rac, or wt-JNK promotes apoptosis
in BR cells (Fig. 6), expression of dn-ASK1, dn-Rac, dn-JNKK, or dn-JNK
only transiently inhibits paclitaxel-induced apoptosis (Table I and
Fig. 2). Protection from paclitaxel-induced DNA fragmentation by
expression of these dn signaling proteins disappeared when cells were
treated for 24-48 h (Fig. 5A). These results are in
agreement with our previous report that paclitaxel-induces JNK/SAPK
activation in BR cells is transient with a peak at 2-4 h and declines
afterward (14). Since suppression of the JNK/SAPK signaling cascade
does not alter cell cycle profiles nor interfere with the
paclitaxel-induced mitotic arrest that peaks at 24 h of paclitaxel
treatment (Fig. 4), G2/M block of the cell cycle may mainly
account for the later phase of paclitaxel-induced apoptosis. Our
results further suggest that the late phase of paclitaxel-induced apoptosis is independent of JNK/SAPK activity. Several recent studies
have suggested that catastrophic activity of Cdks may be a terminal
effector in apoptotic pathway (63, 64).
Results in this study do not support the hypothesis that activated
JNK/SAPK is required for phosphorylation of Bcl-2 in paclitaxel-treated cells (21). Bcl-2 is known to protect cells from apoptosis (65) and
paclitaxel has been shown to induce both phosphorylation of Bcl-2 and
apoptosis (11, 12, 66, 67). However, the roles of Bcl-2 phosphorylation
in promoting (11, 12, 67-69) or inhibiting apoptosis (70, 71) remain controversial.
Some reports suggest that Bcl-2 may act upstream of JNK/SAPK (72, 73).
In BR cells, however, JNK/SAPK activation peaked at 2 h (14),
while Bcl-2 phosphorylation and the G2/M arrest of the cell
cycle required 12-16 h of paclitaxel treatment.2 These
temporal differences suggest that phosphorylated Bcl-2 is unlikely to
act upstream of JNK/SAPK. The observations that inhibition of JNK/SAPK
did not interfere with paclitaxel-induced G2/M arrest and
Bcl-2 phosphorylation (Figs. 4 and 8) suggest that paclitaxel-activated
JNK/SAPK is independent of Bcl-2 phosphorylation occurring in
paclitaxel-treated cells. Recent reports further demonstrate that
phosphorylation of Bcl-2 occurs only in cells blocked at
G2/M phase after paclitaxel treatment (66, 74). Therefore,
JNK/SAPK activation and Bcl-2 phosphorylation may reside in distinct,
independent pathways. Instead of the JNK/SAPK cascade, PKA activation
has been suggested to account for Bcl-2 phosphorylation in cells with
microtubule damages (75). The role(s) of Bcl-2 phosphorylation in
regulating apoptosis in general and in paclitaxel-induced apoptosis in
particular apparently requires further clarification.
Our results, for the first time, identify a Bcl-2
phosphorylation-independent role of JNK/SAPK in promoting
paclitaxel-induced apoptosis and demonstrate that multiple mechanisms
are involved in apoptosis resulting from microtubule damage, including
a JNK/SAPK-dependent early phase and a JNK/SAPK-independent
late phase.
 |
ACKNOWLEDGEMENTS |
We thank Drs. A. T. Ichiki, J. Merryman,
R. A. Popp, D. S. Torry, and W. D. Wicks of the
University of Tennessee for discussions and advice. Technical
assistance from R. B. Andrews, L. E. Barns, E. Bukovska,
J. S. Foster, and C.-L. Wang is gratefully acknowledged. We are
indebted to Drs. J. Avruch, A. Hall, E. Harlow, G. L. Johnson, M. Karin, and Z.-G. Liu for cDNA clone and expression vectors and
appreciate Dr. R. Schmied of Calbiochem for helpful suggestions on the
caspase-3 assay. T.-H. W. is grateful to Dr. M. R. Caudle, Dean of the Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, for encouragement and support.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants AA-08328 and CA-68538 (to J. W.) and National Science
Council (Taiwan) Grant NSC-86-2314-B-182-077 (to H.-S. W.).

To whom correspondence should be addressed: Dept. of Obstetrics
and Gynecology, University of Tennessee Medical Center, 1924 Alcoa
Highway, Knoxville, TN 37920. Tel.: 423-544-8960; Fax: 423-544-6863; E-mail: mcf7{at}msm.com.
2
T-H. Wang, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
JNK, c-Jun
N-terminal kinase;
7-AAD, 7-amino actinomycin D;
ASK1, apoptosis
signal-regulating kinase 1;
Bcl-2, oncoprotein identified in
B-cell leukemia/lymphoma-2;
Cdk, cyclin-dependent kinase;
CMV, cytomegalovirus;
dn, dominant-negative;
ELISA, enzyme-linked
immunosorbent assay;
HA, hemagglutinin epitope of influenza virus;
JNKK, JNK kinase;
PARP, poly(A)DP-ribose polymerase;
PE,
phycoerythrin;
pEGFP, plasmid for enhanced green fluorescent protein;
PKA, protein kinase A;
SAPK, stress-activated protein kinase;
wt, wild type;
GST, glutathione S-transferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-]propanesulfonic
acid;
Me2SO, dimethyl sulfoxide.
 |
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