Oncogene (1999) 18, 377 ± 384
ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00
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Di€erential regulation of mitogen-activated protein kinases by
microtubule-binding agents in human breast cancer cells
Alexander A Shtil1, Sandhya Mandlekar2, Rong Yu2, Robert J Walter3, Karen Hagen1,
Tse-Hua Tan4, Igor B Roninson1 and Ah-Ng Tony Kong2
1
Department of Molecular Genetics, College of Medicine, 2Department of Pharmaceutics and Pharmacodynamics, Center for
Pharmaceutical Biotechnology, College of Pharmacy; University of Illinois at Chicago, Chicago, Illinois 60607; 3Department of
Surgery, Cook County Hospital, Chicago, Illinois 60612; 4Department of Microbiology and Immunology, Baylor College of
Medicine, Houston, Texas 77030, USA
Drug design targeted at microtubules has led to the
advent of some potent anti-cancer drugs. In the present
study, we demonstrated that microtubule-binding agents
(MBAs) taxol and colchicine induced immediate early
gene (c-jun and ATF3) expression, cell cycle arrest, and
apoptosis in the human breast cancer cell line MCF-7.
To elucidate the signal transduction pathways that
mediate such biological activities of MBAs, we studied
the involvement of mitogen-activated protein (MAP)
kinases. Treatment with taxol, colchicine, or other
MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in
MCF-7 cells. In contrast, p38 was activated only by
taxol and none of the MBAs changed the activity of
extracellular signal-regulated protein kinase 2 (ERK2).
Activation of JNK1 or p38 by MBAs occurred
subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds.
Furthermore, baccatine III and b-lumicolchicine, inactive
analogs of taxol and colchicine, respectively, did not
activate JNK1 or p38. These results suggest that
interactions between microtubules and MBAs are
essential for the activation of these kinases. Pretreatment
with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicininduced JNK1 activity, but had no e€ect on JNK1
activation by MBAs, excluding a role for oxidative
stress. However, BAPTA/AM, a speci®c intracellular
Ca2+ chelator, attenuated JNK1 activation by taxol but
not by colchicine, and had no e€ect on microtubule
changes induced by taxol. Thus, stabilization or
depolymerization of microtubules may regulate JNK1
activity via distinct downstream signaling pathways. The
di€erential activation of MAP kinases opens up a new
avenue for addressing the mechanism of action of antimicrotubule drugs.
Keywords: taxol; colchicine; MAP kinases; microtubules; breast cancer
Correspondence: A-NT Kong
The ®rst two authors contributed equally to this work
Received 23 February 1998; revised 17 August 1998; accepted 17
August 1998
Introduction
Microtubules, the cytoskeletal structures formed by the
polymerization of tubulin heterodimers, play a crucial
role in many biological processes including mitosis,
cell-cell communication, intracellular transport, cell
growth, and programmed cell death or apoptosis
(Dustin, 1984). Given the pivotal importance of
microtubules in cellular function, they have been the
targets for the designing of e€ective anti-cancer drugs,
such as taxol and the vinca alkaloids. By interfering
with the dynamics of microtubule assembly, microtubule-binding agents (MBAs) exert profound e€ects
on cellular processes. It has been shown that MBAs
induce gene expression (Karin et al., 1995), cause cell
cycle arrest (Jordan et al., 1991) and initiate apoptosis
(Bhalla et al., 1993). However, the signal transduction
pathways that mediate the cellular responses to MBAs
remain unknown.
Mitogen-activated protein kinases (MAPKs), characterized as proline-directed serine/threonine kinases
(Cobb and Goldsmith, 1995), are important cellular
signaling components which convert various extracellular signals into intracellular responses through serial
phosphorylation cascades (Marshall, 1994). In mammalian cells, three distinct MAPK cascades have been
identi®ed (Cano and Mahadevan, 1995). These include
extracellular signal-regulated protein kinases (ERKs),
c-jun N-terminal kinases or stress-activated protein
kinases (JNKs/SAPKs), and p38. Each MAP kinase
cascade consists of a module of three kinases: a
MAPKKK (mitogen-activated protein kinase kinase
kinase) which phosphorylates and activates a MAPKK
(mitogen-activated protein kinase kinase), which, in
turn, phosphorylates and activates a MAPK (Marshall,
1994). The ERK signaling cascade can be triggered by
growth factors and phorbol esters through Rasdependent activation of Raf-MEK-ERK pathway
(Stokoe et al., 1994). Activation of ERK can also be
achieved via certain G-protein-coupled receptors and
protein kinase C (Burgering and Bos, 1995). The JNK
cascade is operated by a parallel signaling module
consisting of MEKK1 (mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase
kinase 1), MKK4 (mitogen-activated protein kinase
kinase 4) and JNK (Kyriakis et al., 1994). Unlike
ERK, the JNK cascade is only modestly activated by
growth factors or phorbol esters and instead is strongly
activated by stress signals such as UV light (Hibi et al.,
1993), g-irradiation (Chen et al., 1996), inhibitors of
protein synthesis (Cano et al., 1994), ceramide (Verheij
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
378
et al., 1996), DNA-damaging drugs (Yu et al., 1996a),
pro-in¯ammatory cytokines (Raingeaud et al., 1995),
or chemopreventive agents (Yu et al., 1996b). Similar
to JNK, p38 kinase is preferentially activated by many
stress stimuli via the MEKK1-MKK3 cascade
(Kyriakis and Avruch, 1996). Activation of MAPK
pathways culminates in the phosphorylation of several
transcription factors, such as c-Myc, p62TCF/Elk-1, cJun, ATF2, and ATF3 which ultimately leads to
changes in the expression of various genes (Karin,
1995). Recent studies suggest that MAP kinases also
regulate the cell cycle and apoptosis (Xia, 1995).
In the present study, we demonstrate that MBAs
activated JNK1 in human breast cancer cell lines in a
time- and dose-dependent manner, whereas p38 MAP
kinase was activated only by the microtubule-stabilizing agent taxol. The activation of both JNK1 and p38
required binding of the drugs to tubulin. Unlike JNK1
and p38, however, activity of ERK2 was not a€ected
by MBAs. Thus, stabilization or depolymerization of
microtubules by MBAs di€erentially activated MAP
kinase signaling pathways, which may be a novel
mechanism for the regulation of MAP kinases in
response to extracellular stress stimuli.
Results
MBAs induce cell cycle arrest and apoptosis in the
MCF-7 human breast cancer cell line
Previous studies have shown that treatment with
MBAs caused cell cycle arrest and apoptosis in some
cancer cell lines (Jordan et al., 1991, Bhalla et al.,
1993). To validate our system, we examined the e€ects
of MBAs on the cell cycle and apoptosis in MCF-7
human breast cancer cells. As shown in Table 1, few
apoptotic cells were detected after 12 h of treatment
with taxol or colchicine. However, prolonged treatment
(24 h) with 1 mM taxol and 2.5 mM colchicine induced a
signi®cant amount of apoptotic cell death (41.7% in
taxol- and 50.3% in colchicine-treated cells). Under
these conditions, taxol and colchicine also caused
signi®cant accumulation of cells with 4n DNA
content, which may represent both G2/M-arrested
cells and polyploid cells arrested in G1, as described
most recently by Lanni and Jacks (1998). Similar
results were obtained when cells were treated with low
concentrations of taxol (100 nM) and colchicine
(250 nM) for 24 h.
Taxol and colchicine up-regulate mRNA levels of c-jun
and ATF3
To test whether changes in microtubule dynamics lead
to alterations in gene expression, we examined the
e€ects of taxol and colchicine on the induction of the
immediate early genes, c-jun and ATF3, which are
known to regulate many other genes. Treatment of
MCF-7 cells with 1 mM taxol or 2.5 mM colchicine
resulted in elevated steady-state levels of c-jun and
ATF3 mRNAs, as determined by RT ± PCR (Figure 1a
and b). The increase of c-jun and ATF3 mRNAs was
time-dependent and maximal induction was seen
between 1.5 and 3 h of treatment.
MBAs di€erentially activate MAP kinases
To search for the potential signal transduction pathways that regulate the above biological activities of
MBAs, we ®rst studied the involvement of JNK1, an
important stress-response signaling kinase. Both
concentration-response and kinetics of JNK1 activation by MBAs were determined in MCF-7 cells. Cells
were harvested after treatment with MBAs and the
endogenous activity of JNK1 was determined by using
an immunocomplex kinase assay. As shown in Figure
2a, taxol activated JNK1 in a dose-dependent manner.
The induced JNK1 activity appeared at a concentration of 0.5 mM and then gradually increased with
increasing taxol concentrations. JNK1 activation by
taxol was also time-dependent (Figure 2b). One
micromolar taxol induced transient JNK1 activation
such that JNK1 activity began to increase after 30 min
of treatment, peaked at 3 h, and rapidly returned to
basal levels after 4 h. Colchicine also activated JNK1
in MCF-7 cells (Figure 2c) but unlike taxol, colchicineinduced JNK1 activity was sustained and continued to
increase even after 8 h of treatment (Figure 2d). When
Western blotting was performed, no change in the level
of JNK1 protein was observed throughout the
treatment period, indicating that the sustained activation of JNK1 resulted from phosphorylation of
preexisting JNK1 molecules rather than de novo
protein synthesis (data not shown). Treatment of
MCF-7 cells with other MBAs, including nocodazole,
podophyllotoxin, or vincristine also caused an increase
of JNK1 activity (Figure 2e). Furthermore, these
agents induced JNK1 activation in BT-20, a human
estrogen receptor-negative breast cancer cell line, to a
similar extent as that seen in MCF-7 cells (data not
Table 1 E€ect of microtubule-binding agents on cell cycle distribution
Treatmenta
Vehicle (control)
Taxol, 1 mM, 12 h
Taxol, 1 mM, 24 h
Colchicine, 2.5 mM, 12 h
Colchicine, 2.5 mM, 24 h
Taxol, 0.1 mM, 24 h
Colchicine, 0.25 mM, 24 h
C=2N
53.4
40.2
21.8
36.4
20.1
18.4
16.7
Cell DNA content (C)b
CN5C54N
C=4N
34.2
18.1
13.4
15.2
11.2
25.9
16.8
10.9
32.6
23.1
35.9
18.4
20.8
22.7
Apoptotic cells
1.5
9.1
41.7
12.5
50.3
34.9
43.8
a
MCF-7 cells were treated for 12 or 24 h with taxol or colchicine as shown in the table. Cell cycle
analysis was performed as described under Materials and methods. The values represent % of cells
(mean of three experiments) with di€erent DNA content. bCells with DNA content equal to 2N are
G1 cells, cells with DNA content between 2N and 4N are S cells; cells with 4N DNA content may
consist of G2/M cells and polyploids at G1 (Lanni and Jacks, 1998)
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
shown). These results suggest that JNK1 activation is a
general response to MBAs.
These data prompted us to investigate whether
other members of the MAP kinase family are also
activated by MBAs. MCF-7 cells were either treated
with 1 mM taxol or 2.5 mM colchicine for the indicated
time periods. The activities of p38 and ERK2 were
determined by immunocomplex kinase assay. As
shown in Figure 3a, taxol induced a time-dependent
p38 activation, whereas colchicine had no e€ect on
p38 activity. Furthermore, neither agent stimulated
ERK2 activity in MCF-7 cells (Figure 3b). Taken
together, these data suggest that MAP kinases are
di€erentially regulated by MBAs.
Figure 1 Taxol and colchicine increase the steady-state levels
mRNAs of c-jun and ATF3. MCF-7 cells were treated with 1 mM
taxol (a) or 2.5 mM colchicine (b) for indicated times. The
induction of c-jun and ATF3 was determined by RT ± PCR.
Data shown are the representative of three independent
experiments with similar results
379
JNK1 activation requires interaction of MBAs with
microtubules
To provide insight into the regulation of MAP
kinases by MBAs, we tested whether interactions
between microtubules and MBAs are critical for
MAP kinase activation. To do so, we ®rst studied
the time course of taxol- or colchicine-induced
tubulin rearrangement. As shown in Figure 4b,
treatment of MCF-7 cells with 2.5 mM colchicine for
30 min caused substantial shortening and disruption
of microtubules as compared with untreated control
cells (Figure 4a). With increasing treatment time,
further microtubule disruption occurred until the
microtubule network virtually collapsed (Figure 4c).
In contrast to cells treated with colchicine, cells
treated with 1 mM taxol exhibited long curving
microtubules extending to the cell margins and
dense bundles of microtubules coursing around the
nucleus and out into the cytoplasm (Figure 4d and
Figure 2 Taxol, colchicine, and other mirotubule-binding agents
stimulate JNK1 activity in a dose- and time-dependent manner in
MCF-7 cells. Cells were treated with drugs as indicated. JNK1
activity, expressed as fold induction over control levels, was
measured by in vitro immunocomplex kinase assay as described in
Materials and methods. (a) Dose response of JNK1 activation by
2.5 h of treatment with taxol; (b) Time course of JNK1 activation
by 1 mM taxol; (c) Dose response of JNK1 activation by 2.5 h of
treatment with colchicine; (d) Time course of JNK1 activation by
2.5 mM colchicine; (e) JNK1 activation by treatment with 1 mM of
nocodazole (NOC), podophyllotoxin (POD), vincristine (VIN) for
2 h. The blots shown are from one of three independent
experiments with similar results
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
380
1
1
1
1
1
1
1
1
Figure 3 Taxol and colchicine di€erentially activate p38 but
have no e€ect on ERK2 activity. After treatment with 1 mM taxol
or 2.5 mM colchicine for indicated time periods, cells were
harvested and the activities of p38 (a) and ERK2 (b) were
measured by in vitro immunocomplex kinase assay as described in
Materials and methods. The experiments were repeated three
times
e). However, in both cases, signi®cant rearrangement
of microtubule structure was evident within 30 min of
treatment, preceding drug-induced JNK1 activation as
seen in Figure 2b and d. In addition, doxorubicin, a
DNA damaging agent, did not a€ect the integrity of
microtubules (Figure 4f).
To provide further evidence into the role of
interactions between microtubules and MBAs in JNK1
or p38 activation, we measured the JNK1 or p38 activity
in MCF-7 cells treated with 1 mM baccatine III, an
inactive analog of taxol, or 2.5 mM b-lumicolchicine, a
colchicine analog which lacks tubulin-binding activity.
As shown in Figure 5a and b, neither agent induced
JNK1 or p38 activation. In preliminary experiments, we
found that pretreatment of MCF-7 cells with 100 nM
taxol prevented microtubule depolymerization by 2.5 mM
colchicine (data not shown). Accordingly, we examined
the e€ect of pretreatment with 100 nM taxol on
colchicine-induced JNK1 activity. As expected, pretreatment with taxol attenuated JNK1 activation by
colchicine (Figure 6). Interestingly, at 100 nM, taxol
alone caused microtubule stabilization, but did not
activate JNK1 (Figure 7a). Further morphological
studies revealed that the microtubule rearrangement
induced by 100 nM taxol was delayed and mild as
compared with that induced by 1 mM taxol. Similar
results were also obtained with colchicine, which, at low
concentration (0.25 mM), induced a delayed and modest
change in microtubule structure and did not activate
JNK1 even up to 24 h (Figure 7b). Taken together, these
data suggest that tubulin-binding and disruption of the
normal microtubule cytoskeleton are crucial for JNK1
or p38 activation by MBAs.
Figure 4 Colchicine and taxol induce morphological changes of
the microtubule network in a time-dependent fashion. Following
culture on coverslips for 40 h, MCF-7 cells were either untreated
(a) or treated with 2.5 mM colchicine for 30 min (b) and 1.5 h (c),
or with 1 mM taxol for 30 min (d) and 1.5 h (e), or treated with
doxorubicin (3.2 mM) for 2 h (f). After treatment, the morphological changes of microtubules were visualized by indirect
immuno¯uorescence assay as detailed in Materials and methods.
Each treatment was done in duplicate. Ten ®elds were examined
in each group. Magni®cation bar, 10 mm
Intracellular calcium chelator blocks JNK1 activation by
taxol, whereas antioxidants have no e€ect
JNK1 activation by various stress stimuli has been
shown to be mediated by the generation of reactive
oxygen intermediates (Cadwallader et al., 1997; Assefa et
al., 1997). To determine the role of oxidative stress in
MBA-induced JNK1 activation, we used several potent
antioxidants, N-acetyl-L-cysteine (NAC), ascorbic acid,
and vitamin E. NAC is a precursor of glutathione, an
abundant intracellular antioxidant, and is also a
scavenger of many di€erent types of reactive free
radicals. Ascorbic acid is a more potent free radical
scavenger than NAC, because of its lower reduction
potential. Vitamin E is a lipid soluble antioxidant, which
e€ectively scavenges lipid-related free radicals. Thus, the
inhibitory e€ect of these antioxidants would implicate
the involvement of oxidative stress. As shown in Figure
8a, pretreatment of MCF-7 cells with NAC (20 mM),
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
381
A.
B.
Figure 7 Low concentrations of taxol and colchicine do not
activate JNK1. MCF-7 cells were treated with 100 nM taxol (a) or
100 nM colchicine (b) for di€erent times. The JNK1 activity was
determined by in vitro immunocomplex kinase assay. The
experiment was repeated twice
Figure 5 Inactive analogs of taxol and colchicine do not activate
JNK1 or p38. After treatment of MCF-7 cells with 1 mM
baccatineIII (inactive analog of taxol) or 2.5 mM b-lumicolchicine
(inactive analog of colchicine) for indicated time periods, the
activity of JNK1 (a) or p38 (b) was determined by in vitro
immunocomplex kinase assay. Data shown are representative of
three independent experiments with similar results
Figure 6 Pretreatment with low concentration of taxol (100 nM)
prevents JNK1 activation by colchicine. MCF-7 cells were
pretreated with 100 nM taxol for 30 min and then challenged
with 2.5 mM colchicine for 2 h. JNK1 activity was measured by in
vitro immunocomplex kinase assay. The experiment was repeated
three times
ascorbic acid (100 mM) and vitamin E (1 mM), which by
themselves had no e€ect on JNK1 basal activity, strongly
inhibited JNK1 activation by hydrogen peroxide (H2O2)
as well as by doxorubicin, which is known to generate
reactive oxygen species (Bachur et al., 1979) and activate
JNK1 (Yu et al., 1996a; Osborn and Chambers, 1996).
However, these antioxidants had no e€ect on taxol- or
colchicine-induced JNK1 activity, suggesting that
microtubule-binding drugs activate JNK1 via an
oxidative stress-independent mechanism (Figure 8b).
When cells were pretreated with a speci®c intracellular
calcium chelator, BAPTA/AM, taxol-induced JNK1
activity was greatly reduced, but JNK1 activation by
colchicine remained intact (Figure 8c). Furthermore, the
presence of BAPTA/AM did not a€ect microtubule
rearrangement induced by both agents (data not shown).
Thus, it is tempting to speculate that drug-induced
microtubule stabilization or depolymerization may
regulate JNK1 activity via distinct downstream signaling pathways.
Discussion
Microtubules are highly labile cellular structures, which
can undergo rapid assembly and disassembly and this
dynamic behavior strongly in¯uences microtubule
function. Pharmacological or other modulation of the
structure-function dynamics of microtubules may result
in cellular stress. In the present study, we demonstrated
that both microtubule-stabilizing (taxol) and -depolymerizing (colchicine, vincristine, nocodadazale, and
podophyllotoxin) agents stimulated JNK1 activity in
human breast cancer cells. However, p38 was activated
only by the stabilizing drug, taxol, and the activity of
ERK2 was not a€ected by any of the agents tested.
Thus, microtubule-binding agents di€erentially activated MAP kinases, which provides a molecular basis
for understanding the mode of action of antimicrotubule drugs.
Activation of JNK1 or p38 by MBAs requires
interactions between the microtubules and MBAs. This
is evidenced by the following results: ®rst, drug-induced
morphological changes of microtubules preceded JNK1
or p38 activation. Second, MBAs did not interact with
kinase molecules directly, because JNK1 or p38 was not
activated by these agents in a cell-free system (data not
shown). Furthermore, pretreatment of cells with a low
concentration of taxol prevented colchicine-induced
depolymerization of microtubules, and also inhibited
JNK1 activation by colchicine. Most importantly,
baccatine III and b-lumicolchicine, non-binding analogs
of taxol and colchicine, respectively, had no e€ect on
JNK1 or p38 activity. However, the question remains as
to how interfering with microtubule dynamics triggers
downstream signaling events leading to the activation of
JNK1 and p38. Previous studies suggested that reactive
oxygen species (ROS) might serve as general mediators
of JNK activation by many environmental stresses and
physiological stimuli, such as UV-C (Adler et al., 1995),
g-irradiation (Chen et al., 1996), arsenite (Liu et al.,
1996), and in¯ammatory cytokines (Sluss et al., 1994). In
this study, we showed that ROS also mediated JNK1
activation by a potent chemotherapeutic drug, doxorubicin, as evidenced by the inhibitory e€ects of
antioxidants, NAC, ascorbic acid and vitamin E.
However, these antioxidants had no e€ect on JNK1
activation by taxol or colchicine. Therefore, MBAs may
activate JNK1 signaling pathway through a distinct
mechanism. It is also intriguing to note that, although
both taxol and colchicine activate JNK1, they may
regulate JNK1 activity through di€erent pathways
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
382
Figure 8 Antioxidants do not inhibit JNK1 activation by taxol
and colchicine, whereas intracellular calcium chelator preferentially blocks taxol-induced JNK1 activity. (a) E€ect of antioxidants on JNK1 activation by Doxorubicin (Dox) and H2O2.
Cells were pretreated with NAC (20 mM), ascorbic acid (100 mM),
or vitamin E (1 mM) for 1 h and then challenged with 3.2 mM Dox
or 800 mM H2O2 for 2 h. JNK1 activity was assayed as described
above; (b) E€ect of antioxidants on JNK1 activation by taxol and
colchicine. Cells were pretreated with antioxidants as above and
challenged with 1 mM taxol or 2.5 mM colchicine for 2 h before
assay for JNK1 activity; (c) E€ect of intracellular calcium
chelator BAPTA/AM on JNK1 activation by taxol and
colchicine. Cells were pretreated with 10 mM BAPTA/AM for
1 h, and then challenged with 1 mM taxol or 2.5 mM colchicine for
2 h. JNK1 activity was assayed as described in Materials and
methods. The experiments were performed three times with
similar results
because taxol and colchicine stimulated JNK1 activity
with di€erent kinetics. Activation of JNK1 by taxol was
transient, whereas the activation of JNK1 by colchicine
was sustained. Furthermore, a speci®c intracellular
calcium inhibitor, BAPTA/AM, inhibited JNK1 activation by taxol but not by colchicine. Identi®cation of such
downstream e€ectors of signaling cascades initiated
through stabilization and depolymerization of microtubules, which lead to JNK1 activation warrants further
study.
Activation of MAP kinases results in phosphorylation
of many cytosolic as well as nuclear substrates (Cobb
and Goddsmith, 1995). Activated JNK1 is known to
phosphorylate transcription factors, c-jun, ATF2, and
Elk-1, leading to transactivation of many genes,
including c-jun itself (Karin, 1995). Activated p38 can
also phosphorylate many transcription factors, such as
ATF2 and Elk-1, thereby enhancing their transcriptional
activity (Karin, 1995). Activation of JNK1 or p38 by
MBAs would, therefore, be expected to induce gene
expression. Indeed, treatment of MCF-7 cells with taxol
or colchicine increased mRNA levels of the immediate
early genes, c-jun and ATF3. The dose response and the
time course of c-jun and ATF3 induction correlated well
with those of JNK1 activation by these two agents.
Consistent with our results, previous studies have shown
that rearrangement of microtubules induced phosphorylation of c-jun and transcriptional activity of AP-1
(Manie et al., 1993), which may contribute to the upregulation of AP-1-dependent genes, such as urokinasetype plasminogen activator, stromelysin, collagenase,
and a- and b-actin (Unemori and Werb, 1986; Matrisian
et al., 1986; Buckler et al., 1988; Sympson and
Geoghegan, 1990). Recently, nuclear factor kappa B
(NF-kB) has been shown to be activated by JNK1 or its
upstream activator MEKK1 (Meyer et al., 1996; Lee et
al., 1997). Thus, activation of JNK1 may also provide a
plausible mechanism for the induction of NF-kBdependent genes by microtubule-depolymerizing agents
(Rosette and Karin, 1995).
Activation of JNK1 has been shown to be a
common phenomenon in apoptotic cell death. This
activation is required for apoptosis induction by
growth factor withdrawal (Xia et al., 1995), radiation
(Chen et al., 1996), heat shock (Zanke et al., 1996), and
ceramide (Verheij et al., 1996). Recently, Yang et al.
(1997) reported that JNK1 activation also mediated
Fas-induced apoptosis. On the other hand, JNK may
not be essential for tumor necrosis factor- or
dexamethasone-mediated apoptosis (Liu et al., 1996;
Chauhan et al., 1997). Thus, the importance of JNK1
activation seems to vary, depending on the nature of
apoptosis-inducing agents. In the present study, we
found that higher concentrations of taxol or colchicine
were required to activate JNK than to induce cell
death. For example, treatment of cells with 100 nM of
taxol or colchicine caused a signi®cant amount of cell
death (Table 1), but did not activate JNK1 even after
24 h of treatment (Figure 7a and b). This suggests that
taxol or colchicine can induce cell death without
activating JNK. Since the action of these drugs is
highly dose-dependent, and we have seen enhanced cell
death upon treatment with 1 mM taxol or with 2.5 M
colchicine, it would be interesting to determine whether
JNK1 activation at such concentrations potentiates
taxol- or colchicine-induced cell death.
In summary, our ®ndings suggest that an intracellular signaling machinery may be involved in sensing
the integrity of the microtubule cytoskeleton and in
initiating cellular responses in reaction to changes in
microtubule dynamics. Tubulin is known to have
GTPase activity and to act as a GTP-binding protein.
Therefore, it is possible that tubulin itself may function
as an activator of the JNK pathway as seen with Rac1/
Cdc42 (Coso et al., 1995). Alternatively, microtubules
may interact with other GTP-binding proteins, as actin
does (Symons, 1996), to initiate downstream signaling.
Future studies focusing on the downstream e€ectors
and the physiological consequences of JNK1 or p38
activation may well unravel a novel mechanism for the
regulation of MAP kinases and for the chemotherapeutic action of anti-microtubule drugs.
Materials and methods
Cell culture and drugs
MCF-7 and BT-20 human breast cancer cells (American
Type Culture Collection, Rockville, MD, USA) were
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
propagated in Dulbecco's modi®ed Eagle's medium supplemented with 10% fetal bovine serum, 0.1 mM non-essential
amino acids, 2 mM L-glutamine (Gibco BRL, Grand Island,
NY, USA), 100 U/ml penicillin and 50 U/ml streptomycin at
378C, in a humidi®ed atmosphere containing 5% CO2. Cells
were routinely tested and found to be negative for
Mycoplasma. For experiments with MAP kinase activation,
cells were incubated overnight in medium without serum and
then treated with the pharmacological agents. The experiments with BAPTA/AM (Calbiochem, La Jolla, CA, USA)
were performed in Ca2+-free Eagle's medium (Sigma
Chemical Co., St Louis, MO, USA). Taxol (paclitaxel),
colchicine, vincristine, podophyllotoxin, nocodazole, and blumicolchicine (Sigma) were dissolved in ethanol as 10006
stock solutions and kept at 7208C. NAC (Sigma) was
dissolved in culture medium prior to the experiments and pH
was adjusted to 7.2.
In vitro immunocomplex assay of JNK1, ERK and p38 kinase
activity
After treatment with drugs, cells were harvested, washed
three times with ice-cold PBS, pH 7.2 and lysed in 0.7 ml
of lysis bu€er (10 mM Tris-HCl, pH 7.1, 50 mM NaCl,
30 mM inorganic sodium pyrophosphate, 50 mM NaF,
100 mM sodium orthovanadate, 2 mM iodoacetic acid,
5 mM ZnCl2, 1 mM phenylmethylsulfonyl ¯uoride, and
0.5% Triton X-100). The lysates were homogenized by
passing through a 23 G needle three times, incubated on ice
for 15 min. and centrifuged (12 000 r.p.m., 10 min at 48C).
The supernatants were transferred to fresh tubes and JNK1
activity was determined as described (Yu et al., 1996a).
Brie¯y, equal amounts of total protein, as determined by
the Bio-Rad Protein Assay, were incubated with rabbit
anti-JNK1 serum (Ab 101) (Meyer et al., 1996) pre-bound
to protein A- Sepharose 4B (Zymed Laboratories, San
Francisco, CA, USA) for 2 h at 48C. The immunocomplexes were then washed twice with lysis bu€er and twice
with kinase assay bu€er (20 mM HEPES, pH 7.9, 10 mM
MgCl2, 2 mM MnCl2, 50 mM b-glycerophosphate, 10 mM pnitrophenyl phosphate, and 100 mM sodium orthovanadate). The kinase reaction was initiated by resuspending
the immunocomplexes in 30 ml of kinase assay bu€er
containing 10 mg of GST-c-jun fusion protein (amino acids
1 ± 79) (Hibi et al., 1993), 2 mCi [g-32P]ATP (3000 Ci/mmol,
NEN-Dupont, Boston, MA, USA) and 20 mM ATP. After
incubation for 30 min. at 308C the reaction was terminated
by adding 10 ml of 46 Laemmli bu€er and heating to 958C
for 5 min. The samples were resolved in 10% SDS ± PAGE.
The phosphorylation of GST-c-jun fusion protein was
visualized by autoradiography and quantitated on a
PhosphoImager (AMBIS, Inc., San Diego, CA, USA).
The assays for ERK2 and p38 kinase activities were
performed according to the procedures described above for
the JNK1 assay with the following modi®cations. After
immunoprecipitation of the lysates with anti-ERK2 or p38
antibody (Santa Cruz Biotech., Santa Cruz, CA, USA), the
kinase activity was determined using 10 mg of myelin basic
protein (Sigma) or 2 mg GST-ATF2 as the substrate for
ERK2 and p38, respectively. Reactions were performed at
308C for 15 min and the phosphorylated products were
resolved in 13.5% SDS ± PAGE and quantitated as described
above.
RNA isolation and RT ± PCR
Total RNA was isolated with TRIzol reagent (Gibco) as
recommended by the manufacturer. RNA (0.5 ± 1 mg) was
treated with 1 U of RNase-free DNase I (Epicentre Tech.,
Madison, WI, USA) for 30 min at 378C followed by
inactivation of the enzyme at 708C for 15 min. RNA was
then reverse transcribed using the Superscript Preamplifi-
cation System (Gibco). Complementary DNA was treated
with 0.1 U/ml E.coli RNase H at 378C for 20 min. PCR was
performed with amounts of cDNAs corresponding to 50 ng
of total RNA, 200 ng/ml of each amplimer (Integrated
DNA Tech., Coralville, IA, USA), 200 nM of each
deoxynucleotide triphosphate, 1 mCi [a-32P]deoxycytidine
triphosphate (Amersham, Arlington Heights, IL, USA,
3000 Ci/mmol), 1.5 mM MgCl2 and 1 U of Taq DNA
polymerase (Gibco). In preliminary experiments, the
optimal numbers of PCR cycles were found to be 23 for
c-jun, 26 for ATF3, and 26 for b2-microglobulin (internal
standard) cDNAs. These numbers yielded PCR products
within the linear exponential range. Each cycle consisted of
30 s at 948C, followed by 30 s at 608C and 1 min at 728C.
The sequence of the primers were presented elsewhere (Yu
et al., 1996a). Since c-jun is an intronless gene (Hattori et
al., 1988) very small amounts of DNA present in the RNA
preparations could be a source of false-positive signals in
PCR. Preliminary PCR was carried out with the omission
of the reverse transcription step, i.e., DNase-treated RNA
was used directly as the template. No detectable signals
were visualized with non-reverse transcribed RNAs after 30
cycles (data not shown). PCR products were ampli®ed on a
OmniGene Temperature Cycler (Hybaid Ltd, Woodridge,
NJ, USA) in separate tubes (Yu et al., 1996a, Komarov et
al.,1996), mixed and resolved in 7.5% PAGE. The gels
were dried and exposed to Kodak X-ray ®lm for 2 h at
7708C.
Immunocytochemical detection of tubulin
MCF-7 cells were grown on glass coverslips for 36 h,
treated with either taxol, colchicine, or doxorubicin for
indicated times (see Results), washed with solution A (PBS
containing 0.1 mM CaCl2 and 1 mM MgCl2, pH 7.2), ®xed
with bu€ered 3% paraformaldehyde (10 min) and permeabilized with 0.5% Triton X-100 (1 min). Non-speci®c
binding was blocked by incubating in solution A containing 5% normal goat serum, 1% bovine serum albumin, and
0.5% Tween-20. Cells were then incubated with mouse
anti-rat b-tubulin monoclonal antibody (TUB2.1; Sigma)
diluted 1 : 200 in solution A for 1 h, washed, and then
incubated with FITC-conjugated rabbit anti-mouse IgG
(Chemicon, Temecula, CA, USA; diluted 1 : 500). Cells
were examined using a Zeiss Axioscop microscope
equipped with a 100 W mercury epi¯uorescence illuminator and ®lter sets for ¯uorescein and for propidium iodide
(PI). Cells were photographed using a 1006 oil immersion
lens with either Kodak Tri-X or Pro 400MC color ®lm (400
ASA).
Cell cycle analysis
Exponentially growing MCF-7 cells (106 per treatment)
were treated with colchicine or taxol, washed with ice-cold
PBS, and resuspended in staining solution (0.1% sodium
citrate, 0.3% Nonidet P-40, 100 mg/ml RNase A and 50 mg/
ml propidium iodide, PI). The cell lysates were vortexed at
high speed for 1 min and incubated on ice for 45 min.
DNA content (PI ¯uorescence) was measured using an
EPICS ELITE ESP ¯ow cytometer (Coulter Corp.,
Hialeah, FL, USA) equipped with an argon laser for
488 nm excitation. The PI ¯uorescence was collected after
passage through the standard optics and a 610 nm
bandpass ®lter. Cell cycle analyses were performed using
the MULTICYCLE Av Program (Phoenix Flow Systems,
San Diego, CA, USA).
Abbreviations
ATF, activation transcription factor; BAPTA/AM, 1,2-bis(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-
383
Activation of MAP kinases by microtubule-binding agents
AA Shtil et al
384
(acetoxymethyl)-ester; ERK, extracellular signal-regulated
protein kinase; GST, glutathione S-transferase; JNK, c-jun
N-terminal kinase; MAPK, mitogen-activated protein
kinase; MBA, microtubule binding agent; NAC, N-acetylL-cysteine; ROI, reactive oxygen intermediates
Acknowledgements
The authors are grateful to Drs V Gelfand and T Ignatova
for critical discussion of the manuscript. This work was
supported by National Institutes of Health grants
R37CA40333 (to IBR), R01-AI38649 (to T-HT) and R01ES06887 (to ANTK). AAS is a Fellow of the Oncology
Research Faculty Development Program of the National
Cancer Institute.
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