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INTRODUCTION |
Apoptosis is a cell death process important in development and
tissue homeostasis (1, 2). Dysregulation of apoptosis contributes to
human diseases including cancer (1). The treatment of many cancers
involves chemotherapeutic drugs that are genotoxins (damage DNA) and
microtubule-interfering toxins (3-5). Examples of each are etoposide,
a topoisomerase inhibitor that functions as an effective genotoxin (4,
6), and taxol, a microtubule stabilizer that effectively arrests cells
in the G2/M phase of the cell cycle (7). Both genotoxins
and microtubule-interfering drugs induce apoptosis in tumor cells (6,
8-11).
It is generally believed that the caspase cleavage of specific proteins
results in the irreversible commitment of cells to undergo apoptosis
(12-14). Caspases are regulated in part by the anti-apoptotic protein,
Bcl2 (15). Overexpression of Bcl2 inhibits the activation of caspases
and blocks genotoxin and taxol-induced apoptosis (13, 15-17). The
anti-apoptotic action of Bcl2 is believed to involve its interaction
with Apaf1, the Ced4 homologue that binds to and controls the
activation of caspase-9 (18-20). Alternatively, Bcl2 interaction with
Bax prevents the pro-apoptotic action of Bax by forming Bcl2-Bax dimer
(15).
We have recently demonstrated that caspases turn off survival signals
in addition to activating death signals (21).
MEKK11 is among the signal
transduction proteins cleaved by caspases early in the apoptotic
response (21, 22). Caspase-3, also referred to as CPP32, cleaves MEKK1
at Asp874, releasing a COOH-terminal 91-kDa fragment that
encodes the MEKK1 kinase domain (22). Transient overexpression of MEKK1
results in the cleavage of MEKK1 generating the 91-kDa kinase fragment. The cleavage of MEKK1 and activation of its kinase activity leads to
further activation of caspases. Kinase inactive MEKK1 effectively reduces caspase activation induced by genotoxins (22). This suggests
that MEKK1 cleavage is involved in the amplification of caspases and
thus contributes to apoptosis.
Herein, we show that kinase inactive MEKK1 blocks etoposide-induced
apoptosis but fails to block microtubule-interfering toxin-induced apoptosis. Treatment with etoposide resulted in cleavage of MEKK1 that
was not detected following taxol or vinblastine treatment of cells.
Thus, MEKK1 contributes to genotoxin-induced apoptosis, whereas
microtubule-interfering toxins induce apoptosis independent of MEKK1
cleavage. Our findings illustrate the existence of two different
signaling pathways, one responsive to DNA damage and the other to
microtubule toxins that differentially involve
caspase-dependent MEKK1 cleavage.
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EXPERIMENTAL PROCEDURES |
Materials--
1-
-D-Arabinofuranosylcytosine
(Ara-c), etoposide, taxol, and vinblastine were purchased from Sigma.
Ara-c was dissolved in phosphate-buffered saline, whereas etoposide,
taxol, and vinblastine were dissolved in dimethyl sulfoxide
(Me2SO). Anti-MEKK1 antiserum (c-22) was purchased from
Santa Cruz, and anti-poly(A) ribose polymerase (PARP) antiserum was
purchased from Upstate Biotechnology.
Cells--
Jurkat cells were cultured in RPMI 1640 medium (Life
Technologies Inc.) supplemented with 100 units/ml
penicillin/streptomycin (Gemini Bio-Products) and containing 10% fetal
bovine serum (Summit Biotechnology) (RPMI-c). Human embryonic kidney
293 cells (HEK293) were grown in Dulbecco's modified Eagle's medium
(Life Technologies Inc.) supplemented with 100 units/ml
penicillin/streptomycin (Gemini Bio-Products) and containing 10%
bovine calf serum (Hyclone).
Immunoblots--
Jurkat cells were incubated with 10 µm
Ara-c, 10 µM etoposide, 10 µM taxol, or 1 µM vinblastine. HEK293 cells were incubated with 100 µM etoposide or 10 µM taxol. Lower
concentrations showed reduced apoptosis in a dose-response curve (data
not shown). The cells were lysed as described previously (29).
Measurement of Caspase Activities--
Cells were lysed in 50 mM Tris, pH 7.4, 1 mM EDTA, 10 mM
EGTA, and 10 µM digitonin for 10 min at 37 °C. Lysates
(120 µg) were incubated with 5 µM DEVE-AMC (Bachem) in
1 ml of 50 mM Tris, pH 7.4, 1 mM EDTA, 10 mM EGTA for 30 min at 37 °C. Fluorescence was monitored
with an excitation wavelength of 380 nm and an emission wavelength of
460 nm.
Measurement of Apoptosis--
Jurkat cells (1-2 × 106) were resuspended in 100 µl of incubation buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 5 mM CaCl2) containing 1 µg/ml propidium iodide
(Sigma) and a dilution of 1:50 Annexin V-Flos solution (Roche Molecular
Biochemicals) and incubated on ice for 15 min. Four hundred µl of
incubation buffer was then added, and the cells were sorted on a flow
cytometer using 488-nm excitation and a 515-nm band-pass filter for
fluorescence detection and a filter >560 nm for propidium iodide
detection. Apoptotic cells were defined as green fluorescent positive
and propidium iodide negative. HEK293 cells were stained with acridine orange (100 µg/ml) and ethidium bromide (100 µg/ml) in
phosphate-buffered saline as described previously (22). The percentage
of apoptotic cells was determined from cells containing normal DNA
staining compared with cells with condensed DNA.
MEKK1 in Vitro Kinase Assay--
MEKK1 was immunoprecipitated
from cell lysates (500 µg) with antibodies raised against specific
sequences of MEKK1 (22). The immunoprecipitates were used in an
in vitro kinase assay with recombinant kinase, inactive SEK1
(SEK1 K-M), as described previously (22). The samples were analyzed by
SDS-polyacrylamide gel electrophoresis after which the gel was fixed in
methanol. The extent of MEKK1 autophosphorylation and SEK1 K-M
phosphorylation was determined on a PhosphorImager (Molecular Devices).
JNK Assay--
JNK activity was measured by a solid-phase kinase
assay using glutathione S-transferase
(GST)-c-Jun1-79 (GST-Jun) bound to glutathione-Sepharose
4B beads to affinity purify JNK from cell lysates as described
previously (23). Quantitation of the phosphorylation of GST-Jun was
performed with a PhosphorImager (Molecular Devices). In other studies,
similar results were obtained by immunoprecipitation of JNK from cell
lysate as with GST-Jun beads.
Transfections--
HEK293 cells were transfected with the
plasmid pcDNA3 containing the open reading frame of Bcl2.
Transfections were performed using LipofectAMINE (Life Technologies,
Inc.). After 2 days, G418 (1.5 mg/ml) was added to the cells, and
clones were isolated and analyzed for Bcl2 overexpression. Positive
clones overexpressing Bcl2, detected by Western blotting, relative to
cells transfected with vector alone were isolated. Transient
transfection of pcDNA3 containing the open reading frame of MEKK1
was performed using LipofectAMINE.
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RESULTS |
MEKK1 Involvement in Apoptosis--
In HEK293 cells, the genotoxin
etoposide, but not the microtubule toxin taxol, induced the cleavage of
the 196-kDa MEKK1 protein at concentrations that maximize the apoptotic
response indicated by the loss of full-length MEKK1 (Fig.
1A). The cleavage fragments of
endogenous MEKK1 were not detected consistent with the other cleaved
proteins in HEK293 cells (21, 22). Indeed, total 196-kDa MEKK1 protein
levels actually increased in response to taxol over the same 48-h time
course where etoposide treatment caused a loss of MEKK1 protein.
Differences in MEKK1 protein levels was not because of differences in
general protein amounts because protein levels of MAPK phosphatase 1 (MKP1), that is not cleaved in response to apoptotic stimuli (21),
remained similar in HEK293 cells following treatment with taxol or
etoposide. Caspase activation and apoptotic response by taxol, however,
was similar in magnitude to that for etoposide (Fig. 1B). A
second microtubule-disrupting agent, vinblastine, also failed to induce
cleavage of full-length MEKK1 in HEK293 cells (data not shown) but
induced caspase activity and an apoptotic response similar to taxol and
etoposide (Fig. 1B).

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Fig. 1.
Cleavage of MEKK1 following treatment of
genotoxins or microtubule-interfering toxins. A, HEK293
cells were treated with 100 µM etoposide (i)
or 10 µM taxol (ii) for the times indicated.
Cells were Western blotted using -MEKK1 antibodies (1:100) or
-MKP-1 antibodies (1:200) as described under "Experimental
Procedures." The level of expression of MEKK1 and MKP-1 was
determined by densitometry (arbitrary units). B, HEK293
cells were treated with 100 µM etoposide, 10 µM taxol, and 1 µM vinblastine for the
indicated times. B, i, cells were lysed in
caspase lysis buffer, and caspase activity is shown as caspase
fluorescence units, determined by cleavage of a fluorescent DEVD
peptide and quantitated by fluorimetry; ii, cells were
stained with acridine orange and counted using a fluorescent microscope
to quantitate condensed DNA. Cells having condensed DNA were scored as
apoptotic (total of 300 cells were scored). All experiments were done
in triplicate.
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The difference between DNA-damaging drugs and microtubule toxins
inducing MEKK1 cleavage was also observed in Jurkat cells. The
DNA-damaging drugs Ara-c and etoposide induce MEKK1 cleavage, shown by
loss of the 196-kDa enzyme (Fig.
2A), activation of caspases, and apoptosis (Fig. 2B); however, Arc-c was less effective
at inducing caspase activity and apoptosis as compared with etoposide. We have demonstrated previously that MEKK1 cleavage is
caspase-dependent (22). In contrast, taxol and vinblastine
treatment of Jurkat cells resulted in an initial increase in MEKK1
expression similar to that observed with HEK293 cells. Over time, MEKK1
protein levels in response to taxol diminished to that or somewhat
lower levels from that of control cells. This contrasts to HEK293 cells
where MEKK1 protein remained elevated following exposure of cells to taxol. Taxol and vinblastine stimulated caspase activity and induced apoptosis in Jurkat cells (Fig. 2B) similar to that observed
in HEK293 cells. These findings demonstrate a difference in the
cleavage and loss of the 196-kDa MEKK1 enzyme during the apoptotic
response of two cell types to DNA damage or microtubule poisoning.

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Fig. 2.
Cleavage of MEKK1 in Jurkat cells.
A and B, Jurkat cells were treated with
genotoxins: i, 10 µM Ara-c; ii, 10 µM etoposide, or microtubule-interfering toxins;
A, iii, 10 µM taxol; iv,
1 µM vinblastine for the times indicated. A,
cells were Western blotted with either -MEKK1 (1:100) or -PARP
(1:100) antibodies as described under "Experimental Procedures."
The amount of full-length MEKK1 was determined by densitometry as shown
as densitometry arbitrary units. B, i, caspase
activity as measured as in Fig. 1B. ii, annexin V
conjugated with fluorescein isothiocyanate was added to cells, and the
amount of annexin V positive cells (apoptotic cells) was determined by
fluorescence-activated cell sorter analysis. , Ara-c; , taxol;
, etoposide; , vinblastine.
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Although MEKK1 is not significantly proteolyzed in response to taxol or
vinblastine, other known caspase substrates were cleaved in response to
these drugs. PARP is a well characterized caspase substrate that is
cleaved early in the apoptotic response (24). Both the genotoxins
(Ara-c and etoposide) and microtubule toxins (taxol and vinblastine)
induced PARP cleavage (Fig. 2A) corresponding to increased
caspase activity and apoptosis (Fig. 2B) in Jurkat cells.
Similar results were found in HEK293 cells (data not shown). The
findings indicated that, in contrast to PARP, the degradation of MEKK1
is differentially regulated by DNA damage versus microtubule poisoning.
We have demonstrated that HEK293 cells that express a kinase-inactive
mutant of MEKK1 have a significantly reduced apoptotic response to
genotoxins such as etoposide (22). Treatment of HEK293 cells expressing
the kinase-inactive inhibitory mutant of MEKK1 with microtubule toxins
gave an apoptotic response similar to control HEK293 cells, measured by
acridine orange (Fig. 3A) or
propidium iodide staining (Fig. 3B). In contrast,
etoposide-induced apoptosis was significantly inhibited
(p < 0.05) in the HEK293 cells expressing
kinase-inactive MEKK1 relative to control cells (Fig. 3).

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Fig. 3.
Apoptosis of HEK293 cells expressing kinase
inactive MEKK1 following treatment with etoposide or
microtubule-interfering agents. HEK293 cells either expressing
vector alone ( ) or MEKK1 K-M ( ) were treated with 100 µM etoposide, 10 µM taxol, or 1 µM vinblastine for 24 h. A, the
percentage of apoptotic cells was determined as in Fig. 1B.
B, cells were stained with propidium iodide, and the extent
of sub-G1 peak (less than 2N DNA content) was determined by
fluorescence-activated cell sorter analysis. * represents the
p value for etoposide-treated cells compared with control
(p < 0.05), and error bars represent the
standard deviation of three separate experiments in both panels
A and B. C, cells were visualized by
digital confocal microscopy using a ×40 water objective.
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Morphological analysis substantiates the differences in apoptotic
potential of cells expressing kinase-inactive MEKK1 (Fig. 3C). In control cells, transfected with vector alone,
treatment with etoposide, taxol, or vinblastine caused membrane
blebbing and cell rounding. In cells expressing the mutant
kinase-inactive MEKK1 protein, the morphological changes induced by
etoposide but not taxol or vinblastine were inhibited. This finding
demonstrates that kinase-inactive MEKK1 inhibits morphological changes
associated with apoptosis induced by etoposide but not taxol or
vinblastine. The morphological findings are consistent with the
biochemical results that MEKK1 cleavage does not significantly
contribute to microtubule toxin-induced apoptosis.
Dissociation of Taxol-stimulated MEKK1 Activity and JNK
Activation--
MEKK1 has been shown to regulate JNK activity in a
variety of cell types (25-28). Exposure of HEK293 cells to
taxol-activated MEKK1 (Fig.
4A), as measured by MEKK1
autophosphorylation and MEKK1 catalyzed phosphorylation of recombinant
SEK1 (22, 23, 25, 28, 29). Etoposide activates MEKK1 to an extent
similar to that of taxol (Fig. 4A). However, taxol treatment
of HEK293 cells did not significantly activate JNK (Fig.
4B); etoposide in the same experiment activated JNK. Jurkat
cells gave a similar result where etoposide and taxol activated MEKK1
(data not shown), but only etoposide significantly activated JNK (Fig.
4B). In human breast carcinoma epidermal T47D cells and
mouse embryonic stem cells that we have similarly characterized, taxol
activates the JNK pathway but to a significantly lower magnitude than
other stress stimulants including DNA damaging drugs, irradiation, and microtubule-disrupting drugs such as nocodazole (data not shown). Thus,
taxol stabilization of microtubules appears to significantly dissociate
the activation of MEKK1 from the JNK pathway.

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Fig. 4.
MEKK1 and JNK kinase activity following taxol
treatment. A, HEK293 cells were treated with 10 µM taxol or 100 µM etoposide for the times
indicated. Following treatment of cells, a MEKK1 kinase assay was
performed using GST-kinase inactive SEK1 as a substrate as described
under "Experimental Procedures." MEKK1 autophosphorylation and
phosphorylation of kinase inactive SEK1 was determined as a fold
increase over control. Error bars represent the standard
deviation of three separate experiments. B, HEK293 cells and
Jurkat cells were treated with 100 or 10 µM etoposide,
respectively, or 10 µM taxol for times indicated. A JNK
assay was performed as described under "Experimental Procedures,"
and the extent of GST-c-Jun phosphorylation was determined. All
experiments were in done in triplicate.
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Bcl2 Blocks Apoptosis Downstream of MEKK1 Cleavage--
An
indicator of microtubule toxin treatment is the phosphorylation of Bcl2
(30-33) because of either interference with microtubule structure or
blockage of cells in the G2/M checkpoint of the cell cycle.
In HEK293 cells, taxol and vinblastine but not etoposide stimulated the
phosphorylation of Bcl2 as characterized by the shift in mobility by
SDS-polyacrylamide gel electrophoresis of the phosphorylated Bcl2
species (Fig. 5A). Expression
of the mutant kinase-inactive MEKK1, which suppressed etoposide but not
taxol-induced apoptosis (Fig. 3), had no effect on taxol or
vinblastine-stimulated Bcl2 phosphorylation (Fig. 5A). In
addition to Bcl2 phosphorylation, taxol also activates Cdc2 kinase
activity that has been proposed to be involved in taxol-induced
apoptosis (34). Expression of the kinase-inactive MEKK1 failed to
inhibit taxol-induced Cdc2 kinase activity as assessed by Cdc2
phosphorylation of histone (data not shown). Thus MEKK1 is not upstream
of either Cdc2 kinase or Bcl2 phosphorylation.

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Fig. 5.
Role of Bcl2 in taxol and MEKK1-induced
caspase activity and apoptosis. A, HEK293 cells were
treated with 10 µM taxol, 1 µM vinblastine,
or 100 µM etoposide; HEK293 cells stably expressing
vector alone or MEKK1 K-M were treated with 10 µM taxol
or 1 µM vinblastine. The cells were Western blot analyzed
with -Bcl2 antibodies (1:100) as described under "Experimental
Procedures." Bcl2 expression following taxol and vinblastine
treatment was similar in both vector alone and MEKK1 K-M expressing
cells. The arrow denotes gel-shifted Bcl2 shown to correlate
with phosphorylation. B, HEK293 cells expressing vector
alone or Bcl2 were treated with 10 µM taxol for 48 h, and the percentage of apoptotic cells were determined as in Fig.
1B. Bcl2 expression levels were increased 5-fold over
endogenous levels in HEK293 cells (data not shown). C, cells
expressing vector alone or Bcl2 were treated with 10 µM
taxol for 48 h, and the fold increase in caspase activity was
determined as in Fig. 1B. D, HEK293 cells having
empty vector or stably expressing Bcl2 were transfected with
full-length MEKK1 (FL MEKK1) or the 91-kDa kinase fragment
(91 kDa MEKK1) for 48 h. The percentage of apoptotic
cells was determined as in Fig. 1B. E, HEK293
cells expressing vector or Bcl2 were transfected with plasmid encoding
full-length MEKK1 for 48 h. Caspase activity was determined as in
Fig. 1B. Error bars represent standard deviations
of three separate experiments.
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Stable overexpression of Bcl2 in HEK293 cells effectively inhibited
apoptosis and caspase activation in response to taxol (Fig. 5,
B and C). Bcl2 overexpression also inhibited
etoposide-induced apoptosis (not shown). Interestingly, Bcl2
overexpression also inhibited apoptosis induced by overexpression of
full-length MEKK1 or the 91-kDa MEKK1 kinase fragment (Fig.
5D). The increased caspase activity observed with MEKK1
expression was also inhibited by Bcl2 overexpression (Fig.
5E). The findings demonstrate that Bcl2 inhibits
MEKK1-dependent and independent apoptosis in HEK293 cells.
Surprisingly, in Bcl2 overexpressing HEK293 cells, exposure to
etoposide induced the cleavage and loss of endogenous MEKK1. In
contrast, the cleavage of Cbl or PARP, both caspase substrates, was
inhibited by Bcl2 overexpression (21, 24) (Fig.
6A). The cleavage of MEKK1 was
dependent on caspases in control and Bcl2 overexpressing cells because
p35, the baculovirus inhibitor of caspases (35), blocked MEKK1
cleavage. These finding suggest that Bcl2 blocks MEKK1-induced
apoptosis and that Bcl2 functions downstream of MEKK1 in the
pathway leading to MEKK1-dependent apoptosis.

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Fig. 6.
Western blot of MEKK1 following transfection
of MEKK1 or treatment with etoposide. A) HEK293 cells
overexpressing either vector alone or Bcl2 were treated with 100 µM etoposide for 24 h. The cells were Western
blotted with -MEKK1 antibodies or -Cbl antibodies (1:100) as
described under "Experimental Procedures." Densitometry was
preformed to quantitate MEKK1 and Cbl expression and shown as
densitometry units. B) HEK293 having empty vector or stably expressing
Bcl2 were transfected with control vector, plasmid encoding HA-tagged
full-length MEKK1 or MEKK1 and the anti-apoptotic viral protein p35 for
24 h. The cells were lysed and Western blotted with -HA
antibodies (1:1000). Full-length MEKK1 is indicated by FL MEKK1 whereas
the cleavage fragments A and B represent the amino-terminal fragments
of MEKK1 encoding the HA-epitope tag. All experiments were done in
triplicate.
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DISCUSSION |
Because of their different mechanisms of action, genotoxins and
microtubule-interfering toxins are used in combination therapies for
the treatment of several different cancers (1, 9, 11). Both genotoxins
and microtubule toxins generally induce death of cells by apoptosis
(8). Based on the markedly different mechanisms of action, the
pro-apoptotic pathways regulated by genotoxins and microtubule toxins
to initiate apoptosis are predicted to be different. Our findings
demonstrate that both types of chemotherapeutic drugs (22) activate
MEKK1. Strikingly, only genotoxins like etoposide, and not the
microtubule toxins taxol or vinblastine, significantly induce the
cleavage of MEKK1 even though treatment of cells with each drug
activates the kinase activity of 196-kDa MEKK1 (data not shown). Thus,
genotoxins and microtubule drugs selectively and differentially
regulate the cleavage of specific caspase substrates. These results
define the differential regulation of caspase-dependent
cleavage of proteins during the apoptotic response to clinically
relevant chemotherapeutic drugs. A likely explanation for this
observation is that either MEKK1 or the caspase that cleaves MEKK1 is
differentially localized in the cell with etoposide versus
taxol treatment. The caspase that cleaves MEKK1 in response to
etoposide treatment is unable to cleave MEKK1 in taxol-treated cells
even though other proteins including Cbl and PARP are cleaved.
Several reports suggest that taxol-induced apoptosis is because of JNK
activation. In OVCA 420 cells, expression of MEKK1 kinase inactive
mutant effectively blocked JNK activity and blocked taxol-induced
apoptosis (36-38). In contrast, both Jurkat and HEK293 cells activate
MEKK1 activity following taxol treatment but failed to activate JNK. In
addition, expression of the kinase inactive MEKK1 in HEK293 cells
failed to block taxol-induced apoptosis. Murine embryonic stem cells
activate JNK following treatment with the microtubule toxin nocodazole.
This response is absent in embryonic stem cells lacking MEKK1; however,
the cells still undergo apoptosis (38). Thus JNK activation in response
to taxol is not required to induce apoptosis.
Overexpression of Bcl2 did not block the caspase-dependent
cleavage of MEKK1. In contrast, cleavage of Cbl and PARP was inhibited. Western blotting of caspase-3 in Bcl2 overexpressing cells indicates that caspase 3 is activated determined by the loss of the inactive procaspase form of caspase 3 following etoposide treatment. Caspase-9 activation, however, was blocked by overexpression of
Bcl2.2 Taken together, the
data show that cleavage of MEKK1 possibly by caspase 3 in the presence
of Bcl2 overexpression does not induce apoptosis. Thus, the ability of
Bcl2 overexpression to block MEKK1-induced apoptosis, and amplification
of caspase activity implies that caspases activated in response to
MEKK1 activation and subsequent cleavage are functionally downstream of Bcl2.
Our results show that altered regulation of Bcl2, Cdc2 kinase activity,
or related regulatory proteins in response to microtubule toxins
effectively bypasses the role of MEKK1 in amplifying caspase activation. The different apoptotic signaling pathways for etoposide and taxol probably contribute to their greater effectiveness in chemotherapy in combination than either alone.
The importance of these findings is that we have identified for the
first time the differential involvement of a pro-apoptotic kinase,
activated 91-kDa MEKK1, in chemotherapy-induced apoptosis. Thus, we
have defined a kinase whose regulation influences the action of
specific classes of chemotherapeutic drugs. This implies that it will
be possible to define pro- and anti-apoptotic components of the cell
death pathway, such as MEKK1, that selectively regulate the apoptotic
potential of specific drugs. In the future, it may be possible to alter
the activity of such kinases to enhance the apoptotic potential of
tumor cells. Such advances in apoptotic signal transduction therapy
will allow chemotherapy and anti-angiogenic therapy to have
significantly greater efficacy in ablating human tumors.