(Received for publication, October 2, 1996, and in revised form, February 19, 1997)
From the Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206
Paclitaxel can induce tumor necrosis factor (TNF)
and interleukin-1 gene expression, similar to lipopolysaccharides.
Since lipopolysaccharide-induced expression of TNF is related to
activation of NF-B, we determined whether NF-
B could be activated
by paclitaxel. In the human lung adenocarcinoma cell line A549,
paclitaxel activated NF-
B in a dose-dependent manner
with maximal activation after 2-4 h. Since paclitaxel could
up-regulate TNF and interleukin-1 secretion and subsequent NF-
B
activation could be caused by these cytokines, the effect of two other
groups of anticancer drugs including vinca alkaloids (vinblastine and
vincristine) and anthracyclines (daunomycin and doxorubicin), neither
of which induce TNF or interleukin-1 gene expression, were examined.
Like paclitaxel, vinblastine, vincristine, daunomycin, and doxorubicin
each caused activation of NF-
B. Therefore, it is unlikely that
activation of NF-
B caused by these agents or by paclitaxel is
mediated via cytokine up-regulation. Furthermore, actinomycin D and
cycloheximide, inhibitors of transcription and translation,
respectively, did not inhibit paclitaxel-induced NF-
B activation.
Several other transcription factors such as AP-1, AP-2, CREB, SP-1, or
TFIID were not activated by antineoplastic agents demonstrating
specificity of NF-
B activation. The involvement of both subunits in
the NF-
B DNA binding complex was demonstrated by its abrogation by
anti-p65 and by supershift by anti-p50 antibodies. Since protein
phosphorylation is implicated in the activation of NF-
B, the effect
of anticancer drugs on protein kinase C activity was measured.
Vincristine, daunomycin, and paclitaxel significantly increased protein
kinase C activity, and vinblastine and doxorubicin caused similar
trends. Following treatment with antineoplastics (1-4 h), cytoplasmic
I
B
degradation occurred concomitantly with translocation of p65
to the nucleus. Specific protein kinase C inhibitors
(bisindolylmaleimide (GF109203X) and calphostin C) blocked the
activation of NF-
B by each compound. Hence, protein kinase C
activation may contribute to NF-
B activation by antineoplastic agents.
Paclitaxel, a diterpene compound, was originally isolated from the
stem bark of Taxus brevifolia and shown to have
antiproliferative activity against various cultured cells as well as
antineoplastic activity in tumor patients (1). These effects of
paclitaxel appear to be related to its ability to bind to tubulin, to
promote microtubule assembly, and to stabilize microtubules by bundle formation (2-4). Recently, Ding et al. (5). have found
paclitaxel to exhibit cell cycle-independent, endotoxin-like effects on
murine macrophages. Paclitaxel, like endotoxin (lipopolysaccharide;
LPS),1 causes murine macrophages to
down-regulate TNF receptors and initiate synthesis and secretion of
TNF. In a similar fashion, paclitaxel can induce expression of both
IL-1 and IL-1
(6). In addition, paclitaxel also induces tyrosine
phosphorylation of microtubule associated protein kinases (7).
Likewise, paclitaxel enhances
-interferon induction of nitric oxide
synthase and secretion of nitric oxide (6), a macrophage tumoricidal
factor. The pathways linking these responses to paclitaxel are believed
to be similar to those causing such responses to LPS (5). Thus, an
intracellular target affected by paclitaxel might be involved in
actions of LPS in macrophages and other cells. Determination of which
intracellular molecule paclitaxel and LPS affect in common could
provide further insight into the actions of LPS on mammalian cells and
participation of the cytoskeleton in responses of cells to their
environment. One potential intracellular target of these compounds is
the transcription factor NF-
B. NF-
B, named for its ability to
recognize a
light chain immunoglobulin gene regulatory element, can
participate in the regulation of numerous genes (reviewed in Baeuerle
(8)). Under basal conditions, it usually exists as a heterodimer of 50- and 65-kDa subunits bound to an inhibitor protein I
B in the cytoplasm. Various stimuli cause I
B to dissociate from the complex allowing the heterodimer to migrate to the nucleus and activate gene
expression. Because NF-
B is involved in several gene expression phenomena caused by LPS (9), we sought to determine if NF-
B is
activated by paclitaxel in the pulmonary adenocarcinoma cell line
A549.
Herein, we report that paclitaxel can cause NF-B activation. Because
paclitaxel can induce cytokines such as TNF and IL-1, which could then
activate NF-
B (8), we assessed the effect of additional classes of
antitumor drugs, vinca alkaloids, and anthracyclines, which do not
induce TNF or IL-1 secretion. The mechanism of action of the vinca
alkaloids is related to the inhibition of microtubule formation in the
mitotic spindle resulting in an arrest of the dividing cells at
metaphase (10). By contrast, the mechanism of action of anthracyclines
is related to their intercalation into DNA (11) and inhibition of RNA
transcription (12). This interaction with DNA confers the cytotoxic
effect of these agents. These drugs also cause DNA strand breaks that appear to be mediated by DNA topoisomerase II (13). Thus, the mode of
action, structure, and function of paclitaxel is entirely different
than that of vinca alkaloids and anthracyclines. Nonetheless, each of
these classes of antineoplastics shared the property of NF-
B
activation.
Because protein kinases are involved in NF-B activation by a variety
of agents (14, 15), we investigated a potential role for these proteins
in NF-
B activation by antineoplastics. Protein kinase C (PKC) is a
calcium and phospholipid-dependent protein kinase that
plays an important role in signal transduction pathways regulating cell
growth (16). In some drug-resistant cell lines, elevation of PKC
activity occurs in response to antitumor drugs (17). Therefore, the
effect of antitumor drugs on the activity of protein kinase C was
examined. In addition, the effects of calphostin C, a specific
inhibitor of PKC, which binds to its regulatory region (18), and the
bisindolylmaleimide GF109203X, which specifically inhibits by binding
to the catalytic region of PKC, were assessed relative to the
activation of NF-
B by anti-tumor drugs.
Vinblastine (Velban®, Lilly) and vincristine (Oncovin®, Lilly) were obtained commercially. Paclitaxel (Taxol®) was obtained from Bristol-Meyers Squibb Company. Doxorubicin hydrochloride, daunomycin hydrochloride, and cremophor EL were obtained from Sigma. Anti-p50 and anti-p65 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other materials were obtained in the highest available grade.
Cell Culture and TreatmentsA549 cells (adenocarcinoma cells) were grown in F12-K (Kaigan's modified) supplemented with 10% fetal calf serum and 100 units of penicillin/streptomycin. Confluent monolayers were treated with various concentration of drugs for various time periods as indicated in the figure legends. Cell viability was determined by the trypan blue exclusion method. Cell viability was unchanged by any of the agents studied over the time courses (up to 4 h) and at the dosages studied. Paclitaxel was dissolved in Me2SO.
Nuclear Extract PreparationNuclear extracts were prepared
by the method of Staal et al. (20) with the following
modifications. Approximately 107 cells were washed in 10 ml
of phosphate-buffered saline and centrifuged (1,500 × g for 5 min). The pellet was resuspended in
phosphate-buffered saline (1 ml), transferred into an Eppendorf tube,
and centrifuged again (16,000 × g; 15 s).
Phosphate-buffered saline was removed, and the cell pellet was
resuspended in 400 µl of buffer A (10 mM HEPES, pH 7.8, 10 mM KCl, 0.1 mM EDTA, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride,
leupeptin (0.5 mg/ml), antipain (0.3 mg/ml)) by gentle pipetting. The
cells were allowed to swell on ice (15 min), after which 25 µl of
10% Nonidet P-40 (Sigma) were added, and the tube was vortexed
vigorously for 10 s. The homogenate was centrifuged for 30 s
in a microcentrifuge. The nuclear pellet was resuspended in buffer C
(20 mM HEPES, pH 7.8, 0.42 M NaCl, 5 mM EDTA, 5 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, 10% (v/v) glycerol), and the tube was
rocked gently at 4 °C for 30 min on a shaking platform. The nuclear
extract was centrifuged for 10 min in a microcentrifuge at 4 °C, and
the supernatant was frozen at 70 °C in aliquots until
electrophoretic mobility shift assay (EMSA) was done. Protein was
quantified by Bradford assay (Bio-Rad) (21).
For the EMSA the
following specific oligonucleotides were obtained from Promega and
end-labeled using T4 polynucleotide kinase (Life
Technologies, Inc.) and [-32]P ATP (ICN) in 10 × kinase buffer (0.5 M Tris-HCl, pH 7.5, 0.1 M
MgCl2, 50 mM dithiothreitol, 1 mM
spermidine, and 1 mM EDTA). For competition studies, 3.5 pmol of unlabeled oligonucleotides were used. Oliognucleotides were:
NF-
B, 5
AGT TGA GGG GAC TTT CCC AGG C 3
; AP-1, 5
CGC TTG ATG AGT
CAG CCG GAA 3
; SP1, 5
ATT CGA TCG GGG CGG GGC GAG C 3
; AP-2, 5
GAT
CGA ACT GAC CGC CCG CGG CCC GT 3
; CREB, 5
AGA GAT TGC CTG ACG TCA GAG
AGC TAG 3
; TFIID, 5
GCA GAG CAT ATA AGG TGA GGT AGG A 3
. 1.75 pmol was end-labeled with [
-32P]ATP (ICN) using T4
polynucleotide kinase (Life Technologies, Inc.) in 10 × kinase
buffer. The labeled double-stranded oligonucleotide was separated from
free [
-32P]ATP using a G-50 Sephadex spin column (5 Prime
3 Prime, Inc., Boulder, CO). The nuclear
protein-32P-labeled oligonucleotide complex was separated
from free 32P-labeled oligonucleotide by electrophoresis
through a 6% native polyacrylamide gel in a running buffer of
0.25 × TBE (5 × TBE = 500 mM Tris, pH 8.0, 450 mM borate, 5 mM EDTA). The binding reaction
was performed according to a modified method of Garner and Revzin (22).
The reaction mixture without labeled oligonucleotide was preincubated
for 15 min at 4 °C followed by a 20-min incubation after addition of
labeled oligonucleotide at room temperature. The binding reaction
contained 10 µg of sample protein, 5 µl of 5 × incubation
buffer (20% glycerol, 5 mM MgCl2, 5 mM EDTA, 5 mM dithiothreitol, 500 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.4 mg/ml calf
thymus DNA). The binding reaction also contained poly(dI-dC) (2 µg;
Pharmacia Biotech Inc.) in a total reaction volume of 25 µl.
For supershift assay some of the binding reactions contained 150 ng of anti-p50 or anti-p65 (Santa Cruz Biotechnology) along with 2 µg of poly(dI-dC) (Pharmacia). Binding reactions were incubated at room temperature for 15 min followed by additional incubation for 3 h at 4 °C.
Western Blot Detection of INuclear extract
was prepared as described earlier. Post nuclear supernatant was treated
as the cytoplasmic extract. Nuclear or cytoplasmic extracts (25 µg)
were separated by electrophoresis in a 12% polyacrylamide gel under
denaturing and reducing conditions. Protein was transferred to a
nitrocellulose membrane (Hybond-ECL, Amersham Corp.) and immunoblotted
with monoclonal anti-IB
or anti-p65 (Santa Cruz Biotechnology)
antibodies, and visualized by the ECL-system (Amersham) using
anti-rabbit horseradish peroxidase IgG (Sigma).
PKC activity was determined using PKC assay kit of Life Technologies, Inc. based on the measurement of the phosphorylation of acetylated myelin basic protein as described by Yasuda et al. (23). Briefly, 5 × 106 cells were washed in phosphate-buffered saline followed by scraping of the monolayers in 0.5 ml of buffer A (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 µg/ml each of aprotinin and leupeptin). Cells were homogenized with 10-15 strokes of a precooled Dounce homogenizer with a B-type pestle at 4 °C. Following incubation of the cell homogenate with buffer A for 30 min, the lysate was centrifuged, and the supernatant was applied to a DEAE ion exchange column (Bio-Rad). Partially purified PKC was eluted with 5 ml of buffer C (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM 2-mercaptoethanol, 0.2 M NaCl). The DEAE eluate (25 µl) was assayed for PKC activity using 50 µM acetylated myelin basic protein as a substrate. Activity of PKC was expressed as pmol/min/5 × 106 cells.
Activation of NF-B by Paclitaxel
To investigate whether paclitaxel induces
NF-B activation, we treated A549 cells with various concentration of
paclitaxel. Nuclear extracts were examined for NF-
B binding activity
by EMSA using consensus NF-
B binding sequences. As shown (Fig.
1A), paclitaxel increased NF-
B activation
in a dose-dependent manner (lanes 6-9). As
little as 7 µM paclitaxel activated NF-
B.
Time Course
Paclitaxel treatment resulted in a rapid
activation of NF-B binding activity. NF-
B binding activity was
detectable after 1 h, and continued to increase through 4 h
(Fig. 1B). Thus, induction of NF-
B by paclitaxel is a
relatively rapid response.
Effect of Actinomycin D and Cycloheximide on NF-B Activation by
Paclitaxel
To determine whether secretion of TNF- or IL-1 by paclitaxel
(5, 6) is responsible for its activation of NF-
B, cells were
pretreated with indicated concentrations of actinomycin D or
cycloheximide (10 min) followed by exposure to paclitaxel (2 h). As
demonstrated (Fig. 2), neither actinomycin D nor
cycloheximide inhibited the activation of NF-
B. Instead there was a
potentiation of activation of NF-
B by paclitaxel following
actinomycin D or cycloheximide pretreatment. In addition, both
cycloheximide and actinomycin D alone activated NF-
B. Thus,
activation of NF-
B by paclitaxel persisted despite inhibition of
transcription or of new protein synthesis.
Effect of Dexamethasone on NF-B Activation by Paclitaxel
In macrophages, paclitaxel has been reported to induce TNF- and
IL-1 production (5, 6). Production of these cytokines is inhibitable by
dexamethasone. Therefore, to rigorously evaluate a potential role for
TNF-
and IL-1 in paclitaxel-induced activation of NF-
B, we
pretreated the cells with dexamethasone (1 µM) for 16 h followed by paclitaxel treatment (2 h). By EMSA, there was no
inhibition of NF-
B activation by paclitaxel due to dexamethasone pretreatment (Fig. 2, lanes 7-10). This indicates that
TNF-
or IL-1 could not be responsible for initial activation of
NF-
B by paclitaxel.
Activation of NF-B by Vinblastine and Vincristine
Vinblastine and vincristine treatment resulted
in activation of NF-B in a dose-dependent manner (Fig.
3A). This activation was specific as
demonstrated by competition reaction with unlabeled oligonucleotide.
Activation of NF-
B by vinblastine was strikingly dose-dependent. Similarly, vincristine induced activation
of NF-
B and demonstrated a dose dependence up to 50 µM. However, an increase in the vincristine concentration
to 100 µM (Fig. 3A, lane 9) did not
cause a further increase in the activation of NF-
B. Control cells
were treated with the diluent for both agents (mannitol dissolved in
distilled water; 100 mg/ml).
Time Course
A549 cells were incubated with vinblastine (50 µM) or vincristine (50 µM) for various
intervals (30 min, and 1, 2, 3, or 4 h). As demonstrated in Fig.
3B, NF-B activation was increased in a
time-dependent manner. Vinblastine and vincristine both
activated NF-
B after 30 min through 4 h. Vinblastine-induced
activation was time-dependent. Vincristine-induced
activation differed only in that it was of relatively delayed onset
(Fig. 3B, lanes 9-11).
Activation of NF-B by Doxorubicin and Daunomycin
Daunomycin activated NF-B in a
dose-dependent manner. Maximal activation occurred at a
concentration of 5 µM (Fig. 4A,
lane 4). Further increases in the concentration of
daunomycin caused decreased NF-
B activation (Fig. 4B,
lanes 3-5). This is perhaps not surprising since the toxic
effect of these drugs at higher concentrations has been well documented
(24, 25). Doxorubicin showed a similar effect on NF-
B activation.
Maximal activation occurred at 30 µM (Fig. 4B,
lane 7). Additional increases in concentration did not
potentiate NF-
B activation further. Thus, daunomycin-induced NF-
B
activation is dose-dependent up to 5 µM and
that due to doxorubicin is dose-dependent up to 30 µM.
Time Course
In addition, the drugs increased NF-B
activation in a time-dependent manner (Fig. 4C),
with continued activation through 4 h. Specifity of NF-
B
activation was confirmed by competition studies with unlabeled
oligonucleotides.
Effect of Anti-p50 and Anti-p65 Antibodies on Activation of NF-B
by Antineoplastic Agents
Each of the antineoplastic agents activated NF-B, and anti-p65
eliminated DNA binding of NF-
B induced by each drug (Fig. 5). Anti-p50 supershifted NF-
B binding to a
considerable extent. Thus, antineoplastic agents caused the
translocation of p50-p65 heterodimer to the nucleus, and both subunits
appear to be present in the
B-binding complex. However, p65 appears
to be primarily responsible for binding to DNA since anti-p65 abolished
DNA binding of NF-
B.
Activation of NF-B by Antineoplastic Agents Is Specific
To determine specificity of activation of NF-B by
antineoplastic agents, EMSA was performed with probes for AP-1, AP-2,
CREB, TFIID, and SP-1 binding factors. As shown (Figs.
6, 7, 8), none of these
transcription factors was activated. Thus, activation of NF-
B by
anticancer drugs appears to be a specific response.
Antineoplastic Agents Cause IB
Degradation and p65
Translocation
Among the events that occur during activation of NF-B are the
degradation of the I
B inhibitory unit and migration of the p65
subunit to the nucleus. To investigate whether the anticancer drugs
cause the degradation of I
B, we prepared cytoplasmic extracts from
the anticancer drug-treated cells and examined the extract using gel
electrophoresis followed by Western blot using I
B-specific polyclonal antibodies. Each of the anticancer drugs caused degradation of I
B (Fig. 9). Paclitaxel treatment for 1 h
resulted in I
B degradation, which was complete after 2-3 h.
Although there were minor variations in the kinetics with which I
B
disappeared from the cytoplasm and was later restored following
treatment with the other anti-cancer drugs, the effect of each was
similar.
When we examined the appearance of p65 in the nuclear extracts of cells
stimulated with anti-cancer drugs or untreated controls, p65 was not
detected in nuclear extracts of control cells (Fig. 10). Paclitaxel treatment caused appearance of p65
after 2 h which continued through 4 h. Again, minor
differences in the rate of appearance of p65 in the nucleus occurred,
but each agent caused this effect in a manner which paralleled the
associated activation of NFB measured by EMSA.
Effect of Antineoplastic Drugs on Protein Kinase C Activity
Since protein kinase C is implicated in activation of NF-B by a
variety of stimuli, we evaluated the effect of anticancer drugs on PKC
activity. A549 cells were treated with indicated concentrations of
anti-tumor drugs for 30 min. Following incubation, cells were harvested
and assayed for PKC activity as described. Vincristine-, daunomycin-,
and paclitaxel-treated cells demonstrated significant increases in PKC
activity (Table I). Vinblastine and doxorubicin
increased PKC activity by 62 and 90%, although these changes were not
statistically significant.
|
Effect of Bisindolylmaleimide and Calphostin C, Specific Inhibitors
of Protein Kinase C, on NF-B Activation by Antineoplastic Agents
GF109203X, a bisindolylmaleimide derivative, is a specific
inhibitor of PKC (19). GF109203X (15 or 25 µM) inhibited
activation of NF-B by each of the antineoplastic agents (Fig.
11A). This effect was potent and consistent.
Calphostin C, a specific inhibitor of PKC, which binds its regulatory
region, also inhibited NF-
B activation by each of the antineoplastic
agents (Fig. 11B).
In the present investigation, we demonstrated that paclitaxel
induces NF-B activation in the pulmonary adenocarcinoma cell line
A549 in a dose- and time-dependent manner. Extension of
these studies revealed that compounds from two additional classes of antineoplastic agents also caused NF-
B activation. Thus, this appears to be a generalized effect of antiproliferative drugs and
suggests that a number of perturbations of cellular homeostasis can
activate NF-
B. This is not altogether surprising in view of the wide
variety of noxious stimuli now recognized to cause NF-
B
activation.
In macrophages, paclitaxel can induce TNF- or IL-1 gene expression
(5, 6). Thus, it is possible that paclitaxel-induced TNF-
could then
activate NF-
B. To evaluate this possibility we pretreated cells with
the transcriptional inhibitor actinomycin D or the translational
inhibitor cycloheximide. Neither of these inhibited activation of
NF-
B by paclitaxel. Instead, there was superinduction of NF-
B by
paclitaxel in the presence of actinomycin D or cycloheximide. Hence,
this activation of NF-
B by paclitaxel is not dependent on new
synthesis of mRNA or protein, which excludes the possibility that
the activation of NF-
B by paclitaxel could have been
cytokine-mediated. To further evaluate a potential role of cytokines in
paclitaxel-induced NF-
B activation, we examined the effect of
dexamethasone which inhibits induction of TNF and IL-1 by paclitaxel.
As shown under "Results," dexamethasone pretreatment did not
inhibit NF-
B activation by paclitaxel. We also evaluated the effect
of other antitumor drugs that do not induce cytokine gene expression
and which are different in structure and mode of action than
paclitaxel. These included vinblastine, vincristine, daunomycin, and
doxorubicin. Each of these drugs induced NF-
B activation in a dose-
and time-dependent manner. Thus, although autocrine
mechanisms cannot be altogether excluded, the data do not support a
role for cytokine up-regulation in the initial activation of NF-
B by
anti-tumor drugs. Nonetheless, it remains quite plausible that
autocrine mechanisms such as induction of cytokines could amplify
NF-
B activation in vivo once the process is initiated by
these agents.
As demonstrated by supershift assay (Fig. 5), p50/p65 heterodimer is
involved in the activation of NF-B by anti-tumor drugs. When
activated nuclear extract was probed with consensus oligonucleotides for transcription factors such as AP-1, CREB, SP-1, or TFIID, no DNA
binding was observed (Figs. 6, 7, 8), suggesting that the activation of
NF-
B by anti-tumor drugs is specific. Activation of NF-
B is
dependent on the detachment and degradation of inhibitor protein I
B.
To further investigate the mechanism of NF-
B activation, we probed
the cytoplasmic extracts of antitumor drug-stimulated cells with
antibody to I
B in a Western blot analysis (Fig. 9). As demonstrated,
I
B was rapidly detached from the complex and subsequently degraded.
This phenomenon occurred concomitantly with appearance of p65 in the
nuclear extract (Fig. 10).
Since removal of IB from the NF-
B complex could be achieved by
phosphorylation of I
B, we suspected a role for a protein kinase in
the activation of NF-
B by antitumor drugs. Protein kinase C has been
shown to be involved in the activation of NF-
B by a variety of
agents (15, 16). Thus, when we measured PKC activity after 30 min of
stimulation with antitumor drugs, an increased activity was observed.
This activity was significantly higher in paclitaxel-, vincristine-,
and daunomycin-stimulated cells than in unstimulated cells. Similar
trends in cell PKC activity occurred following treatment with
vinblastine and doxorubicin (Table I). To further ascertain the role of
PKC in the activation process, we incubated cells with specific
inhibitors of PKC, including calphostin C or bisindolylmaleimide
GF103209X. Calphostin C is a specific inhibitor which binds to the
regulatory region of PKC (18). On the other hand, GF109203X is a
specific inhibitor which binds to the catalytic domain of PKC (19). As
demonstrated, both compounds inhibited activation of NF-
B by
antineoplastic agents (Fig. 11). Thus, a role for PKC in the activation
of NF-
B by antitumor drugs is demonstrated.
It has been reported that NF-B can be activated by
microtubule-depolymerizing agents (26). In that study, such activation could be abrogated by microtubule-stabilizing agents (26), suggesting a
possible role for cytoskeletal disorganization as a signaling event for
the activation of NF-
B. However, in the present report we have shown
that paclitaxel, which is a microtubule-stabilizing anti-tumor agent,
vinblastine and vincristine, which are microtubule-depolymerizing agents, and daunomycin and doxorubicin, which are drugs that
intercalate into DNA and inhibit RNA synthesis, all could induce
NF-
B. Although these drugs are structurally and functionally
dissimilar, they share a common property, which is activation of
protein kinase C. Thus, since these drugs have effects other than
interfering with microtubule assembly, it is possible that they induce
NF-
B by mechanisms distinct from microtubule-associated mechanisms. As demonstrated, one such potential mechanism is through the increased activity of protein kinase C.