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INTRODUCTION |
The trichothecenes are a structurally related family of low
molecular weight mycotoxins synthesized by various Fusarium
species. The ability of trichothecenes to inhibit the growth of rapidly proliferating cells in vitro and to selectively target
tissues with a high mitotic index (e.g. bone marrow,
gastrointestinal epithelium) prompted the selection of a representative
compound (i.e. diacetoxyscirpenol) for testing in phase I
and phase II clinical trials in human cancers (1-5). Trichothecenes
inhibit the peptidyltransferase reaction by binding to the 60 S
ribosomal subunit in eukaryotic cells (6-10), and their
antiproliferative activity has been presumed to be a consequence of
inhibition of protein synthesis. The recent discovery that
peptidyltransferase inhibitors can trigger a ribotoxic stress response
that activates JNK11 (11), a
stress-activated MAP kinase that signals the cellular response to
stress (12-17), suggests that the toxicity of trichothecenes might not
be a simple function of translational arrest. Anisomycin, a
peptidyltransferase inhibitor that competes with trichothecenes for
binding to the 60 S ribosomal subunit (6, 7, 9, 10), is a strong
activator of JNK and p38 MAP kinases (11-13, 15). Anisomycin also
induces rapid apoptosis in human lymphoid cells (18) (in marked
contrast to the delayed apoptosis induced by many protein synthesis
inhibitors that do not activate JNK and p38 MAP kinases,
e.g. puromycin, emetine) (19, 20), suggesting that the
toxicity of compounds that target the peptidyltransferase site is
multifactorial. The role of JNK/p38 kinase activation in the induction
of apoptosis is controversial. Although JNK activation is required for
the induction of apoptosis in some experimental systems (21-24), it is
not required in other systems (25-28). It appears that the functional
response to JNK activation can differ in different cell types exposed
to different apoptotic stimuli. The precise relationship between
anisomycin-induced translational arrest, JNK activation, and apoptosis
is not known.
Deacetylation of anisomycin markedly inhibits its ability to bind to
ribosomes, arrest translation (29, 30), activate JNK/p38 kinases, and
induce apoptosis (see below), suggesting that the functional effects of
anisomycin may be related to ribosome binding. The rich structural
diversity of the trichothecenes has the potential to further dissect
the relationship between these ribosome initiated functional responses.
Individual trichothecenes differ significantly in their ability to
induce translational arrest (see below). The structural features that
determine these functional differences are centered around chemical
sidegroups that modify the C7 and C8 positions of a pentane ring common
to all trichothecenes (see Fig. 1A and Table I). For
example, diacetoxyscirpenol is a potent inhibitor of protein
translation, whereas 3-acetyldiacetoxyscripentriol (produced by
acetylation of the C6 position of the pentane ring) is not (Table I).
Structural features that affect the ability of individual
trichothecenes to interact with the ribosomal peptidyltransferase site
are likely to determine the distinct functions of these compounds. As
such, a structure:function analysis comparing the ability of individual
trichothecenes to inhibit protein synthesis, activate JNK/p38 kinases
and induce apoptosis might improve our understanding of how ribosome
binding regulates these diverse functions. The results of this type of
analysis suggest that cooperation between JNK/p38 kinase activation
and translational arrest is important for the induction of rapid
apoptosis by selected inhibitors of protein synthesis. Consequently,
the relative ability of individual trichothecenes to trigger
translational arrest and JNK/p38 kinase activation are likely to
determine their efficacy as antineoplastic drugs. Importantly, the one
trichothecene that has been tested for antineoplastic activity in
clinical trials (1-5) is a relatively weak activator of JNK/p38
kinases (see below). Classification of natural or synthetic
trichothecenes using the functional parameters defined in this study
might facilitate selection of the most promising compounds for testing
in clinical trials.
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EXPERIMENTAL PROCEDURES |
Materials
Trichothecenes and other protein synthesis and protease
inhibitors were obtained from Sigma unless indicated otherwise. Stock solutions for all compounds were prepared in Me2SO at 3.3 mM, except for emetine, which was dissolved in water at 10 mg/ml.
Cell Treatments
The Jurkat human T-lymphoid cell line was grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 500 units/ml penicillin and 500 µg/ml streptomycin. For various treatments, cells were collected at 1.0-1.5 × 106/ml
and resuspended at 1.0 × 107/ml in the fresh growth
medium. Anisomycin (3.8 µM), trichothecenes (10 µM), or other protein synthesis inhibitors (or equivalent volumes of solvents for control samples) were added in a volume not
exceeding 1% of the total culture volume and incubated at 37 °C for
the indicated times. For treatments with two reagents, cells were
incubated with the first reagent (or solvents for control samples) for
30 min at 37 °C before addition of the second reagent (or solvents
for control samples) and continued culture at 37 °C for indicated
periods of time. Cells were collected by centrifugation at 2,000 × g for 1 min at 4 °C, washed twice with ice-cold
phosphate-buffered saline and then frozen in liquid nitrogen for
storage at
80 °C until further analysis.
Apoptosis Induction Assays
DNA Fragmentation Assay--
After 3 h of treatment with
various agents, 5 × 106 cells per treatment were
lysed in 0.5 ml of 10 mM Tris (pH7.5), 1% Triton X-100, 5 mM EDTA, incubated on ice for 10 min, vortexed for 5 s, and lysates were clarified for 5 min at 4 °C in an Eppendorf microcentrifuge at the top speed. 0.45 ml of the supernatants were
extracted once with an equal volume of phenol/chloroform (1:1) and
aqueous phases were adjusted to 0.5 M NaCl and precipitated with equal volumes of isopropanol, followed by overnight incubation at
20 °C. Precipitates were collected by centrifugation (10 min) at
4 °C in an Eppendorf microcentrifuge at the top speed, pellets were
washed with 70% ethanol, air dried and resuspended in 40 µl of 10 mM Tris (pH 7.5), 1 mM EDTA, 50 µg/ml RNase
A. Following a 30 min incubation at 37 °C, 10 µl aliquots were
separated on 1.2% agarose gels in TAE buffer as described (31).
Fluorescent Assay of Caspase-3 Activity--
DEVD-specific
caspase activity was determined as described (32) with modifications;
107 cells were resuspended in 0.1 ml of lysis buffer (20 mM HEPES, pH 7.1, 1% Triton X-100, 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/ml pepstatin, 10 µg/ml leupeptin, 2 µg/ml aprotinin, 25 µg/ml N-acetyl-leu-leu-norleucinal (Calbiochem), incubated on ice
for 10 min, vortexed for 5 s, and lysates were clarified for 10 min at 4 °C in an Eppendorf microcentrifuge at the top speed.
DEVD-specific caspase activity was determined in triplicate by mixing
10 µl of supernatants (50 µg of protein) with 0.2 ml of reaction
buffer (100 mM HEPES (pH 7.1), 10% sucrose, 0.1% CHAPS,
10 mM dithiothreitol, 0.1 mg/ml bovine serum albumin with 2 µM DEVD-AMC) and incubating at 30 °C for 20 min. The
DEVD-specific caspase activity was calculated by measuring fluorescence
of released AMC using CytoFluor 4000 MultiWell Plate Reader (PerSeptive
Biosystems, Framingham, MA) with excitation at 360 nm and emission at
460 nm.
Caspase-3 and Poly(ADP)ribose Polymerase (PARP)
Cleavage--
The cell lysates used for enzymatic assay of caspase-3
(see above) were also subjected to Western blotting analysis with
caspase-3 (CPP32)-specific antibodies (PharMingen, San Diego, CA)
according to the manufacturer's instructions and with PARP-specific
monoclonal antibodies C-2-10 as described (33).
JNK Kinase Assays
JNK kinase activity was assayed as described previously (34)
with slight modifications. Aliquots of 1.0 × 107
cells were lysed in 200 µl of lysis buffer A (20 mM
HEPES, pH 7.1, 1% Triton X-100, 50 mM KCl, 5 mM EDTA, 5 mM EGTA, 50 mM
-glycerophosphate, 2 mM dithiothreitol, 1 mM
Na3VO4, 50 mM NaF, 50 nM calyculin A, 10 µg/ml leupeptin, 1 µg/ml aprotinin,
1 µg/ml pepstatin A, 1 µg/ml antipain, 250 mg/ml benzamidine, and
20 µg/ml phenylmethylsulfonyl fluoride), incubated on ice for 10 min,
vortexed for 10 s, and clarified by centrifugation at 15,000 × g for 5 min at 4 °C. 5 µl of supernatants were added
to a 25-µl reaction volume in 40 mM HEPES, pH 7.1, 25 nM calyculin A, 1 mM
Na3VO4, 10 mM MgCl2, 50 µM ATP including 10-20 µCi of
[
-32P]ATP (NEN Life Science Products) and 1 µg of
glutathione S-transferase/ c-Jun (1-135) as a substrate.
After 20 min incubation at 30 °C, the reactions were stopped by
adding 10 µl of 4× SDS loading buffer with 2-mercaptoethanol (35)
and boiling for 5 min. One-third of each reaction was separated on an
SDS-polyacrylamide gel (35), blotted onto polyvinylidene difluoride
membrane (Immobilon P, Millipore, Bedford, MA), exposed to x-ray film
and subsequently quantitated using a Bio-Rad model GS-525 PhosphorImager.
Activation of Stress-activated Kinase p38
Activation of kinase p38 was assayed as described previously
(36) by Western blotting with antibody 9211 (New England Biolabs, Beverly, MA), recognizing the activated (phosphorylated) form of p38
kinase. Duplicate filters were probed with antibody 9212, recognizing
both phosphorylated and unphosphorylated forms of p38 to verify equal loading.
Protein Synthesis Inhibition Assays
Protein synthesis was assayed by measuring the incorporation of
labeled amino acids into cellular proteins, essentially as described by
Ausbel et al. (37) with the following modifications: Jurkat
cells were grown and collected as described above, washed once with
Hanks' balanced salt solution and resuspended at 2 × 106/ml in cysteine- and methionine-free RPMI 1640 medium
supplemented with 10% dialyzed heat-inactivated fetal bovine serum,
incubated for 15 min at 37 °C and treated in triplicate with protein
synthesis inhibitors (or with corresponding solvents for control
samples) for 20 min at 37 °C before the addition of 50 µCi/ml of
35S-labeled methionine/cysteine mixture (NEN Life Science
Products) and incubation for an additional 20 min at 37 °C. Cells
were then centrifuged at 2,000 × g for 1 min at
4 °C, washed twice with ice-cold phosphate-buffered saline and
solubilized in lysis buffer (200 µl/106 cells; 10 mM Tris, pH 7.2, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 20 µg/ml chymostatin, 3 µg/ml leupeptin, 14 µg/ml pepstatin A, 1.7 mg/ml benzamidine, and
10 µg/ml aprotinin). After a 10-min incubation on ice, cell lysates
were vortexed for 10 s and clarified by centrifugation at
15,000 × g for 10 min. 50 µl of the supernatants
were mixed with 500 µl of 100 µg/ml bovine serum albumin, and
proteins were precipitated by the addition of 500 µl of 20%
trichloroacetic acid. After 20 min on ice, precipitated proteins were
collected by filtration through glass microfiber filters (GF/C,
Whatman), washed with 10 ml of 10% trichloroacetic acid and 5 ml of
ethanol, and air dried. Incorporation of radiolabeled amino acids into
cellular proteins was quantitated by liquid scintillation counting in a
Packard 1600 TR counter.
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RESULTS |
Activation of JNK and p38 MAP Kinases by
Trichothecenes--
We compared the relative ability of anisomycin
(3.8 µM) and trichothecene mycotoxins (10 µM) to activate JNK (Fig.
1B) and p38 (Fig.
1C) MAP kinases in Jurkat T cells. Within each structural subfamily (i.e. derivatives of nivalenol, scirpenol and T-2
toxin, see Fig. 1A), we identified trichothecenes that
induce strong (e.g. nivalenol, scirpentriol, and T-2 triol),
intermediate (e.g. acetyldeoxynivalenol, acetoxyscirpenol,
and HT-2), or weak (e.g. verrucarin, T-2 toxin) activation
of JNK/p38 kinases. The different activity of these compounds cannot be
explained by differential cell permeability, as several trichothecenes
(e.g. deoxynivalenol and 3-acetyldeoxynivalenol; T-2 triol
and acetyl-T-2 toxin) that differ dramatically in their ability to
activate JNK/p38 kinases (Fig. 1), are similarly potent inhibitors of
protein synthesis (see Table I, and
below). The strong correlation between the ability of individual
trichothecenes to activate JNKs and p38 (compare Fig. 1,
B and C), suggests that both of these MAP kinases are activated via the same mechanism, the ribotoxic stress response. Therefore, our data indicate that structural differences between individual trichothecenes can influence their ability to trigger the
ribotoxic stress response.

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Fig. 1.
Activation of JNKs and p38 stress-activated
MAP kinases by trichothecenes. A, structures of the
individual trichothecenes used in this study (*, macrocyclic ring;
**, (CH3)2CHCH2CO; NA, not
applicable); B, activation of JNKs by trichothecenes. Jurkat
cells were treated for 2 h with various members of each of the
three trichothecene subfamilies (10 µM; lanes
3-15), anisomycin (3.8 µM), or Me2SO
(control lane). Quantitation of the relative intensity of
glutathione S-transferase/c-Jun phosphorylation was
performed as described under "Experimental Procedures."
C, activation of p38 stress-activated MAP kinase by three
trichothecene subfamilies (10 µM). Cell lysates were
Western blotted with antibodies recognizing phosphorylated p38 (pp38),
as described under "Experimental Procedures."
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Table I
Inhibition of protein synthesis by various trichothecenes
The level of protein synthesis after a 20 min treatment with individual
trichothecenes (all at 10 µM) was determined as described
under "Experimental Procedures." Mean ± S.D. is indicated in
parentheses. Also included data from Fig. 2C (caspase
activation) and Fig. 1B (JNK activation) for comparison.
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Induction of Apoptosis by Trichothecenes--
During our analysis
of JNK activation by various trichothecenes, we noticed that many
trichothecenes induce what appears to be a typical apoptotic cell death
in Jurkat cells. The relative ability of individual trichothecenes to
induce various manifestations of apoptosis was assessed by monitoring
internucleosomal DNA fragmentation (Fig.
2A), processing of
pro-caspase-3 (Fig. 2B), activation of DEVD-specific
caspases (Fig. 2C), and cleavage of one of the major caspase-3 substrates, PARP (Fig. 2D). This analysis
identified trichothecenes within each structural subfamily that are
strong (e.g. deoxynivalenol, scirpentriol, and T-2 triol),
intermediate (e.g. nivalenol, diacetoxyscirpentriol, HT-2),
and weak (e.g. 3-acetyldeoxynivalenol, varrucarin, T-2)
inducers of apoptosis. Comparison of results presented in Figs. 1 and 2
reveals that activation of JNK/p38 kinases is not sufficient for the
induction of apoptosis (see also Table I). Thus trichothecenes that
similarly activate JNK/p38 kinases (e.g. T-2 triol and T-2
tetraol, Fig. 1) can differ significantly in their ability to induce
apoptosis as measured by caspase-3 activation (Fig. 2, B and
C). Nevertheless, the most potent apoptotic trichothecenes
strongly activate JNK/p38 kinases, suggesting that kinase activation
might contribute to the efficient induction of rapid apoptosis.

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Fig. 2.
Apoptosis induction by trichothecenes.
A, trichothecenes induce internucleosomal DNA fragmentation.
Jurkat cells were incubated for 3 h with the indicated
trichothecenes (10 µM) or with Me2SO
(control lane), anisomycin (3.8 µM), or
staurosporine (5 µM), and extranuclear DNA was collected
and separated on an agarose gel as described under "Experimental
Procedures." B, trichothecenes induce caspase-3 cleavage.
Cells were treated with the indicated trichothecenes (10 µM) as described under "Experimental Procedures," and
25 µg of the lysates were analyzed by Western blotting using
polyclonal antibodies to caspase-3 (PharMingen). C,
trichothecenes induce caspase-3 activation. Cells were treated with the
indicated trichothecenes (10 µM) as in Fig. 2B, and DEVD-specific
caspase activity in 50 µg of lysates was measured as described under
"Experimental Procedures." D, trichothecenes induce PARP
cleavage. Cells were treated with the indicated trichothecenes (10 µM) as in Fig. 2B, lysed as described under
"Experimental Procedures" (enzymatic assay for caspase-3 activity),
and 25 µg of each lysate was analyzed by Western blotting using
monoclonal antibodies to PARP. Arrows indicate PARP, 116-kDa
native PARP polypeptide; PARP*, 89-kDa fragment generated by caspase-3
digestion (56, 57).
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The sequential activation of stress-induced MAP kinases and caspases
differs in different experimental systems (25, 28, 38-40). Because of
the strong correlation between JNK activation and trichothecene-induced
apoptosis (compare Figs. 1 and 2; summarized in Table I), we determined
the temporal order of JNK and caspase-3 activation by several
trichothecenes and anisomycin (Fig. 3). The kinetics of JNK activation by anisomycin and T-2 triol are similar,
with each drug producing maximal activation within 15 min (Fig.
3A) followed by detectable caspase activation at 1-2 h
(Fig. 3, B and C). This sequential order of JNK
and caspase-3 activation is even better illustrated by T-2 tetraol,
which has a slower rate of cellular uptake than T-2 toxin (10, 41) and therefore requires 2 h for maximal activation of JNK (Fig.
3A), and 3 h for detectable caspase activation (Fig.
3B). Therefore, in response to trichothecenes and
anisomycin, JNK activation precedes caspase-3 activation,
distinguishing this process from a similarly rapid Fas-induced
apoptosis in which JNK/p38 kinases are activated after caspase-3 during
the later stages of cell death (38-40). A more direct test of a role
for JNKs in the activation of caspases would be provided by the
demonstration that dominant negative JNK inhibits trichothecene-induced
caspase activation. Unfortunately, the low transfection efficiency of
Jurkat cells prevented us from obtaining useful information from
this experiment.

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Fig. 3.
Kinetics of JNKs and caspase-3 activation.
A, cells were treated for the indicated times with
anisomycin (3.8 µM), T-2 triol (10 µM), or
T-2 tetraol (10 µM) before preparing lysates for
quantitation of JNK activity as described under "Experimental
Procedures." B, cells were treated for the indicated times
with anisomycin (3.8 µM), T-2 triol (10 µM), or T-2 tetraol (10 µM) before
preparing lysates for quantitation of caspase-3 cleavage. The 19-kDa
caspase-3 cleavage product is indicated by an arrow on the
left. C, cells were treated for 0, 2, 4, or
6 h with 10 µM T-2 tetraol or Me2SO
(control) and caspase-3 activity was determine as described
under "Experimental Procedures."
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Inhibition of Protein Synthesis by Trichothecenes--
Activation
of JNK/p38 kinases can signal cell survival or induce cell death in
different cell types under different conditions (42). Inhibitors of
protein synthesis can promote the induction of apoptosis in response to
inflammatory cytokines that activate JNK/p38 kinases (e.g.
Fas-ligand, tumor necrosis factor-
), suggesting that the survival
pathway, but not the death pathway, requires new protein synthesis (43,
44). The possibility that differential inhibition of protein synthesis
might determine the functional response to trichothecene-induced
JNK/p38 kinase activation prompted us to compare the ability of
individual trichothecenes to inhibit protein synthesis in Jurkat cells.
Table I compares the ability of individual trichothecenes to inhibit
protein synthesis, activate caspase-3, and activate JNK. Although there
is no obvious correlation between any two trichothecene-induced effects
(e.g. protein synthesis inhibition versus JNK
activation; protein synthesis inhibition versus caspase
activation; JNK activation versus caspase activation), the
tendency for apoptotic trichothecenes to strongly inhibit protein
synthesis and strongly activate JNKs (Table I) suggests that these two
effects might cooperate in the induction of apoptosis. To test this
possibility, we measured the dose-dependent inhibition of
protein synthesis produced by trichothecenes that strongly activate JNK
kinases (>9-fold activation; Table I) and derived IC50
values for this functional response (Fig.
4A). We then compared the
activation of caspase-3 at a trichothecene concentration (10 µM) at which each of these compounds maximally activate
JNK1 (determined in dose-response experiments using the assay described
in Fig. 1B). Under these conditions, caspase activation is a
linear function of IC50 (Fig. 4B), suggesting
that inhibition of protein synthesis and activation of JNK/p38 kinases
both contribute to the activation of caspase-3. Although the assay for
JNK activation is not sufficiently quantitative to allow a similar
analysis of trichothecenes that strongly inhibit protein synthesis,
we compared the ability of these trichothecenes to activate caspase-3
at a concentration (10 µM) that inhibits protein
synthesis by >95% (see Table I). Under these conditions, caspase
activation is a linear function of JNK activation (Fig. 4C),
again suggesting that trichothecene-induced translational arrest and
JNK/p38 kinase activation cooperate in the induction of apoptosis.

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Fig. 4.
Caspase activation is affected by both
translational arrest and JNK activation. A, dose response of
inhibition of protein synthesis by strong activators of JNK1. Jurkat
cells were treated with trichothecenes for 20 min before measuring the
incorportation of [35S]methionine as described under
"Experimental Procedures." The percent inhibition of protein
synthesis was calculated in relation to an untreated control sample.
Error bars represent the standard deviation from triplicate samples.
B, caspase activation by trichothecenes that strongly
activate JNK1 as a function of IC50 for protein
synthesis inhibition. IC50 values for protein synthesis
inhibition were calculated from Fig. 4A. Caspase activation produced by 10 µM T-2 triol, scirpentriol, diacetylverrucarol,
nivalenol, or T-2 tetraol as a function of IC50 was
analyzed by a best fit simple polynomial, and the regression
coefficient was calculated using the Cricket graph program (Cricket
Software, Malvern, PA). C, the relative ability of
trichothecenes (scirpentriol, T-2 triol, diacetoxyscirpentol,
acetyldeoxynivalenol, HT-2, acetyl T-2, T-2 toxin, verrucarin, all at
10 µM) that strongly inhibit protein synthesis (>95%
inhibition, Table I) to activate JNKs and caspase-3 were compared. The
best fit simple polynomial and regression coefficient were calculated
using the Cricket graph program.
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Deacetylanisomycin, T-2 Toxin, and Verrucarin Block the Activation
of JNK and Caspase-3 by Both Anisomycin and Apoptotic
Trichothecenes--
The observation that translational arrest and
JNK/p38 kinase activation are independently triggered by individual
trichothecenes led us to question whether binding to a common ribosomal
site is required for trichothecene- and anisomycin-induced
activation of JNK/p38 and caspase-3. If activation of JNK/p38 kinases
requires ribosome binding, inactive anisomycin derivatives or
trichothecenes that inhibit translation without activating JNK/p38
kinases (e.g. T-2 toxin, verrucarin, see Table I) might
inhibit the function (i.e. JNK/p38 kinase and caspase-3
activation) of apoptotic trichothecenes and/or anisomycin.
Deacetylanisomycin (DA) is an anisomycin analog that enters cells,
binds to ribosomes, and inhibits protein synthesis (albeit with
10,000-fold lower potency than anisomycin; data not shown). When used
at a concentration that inhibits protein synthesis by 65% (300 µg/ml), it fails to activate JNKs on its own, and inhibits activation
of JNKs by T-2 triol (10 µM), T-2 tetraol (10 µM), and anisomycin (3.8 µM) (Fig.
5A). At similar
concentrations, DA also inhibits anisomycin-induced translational
arrest in Jurkat cells, as well as in rabbit reticulocyte lysates,
suggesting that its functional effects are a consequence of ribosome
binding (data not shown). T-2 toxin (10 µM) (Fig.
5A) and verrucarin (10 µM) (data not shown)
similarly inhibit the activation of JNKs by these compounds.
Pretreatment with either DA (300 µg/ml), T-2 toxin (10 µM), or verrucarin (10 µM) also prevents
caspase-3 activation in Jurkat cells cultured with apoptotic
trichothecenes (T-2 triol, diacetylverrucarol, and deoxynivalenol, Fig.
5B). The concentrations of anisomycin, T2-toxin and
verrucarin used in these experiments is at or above the
IC50 for binding to free ribosomes (approximately 10 µM, 0.6 µM and 10 µM,
respectively) (10), consistent with a role for competitive displacement
at the level of ribosome binding. The suggestion that DA and
nonapoptotic trichothecenes can occupy to the peptidyltransferase site
and inhibit the function of anisomycin and apoptotic trichothecenes is
consistent with a role for ribosome binding in the activation of
JNK/p38 kinases and, subsequently, caspase-3.

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Fig. 5.
Deacetylanisomycin (DA) and T-2 toxin block
anisomycin- and trichothecene-induced JNK and caspase-3 activation.
A, DA and T-2 toxin block JNK activation. Cells were
pretreated for 30 min with Me2SO (lanes 1-4),
300 µg/ml DA (lanes 5-8), or 10 µM T-2
toxin (lanes 9-12) and then treated with Me2SO
(control lanes), 10 µM T-2 triol, 10 µM T-2 tetraol, or 3.8 µM anisomycin; the
JNKs activity was determined as described under "Experimental
Procedures." B, effect of DA, T-2, or verrucarin
pretreatments on caspase-3 activation. Cells were pretreated with
either Me2SO (Nothing series), or 300 µg/ml
DA, 10 µM T-2 toxin, or 10 µM verrucarin
for 30 min and then treated with 3.8 µM anisomycin or 10 µM of indicated trichothecenes or Me2SO
(control) for 3 h. Caspase-3 activity was determined as
described under "Experimental Procedures." C, high
concentrations of anisomycin overcome the T-2 toxin-induced inhibition
of JNK activation. Cells were pretreated for 30 min with either
Me2SO (lanes 1-4) or T-2 toxin (10 µM) (lanes 5-8), before the addition of
Me2SO (lanes 1 and 5) or the
indicated concentrations of anisomycin. After a further 2-h incubation,
cell lysates were prepared for quantitation of JNKs activity as
described under "Experimental Procedures."
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Activation of the Ribotoxic Stress Response by Anisomycin Does not
Require Active Translation--
Activation of JNK/p38 kinases in
response to ribotoxic stress has been proposed to require on-going
protein translation (11). If this is true, the profound translational
arrest induced by T-2 toxin and verrucarin could account for the
inhibition of JNK/p38 kinase activation observed in Fig. 5A.
Translational arrest could not, however, account for the inhibition of
JNK/p38 kinase activation induced by DA, a compound that only partially
inhibits protein synthesis under the conditions employed. We therefore
compared the ability of anisomycin to activate JNKs in Jurkat cells
pre-incubated in the absence or presence of T-2 toxin (10 µM, 30 min, conditions that reduced protein synthesis to
<98% of control levels). The ability of T-2 toxin to prevent
anisomycin-induced JNK activation could be overcome at high
concentrations of anisomycin (Fig. 5C), suggesting that
displacement of T-2 toxin from the peptidyltransferase site might allow
anisomycin to activate JNK/p38 kinases in the absence of protein synthesis.
Further evidence that ribosome binding is required for
peptidyltransferase inhibitors to activate JNK/p38 kinases and induce apoptosis was provided by analyzing the effects of structurally unrelated compounds that compete with trichothecenes for ribosome binding. Emetine was previously reported to block JNK activation by
anisomycin (11). We found that emetine blocks anisomycin- and T-2
tetraol-induced JNK activation (Fig.
6A, lanes 5-8), as well as JNK activation induced by other trichothecenes (nivalenol, fusarenon, and trichothecin, data not shown), suggesting that emetine
may block the access of anisomycin or trichothecenes to a common
ribosomal binding site. Interestingly, emetine does not block T-2
triol-induced JNK activation (Fig. 6A, lane 6).
Although this reinforces our conclusion that active translation is not required for trichothecene-induced JNK activation, it is surprising because T-2 triol differs from T-2 tetraol only in the addition of a
3-methylbutyryloxy group (R4 in Fig. 1A) at the C8
position.

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Fig. 6.
Effects of protein synthesis inhibitors
on anisomycin- and trichothecene-induced JNK and caspase-3
activation. A, effect of protein synthesis inhibitors
emetine and harringtonine on trichothecene- and anisomycin-induced JNK
activation. Cells were pretreated for 30 min with Me2SO
(lanes 1-4), emetine (180 µM) (lanes
5-8), or harringtonine (30 µM) (lanes
9-12), and then treated with either Me2SO
(control lanes), T-2 triol (10 µM), anisomycin
(3.8 µM), or T-2 tetraol (10 µM) as
indicated; after 2 h, the JNKs activity was determined as
described under "Experimental Procedures." B, effect of
protein synthesis inhibitor pretreatments on caspase-3 activation.
Cells were pretreated with either Me2SO (Nothing
series), 180 µM emetine, or 33 µM
harringtonine for 30 min and then treated with 3.8 µM
anisomycin, 10 µM T-2 triol, or Me2SO
(control) for 2 h. Caspase-3 activity was determined as
described under "Experimental Procedures."
|
|
Harringtonine, a plant alkaloid that is structurally unrelated to
either anisomycin or trichothecenes, competes with these compounds for
binding to the ribosomal peptidyltransferase site (45, 46). As shown in
Fig. 6A, harringtonine weakly activates JNKs on its own, but
efficiently blocks both anisomycin- and trichothecene-induced JNK
activation (Fig. 6A, lanes 9-12). Both emetine
and harringtonine also inhibit caspase activation by apoptotic
trichothecenes (Fig. 6B). (Here, the inability of emetine to
prevent JNK activation by T-2 triol is consistent with in its relative
inability to block caspase activation by this trichothecene.) Because
extended treatment with many inhibitors of protein synthesis can induce
apoptosis (19, 20, 47), it is not surprising that treatment with
emetine or harringtonine alone induced a low level of caspase-3
activation (Fig. 6B); however, the ability of both emetine
and harringtonine to block anisomycin- or trichothecene-induced
caspase-3 activation suggests that the mechanisms of apoptosis
induction by anisomycin/trichothecenes on one hand, and emetine or
harringtonine, on the other, are different.
 |
DISCUSSION |
Activation of JNK1 by protein synthesis inhibitors that bind to or
alter the structure of 28 S ribosomal RNA (e.g. blasticidin S, gougerotin, anisomycin, ricin toxin, sarcin toxin) led Iordanov et al. (11) to propose the existence of a ribotoxic stress
response in eukaryotic cells. The ability of ribosomes to sense
cellular stress and activate signaling pathways that alter cellular
function has been well characterized in prokaryotes. In response to
amino acid starvation, prokaryotic ribosomes produce guanosine
3',5'-bispyrophosphate (ppGpp), a nucleoside analogue that arrests
transcription of genes encoding translation factors, a response that
promotes survival under starvation conditions (48). By promoting the
activation of JNK and p38 MAP kinases, eukaryotic ribosomes might
similarly induce the transcription of genes that regulate the cellular
response to stress. Our results provide support for this concept by
showing that selected trichothecenes, compounds that target the
ribosomal peptidyltransferase site (6, 7, 9, 10, 49), also activate JNK
and p38 MAP kinases. The rich structural diversity of the
trichothecenes allowed us to carry out a limited structure:function analysis of compounds that target this functional domain on the large
ribosomal subunit. By comparing the ability of individual trichothecenes to inhibit protein synthesis, activate JNK/p38 kinases,
and induce apoptosis, we have grouped these compounds into distinct
functional classes. Most trichothecenes strongly inhibit protein
synthesis (Table I). Among these, induction of apoptosis is linearly
correlated with the ability to activate JNK and p38 MAP kinases (Fig.
4B). Trichothecenes that inhibit protein synthesis without
activating JNKs (e.g. acetyl T-2, T-2 toxin, and
verrucarin), induce a 3-4-fold increase in caspase activation,
indicating that JNK/p38 kinases may not be necessary for low level
caspase activation. Among trichothecenes that strongly activate JNK/p38
kinases, induction of apoptosis is linearly correlated with the ability
to inhibit protein synthesis. Taken together, these results reveal that
the ability of individual trichothecenes to induce rapid apoptosis may
require both translational arrest and JNK/p38 kinase activation.
The ability of emetine to inhibit anisomycin-, palytoxin (a modulator
of Na/K ATPase function), and UV-induced JNK1 activation led Iordanov
et al. (11, 50, 51) to conclude that active translation is
required to trigger the ribotoxic stress response. Our results showing
that high concentrations of anisomycin can overcome the T-2
toxin-induced inhibition of JNK activation (Fig. 5C) require
a re-examination of this conclusion. T-2 toxin inhibits protein
synthesis by >98% under these conditions, indicating that anisomycin
can activate JNKs in the absence of active translation. The further
observation that T-2 triol (but not T-2 tetraol or anisomycin)
activates JNKs in cells rendered translationally incompetent by
pretreatment with emetine (producing >98% inhibition of protein synthesis) is also not consistent with a requirement for active ribosomes in this process (see Fig. 6A). Although the
binding site of emetine is located on the small ribosomal subunit, its close proximity to the trichothecene binding site on the large ribosomal subunit allows it to compete with T-2 toxin for ribosome binding (49, 52, 53). The ability of emetine to block T-2 tetraol, but
not T-2 triol-induced JNK activation suggests that emetine can
interfere with the binding of the tetraol, but not the triol to the
peptidyltransferase site on the large ribosomal subunit. The ability of
emetine to also inhibit palytoxin and UV-induced JNK activation further
suggests that emetine either stabilizes ribosomal structure, or
prevents the binding of a natural ribosomal ligand involved in the
ribotoxic stress response (alternatively, the activation of JNK/p38
kinases by palytoxin and UV irradiation may be dependent upon protein
synthesis). Pactomycin, another compound that inhibits the ribotoxic
stress response (11), also binds to the small ribosomal subunit.
Although it is not known to interfere with the binding of anisomycin or
trichothecenes, photoaffinity labeling experiments indicate that it
also interacts with the large ribosomal subunit (54, 55), suggesting
that it could block access to the anisomycin- and trichothecene-binding site.
The identification of trichothecene derivatives (e.g. T-2
tetraol and, to a lesser degree, 3-acetyldiacetoxyscirpenol, Table I)
that activate JNK and p38 kinases without significantly affecting protein synthesis suggests that different parts of the trichothecene molecule are responsible for translational arrest and JNK/p38 kinase
activation. The functional effects of these compounds could result from
the displacement of ribosome-binding molecules (e.g. elongation factors, charged tRNAs, mRNAs, etc.), the induction of a
conformational change in the ribosome itself, or both. The ability of
plant toxins such as ricin and sarcin to activate JNK1 following the
catalytic depurination or cleavage of 28 S RNA (11) suggests that a
conformational change in the ribosome can result in JNK/p38 kinase
activation. How this conformational change leads to JNK1 activation
remains to be determined. The ability of some trichothecenes, but not
others, to activate JNK/p38 kinases suggests that the presence or
absence of side groups that interact with ribosome target sites or
displace the binding of ribosome-associated molecules might determine
whether JNK/p38 kinases are activated. The effect that various
modifications of trichothecene structure have on JNK activation seems
to be a direct one, because these modifications do not affect the
ability of most trichothecenes to enter cells and rapidly inhibit
translation (Table I). Although the variable R1-R4 trichothecene side
groups (Fig. 1A) must determine their relative ability to
inhibit protein synthesis and activate JNK/p38 kinases, there is no
obvious correlation between the structure of these groups and
trichothecene function.
In conclusion, our results suggest that: (i) the functional effects of
trichothecenes are initiated by ribosome binding; (ii) trichothecene-induced translational arrest and JNK/p38 kinase activation are independent dissociable events; and (iii) translational arrest and JNK/p38 kinase activation appear to cooperate in the induction of apoptosis. Consequently, classification of trichothecenes based on their relative ability to inhibit protein synthesis, activate
JNK/p38 kinases, and induce apoptosis might facilitate the
selection of natural and synthetic compounds for clinical trials in
human cancers.