Trichothecene Mycotoxins Trigger a Ribotoxic Stress Response That Activates c-Jun N-terminal Kinase and p38 Mitogen-activated Protein Kinase and Induces Apoptosis
1999; Elsevier BV; Volume: 274; Issue: 20 Linguagem: Inglês
10.1074/jbc.274.20.13985
ISSN1083-351X
AutoresVictor Shifrin, Paul Anderson,
Tópico(s)NF-κB Signaling Pathways
ResumoThe trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g.anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis. The trichothecene family of mycotoxins inhibit protein synthesis by binding to the ribosomal peptidyltransferase site. Inhibitors of the peptidyltransferase reaction (e.g.anisomycin) can trigger a ribotoxic stress response that activates c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases, components of a signaling cascade that regulates cell survival in response to stress. We have found that selected trichothecenes strongly activate JNK/p38 kinases and induce rapid apoptosis in Jurkat T cells. Although the ability of individual trichothecenes to inhibit protein synthesis and activate JNK/p38 kinases are dissociable, both effects contribute to the induction of apoptosis. Among trichothecenes that strongly activate JNK/p38 kinases, induction of apoptosis increases linearly with inhibition of protein synthesis. Among trichothecenes that strongly inhibit protein synthesis, induction of apoptosis increases linearly with activation of JNK/p38 kinases. Trichothecenes that inhibit protein synthesis without activating JNK/p38 kinases inhibit the function (i.e. activation of JNK/p38 kinases and induction of apoptosis) of apoptotic trichothecenes and anisomycin. Harringtonine, a structurally unrelated protein synthesis inhibitor that competes with trichothecenes (and anisomycin) for ribosome binding, also inhibits the activation of JNK/p38 kinases and induction of apoptosis by trichothecenes and anisomycin. Taken together, these results implicate the peptidyltransferase site as a regulator of both JNK/p38 kinase activation and apoptosis. The trichothecenes are a structurally related family of low molecular weight mycotoxins synthesized by various Fusariumspecies. 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 (1Bukowski R. Vaughn C. Bottomley R. Chen T. Cancer Treat. Rep. 1982; 66: 381-383PubMed Google Scholar, 2Adler S. Lowenbraun S. Birch B. Jarrell R. Garrard J. Cancer Treat. Rep. 1984; 68: 423-425PubMed Google Scholar, 3DeSimone P. Greco F. Lessner H. Bartolucci A. Am. J. Clin. Oncol. 1986; 9: 187-188Crossref PubMed Scopus (13) Google Scholar, 4Goodwin W. Stephens R. McCracken J. Groppe C. Cancer Treat. Rep. 1983; 67: 285-286PubMed Google Scholar, 5Thigpen J. Vaughn C. Stuckey W. Cancer Treat. Rep. 1981; 65: 881-882PubMed Google Scholar). Trichothecenes inhibit the peptidyltransferase reaction by binding to the 60 S ribosomal subunit in eukaryotic cells (6Jimenez A. Vazquez D. Eur. J. Biochem. 1975; 54: 483-492Crossref PubMed Scopus (28) Google Scholar, 7Jimenez A. Sanchez L. Vazquez D. Biochim. Biophys. Acta. 1975; 383: 427-434Crossref PubMed Scopus (71) Google Scholar, 8Carter C. Cannon M. Smith K. Biochem. J. 1976; 154: 171-178Crossref PubMed Scopus (20) Google Scholar, 9Cannon M. Jimenez A. Vazquez D. Biochem. J. 1976; 160: 137-145Crossref PubMed Scopus (35) Google Scholar, 10Middlebrook J. Leatherman D. Biochem. 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Sanchez L. Vazquez D. Biochim. Biophys. Acta. 1975; 383: 427-434Crossref PubMed Scopus (71) Google Scholar, 9Cannon M. Jimenez A. Vazquez D. Biochem. J. 1976; 160: 137-145Crossref PubMed Scopus (35) Google Scholar, 10Middlebrook J. Leatherman D. Biochem. Pharmacol. 1989; 38: 3103-3110Crossref PubMed Scopus (37) Google Scholar), is a strong activator of JNK and p38 MAP kinases (11Iordanov M. Pribnow D. Magun J. Dinh T. Pearson J. Chen S. Magun B. Mol. Cell. Biol. 1997; 17: 3373-3381Crossref PubMed Google Scholar, 12Cano E. Hazzalin C. Mahadevan L. Mol. Cell. Biol. 1994; 14: 7352-7362Crossref PubMed Scopus (275) Google Scholar, 13Cano E. Doza Y. Ben-Levy R. Cohen P. Mahadevan L. Oncogene. 1996; 12: 805-812PubMed Google Scholar, 15Kyriakis J. Banerjee P. Nikolakaki E. Dai T. Rubie E. Ahmad M. Avruch J. Woodgett J. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2450) Google Scholar). Anisomycin also induces rapid apoptosis in human lymphoid cells (18Polverino A. Patterson S. J. Biol. Chem. 1997; 272: 7013-7021Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) (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) (19Kochi S. Collier R. Exp. Cell Res. 1993; 208: 296-302Crossref PubMed Scopus (144) Google Scholar, 20Sugita M. Morita T. Yonesaki T. Zool. Sci. 1995; 12: 419-425Crossref PubMed Scopus (14) Google Scholar), 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 (21Xia Z. Dickens M. Raingeaud J. Davis R. Greenberg M. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5074) Google Scholar, 22Chen Y. Wang W. Kong A. Tan T. J. Biol. 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A. 1998; 95: 5595-5600Crossref PubMed Scopus (114) Google Scholar). 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 (29Crow T. Forrester J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4490-4494Crossref PubMed Scopus (50) Google Scholar, 30Eskin A. Yeung S. Klass M. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7637-7641Crossref PubMed Scopus (42) Google Scholar), 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. 1 A 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 (1Bukowski R. Vaughn C. Bottomley R. Chen T. Cancer Treat. Rep. 1982; 66: 381-383PubMed Google Scholar, 2Adler S. Lowenbraun S. Birch B. Jarrell R. Garrard J. Cancer Treat. Rep. 1984; 68: 423-425PubMed Google Scholar, 3DeSimone P. Greco F. Lessner H. Bartolucci A. Am. J. Clin. Oncol. 1986; 9: 187-188Crossref PubMed Scopus (13) Google Scholar, 4Goodwin W. Stephens R. McCracken J. Groppe C. Cancer Treat. Rep. 1983; 67: 285-286PubMed Google Scholar, 5Thigpen J. Vaughn C. Stuckey W. Cancer Treat. Rep. 1981; 65: 881-882PubMed Google Scholar) 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.Table IInhibition of protein synthesis by various trichothecenesTrichothecenesProtein SynthesisCaspase-3 ActivationJNK Activation% control-foldScirpentriol3.6 (0.3)16.614.4Diacetylverrucarol7.1 (1.5)9.313.9T-2 triol4.7 (0.4)12.411.9Nivalenol51.6 (5.8)6.911.9T-2 tetraol83.6 (9.8)1.79.0Acetyldiacetoxyscirpenol70.5 (1.8)1.54.3Diacetoxyscirpenol2.4 (0.2)9.43.4Acetyldeoxynivalenol2.2 (0.1)4.82.5HT-21.6 (0.2)8.22.4T-2 tetraoltetraacetate49.4 (2.9)1.81.3Iso-T-211.0 (2.6)1.71.3Acetyl T-22.1 (0.1)3.21.2T-2 toxin1.4 (0.2)4.21.2Verrucarin1.6 (0.2)3.91.2Control100 (7.5)1.01.0The 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. 2 C (caspase activation) and Fig. 1 B (JNK activation) for comparison. Open table in a new tab 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. 2 C (caspase activation) and Fig. 1 B (JNK activation) for comparison. 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. 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. 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 (31Tian Q. Streuli M. Saito H. Schlossman S. Anderson P. Cell. 1991; 67: 629-639Abstract Full Text PDF PubMed Scopus (349) Google Scholar). DEVD-specific caspase activity was determined as described (32Nicholson D. Ali A. Thornberry N. Vaillancourt J. Ding C. Gallant M. Gareau Y. Griffin P. Labelle M. Lazebnik Y. Munday N.A. Raju S.M. Smulson M.E. Yamin T.-T. Yu V.L. Miller D.K. Nature. 1995; 376: 37-43Crossref PubMed Scopus (3837) Google Scholar) 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/mlN-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. 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 (33Budihardjo I. Poirier G. Kaufmann S. Mol. Cell. Biochem. 1998; 178: 245-249Crossref PubMed Scopus (8) Google Scholar). JNK kinase activity was assayed as described previously (34Shifrin V. Davis R. Neel B. J. Biol. Chem. 1997; 272: 2957-2962Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) with slight modifications. Aliquots of 1.0 × 107cells were lysed in 200 μl of lysis buffer A (20 mmHEPES, pH 7.1, 1% Triton X-100, 50 mm KCl, 5 mm EDTA, 5 mm EGTA, 50 mmβ-glycerophosphate, 2 mm dithiothreitol, 1 mmNa3VO4, 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 mmNa3VO4, 10 mm MgCl2, 50 μm ATP including 10–20 μCi of [γ-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 (35Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar) and boiling for 5 min. One-third of each reaction was separated on an SDS-polyacrylamide gel (35Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar), 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 kinase p38 was assayed as described previously (36Gabai V. Meriin A. Mosser D. Caron A. Rits S. Shifrin V. Sherman M. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar) 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 was assayed by measuring the incorporation of labeled amino acids into cellular proteins, essentially as described by Ausbel et al. (37Ausbel F. Brent R. Kingston R. Moore D. Seidman J. Smith J. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1995Google Scholar) 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 of35S-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. We compared the relative ability of anisomycin (3.8 μm) and trichothecene mycotoxins (10 μm) to activate JNK (Fig.1 B) and p38 (Fig.1 C) MAP kinases in Jurkat T cells. Within each structural subfamily (i.e. derivatives of nivalenol, scirpenol and T-2 toxin, see Fig. 1 A), 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. 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.2 A), processing of pro-caspase-3 (Fig. 2 B), activation of DEVD-specific caspases (Fig. 2 C), and cleavage of one of the major caspase-3 substrates, PARP (Fig. 2 D). 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 2reveals 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 andC). Nevertheless, the most potent apoptotic trichothecenes strongly activate JNK/p38 kinases, suggesting that kinase activation might contribute to the efficient induction of rapid apoptosis. The sequential activation of stress-induced MAP kinases and caspases differs in different experimental systems (25Goillot E. Raingeaud J. Ranger A. Tepper R. Davis R. Harlow E. Sanchez I. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3302-3307Crossref PubMed Scopus (246) Google Scholar, 28Deak J. Cross J. Lewis M. Qian Y. Parrott L. Distelhorst C. Templeton D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5595-5600Crossref PubMed Scopus (114) Google Scholar, 38Juo P. Kuo C. Reynolds S. Konz R. Raingeaud J. Davis R. Biemann H. Blenis J. Mol. Cell. Biol. 1997; 17: 24-35Crossref PubMed Scopus (282) Google Scholar, 39Rudel T. Zenke F. Chuang T. Bokoch G. J. Immunol. 1998; 160: 7-11PubMed Google Scholar, 40Rudel T. Bokoch G. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (606) Google Scholar). 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.3 A) 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 (10Middlebrook J. Leatherman D. Biochem. Pharmacol. 1989; 38: 3103-3110Crossref PubMed Scopus (37) Google Scholar, 41Middlebrook J. Leatherman D. J. Pharmacol. Exp. Ther. 1989; 250: 860-866PubMed Google Scholar) and therefore requires 2 h for maximal activation of JNK (Fig.3 A), and 3 h for detectable caspase activation (Fig.3 B). 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 (38Juo P. Kuo C. Reynolds S. Konz R. Raingeaud J. Davis R. Biemann H. Blenis J. Mol. Cell. Biol. 1997; 17: 24-35Crossref PubMed Scopus (282) Google Scholar, 39Rudel T. Zenke F. Chuang T. Bokoch G. J. Immunol. 1998; 160: 7-11PubMed Google Scholar, 40Rudel T. Bokoch G. Science. 1997; 276: 1571-1574Crossref PubMed Scopus (606) Google Scholar). 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. Activation of JNK/p38 kinases can signal cell survival or induce cell death in different cell types under different conditions (42Anderson P. Microbiol. Mol. Biol. Rev. 1997; 61: 33-46Crossref PubMed Scopus (165) Google Scholar). 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-α), suggesting that the survival pathway, but not the death pathway, requires new protein synthesis (43Leist M. Gantner F. Bohlinger I. Germann P. Tiegs G. Wendel A. J. Immunol. 1994; 153 (and references therein): 1778-1788PubMed Google Scholar,44Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4578) Google Scholar). 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 IC50values for this functional response (Fig.4 A). 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. 1 B). Under these conditions, caspase activation is a linear function of IC50 (Fig. 4 B), 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
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