WO1999040912A1 - Method of stimulating apoptosis using trichothecene mycotoxins - Google Patents

Abstract

The present invention is directed to methods for modulating apoptosis both in vitro and in vivo using trichothecene mycotoxins. It is also directed to a method for identifying agents that induce apoptosis without inhibiting protein synthesis.

Description

Method of Stimulating Apoptosis Using Trichothecene Mycotoxins

Field of the Invention

The present invention is directed to methods useful in treating pathological conditions, particularly cancer, associated with abnormalities in cellular apoptosis. The agents used in these methods are trichothecene mycotoxins.

Background of the Invention

The trichothecenes are a structurally related family of low molecular weight mycotoxins synthesized by various species of Fusarium. The ability of the trichothecenes to inhibit the growth of rapidly proliferating cells in vitro and to selectively target tissues with a high mitotic index led to the clinical testing of diacetoxyscirpenol as a treatment of human cancer (Bukowski et al., Cancer Treat. Rep. (5(5:381-383 (1982); Adler, et al, Cancer Treat. Rep. (5S423-425 (1984); DeSimone, et al., Am. J. Clin. Oncol. 9:187-188 (1986); Goodwin, et al, Cancer Treat. Rep. (57:285-286 (1983); Thigpen, etal, Cancer Treat. Rep. 55:881-882 (1981)). Unfortunately, toxicity proved to be a major problem. Trichothecenes inhibit the peptidyl transferase reaction by binding to the 60S ribsomal subunit in eukaryotic cells and their antiproliferative activity has been presumed to be a consequence of inhibition of protein synthesis. Because the same mechanism appeared responsible for both therapeutic action and toxicity, interest in trichothecenes as a cancer treatment largely subsided. The identification of trichothecenes that inhibit cancer cell proliferation but which have substantially reduced toxicity would represent a significant advance in clinical medicine.

Summary of the Invention

The present invention is based, in part, on the discovery that, contrary to accepted belief, the effect of trichothecenes on cell survival does not depend upon their inhibiting protein synthesis. Instead, it appears that the antiproliferative effect is due to the activation of MAP kinases inducing cellular apoptosis. By carefully screening compounds for high apoptotic activity and low inhibition of protein translation, trichothecenes suitable for the clinical treatment of cancer may be identified.

In its first aspect, the present invention is directed to a method of treating a patient for a condition associated with an abnormally low level of cellular apoptosis by administering a 2 trichothecene mycotoxin in an amount and for a duration sufficient to significantly increase the rate of apoptosis. In order to determine whether a specific group of cells is undergoing excessive apoptosis, they may be stained, microscopically examined, and the results compared with comparable cells known to be normal. Other assays, e.g., those described in the Examples section, may also be used. A "significant increase" is one that is statistically significant according to scientifically accepted standards. Preferably the condition treated is cancer and the trichothecene induces the death of cancerous cells. Preferred trichothecenes are those that do not substantially inhibit protein synthesis. As used herein, a trichothecene does not substantially inhibit protein synthesis if, when tested at a concentration of 10 μM in the in vitro assay described in the Examples section herein, protein synthesis is maintained at a level of at least

70% of that seen in untreated control cells. The preferred trichothecenes are T-2 tetraol and 3- acetyldiacetoxyscirpentriol.

In a second aspect, the invention is directed to a method for assaying a trichothecene mycotoxin for its potential as a clinically useful anticancer therapeutic by determining both its apoptotic activity and its effect on protein synthesis. Any method for evaluating these properties may be used but the caspase-3 and protein synthesis assays described in the Examples section herein are generally preferred. The higher the ratio of apoptotic to protein synthesis inhibitory activity, the greater the potential that the compound has as a clinically useful drug. For example, if a compound assayed at a concentration of 10 μM induces a 2-fold increase in apoptotic activity and inhibits protein synthesis by 20% relative to controls, this would be preferred over a compound that induced a 2-fold increase in apoptotic activity but inhibited protein synthesis by 40%.

The invention is also directed to a method for inducing apoptosis in cultured cells by contacting the cells with a solution that contains one or more trichothecene mycotoxins. Preferred compounds for use in the assay are deoxynivalenol, scirpentriol and T-2 triol. Other compounds that may be used include nivalenol, diacetoxyscirpentriol, HT-2 and diacetylverrucarol. The concentration of mycotoxins can be adjusted to optimize apoptosis using methods that are standard in the art. Studies performed in vitro can be used to help identify cells, e.g., cells obtained in biopsy samples, that are responsive to the mycotoxins. 3

The present invention is also directed to a method for assaying a test compound for an ability to induce cellular apoptosis by determining the extent to which the compound interferes with the interaction between trichothecene mycotoxins, or similar compounds, and ribosomes. One way to carry out this method is to perform binding assays in which ribosomes or a ribosomal fragment containing the trichothecene mycotoxin binding site are incubated with a detectably labeled ligand known to modulate apoptosis by ribosomal interaction. The results obtained in the absence of test compound are compared with the results obtained from incubating ribosomes with a detectably labeled ligand in the presence of test compound. Results indicating that the test compound is capable of displacing the specific binding of the labeled ligand suggests that the compound might be used to induce apoptosis. Trichothecene mycotoxins that may be labeled and used in the assay include deoxynivalenol, scirpentriol, nevalenol, diacetoxyscirpentriol, HT-2 and diacetylverrucarol. Preferably a compound identified as displacing ligand will also be directly assayed for apoptotic activity and for its effect on protein synthesis.

Brief Description of the Figures

Figure 1 : Figure 1 shows formulas for nivalenol, scirpenol and T-2 toxin subfamilies of trichothecenes. The arrows point out inter-family differences at the C7 and C8 side chains. For structural features of R1-R4 side chains see Figure 2 A.

Figure 2: Figure 2A shows trichothecenes used in experiments discussed herein. With respect to JNK activation, the symbol + indicates more than a five-fold activation; the symbol +/- indicates a 2 to 5 fold activation; and the symbol - indicates less than a 2-fold activation. With respect to protein synthesis, the symbol + indicates strong inhibition, with protein synthesis occurring at a level below 15 percent of control; the symbol +/- indicates weak inhibition, with protein synthesis occurring at between 15 and 70 percent of the control level; and the symbol - indicates very weak inhibition with protein synthesis occurring at 70 percent or more of the control level. Figure 2B shows the effect on protein synthesis of compounds other than trichothecenes. 4

Detailed Description of the Invention

Programmed cell death, apoptosis, is a normal aspect of animal development and tissue homeostasis. The process serves to regulate cell number, facilitate morphogenesis, remove harmful or abnormal cells, and eliminate cells that have already performed their function. Unfortunately, abnormal changes in the rate of cellular apoptosis sometimes occur and have been associated with a number of pathological conditions, including cancer.

The present invention is concerned with agents, the trichothecene mycotoxins, that interact with ribosomes and induce apoptotic activity. These agents may be used in vitro to determine whether cells are responsive and in experiments designed to elucidate the pathways by which apoptosis is controlled. In order to determine the extent to which apoptotic activity is proceeding, samples of cultured cells may be harvested, pelleted, and mixed with acridine orange diluted in phosphate-buffered saline. The percentage of cells with apoptotic morphology (nuclear and cytoplasmic condensation, nuclear fragmentation, membrane blebbing, apoptotic body formation) may then be analyzed microscopically. Alternatively, cells may be stained with propidium iodine as described by Nicoletti et al. (J. Immunol. Methods 139:271-279

(1991)). The percentage of apoptotic cells may then be quantitated using Flow Cytometry with apoptotic cells being distinguished from non-apoptotic intact cells by a decreased DNA content as reflected by lower propidium iodine staining intensity. Other methods for measuring cellular apoptosis are described in the Examples section and may also be used.

Agents inducing apoptosis such as the trichothecene mycotoxins may also be delivered in vivo to a patient suffering from a condition characterized by abnormal cellular apoptosis. Biopsy samples obtained from the patient can be used to determine the percentage of cells that are apoptotic and, by comparing the results with similar tissue samples from normal individuals, a determination can be made as to whether abnormal apoptosis is occurring. Typically, a physician should begin by administering a low dose of therapeutic agent and then determine whether any improvement has been observed in a patient's condition. For example, an improvement in a cancer patient would be evidenced by a reduction in tumor growth, a reduction in tumor size, a reduction in the number of metastases associated with a tumor, etc. If no response is seen at the initial dosage, it may then be raised until a therapeutic effect is achieved or side effects become unacceptable. 5

Preferred trichothecene mycotoxins induce apoptosis but have relatively little effect on protein synthesis. A physician may begin by initially administering an agent at a dosage of, for example, 1 nmol/kg/day and increase the dosage over a period of weeks up to, for example, 1 μmol/kg/day. During this time, the symptoms of the patient would be periodically evaluated. If no improvement was observed over, for example, a period of three months, drug administration may be discontinued. These are simply guidelines, since the actual dosage will be carefully selected and titrated by the attending physician based upon clinical factors unique to each patient. The optimal daily dose will be determined by methods known in the art and will be influenced by factors such as the age of the patient, disease state, side effects associated with the particular agent being administered, and other clinically relevant factors.

The present methods are not limited to any particular dosage form or route of administration. Although oral administration will generally be most convenient, the invention is compatible with parenteral, transdermal, sublingual, buccal, or implantable routes of administration as well. Agents may be given in a substantially purified form or, preferably, as part of a pharmaceutical composition containing one or more excipients or flavoring agents.

Compositions may also include other active ingredients for the treatment of patients. The preparations may be solid or liquid and take any of the pharmaceutical forms presently used in medicine, e.g., tablets, gel capsules, granules, suppositories, transdermal compositions, or injectable preparations.

The active ingredient or ingredients may be incorporated into dosage forms in conjunction with the vehicles that are commonly employed in pharmaceutical preparations, e.g. , talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Methods for preparing appropriate formulations are well known in the art (see, e.g.. Remington's Pharmaceutical Sciences. 16th ed., A. Oslo ed., Easton, PA (1980)).

In order to determine the effect of a treatment on disease, patients should be evaluated on a regular basis over an extended period of time. It may take several weeks for the full therapeutic effect of a treatment to become apparent. The effect of treatment on apoptotic activity can be determined by obtaining biological samples from the patient and then staining 6 them in one of the manners discussed above. The effect of treatment on parameters such as tumor size, tumor growth and tumor metastasis may be determined using standard radiological procedures.

In another aspect, the present invention is directed to a method for assaying a test compound to determine if it will induce cellular apoptosis. In binding assays, isolated ribosomes, or ribosomal fragments containing the site at which trichothecene mycotoxins interact, are incubated together with a ligand known to bind to this site and with the compound being tested. Preferably, the ligand will be a detectably labeled trichothecene mycotoxin. After binding is complete, ribosomes are separated from the solution containing the ligand and test compound, e.g., by centrifugation, and the amount of binding that has occurred is determined.

Non-specific binding may be determined by carrying out the binding reaction in the presence of a large excess of unlabeled ligand. For example, labeled ligand may be incubated with ribosome and test compound in the presence of a thousandfold excess of unlabeled ligand. Nonspecific binding should be subtracted from the total binding, i.e., binding in the absence of unlabeled ligand, to arrive at the specific binding for each sample tested. Other steps such as washing, stirring, shaking, filtering and the like may be included in the assays as necessary. It is highly desirable that compounds identified as displacing the binding of ligand to ribosome be re-examined in a concentration range sufficient to perform a Scatchard analysis on the results. This type of analysis is well known in the art and can be used for determining the affinity of a test compound for receptor (see, e.g., Ausubel et al., Current Protocols in

Molecular Biology. 11.2.1-11.2.19 (1993)).

An alternative and/or complimentary procedure is to test compounds for their ability to induce apoptosis but not inhibit protein synthesis. This may be carried out using the protocols described in the Examples section below. 7

Examples

Example 1 : Trichothecene Mycotoxins Regulate a Ribosomal Signaling Pathway that Activates JNK/p38 MAP Kinases

A. Experimental Procedures Materials

Trichothecenes and other protein synthesis and protease inhibitors were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Stock solutions were prepared in DMSO at 3.3 mM, or in water at 10 mg/ml (puromycin, emetin and cycloheximide).

Cell treatments Jurkat T-lymphoid human cells were grown in RPMI 1640 medium supplemented with

10%) heat-inactivated fetal bovine serum, 500 U/ml of penicillin and 500 μg/ml of streptomycin. For various treatments, cells were collected at 1.0-1.5 x lOVml and resuspended at 1.0 x 10⁷/ml in the fresh growth media. Anisomycin, trichothecenes or other protein synthesis inhibitors (or equivalent volumes of solvents for control samples) were added in a volume not exceeding 1% of a total culture volume and incubated at 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 in culture at 37°C for an additional 2 hrs. Cells were collected by centrifugation at 2000x g for 1 min at 4°C, and washed twice with ice-cold phosphate-buffered saline (PBS) before freezing cell pellets in dry-ice ethanol for storage at -80 °C until further analysis.

Activation of stress-activated kinasep38

Activation of kinase p38 was assayed as described previously (Gabai, et al., J. Biol.

Chem. 272:18033-18037 (1997)) 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 (New England Biolabs), recognizing both phosphorylated and unphosphorylated forms of p38 to verify equal loading. 8

.INK kinase assays

JNK kinase activity was assayed as described previously (Shifrin, et al, J. Biol Chem. 272:2957-2962 (1997)) with slight modifications. Aliquots of 1.0 x IO⁷ frozen cells were lysed in 200 μl of lysis buffer A (20 mM HEPES, pH 7.1, 1% Triton X-100, 50 raM KC1, 5 mM EDTA, 5 mM EGTA, 50 mM β-glycerophosphate, 2 mM DTT, 1 mM Na₃PO₄, 50 mM NaF,

50 nM calyculin A, 10 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml antipain, 250 mg/ml benzamidine and 20 μg/ml PMSF), incubated on ice for 10 min, vortexed for 10 sec and clarified by centrifugation at 15,000x g for 5 min at 4°C. 5μl of supematants were added to a 25 μl reaction volume in 40 mM HEPES pH 7.1, 25 nM calyculin A, 1 mM Na₃PO₄, 10 mM MgCl₂, 50 μM ATP including 10-20 μCi of [γ]32P-ATP (NEN, Boston, MA) and 1 μg of GST- cJun (1-135) as a substrate. After 20 min incubation at 30°C, reactions were stopped by adding lOμl of 4x SDS loading buffer with 2-mercaptoethanol (Harlow et al, Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988)) and boiling for 5 min. One third of each reaction was separated on an SDS-polyacrylamide gel (Harlow et al, Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY (1988)), blotted onto PVDF membrane (Immobilon P, Millipore, Bedford, MA), exposed to X ray film and subsequently quantitated using a BioRad Model GS-525 Phosphor Imager.

Protein synthesis inhibition assays Protein synthesis was assayed by measuring the incorporation of labeled amino acids into cellular proteins, essentially as described in Ausubel et al, (Current Protocols in Molecular Biology. John Wiley and Sons (1995))with the following modifications: Jurkat cells were grown and collected as described above, washed once with Hank's balanced salt solution and resuspended at 2xl0⁶/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, Boston, MA) and incubation for an additional 20 min at 37°. Cells were then centrifuged at 2000X g for 1 min at 4°, washed twice with ice-cold PBS and solubilized in lysis buffer (200 μl per 10⁶ cells; 10 mM Tris pH 7.2, 0.1 % SDS, 1 % Triton

X-100, P/o sodium deoxycholate, 150 mM NaCl, 20 μg/ml chymostatin, 3 μg/ml leupeptin, 14 9 μg/ml pepstatin A, 1.7 mg/ml benzamidine and 10 μg/ml aprotinin). After 10 min incubation on ice, cell lysates were vortexed for 10 seconds and clarified by centrifugation at 15,000x g for 10 min. 50 μl of the supematants were mixed with 500 μl of 100 μg/ml BSA, and proteins were precipitated by the addition of 500 μl of 20%> TCA. After 20 min. on ice, precipitated proteins were collected by filtration through glass microfiber filters (GF/C, Whatman,

Hillsboro, OR), washed with 10 ml of 10%> TCA 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.

In competition experiments, cells were treated as described above, except that treatment with the first agent was for 30 min, and treatment with the second agent and subsequent labeling were for 20 min each. In long-term protein synthesis inhibition experiments cells were treated with inhibitors for 2 firs prior to 20 min labeling as described above.

B. Results and Discussion

Activation of INK andp38 MAP kinases by selected trichothecenes Jurkat T cells were used to compare the relative ability of anisomycin and trichothecene mycotoxins to inhibit protein synthesis (Table 1) and activate IN and p38 SAMKs. The trichothecenes are divided into three structural subfamilies which differ in the side groups at the C7 and C8 positions of the trichothecene molecule (Fig. 1, arrows; see also Fig. 2A for individual structures). Within all three subfamilies (derivatives of nivalenol, scirpenol and T-2 toxin), members were identified that strongly activate SAMKs (e.g., nivalenol, scirpentriol and

T-2 triol), as well as members that more weakly activate SAMKs (e.g., 3-acetyldeoxynivalenol, HT-2). There is a very good correlation between the ability of individual trichothecenes to activate JNKs and p38. Differences in the ability of individual trichothecenes to activate SAMKs could not be explained by differential cell permeability, since several closely related trichothecenes (e.g., deoxynivalenol and 3-acetyldeoxynivalenol; T-2 triol and T-2 toxin) differ dramatically in their ability to activate JNKs and p38, but similarly inhibit protein synthesis (Table 1). The ability of various nivalenol derivatives (which activate SAMKs), but not T-2 toxin or verrucarin A (which do not activate SAMKs) to increase IL-2 expression in activated CD4+T cells (Ouyang, et al. Toxicology 104:187-202 (1995)) further suggests that 10 trichothecene-induced SAMK activation targets a physiologically important component of the stress-activated kinase signaling cascade.

Trichothecene-induced translational arrest is not sufficient to activate SAMKs: although T-2 toxin, acetyl T-2 toxin and T-2 triol are strong protein synthesis inhibitors, only T-2 triol activates SAMKs. Moreover, trichothecene-induced translational arrest is not necessary for

SAMK activation, since T-2 tetraol activates SAMKs without significantly inhibiting protein synthesis. The inability of T-2 tetraol to inhibit protein synthesis was reported previously (Middlebrook, et al, J. Pharmacol. Exp. Ther. 250:860-866 (1989)), where it was estimated that T-2 tetraol is about 300-fold less potent, on a molar basis, than T-2 toxin. This reduced potency is not due to decreased ribosome binding, since T-2 tetraol binds to isolated ribosomes as efficiently as T-2 toxin (Middlebrook et al, J. Pharmacol. Exp. Ther. 250:860-866 (1989)). Taken together, these results suggest that the ability of trichothecenes to inhibit protein synthesis is independent of their ability to activate SAMKs.

The kinetics of JNK activation by anisomycin and T-2 triol are similar, with each drug producing maximal activation within 15 min. The slower kinetics of T-2 tetraol are probably a consequence of its slower rate of cellular uptake (Middlebrook et al. , J. Pharmacol. Exp. Ther. 250:860-866 (1989)). Maximal activation of JNK kinases by T-2 tetraol (which occurs after 2 hours) occurs in the absence of significant protein synthesis inhibition, reaffirming our conclusion that translational arrest is not necessary for trichothecene-induced JNK activation.

Deacetylanisomycin does not activate JNKs and inhibits anisomycin- and trichothecene-induced .JNK activation

Anisomycin has been proposed to activate SAMKs by triggering a ribotoxic stress response (Iordanov, et al, Molecular and Cellular Biology 17:3373-3381 (1997)). The ability of T-2 tetraol to dissociate effects on protein synthesis from effects on SAMK- activation leads to a question of whether ribosome binding is required for trichothecene-mediated activation of

SAMKs. Derivatives of anisomycin or trichothecene that arrest translation without activating

SAMKs should prevent the binding of structurally related SAMK-activating compounds to ribosomes. The ability of these compounds to inhibit SAMK activation would be consistent with a requirement ribosome binding for activation of SAMKs. 11

Deacetylation of anisomycin (see Fig. 2B for a structural formula) was previously reported to eliminate its inhibitory effects on protein synthesis (Eskin, et al, Proc. Nat'l Acad. Sci. USA 57:7637-7641 (1984); Crow, et al, Proc. Nat'l Acad. Sci. USA 57:4490-4494 (1990)). Although deacetylation reduces the ability of anisomycin to inhibit protein synthesis by approximately 10,000-fold, deacetylanisomycin (DA) still induces a dose-dependent translational arrest in Jurkat cells (Table 1). In contrast, DA is unable to activate SAMKs at even the highest concentration tested, thus fulfilling the requirements for a potential competitive inhibitor of S AMK-activating anisomycin/trichothecene compounds.

As an indirect measure of the ability of DA to penetrate the plasma membrane and interact with ribosomes, the effects of DA on anisomycin-induced translational arrest were measured. At a concentration of 1350 μM, DA inhibits protein synthesis by approximately 70%). Pre-treatment with DA significantly impairs the ability of both anisomycin and trichothecenes to inhibit protein synthesis in intact cells, as well as in a reticulocyte lysate-based in vitro translation system. Thus, whereas deacetylation of anisomycin greatly reduces its ability to inhibit protein synthesis and eliminates its ability to activate SAMKs, it does not eliminate its ability to enter cells and interact with ribosomes. The ability of DA to inhibit translational arrest by structurally unrelated compounds that bind to a common site on the large ribosomal subunit (i.e., the trichothecenes) is consistent with a role for DA in the competitive displacement of anisomycin and trichothecenes from this binding site.

Whereas T-2 triol, T-2 tetraol, and anisomycin potently activate JNK in Jurkat cells, pre-treatment of Jurkat cells with DA eliminates JNK activation by these compounds in a dose-dependent manner. Pre-treatment with DA also inhibits nivalenol-, fusarenon- and trichothecene-induced JNK activation. These results suggest that JNK activation by both trichothecenes and anisomycin requires binding to a common ribosomal site, which is blocked by DA pre-treatment.

T-2 toxin inhibits anisomycin- and trichothecene-induced JNK activation independently of translational arrest

If ribosome binding is required for SAMK activation by selected trichothecenes, T-2 toxin, a trichothecene that inhibits protein synthesis without activating SAMKs, should also 12 inhibit the activation of SAMKs by anisomycin and trichothecenes. Indeed, T-2 toxin has previously been shown to block anisomycin-induced JNK activation (Iordanov, et al. , Mol Cell. Biol 77:3373-3381 (1997)). It was found that T-2 toxin also blocks trichothecene- induced JNK activation. Moreover, pre-treatment with either T-2 toxin or verrucarin A (a non-activating trichothecene from the scirpenol/verrucarol subfamily, see Fig. 2 and Table 1) also blocks nivalenol-, fusarenon- and trichothecene-, as well as anisomycin-induced JNK activation. Therefore, non-activating trichothecenes block JNK activation by both JNK- activating trichothecenes and anisomycin.

Previously, the blocking effect of T-2 toxin on anisomycin-induced JNK activation was suggested to be a consequence of T-2-induced translational arrest (Iordanov, et al, Mol. Cell.

Biol. 77:3373-3381 (1997)). In order to verify this explanation, T-2 toxin (10 μM) was used to inhibit protein synthesis in Jurkat cells by >98%> prior to the addition of graded concentrations of anisomycin. Although T-2 toxin inhibits JNK activation at low concentrations of anisomycin, the inhibitory effect is overcome at higher concentrations of anisomycin, indicating that anisomycin can activate JNK in the absence of protein synthesis. These results indicate that

T-2 toxin blocks anisomycin-induced JNK activation not by imposing a translational arrest, but by competing with anisomycin for biding to a common ribosomal site, an effect that can be overcome by increasing the concentration of anisomycin. Taken together, these results strongly suggest that the ability of anisomycin and trichothecenes to inhibit protein synthesis and/or activate SAMKs requires binding to a common ribosomal target. Although the chemical structure of individual compounds determines whether ribosome binding results in translational arrest and or SAMK activation, these functional effects are dissociable.

Protein synthesis inhibitors that bind ribosomes also block trichothecene- and anisomycin-induced JNK activation Competition between anisomycin and trichothecenes, as well as between trichothecenes and harringtonines for binding to a common site on the 60S ribosomal subunit is well documented (Jimenez, et al, Eur. J. Biochem. 54:483-492 (1975); Cannon, et al, Biochem. J. 160:137-45 (1976); Middlebrook, et al, Biochem. Pharmacol. 755:3103-3110 (1989); Fresno, et al, Eur. J. Biochem. 72:323-330 (1977)). Harrington, a plant alkaloid structurally unrelated to either anisomycin or the trichothecenes (Fig. 2B) was tested for its ability to activate JNK 13 and/or interfere with JNK activation induced by anisomycin or trichothecenes. It was found that harringtonine does not activate JNKs, but efficiently blocks both anisomycin- and trichothecene- induced JNK activation. This effect is not a consequence of translational arrest per se, since a similar level of protein synthesis inhibition produced by puromycin (which does not bind to ribosomes, but causes premature termination), does not block anisomycin- or trichothecene- induced JNK activation. Interestingly, translational arrest induced by cycloheximide (which is capable of significant JNK activation on its own) still allows additional JNK activation by trichothecenes and anisomycin, again suggesting that active translation is not required for anisomycin- or trichothecene-induced JNK activation.

Another ribosome-binding protein synthesis inhibitor, emetine, was previously reported to block JNK activation by anisomycin (Iordanov, et al., Mol. Cell. Biol. 17:3373-3381 (1997)). Although emetine's binding site 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 (Leatherman, et al. J. Pharmacol. Exp. Ther. 266:732-740 (1993)). It was found that emetine blocks anisomycin- and T-2 tetraol-induced JNK activation, as well as fusarenon-, nivalenol- and trichothecin-induced JNK activation, suggesting that emetine blocks anisomycin' s or trichothecenes' access to a ribosomal binding site, rather than blocking JNK activation through a general translational arrest. Interestingly, emetine does not block T-2 triol-induced JNK activation. Although this reinforces the 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. 2) at the C8 position. It is possible that this side group somehow stabilizes the binding of T-2 triol to the ribosome, preventing its competitive displacement by emetine.

What is the mechanism whereby DA, T-2 toxin, harringtonine and emetine block JNK activation by anisomycin and trichothecenes? The experiments discussed above clearly demonstrate that the translational arrest imposed by these molecules can be dissociated from their ability to inhibit JNK activation by anisomycin and trichothecenes. At the same time, the ability of non-SAMK activating compounds to inhibit the effect of SAMK-activating compounds strongly suggests a requirement for ribosome binding in SAMK activation. The fact that three structurally distinct compounds (i.e., DA, T-2 toxin and harringtonine) that compete 14 for binding to a common ribosomal site are also inhibitors of SAMK activation makes it improbable that a second intracellular target is responsible for the effects observed. Indeed, the convergent evolution of structurally distinct natural products that target a common ribosomal site points to the importance of this site in the regulation of ribosome function. It is likely that this site regulates not only translation, but also a ribosome-intrinsic stress signaling pathway.

The ability of pactomycin, a protein synthesis inhibitor that prevents coupling of the 60S ribosomal subunit with the 40S initiation complex (Vazquez, Mol. Biol. Biochem. Biophys. 30:1-10 (1979)) emetine and T-2 toxin to inhibit anisomycin-induced SAMK activation led Iordanov, et al. (Mol. Cell. Biol. 7:3373-3381 (1997)) to conclude that active translation is required to trigger the ribotoxic stress response. The results discussed above clearly show that both anisomycin and trichothecenes can activate SAMKs in Jurkat cells rendered translationally incompetent by either T-2 toxin, puromycin, or cycloheximide. Although pactomycin, like emetine, binds to the small ribosomal subunit, it is not known to interfere with the binding of anisomycin or trichothecenes. However, photoaffinity labeling experiments indicate that it also interacts with the large ribosomal subunit (Synetos, et al, Biochim. Biophys. Ada 5(55:249-253

(1986); Tejedor, et al., Biochemistry 24:3667-3672 (19985)), suggesting that it might block access to the anisomycin- and trichothecene-binding site. Alternatively, by preventing the assembly of functional 80S ribosomes, pactomycin might prevent the formation of a functional anisomycin-binding site.

The identification of trichothecene derivatives (e.g., T-2 tetraol and, to a smaller degree,

3-acetyldiacetoxyscirpentriol, Table 1) that activate JNK without significantly affecting protein synthesis suggests that different parts of the trichothecene molecule are responsible for translational arrest and JNK activation. (In the case of the smaller anisomycin molecule, however, the Rl group (see Fig. 2) may be responsible for both translational arrest and JNK 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 SAMKs following the catalytic depurination or cleavage of 28S RNA suggests that a conformational change in the ribosome can result in SAMK activation. The ability of some trichothecenes, but not others, to activate SAMKs suggests that 15 the presence or absence of side groups that interact with ribosome target sites or displace the binding of ribosome-associated molecules might determine whether SAMKs are activated. The effect that various modifications of trichothecene structure have on JNK activation seems to be a direct one, since these modifications do not affect the ability of most trichothecenes to enter cells and rapidly inhibit translation (Table 1).

Although there is no simple correlation between the structure of R1-R4 side groups within trichothecene subfamilies and the ability to activate JNKs, one structural feature that might favor SAMK activation is the presence of a hydroxyl group at the C3 position of the pentane ring (i.e., Rl in Fig. 2 A; witness nivalenol, deoxynivalenol, fusarenon, scirpenetriol, T-2 triol, and T-2 tetraol). Exceptions to this generally are diacetoxyscirpentriol and T-2 triol both of which have acetyl groups at their R2 and R3 positions, modifications that might blunt SAMK activation (witness diacetoxyscirpentriol, 3-acetyldiacetoxyscirpentriol, T-2 tetraol tetraacetate, acetyl T-2; Fig. 2A). Therefore, the electron density and charge distribution of the R1-R3 -containing part of the trichothecene molecule might influence the ability of a given trichothecene to trigger ribosome-generated SAMK-activating signals.

16

Table 1. Inhibition of protein synthesis by various compounds.

Protein synthesis,

Treatment % of control, (+/- SD)

Control 100 (7.5)

Anisomycin, 38μM 1.7 (0.2) Anisomycin, 3.8 μM 5.1 (1.1) Anisomycin, 0.38 μM 26.7 (0.8) Anisomycin, 0.038 μM 101.9 (9.5)

DA, 45 μM 89.5 (6.5) DA, 450 μM 53.8 (13.2)

Nivalenol, lOμM 51.6 (5.8) Deoxynivalenol, 10 μM 6.9 (0.7) 3-Acetyldeoxynivalenol, 10 μM 2.2 (0.1) Fusarenon, 10 μM 3.6 (0.69) Trichotecin, 10 μM 3.1 (0.3)

Verrucarol, 10 μM 92.4 (6.5) Diacetyl verrucarol, 10 μM 7.1 (1.5) Verrucarin A, 10 μM 1.6 (0.2) Scirpenetriol, 10 μM 3.6 (0.34) Diacetoxyscirpentriol, 10 μM 2.4 (0.19) 3-Acetyldiacetoxyscirpentriol, 10 μM 70.5 (1.8)

T-2, 10 μM 1.4 (0.2)

T-2 triol, μM 4.7 (0.4)

T-2 tetraol, 10 μM 83.6 (9.8)

T-2 tetraol tetraacetate, 10 μM 49.4 (2.9)

Acetyl T-2, 10 μM 2.1 (0.1)

HT-2, 10 μM 1.6 (0.1)

Iso-T-2, 10 μM 11.0 (2.6)

Cycloheximide 100 μg/ml (356 μM) 1.8 (0.1)

Puromycin 100 μg/ml (184 μM) 1.0 (0.1)

Emetin 100 μg/ml (180 μM) 1.2 (0.1)

Harringtonine, 10 μM 2.8 (0.1) 17

Example 2: Trichothecene Mycotoxins Trigger a Ribotoxic Stress Response that Activates JNK and p38 MAP Kinases and Induces Apoptosis

A. Experimental Procedures

Except as indicated below, all procedures and materials were the same as for Example 1.

Materials

Trichothecenes and other protein synthesis and protease inhibitors were obtained from Sigma (St. Louis, MO) unless indicated otherwise. Stock solutions were prepared in DMSO at 3.3 mM, or in water at 10 mg/ml (puromycin, emetin and cycloheximide).

Cell treatments Jurkat T-lymphoid human cells were grown in RPMI 1640 medium supplemented with

10%) heat-inactivated fetal bovine serum, 500 U/ml of penicillin and 500 μg/ml of streptomycin. For various treatments, cells were collected at 1.0-1.5 x 107ml and resuspended at 1.0 x 10⁷/ml in the fresh growth media. Anisomycin, trichothecenes or other protein synthesis inhibitors (or equivalent volumes of solvents for control samples) were added in a volume not exceeding 1% of a total culture volume and incubated at 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 in culture at 37 °C for indicated periods of time. Cells were collected by centrifugation at 2000x g for 1 min at 4°C, and washed twice with ice-cold phosphate-buffered saline (PBS) and then frozen in liquid nitrogen for storage at -80 °C until further analysis.

DNA fragmentation assay (first apoptosis induction assay)

After 3 hours of treatment with various agents, 5 x IO⁶ Jurkat human T-lymphoid cells per treatment were lysed in 0.5 ml of 10 mM Tris (pH 7.5), 1% Triton X-100, 5 mM EDTA, incubated on ice for 10 min, vortexed for 5 sec, and lysates were clarified for 5 min at 4°C in an Eppendorf microcentrifuge at top speed. 0.45 ml of the supematants 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 18 microcentrifiige at 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 (Tian, et al, Cell 67:629-39 (1991)).

Fluorescent assay of caspase-3 activity (second apoptosis induction assay)

DEVD-specific caspase activity was determined as described (Nicholson, et al., Nature 376:37-43 (1995)) with modifications: IO⁷ cells were resuspended in 0.1 ml of lysis buffer (20 mM HEPES, pH 7.1, 1% Triton X-100, 10 mM KCI, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 5 μg/ml pepstatin, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 25 μg/ml ALLN (Broehringer Mannheim, Indianapolis, IN)), incubated on ice for

10 min, vortexed for 5 sec, and lysates were clarified for 10 min at 4 °C in an Eppendorf microcentrifuge the top speed. DEVD-specific caspase activity was determined in triplicate by mixing 10 μl of supematants (50 μg of protein) with 0.2 ml of reaction buffer (100 mM HEPES (pH 7.1), 10% sucrose, 0.1% CHAPS, 10 mM DTT, 0.1 mg/ml BSA 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 a CytoFluor 4000 MultiWell Plate Reader (PerSeptive Biosystems, Framingham, MA) with excitation at 360 nm and emission at 460 nm.

Caspase-3 and PARP cleavage (third apoptosis induction assay)

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 manufacturer's instructions and with PARP-specific monoclonal antibodies C-2-10 as described previously (Budihardjo, et al, Mol. Cell. Biochem. 775:245-9

(1988)).

Activation of stress-activated kinase p38 Activation of kinase p38 was assayed as described previously (Gabai, et al, J. Biol.

Chem. 272:18033-18037 (1997)) 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 (New England Biolabs), recognizing both phosphorylated and unphosphorylated forms of p38 to verify equal loading. 19

B. Results

Activation of JNK andp38 MAP kinases by trichothecenes

The relative ability of anisomycin and trichothecene mycotoxins to activate JNK and p38

MAP kinases in Jurkat T cells was compared. Within each structural subfamily (i.e., derivatives of nivalenol, scirpenol and T-2 toxin), trichothecenes were identified 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, are similarly potent inhibitors of protein synthesis (see Table 2 below). The strong correlation between the ability of individual trichothecenes to activate JNKs and p38 suggests that both of these MAP kinases are activated via the same mechanism — the ribotoxic stress response.

Therefore, the data indicate that structural differences between individual trichothecenes can influence their ability to trigger the ribotoxic stress response.

Induction of apoptosis by trichothecenes

During analysis of JNK activation by various trichothecenes, it was 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 intemucleosomal DNA fragmentation, processing of pro-caspase-3, activation of DEVD-specific caspases, and cleavage of one of the major caspase-3 substrates - poly (ADP) ribose polymerase (PARP). 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 revealed that activation of stress kinases is not sufficient for the induction of apoptosis. Thus, trichothecenes that similarly activate stress kinases (e.g., T-2 triol and T-2 tetraol) can differ significantly in their ability to induce apoptosis as measured by caspase-3 activation. Nevertheless, the most potent apoptotic trichothecenes strongly activate stress kinases, suggesting that kinase activation contributes to the efficient induction of rapid apoptosis. 20

The sequential activation of stress-induced MAP kinases and caspases differs in different experimental systems (Goillot, et al, Proc. Nat'l Acad. Sci. USA 94:3302-3307 (1997); Deak, et al, Proc. Nat'l Acad. Sci. USA 95:5595-600 (1998); Juo, et al, Mol. Cell. Biol. 77:24-35 (1997); Rudel, et al, J. Immunol. 7(50:7-11 (1998); Rudel, et al, Science 27(5:1571-4(1997)). Because of the strong correlation between JNK activation and trichothecene-induced apoptosis summarized in Table 2, the temporal order of JNK and caspase-3 activation was determined for several trichothecenes and anisomycin. The kinetics of JNK activation by anisomycin and T-2 triol are similar, with each drag producing maximal activation within 15 min followed by detectable caspase activation at 1 hr. 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

(Middlebrook, et al, Biochem. Pharmacol. 55:3103-3110 (1984); Middlebrook, et α/.,(1989) J. Pharmacol. Exp. Ther. 250:860-866 (1989)), and therefore requires two hours for maximal activation of JNK. Correspondingly, significantly longer incubations with T-2 tetraol are required to induce caspase-3 activation. 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 stress kinases are activated after caspase-3 during the later stages of cell death (Juo, et al, Mol Cell. Biol. 17:24-35 (1997); Rudel, et al, J. Immunol. 160:7-11 (1998); Rudel, et al, Science 27(5:1571-4 (1997)).

Inhibition of protein synthesis by trichothecenes Activation of stress kinases can signal cell survival or induce cell death in different cell types under different conditions. Inhibitors of protein synthesis can promote the induction of apoptosis in response to inflammatory cytokines that activate stress kinases (e.g., Fas-ligand, TNF- ), suggesting that the survival pathway, but not the death pathway, requires new protein synthesis (Leist, et al, J. Immunol. 755:1778-88 (1994); Nagata, Cell 55:355-365 (1997)). Table 2 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 vs. JNK activation; protein synthesis inhibition vs. caspase activation; JNK activation vs. caspase activation), the tendency for apoptotic trichothecenes to strongly inhibit protein synthesis and strongly activate JNKs (Table 2) suggests that these two effects might cooperate in the induction of apoptosis. To confirm a role for protein synthesis inhibition and JNK activation in the activation of apoptotic 21 caspases, a comparison was made of the ability of trichothecenes that strongly activate JNK kinases (>9 fold activation) or strongly inhibit protein translation (>95% inhibition) to activate caspases. This analysis revealed that caspase activation is a dependent, linear function of both protein synthesis inhibition and JNK activation. The results indicate that trichothecene-induced translational arrest and stress kinase activation cooperate in the induction of apoptosis.

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 stress kinase activation are independently triggered by individual trichothecenes led to the question of 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) 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). 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, T-2 tetraol and anisomycin. At similar concentrations, DA also inhibits anisomycin-induced translational arrest in rabbit reticulocyte lysates, suggesting that its functional effects are a consequence of ribosome binding. T-2 toxin and verrucarin similarly inhibit the activation of JNKs by these compounds. Pre-treatment with either DA, T-2 toxin or verrucarin also prevents caspase-3 activation in Jurkat cells cultured with apoptotic trichothecenes (T-2 triol, diacetylverrucarol and deoxynivalenol. The ability of DA and non-apoptotic trichothecenes to bind to the peptidyl transferase site and inhibit the function of anisomycin and apoptotic trichothecenes suggests that ribosome binding is required for the activation of stress kinases and, subsequently, caspase- 3. 22

Activation of the ribotoxic stress response by anisomycin does not require active translation

Activation of stress kinases in response to ribotoxic stress has been proposed to require on-going protein translation (Iordanov, et al, Mol. Cell. Biol. 77:3373-3381 (1997)). If this is true, the profound translational arrest induced by T-2 toxin and verrucarin could account for the inhibition of stress kinase activation observed. Translational arrest could not, however, account for the inhibition of stress kinase activation induced by DA, a compound that only partially inhibits protein synthesis under the conditions employed. A comparison was therefore made of the ability of anisomycin to activate JNKs in Jurkat cells pre-incubated in the absence or presence of T-2 toxin (10 μM, 30 minutes, 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, suggesting that displacement of T-2 toxin from the peptidyl transferase site might allow anisomycin to activate stress kinases in the absence of protein synthesis.

Further evidence that ribosome binding is required for peptidyl transferase inhibitors to activate stress kinases and induce apoptosis was provided by analyzing the effects of structurally unrelated compounds that compete with trichothecenes for ribosome binding. Emetine, previously reported to block JNK activation by anisomycin (Iordanov, et al, Mol. Cell. Biol. 17:3373-3381 (1997)), was found to block anisomycin- and T-2 tetraol-induced JNK activation as well as JNK activation induced by other trichothecenes (nivalenol, fusarenon and trichothecin). This suggests that emetine may block anisomycin's or trichothecenes' access to a common ribosomal binding site. Interestingly, emetine does not block T-2 triol-induced JNK activation. Although this reinforces the 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 at the C8 position.

Harringtonine, a plant alkaloid that is structurally unrelated to either anisomycin or trichothecenes, competes with these compounds for binding to the ribosomal peptidyl transferase site (Fresno, et al, Eur. J. Biochem. 72:323-330 (1977); Hobden, et al, Biochem.

J. 190:765-70 (1980)). It was found that harringtonine weakly activates JNKs on its own, but efficiently blocks both anisomycin- and trichothecene-induced JNK activation. Both emetine 23 and harringtonine also inhibit caspase activation by apoptotic trichothecenes. Here, the ability of emetine to prevent JNK activation by T-2 triol is reflected in its relative inability to block caspase activation by this trichothecene. Since extended treatment with many inhibitors of protein synthesis can induce apoptosis (Kochi, et al, Exp. Cell. Res. 208:296-302 (1993)), it is not surprising that treatment with emetine or harringtonine alone induced a low level of caspase-3 activation. However, the ability of both emetine and harringtonine to block anisomycin- or trichothecene-induced caspase-3 activation suggests that the mechanism of apoptosis induction by anisomycin/trichothecenes on one hand, and emetine or harringtonine, on the other, are different.

C. Discussion

Activation of JNK1 by protein synthesis inhibitors that bind to, or alter the structure of, 28S ribosomal RNA (e.g., blastocidin S, gougerotin, anisomycin, ricin toxin, sarcin toxin) led Iordanov, et al. (Mol. Cell. Biol. 77:3373-3381 (1997)) 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. This response promotes survival under starvation conditions (Cashel, et al, in Escherichia coli and Salmonella Typhimurium: Cellular and Molecular Biology ,(Neidhardt, et al, eds.) vol. 1, pp. 1410-38, American Society for Microbiology, Washington, D.C. (1997)).

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. The results discussed above provide support for this concept by showing that selected trichothecenes, compounds that target the ribosomal peptidyl transferase site (Jiminez, et al, Eur. J. Biochem. 54:483-492 (1975); Jiminez, et al, Biochim. Biophys. Acta 383:427-34

(1975)), also activate JNK and p38 MAP kinases. The rich structural diversity of the trichothecenes allowed for 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, these compounds may be grouped into distinct functional classes. Most trichothecenes strongly inhibit protein synthesis (Table 2). Among these, induction of apoptosis is linearly correlated 24 with the ability to activate JNK and p38 MAP kinases. 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 stress kinases may not be necessary for low level caspase activation. Among trichothecenes that strongly activate stress kinases, induction of apoptosis is linearly correlated with the ability to inhibit protein synthesis. Taken together, this analysis reveals that the ability of individual trichothecenes to induce rapid apoptosis is a function of both translational arrest and stress 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. ((Iordanov, et al, Mol. Cell. Biol. 77:3373-3381 (1997); Iordanov, et al, J. Biol. Chem. 275:15794-803 (1998); Iordanov, et al, J. Biol. Chem. 275:3528-34 (1998)) to conclude that active translation is required to trigger the ribotoxic stress response. The results above showing that high concentrations of anisomycin can overcome the T-2 toxin-induced inhibition of JNK activation require a re- examination of this conclusion. T-2 toxin inhibits protein translation by >98% under these conditions, indicating that anisomycin can activate JNKs in the absence of active translation.

Although it is possible that high concentrations of anisomycin trigger an alternative pathway to JNK activation, low concentrations of anisomycin activate JNKs in cells pre-treated with puromycin, a protein synthesis inhibitor that does not bind to ribosomes, but causes premature termination. The further observation that T-2 triol (but not T-2 tetraol or anisomycin) activates JNKs in cells rendered translationally incompetent by pre-treatment with emetine (producing

>98% inhibition of protein synthesis) is also not consistent with a requirement for active ribosomes in this process. Although emetine's binding site 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 (Leatherman, et al, J. Pharmacol. Exp. Ther. 2(5(5:741-748 (1993); Leatherman, et al, J. Pharmacol. Exp. Ther. 266:732-740

(1993)). 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 peptidyl transferase 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 stress kinases by palytoxin and UV 25 irradiation may be dependent upon protein synthesis). Pactomycin, another compound that inhibits the ribotoxic stress response (Iordanov, et al, Mol. Cell. Biol. 17:3373-3381 (1997)), also binds to the small ribosomal subunit. Although it is not known to interfere with the binding of anisomycin or trichothecenes, photoaffmity labeling experiments indicate that it also interacts with the large ribosomal subunit (Synetos, et al, J, Biochim. Biophys. Acta 838:249-

253 (1986); Tejedor, et al, Biochemistry 24:3667-3672 (1985)), suggesting that it could block access to the anisomycin- and trichothecene-binding site. Alternatively, by preventing the assembly of functional 80S ribosomes, pactomycin might prevent the formation of a functional anisomycin-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 stress 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 carcin to activate JNK1 following the catalytic depurination or cleavage of 28S RNA (Iordanov, et al, Mol. Cell. Biol. 77:3373-3381 (1997)) suggests that a conformational change in the ribosome can result in stress kinase activation. The ability of some trichothecenes, but not others, to activate stress 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 stress kinases are activated. The effect that various modifications of trichothecene structure have on JNK activation seems to be a direct one, since these modifications do not affect the ability of most trichothecenes to enter cells and rapidly inhibit translation.

In conclusion, the results herein suggest that: i) the functional effects of trichothecenes are initiated by ribosome binding, ii) trichothecene-induced translational arrest and stress kinase activation are independent, dissociable events; and iii) translational arrest and stress kinase activation cooperate in the induction of apoptosis. Consequently, classification of trichothecenes based on their relative ability to inhibit protein synthesis, activate stress kinases, 26 and induce apoptosis should facilitate the selection of natural and synthetic compounds for clinical trials in human cancers.

Table 2. Inhibition of protein synthesis, JNK Activation and Induction of Apoptosis

Trichothecenes Protein Caspase-3 JNK synthesis, % activation activation of control -fold -fold

Scirpentriol 3.6 (0.3) 16.6 14.4

Diacetylverrucarol 7.1 (1.5) 9.3 13.9

T-2 triol 4.7 (0.4) 12.4 11.9

Nivalenol 51.6 (5.8) 6.9 11.9

T-2 tetraol 83.6 (9.8) 1.7 9.0

Acetyldiacetoxysciφenol 70.5 (1.8) 1.5 4.3

Diacetoxysciφenol 2.4 (0.2) 9.4 3.4

Acetyldeoxynivalenol 2.2 (0.2) 4.8 2.5

HT-2 1.6 (0.2) 8.2 2.4

T-2 tetraoltetraacetate 49.4 (2.9) 1.8 1.3

Iso-T-2 11.0 (2.6) 1.7 1.3

Acetyl T-2 2.1 (0.1) 3.2 1.2

T-2 toxin 1.4 (0.2) 4.2 1.2

Verrucarin 1.6 (0.2) 3.9 1.2

Control 100.0 (7.5) 1.0 1.0

All references cited herein are fully incoφorated by reference. Having now fully described the invention, it will be understood by those of skill in the art that the invention may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof.

Claims

27What is Claimed is:

1. A method of treating a patient for a condition characterized by an abnormally low level of cellular apoptosis comprising administering to said patient a pharmaceutical composition comprising one or more trichothecene mycotoxins, wherein said pharmaceutical composition is administered in an amount and for a duration sufficient to significantly increase said cellular apoptosis.

2. The method of claim 1, wherein said patient is treated for cancer and said trichothecene mycotoxin is administered in an amount and for a duration sufficient to significantly increase the rate of apoptosis among cancer cells.

3. The method of either claim 1 or 2, wherein said trichothecene mycotoxin, at a concentration of lO╬╝M, does not substantially inhibit protein synthesis in vitro .

4. The method of claim 3, wherein said trichothecene mycotoxin is T-2 tetraol.

5. The method of claim 3, wherein said trichothecene mycotoxin is 3-acetyldiacetoxy- sciφentriol.

6. A method for determining the potential of a trichothecene mycotoxin as a clinically useful anticancer agent, comprising: a) assaying the apoptotic activity of said trichothecene mycotoxin; b) assaying the inhibitory effect of said trichothecene mycotoxin on protein synthesis; and c) comparing the results obtained in steps a) and b), wherein, a higher ratio of apoptotic to inhibitory activity is indicative of a greater potential that the compound will be a clinically useful anticancer agent.

7. The method of claim 6, wherein apoptotic activity is determined by assaying caspase-3 activity in cultured cells. 28

8. The method of claim 7, wherein the inhibitory effect of said compound on protein synthesis is assayed by determining the incoφoration of radioactively labeled amino acids into proteins in cells cultured both in the presence and absence of said trichothecene mycotoxin.

9. A method of inducing apoptosis in cultured cells by contacting said cells with a solution containing one or more trichothecene mycotoxins.

10. The method of claim 1, wherein said trichothecene mycotoxin is selected from the group consisting of deoxynivalenol, sciφentriol and T-2 triol.

11. The method of claim 1, wherein said trichothecene mycotoxin is selected from the group consisting of nivalenol, diacetoxysciφentriol, HT-2 and diacetylverrucarol.

12. A method of assaying a test compound to determine if it will induce cellular apoptosis, said method comprising: a) isolating ribosomes or ribosomal fragments containing the binding site for protein synthesis inhibitors such as trichothecene mycotoxins; b) incubating said ribosomes with a ligand that binds specifically to said binding site and which has been detectably labeled; c) incubating said ribosomes with said detectably labeled ligand in the presence of said test compound; d) determining that said test compound will induce cellular apoptosis if it displaces the specific binding of said detectably labeled ligand.

13. The method of claim 12, wherein said detectably labeled ligand is a trichothecene mycotoxin.

14. The method of claim 13, wherein said trichothecene mycotoxin is selected from the group consisting of deoxynivalenol and sciφentriol.

15. The method of claim 13, wherein said trichothecene mycotoxin is selected from the group consisting of nevalenol, diacetoxysciφentriol, HT-2 and diacetylverrucarol.