Activation of stress-activator protein kinase/c-Jun N-terminal kinase by the non-TPA-type tumor promoter palytoxin.

Previous studies have shown that structurally diverse tumor promoters can modulate protein kinases involved in signal transduction. In this study, we show that palytoxin, a potent non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor promoter, induces a signaling pathway leading to the activation of the stress-activated protein kinases/c-Jun N-terminal kinases (JNK) in Swiss 3T3 fibroblasts. Treatment of cells with doses as low as 0.1 mN palytoxin results in significant activation of JNK. In contrast to epidermal growth factor, which induces a transient activation of JNK in Swiss 3T3 cells, palytoxin causes prolonged enzyme activation. Since stimulation of ion flux appears to play an important role in the mechanism of action of palytoxin in other systems, we investigated the role of sodium and calcium in the activation of JNK: (a) our results show that incubation of Swiss 3T3 cells in a sodium-free medium dramatically reduced the magnitude of JNK activation by palytoxin; and (b) we found that the sodium ionophore gramicidin activates JNK. Together, these results suggest that sodium influx, which is a hallmark of palytoxin action, may play a key role in the activation of JNK by palytoxin. Our results indicate that calcium influx is not necessary or sufficient for palytoxin-induced activation of JNK. In contrast to palytoxin, the TPA-type tumor promoter phorbol 12,13-dibutyrate and the non-TPA-type tumor promoters thapsigargin and okadaic acid do not appear to activate JNK in this system. In contrast to phorbol 12,13-dibutyrate, palytoxin does not activate the p42/p44 mitogen-activated protein kinases. Our results demonstrate that Swiss 3T3 fibroblasts, palytoxin can activate a protein kinase signaling pathway that is distinct from that activated by the prototypical phorbol ester tumor promoters and other potent skin tumor promoters.

N-(4'-hydroxyphenylacetyl)palytoxin: a palytoxin prodrug that can be activated by a monoclonal antibody-penicillin G amidase conjugate.

Palytoxin (PTX), one of the most toxic nonprotein molecules known, is cytotoxic at picomolar concentrations against a wide variety of cell types. In contrast to most cytotoxins, PTX exerts its activity extracellularly. A method for targeting PTX to tumor cells is described in which a monoclonal antibody-enzyme conjugate activates a PTX prodrug at surfaces of tumor cells. The prodrug, N-(4'-hydroxyphenylacetyl)palytoxin (NHPAP), was prepared by reacting PTX with an active ester of 4-hydroxyphenylacetic acid. NHPAP was 1000 times less toxic than PTX to a panel of carcinoma and lymphoma cell lines. The cytotoxic activity of the combination of penicillin G amidase from Escherichia coli with NHPAP was equal to PTX. Two cell lines that were multidrug resistant showed no enhanced resistance to NHPAP +/- penicillin G amidase. Immunologically specific activation of NHPAP took place when H2981 cells (L6 antigen positive) were treated with the monoclonal antibody conjugate L6-penicillin G amidase followed by NHPAP. This system is distinguished from other prodrug activation schemes, since the released drug exerts its activity extracellularly, has high potency, and may be able to overcome the multidrug resistant phenotype.

Inhibition of the actions of palytoxin on the K+ efflux from red cells and on the Na/K-ATPase activity by a monoclonal antibody.

Palytoxin (PTX) binds to the Na/K pump, inhibits the (Na/K)-ATPase and forms Na and K permeable channels in human red cells. Here, we report that a monoclonal antibody raised against a derivative of PTX (Bignami, G.S. et al. (1992) Toxicon 30, 687-700) inhibits these effects. The observations are consistent with a model in which (a) the antibody binds and, thus reduces the concentration of free PTX available to react with the Na/K pump, and, (b) the PTX-antibody complex also binds to the PTX receptor on the Na/K pump but in such a way that a cation permeable channel is not formed, probably by reducing the concentration of free PTX. Using this model, we estimate that the apparent dissociation constant for the binding of PTX to antibody is 0.2 nM.

Biological activity of 26-succinylbryostatin 1.

Bryostatin 1, a macrocyclic lactone, has undergone phase I trials as an anticancer agent. Because of the lipid solubility of this compound it must be delivered either in ethanol or in a PET formulation. During the trial, these vehicles caused a large number of treatment-related side effects. We have synthesized the triethanolamine salt of 26-succinylbryostatin 1 and find that this compound is approx. 100-fold more water soluble than bryostatin 1. Because of the potential for clinical use, we have evaluated the biologic activity of this compound. We find that in a concentration-dependent manner 26-succinylbryostatin 1 is capable of activating protein kinase C (PKC) in vitro and displacing [3H]PDBu from PKC. However, at all concentrations tested the activity was less than the parent compound bryostatin 1. Addition of bryostatin 1 but not 26-succinylbryostatin 1 to U937 leukemic cells in culture stimulated a drop in cytosolic PKC, secondary to translocation of PKC to the membrane. Although 26-succinylbryostatin 1 did not stimulate a drop in the cytosolic levels of PKC, addition to U937 cells activated transcription from an AP-1 enhancer construct and c-Jun protein phosphorylation in a similar fashion to bryostatin 1 and differentiation of U937 cells. Unlike bryostatin 1, 26-succinylbryostatin 1 was unable to cause aggregation of human platelets. Although injection of bryostatin-1 into mice carrying B16 melanoma inhibits tumor growth, there was no significant inhibition of melanoma growth when identical doses of 26-succinylbryostatin 1 were injected. Therefore, 26-succinylbryostatin 1 shares some but not all of the pharmacologic properities of bryostatin 1. This compound can activate protein phosphorylation without lowering cytosolic levels of PKC.

An enzyme immunoassay for the determination of taxol and taxanes in Taxus sp. tissues and human plasma.

A rapid and sensitive indirect competitive inhibition enzyme immunoassay (CIEIA) method has been developed for quantitating taxanes in extracts of Taxus brevifolia Nutt. tissue and in human plasma. High titer rabbit polyclonal antibodies (pAb) were raised against a conjugate of 7-succinyltaxol and keyhole limpet hemocyanin. The presence of taxane analyte competitively inhibited the binding of the rabbit anti-taxane pAbs to a 7-succinyltaxol-bovine serum albumin solid phase coating antigen. The CIEIA detected taxol and cephalomannine in concentrations as low as 0.3 ng/ml (3.5 x 10(-4) microM), but did not detect baccatin III or 10-deacetylbaccatin III at concentrations below 1000 ng/ml (1.7 microM and 1.8 microM, respectively). This method is useful for estimating the taxane content of T. brevifolia extracts and may be useful for monitoring the taxol plasma level of taxol-treated patients.

Monoclonal antibodies to taxanes that neutralize the biological activity of paclitaxel.

Recently it has been proposed that drug-specific neutralizing antibodies may limit side-effects that occur during chemotherapy. These studies were undertaken to determine if monoclonal antibodies, 3C6 specific to paclitaxel and 8A10 specific to taxane diterpenes, are inhibitors of paclitaxel-induced inhibition of proliferation and cellular microtubule and nuclear changes. The results show that 3C6 and 8A10 each inhibit paclitaxel-induced cytotoxicity, microtubular bundling, stabilization from vinblastine-induced microtubule depolymerization and the formation of micronuclei. We conclude that these antibodies effectively neutralize paclitaxel activity in vitro and that they may be useful to determine if antibody-blocking strategies can prevent dose-limiting toxicities.

Pfiesteria: review of the science and identification of research gaps. Report for the National Center for Environmental Health, Centers for Disease Control and Prevention.

In connection with the CDC National Conference on Pfiesteria, a multidisciplinary panel evaluated Pfiesteria-related research. The panel set out what was known and what was not known about adverse effects of the organism on estuarine ecology, fish, and human health; assessed the methods used in Pfiesteria research; and offered suggestions to address data gaps. The panel's expertise covered dinoflagellate ecology; fish pathology and toxicology; laboratory measurement of toxins, epidemiology, and neurology. The panel evaluated peer-reviewed and non-peer-reviewed literature available through June 2000 in a systematic conceptual framework that moved from the source of exposure, through exposure research and dose, to human health effects. Substantial uncertainties remain throughout the conceptual framework the panel used to guide its evaluation. Firm evidence demonstrates that Pfiesteria is toxic to fish, but the specific toxin has not been isolated or characterized. Laboratory and field evidence indicate that the organism has a complex life cycle. The consequences of human exposure to Pfiesteria toxin and the magnitude of the human health problem remain obscure. The patchwork of approaches used in clinical evaluation and surrogate measures of exposure to the toxin are major limitations of this work. To protect public health, the panel suggests that priority be given research that will provide better insight into the effects of Pfiesteria on human health. Key gaps include the identity and mechanism of action of the toxin(s), the incomplete description of effects of exposure in invertebrates, fish, and humans, and the nature and extent of exposures that place people at risk.

A rapid and sensitive hemolysis neutralization assay for palytoxin.

The hemolytic action of palytoxin was exploited to develop a simple, sensitive assay with specificity based on a palytoxin neutralizing monoclonal antibody. Suspensions of murine erythrocytes incubated at 37 degrees C in round-bottom microtiter trays formed visible cell pellets which could be lysed by palytoxin. Hemolysis by palytoxin was time- and temperature-dependent, with a 24 hr detection limit of 1 pg/ml. The assay selectively detected palytoxin in a crude extract of Palythoa tuberculosa.

Antibodies to maitotoxin elicited by immunization with toxin-fragment conjugates.

Maitotoxin is a water-soluble polyether toxin produced by the benthic dinoflagellate, Gambierdiscus toxicus. Toxin fragments generated by periodate oxidation were conjugated to keyhole limpet hemocyanin, and the resulting immunogens were used to elicit maitotoxin-specific antibodies in mice. A competitive immunoassay developed with polyclonal IgG antibodies detected approximately 3 nM purified maitotoxin standard (IC50 approximately 13 nM; 45 ng/ml) but did not detect other polyether marine toxins. These are the first reported maitotoxin-specific antibodies.

Prophylaxis and treatment with a monoclonal antibody of tetrodotoxin poisoning in mice.

The ability of a tetrodotoxin (TTX)-specific monoclonal antibody to confer passive protection against lethal TTX challenge was investigated. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10(-9) M and is about 50-fold less reactive with anhydrotetrodotoxin and unreactive with tetrodonic acid by competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay, but had no effect on the binding of saxitoxin to the sodium channel on rat brain membranes. In prophylaxis studies, mice were administered T20G10 via the tail vein 30 min prior to i.p. TTX challenge (10 micrograms/kg). Under these conditions, 100 micrograms T20G10 protected 6/6 mice, whereas 3/6 mice were protected with 50 micrograms T20G10. Non-specific control monoclonal antibody did not protect against lethality. Therapy studies simulating oral intoxication were performed with mice given a lethal dose of TTX by gavage in a suspension of non-fat dry milk in phosphate-buffered saline. Death occurred within 25-35 min in 6/6 mice not treated with T20G10. However, 500 micrograms T20G10 administered via the tail vein 10-15 min after oral TTX exposure prevented death in 6/6 mice. Lower doses of mAb conferred less protection.

Lophozozymus pictor toxin: a fluorescent structural isomer of palytoxin.

Purified Lophozozymus pictor toxin (LPTX) shares many properties similar to palytoxin (PTX). LPTX and palytoxin isolated from Palythoa caribaeorum (C-PTX) have similar mol. wts of approx. 2680 on ionspray mass spectrometry (MS). In addition, antibodies against PTX could recognize and bind LPTX. Mixed mode high-performance liquid chromatography (HPLC) of LPTX, C-PTX and H-PTX (isolated from Palythoa tuberculosa) showed a major PTX component common to all three with the characteristic PTX-like UV spectrum at a retention time (Rt) of 17 min. However, LPTX exhibits fluorescence but PTX of equivalent toxicity does not. LPTX showed a unique peak at Rt of approx. 22 min on mixed mode HPLC. In addition, LPTX and C-PTX showed different ion fragmentation patterns on MS/MS. These results suggest that LPTX and the palytoxins are structural isomers, containing at least one difference which gives rise to fluorescence in LPTX.

Monoclonal antibody-based enzyme-linked immunoassays for the measurement of palytoxin in biological samples.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa. The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

An enzyme immunoassay for determining plasma concentrations of didemnin B.

Didemnin A was conjugated at the amino terminus of the N-methylleucine residue, via the linkers N-succinimidyl-3-(2-pyridyldithio)-propionate and trans-1,4-maleimidomethyl-cyclohexane carboxylic acid, to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). The didemnin-KLH conjugates were used to hyperimmunize rabbits. The resulting high titer antisera were employed with didemnin-BSA conjugate-coated microtiter plate wells to develop an indirect competitive inhibition enzyme immunoassay (CIEIA) that was fully cross reactive with didemnin B. A CIEIA is described that is capable of detecting the drug in plasma from didemnin B-treated patients at concentrations down to 1-3 ng/ml. This simple, sensitive CIEIA has been employed to demonstrate plasma drug clearance profiles with samples from didemnin B-treated patients.

Evidence for palytoxin as one of the sheep erythrocyte lytic in lytic factors in crude extracts of ciguateric and non-ciguateric reef fish tissue.

The occurrence of palytoxin or its congener in fish extracts has been presented in this study. The presences of hemolytic factors in fish extracts of Hawaiian reef fish and their implication in ciguatera poisoning have been shown by the sheep erythrocyte assay. By use of the anti-palytoxin inhibition assay with fish extracts and sheep red blood cell (RBC), it was shown that palytoxin was one of the major factors in the lysis of sheep erythrocytes. Ouabain, an antagonist of palytoxin for the Na+/K+ ATPase receptor on RBC, also showed inhibition of sheep RBC lysis by fish extracts. From these results, it was concluded that, in part, palytoxin and other palytoxin-related, hemolysin-like factors in fish extracts were responsible for sheep cell hemolysis.

Phase II clinical and pharmacological study of didemnin B in patients with metastatic breast cancer.

Sixteen evaluable patients with metastatic breast cancer were entered into a phase II trial of didemnin B. They received the drug at an initial dose of 5.6 mg/m2 every 21 to 28 days. Major toxicities noted were myalgia and nausea and vomiting while myelosuppression was mild. There were no complete responses; however, two minor responses were observed. The pharmacokinetics of didemnin B were studied in 10 patients who received the drug as 30 to 60 min i.v. infusions. A sensitive competitive inhibition enzyme immunoassay was used to quantitate didemnin B levels. Drug was observed to be rapidly cleared from plasma in a biphasic manner (t1/2 alpha = 0.12 hr, t1/2 beta = 4.8 hr). Although the assay could not identify the presence of specific metabolites, the increase of apparent didemnin B levels in plasma at later time points suggested the formation of unidentified metabolites which cross reacted with the antibody in the analytical procedure. In vitro experiments indicated that didemnin B was not bound to bovine serum albumin and only a minor portion (24%) of drug was found associated with red blood cells.

A monoclonal antibody-based immunoassay for detecting tetrodotoxin in biological samples.

Spleen cells from mice hyperimmunized with a keyhole limpet hemocyanin-tetrodotoxin-formaldehyde conjugate were fused with murine P3X63Ag8.653 myeloma cells. A single hybridoma clone was identified that secretes an IgG1,k monoclonal antibody (MAb), designated T20G10, against tetrodotoxin (TTX), with an estimated affinity of 1.2 x 10(8) L/M. Competitive inhibition enzyme immunoassays (CIEIAs) for detecting TTX were developed using this MAb. A direct CIEIA using alkaline phosphatase-labeled MAb detected TTX with sensitivities at IC50 and IC20 of 6-7 ng/ml and 2-3 ng/ml, respectively. The accuracy of the direct CIEIA was comparable with the high-performance liquid chromatography (HPLC) and the mouse bioassay systems, but the direct CIEIA exhibited greater sensitivity. The direct CIEIA was also more cost effective, as it required less sample preparation, a shorter assay time, and reduced investment in equipment than either of the other assay systems.

Taxane-specific monoclonal antibodies: measurement of taxol, baccatin III, and "total taxanes" in Taxus brevifolia extracts by enzyme immunoassay.

Three monoclonal antibodies with either specificity to taxol or baccatin III, or cross-reactivity with several common taxanes have been prepared and used to develop sensitive competitive-inhibition enzyme immunoassays. The hybridomas producing these monoclonal antibodies were obtained by fusing P3X63Ag8.653 plasmacytoma cells and splenocytes from mice hyperimmunized with keyhole limpet hemocyanin-7-succinyltaxol or -7-succinylbaccatin III conjugates. Direct and indirect competitive inhibition enzyme immunoassays were developed with these monoclonal antibodies and microtiter plates coated with bovine serum albumin conjugates of the complementary hapten. Detection limits for the direct competitive inhibition enzyme immunoassays, conducted in buffer containing 10% MeOH, were 0.6 nM taxol for 3C6 (anti-taxol); 1.1 nM baccatin III for 3H5 (anti-baccatin III); and 0.6 nM taxol or baccatin III for 8A10 (anti-taxane). The immunoassays accurately detected taxol, baccatin III, and "total taxanes" in crude MeOH extracts of Taxus brevifolia bark and in hplc fractions of these extracts.