Abstract
Protein phosphatase 2A (PP2A) plays a central role in essential phosphorylation-dependent signal transduction pathways. It is also a principal target for many natural toxicants (cantharidin, microcystins, diarrhetic shellfish poisons) and a synthetic herbicide (endothall). This study develops a cellular model to explore the toxicology of PP2A inhibitors by use of a [3H]cantharidic acid ([3H]CA) ligand binding assay to quantify interactions at the toxicant site and cell viability to evaluate in vivotoxicity. Mouse neuroblastoma (N1E-115) cells are similar to mouse brain with respect to the affinity (12–15 nM), number (B max, 9–22 pmol/mg protein) and ligand specificity of this binding site. In addition, the competitive potency of ten analogs of CA (including endothall) and two potent diarrhetic shellfish poisons (okadaic acid and calyculin A) is correlated (r 2 = .9) with and therefore predictive of their cytotoxicity. The only exception is microcystin LR which is a potent inhibitor at the binding site but is not cytotoxic, possibly reflecting a lack of cellular uptake. ATP and several other phosphorus-containing bifunctional acids inhibit [3H]CA binding by phosphorylation-independent pathways; pyrophosphate apparently acts as a competitive inhibitor. Mn++ and five other divalent cations are also inhibitors with a unique action of Mn++ at 25 to 50 μM in increasing [3H]CA binding, which suggests a specific role in PP2A function. Neuroblastoma cells are therefore suitable to study the mechanisms by which the toxicant, ATP and Mn++ binding sites regulate PP2A activity and cell physiology.
Footnotes
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Send reprint requests to: Dr. John E. Casida, Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, 114 Wellman Hall, University of California, Berkeley, CA 94720-3112.
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↵1 The project described was supported by grant PO1 ES00049 from the National Institute of Environmental Health Sciences, National Institutes of Health, and by Elf-Atochem North America, Inc. (Philadelphia, PA).
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↵2 Present address: Department of Entomology, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel.
- Abbreviations:
- CA and [3H]CA
- cantharidin and [3H]cantharidic acid as a radioligand, respectively
- DSP
- diarrhetic shellfish poison
- MTT
- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- N1E-115
- mouse neuroblastoma cell line
- PP
- protein phosphatase, includes 1, 2A, 2B and 2C
- PSCP
- 2-phenyl-4H-1,3,2-benzodioxaphosphorin 2-oxide
- EDTA
- ethylenediaminetetraacetic acid
- EGTA
- ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- Received June 25, 1996.
- Accepted November 8, 1996.
- The American Society for Pharmacology and Experimental Therapeutics
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