Comparative cytotoxic effects of two protein phosphatase inhibitors, okadaic acid and calyculin A, on thyroid cells

The cytotoxicity of two inhibitors of protein phosphatases PP1 and PP2A has been investigated on primary cultures of dog thyrocytes. Both compounds, okadaic acid and calyculin A elicited dose- and time- related effects, i.e. apoptosis and necrosis. In addition a pronounced detachment of the cells from the monolayer was also observed. Based on the different patterns of morphological alterations and on the biochemical data, it was concluded that each compound induced different types of cell death; this provides additional evidence that a specific cell type can initiate distinct programs of death depending on the triggering stimulus. To explain the effects recorded when both compounds were added concomitantly, a functional interaction between PP1 and PP2A has been proposed. Finally, all the effects appeared modulated, to different extent, by cycloheximide and by actinomycin D. This supports the view that de novo RNA synthesis is required for the induction of death by these phosphatase inhibitors in these cells.     

Calyculin A and okadaic acid: inhibitors of protein phosphatase activity

Calyculin A and okadaic acid induce contraction in smooth muscle fibers. Okadaic acid is an inhibitor of phosphatase activity and the aims of this study were to determine if calyculin A also inhibits phosphatase and to screen effects of both compounds on various phosphatases. Neither compound inhibited acid or alkaline phosphatases, nor the phosphotyrosine protein phosphatase. Both compounds were potent inhibitors of the catalytic subunit of type-2A phosphatase, with IC50 values of 0.5 to 1 nM. With the catalytic subunit of protein phosphatase type-1, calyculin A was a more effective inhibitor than okadaic acid, IC50 values for calyculin A were about 2 nM and for okadaic acid between 60 and 500 nM. The endogenous phosphatase of smooth muscle myosin B was inhibited by both compounds with IC50 values of 0.3 to 0.7 nM and 15 to 70 nM, for calyculin A and okadaic acid, respectively. The partially purified catalytic subunit from myosin B had IC50 values of 0.7 and 200 nM for calyculin A and okadaic acid, respectively. The pattern of inhibition for the phosphatase in myosin B therefore is similar to that of the type-1 enzyme.     

Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor

This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication. OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv. Cancer. Res. 61 (1993) 143). However, all of its cellular targets have not yet been characterized. The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity. Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP. The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM). The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.     

Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells.

Inhibitors of protein phosphatases 1/2A (okadaic acid and calyculin A) exhibited differential cytotoxicity toward three human leukemia cell lines, in an increasing order of resistance, HL60 less than HL60/ADR less than K562 cells. Cytotoxicity of the toxins was associated with marked mitotic arrest of the cells, characterized by chromatid scattering/overcondensation and abnormal mitotic spindles. In all cases, calyculin A was more potent than okadaic acid. Protein phosphorylation experiments in intact cells revealed that HL60/ADR, the adriamycin-resistant variant, showed a higher overall phosphorylation of nuclear proteins than the drug-sensitive parental HL60, and that phorbol ester (protein kinase C activator) and calyculin A appeared to more specifically stimulate phosphorylation of p66 and p60, respectively. It was suggested that the toxins might be useful in delineating mechanisms underlying certain properties of cancer cells (such as multidrug resistance, mitosis and differentiation) related to protein phosphorylation/dephosphorylation reactions.     

Differing effects of the protein phosphatase inhibitors okadaic acid and microcystin on translation in reticulocyte lysates.

The effects of the cyanobacterial toxin and protein phosphatase inhibitor, microcystin, on translation in rabbit reticulocyte lysates have been studied. Microcystin inhibited translation with similar potency to the protein phosphatase inhibitor okadaic acid. Unlike low concentrations of okadaic acid, however, it inhibited both the initiation and elongation stages. This was demonstrated using EGTA to inhibit the phosphorylation and inactivation of elongation factor eEF-2. A method for detecting changes in eEF-2 phosphorylation was developed. eEF-2 was found to exist as three different species: eEF-2 was largely monophosphorylated in reticulocyte lysates under control conditions, the remainder being unphosphorylated. Okadaic acid and microcystin increased the level of the bisphosphorylated species. The implications of multiple phosphorylation of eEF-2 for the control of translation is discussed. Microcystin was also found to increase the phosphorylation of eIF-2 alpha (and therefore to inhibit initiation) at lower concentrations than okadaic acid, suggesting that the major eIF-2 alpha phosphatase in the reticulocyte lysate is phosphatase-1.     

Mitotic arrest and enhanced nuclear protein phosphorylation in human leukemia K562 cells by okadaic acid, a potent protein phosphatase inhibitor and tumor promoter.

We investigated the effects of the non-phorbol tumor promoter okadaic acid on human leukemia K562 cells. It was found that okadaic acid potently and reversibly inhibited cell growth, with a nearly complete inhibition of thymidine uptake seen at about 10 nM. The cytotoxicity of okadaic acid was characterized by a marked mitotic arrest of the cells exhibiting scattered chromosomes and abnormal anaphase-like structures, a phenomenon distinct from the typical metaphase arrest caused by colchicine. Okadaic acid (10-1,000 nM) greatly stimulated phosphorylation of a number of nuclear proteins in K562 cells. Phosphorylation of many of the same proteins was also stimulated by 12-O-tetradecanoylphorbol-13-O-acetate, a protein kinase C activator. The present findings, consistent with recent reports that okadaic acid is a potent inhibitor of protein phosphatases 1 and 2A (PP1 and PP2A) shown to be essential for normal mitosis, provided evidence for the first time that okadaic acid inhibition of PP1/PP2A resulted in enhanced nuclear protein phosphorylation and subsequent mitotic arrest.     

Proteins in human myeloid leukemia cell line HL60 reacting with retinoic acid monoclonal antibodies

The vitamin A derivative, retinoic acid (RA) has various biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of leukemia cells and inhibits cell growth. There are two pathways for RA action; one via RA nuclear receptors (RARs), and one via acylation of proteins by RA (retinoylation). However, an understanding of which actions of RA occur via RARs and which occur via retinoylation is lacking. Thus, we undertook the examination of HL60 proteins using anti-RA monoclonal antibodies (ARMAs). These ARMAs showed specific binding to proteins in a saturable manner depending on protein and antibody concentration. Proteins eluted by Mono Q anion exchange chromatography and separated using two-dimensional polyacrylamide gel electrophoresis were detected by ARMAs. One of these ARMA-bound proteins in HL60 cells was identified as alpha-actinin. These results indicate that retinoylated proteins in HL60 cells can be recognized by ARMAs and that alpha-actinin modified by RA may play a significant role in RA-induced differentiation, including the promotion of cytomorphology changes.     

Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60

all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.     

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets.

The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.     

Effects of the phosphatase inhibitors,okadaic acid,ATPgammaS, and calyculin A on the dividing sand dollar egg

The effects of the phosphatase inhibitors, okadaic acid (OA), adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and calyculin A (CL-A) on anaphase chromosome movement, cytokinesis, and cytoskeletal structures at cell division were examined by being microinjected into mitotic sand dollar eggs. When OA was injected, chromosome movement was inhibited and, moreover, chromosomes were ejected from the polar regions of the mitotic apparatus. By immunofluorescence, microtubules were observed to be severed in the OA-injected eggs, causing the smooth cell surface to be changed to an irregular surface. When ATPgammaS and CL-A were injected, the effect on cell shape was remarkable: In dividing eggs, furrowing stopped within several seconds after injection, small blebs appeared on the cell surface and became large, spherical or dumbbell cell shapes then changed to irregular forms, and subsequently cytoplasmic flow occurred. Microfilament detection revealed that actin accumulation in the cortex, which was not limited to the furrow cortex, occurred shortly after injection. Cortical accumulation of actin is thought to induce force generation and random cortical contraction, and accordingly to result in bleb extrusion from the cortex. Consequently, the phosphatase inhibitors inhibited the transition from mitosis to interphase by mediating cortical accumulation of actin filaments and/or fragmentation of microtubules.     

Maturation inducer activity and the process of human myeloid leukemia cell differentiation

The process and mechanism of human myeloid leukemia cell differentiation induced by T-cell lymphokine maturation inducer activity was investigated. The maturation inducer activity was purified from conditioned medium of normal peripheral blood lymphocytes and shown to be a 50,000 M.W. protein. The degree of maturation of myeloid cell cultures was directly related to the dosage of the inducer. The interaction of the leukemia cells with the inducer led to initiation of terminal differentiation to monocytic cells. Proliferation cessation of the leukemia cells and the expressions of mature monocytic cells indicated a continuous and multistaged process.     

Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line.

All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor alpha (RARalpha) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation of ASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5'-upstream flanking region of human ASMase gene (-519/+300) conjugated with the luciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RARalpha or the PML/RARalpha hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5'-end (-519/-485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence.     

Purification and physicochemical characterization of a human placental acid phosphatase possessing phosphotyrosyl protein phosphatase activity

A 17-kilodalton (kDa) human placental acid phosphatase was purified 21,400-fold to homogeneity. The enzyme has an isoelectric point of pH 7.2 and a specific activity of 106 mumol min-1 mg-1 using p-nitrophenyl phosphate as a substrate at pH 5 and 37 degrees C. This placental acid phosphatase showed activity toward phosphotyrosine and toward phosphotyrosyl proteins. The pH optima of the enzyme with phosphotyrosine and with phosphotyrosyl band 3 (from human red cells) were between pH 5 and 6 and pH 5 and 7, respectively. The Km for phosphotyrosine was 1.6 mM at pH 5 and 37 degrees C. Phosphotyrosine phosphatase activity was not inhibited by tartrate or fluoride, but vanadate, molybdate, and zinc ions acted as strong inhibitors. Enzyme activity was also inhibited by DNA, but RNA was not inhibitory. It is a hydrophobic nonglycoprotein containing approximately 20% hydrophobic amino acids. The average hydrophobicity was calculated to be 903 cal/mol. The absorption coefficient at 280 nm, E1% 1cm, was determined to be 5.7. The optical ellipticity of the enzyme at 222 nm was -5200 deg cm2 dmol-1, which would correspond to a low helical content. Free sulfhydryl and histidine residues were necessary for the enzyme activity. The enzyme contained four reactive sulfhydryl groups. Chemical modification of the sulfhydryls with iodoacetate resulted in unfolding of the protein molecule as detected by fluorescence emission spectroscopy. Antisera against both the native and the denatured protein were able to immunoprecipitate the native enzyme. However, upon denaturation, the acid phosphatase lost about 70% of the antigenic determinants. Both antisera cross-reacted with a single 17-kDa polypeptide on immunoblotting.     

The human red cell acid phosphatase activity and its statistical evaluation

Summary We examined 396 bloods samples of the five most common phenotypes of the human red cell acid phosphatase (E.C.3.1.3.2). The different enzyme activity of the individual phenotypes was statistically evaluated and an explanation was sought.     

Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line.

Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.