Daxx Shortens Mitotic Arrest Caused by Paclitaxel

Resistance to anti-neoplastic drug paclitaxel is frequent in breast cancer patients. Moststudies of paclitaxel resistance have focused on pathways that elicit cellular response,while little is known about players involved in the acquirement of taxane resistance. Byscreening a cohort of breast cancer cell lines, we observed a correlation between level ofprotein Daxx and response to paclitaxel. Cells lines expressing increased level of Daxxdisplayed a robust paclitaxel response with nearly all cells undergoing micronucleation,while cell lines with low amount of Daxx showed a decrease in micronucleation, andaccumulation in mitosis. At used paclitaxel concentrations, apoptotic levels werenegligible in all cell lines tested. Human cell lines expressing anti-Daxx siRNA as well asDaxx-/- mouse fibroblasts showed similar cellular response to paclitaxel. Importantly,absence or depletion of Daxx resulted in cell survival after paclitaxel treatment, asmeasured by colony formation assay. We conclude that Daxx may be an importantpredictive factor in cellular response to paclitaxel, which emphasizes a critical butunknown function of this protein in mitotic progression, which, when disabled, leads tosurvival advantages upon paclitaxel treatment.     

蛋白磷酸酯酶抑制剂冈田酸对人神经母细胞瘤 SK-N-SH细胞系tau蛋白磷酸化水平的影响

观察蛋白磷酸酯酶-1和蛋白磷酸酯酶-2A的抑制剂冈田酸(okadaicacid,OA)对人神经母细胞瘤系SK-N-SH细胞tau蛋白磷酸化水平的变化,确定tau蛋白过度磷酸化细胞模型的合适剂量和时间。用不同剂量OA与SK-N-SH细胞共温育不同时间,用显微镜观察细胞形态变化,用Western印迹法检测磷酸化tau蛋白和非磷酸化tau蛋白在Ser202位点和Ser404位点磷酸化水平的变化。10～160nmol/LOA与SK-N-SH神经细胞温育3～24h,可引起细胞形态损伤呈剂量依赖性和时间依赖性的变化,起效剂量和时间为10nmol/L和3h。10nmol/LOA与SK-N-SH细胞温育6～24h,磷酸化tau蛋白Ser199/Ser202位点和Ser404位点的表达明显增高,非磷酸化tau蛋白Ser202位点和Ser404位点的表达明显降低,总tau蛋白含量无明显变化。OA可以作为很好的研究tau蛋白过度磷酸化的工具药,10nmol/LOA与SK-N-SH神经细胞共温育6h可以作为制备细胞模型的适宜条件。     

Okadaic Acid, a Protein Phosphatase Inhibitor, Inhibits Nerve Growth Factor-Directed Neurite Outgrowth in PC 12 Cells

The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20-25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50 of approximately 7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF-primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid-sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGE.     

Reversible Activation of Glutamate Transport in Rat Brain Glia by Protein Kinase C and an Okadaic Acid-Sensitive Phosphoprotein Phosphatase

High-affinity L-glutamate (GLU) transport is an important regulator of excitatory amino acid (EAA) concentrations in brain extracellular fluid and may play a key role in excitatory synaptic transmission. In view of evidence that EAA transporters (EAAT) are heterogenous and contain consensus sites for phosphorylation, this investigation was undertaken to contrast the effects of transporter phosphorylation in fractions derived from glia and neurons (synaptosomes) of the adult rat forebrain. Treatment with phorbol-12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), increased the maximal rate of GLU transport in glial plasmalemmal vesicles by greater than 50 percent (237 ± 18 vs. 365 ± 27 pmol/mg protein/90s, p < 0.05) but caused no change in synaptosomes. The effect by PDBu was concentration and time-dependent and was inhibited completely by the PKC inhibitor calphostin C. Inhibition of serine-threonine phosphoprotein phosphatases with okadaic acid produced similar effects which were not additive with PDBu. Together, these results demonstrate that glial EAAT can be regulated by multiple phosphorylation processes.     

Studies on Phosphoprotein Phosphatase in Chick Embryo

Several properties of partially purified phosphoprotein phosphatase from chick embryo are described. The enzyme was active toward casein, phosvitin and phosphopeptone, but not toward low molecular weight phosphate esters including aliphatic and aromatic phos-phomonoesters, a phosphodiester, pyrophosphates and phosphoamides. The enzyme was not activated by reducing compounds. Heavy metal ions and sulfhydryl inhibitors inhibited the enzyme activity, but the inhibited enzyme was partially reactivated with cysteine. Metal chelating agents also inhibited the activity. To the oxalate treated enzyme, Fe⁺⁺ and Co⁺⁺ had a stimulatory effect. Differences in property between phosphoprotein phosphatases of chick embryo and of mammalian tissues are discussed.     

Phosphoprotein Phosphatase Activities in Alzheimer Disease Brain

Abstract: Microtubule-associated protein τ is known to be hyperphosphorylated in Alzheimer disease brain and this abnormal hyperphosphorylation is associated with an inability of τ to promote the assembly of microtubule in the affected neurons. Our previous studies demonstrated that abnormally phosphorylated τ could be dephosphorylated after treatment with alkaline phosphatase, thereby suggesting that the abnormal phosphorylation of τ might in part be the result of a deficiency of the phosphoprotein phosphatase system in patients with Alzheimer disease. In the present study we used ³²P-labeled phosphorylase kinase and poly(Glu.Tyr) 4:1 as substrates to measure phosphoprotein phosphatase activities in Alzheimer disease and control brains. The activities of phosphoseryl/ phosphothreonyl-protein phosphatase types 1, 2A, 2B, and 2C and of phosphotyrosyl-protein phosphatase in frontal gray and white matters from 13 Alzheimer brains were determined and compared with those from 12 age-matched control brains. The activities of type 1 phosphatase and phosphotyrosyl phosphatase in gray matter and of type 2A phosphatase in both gray and white matters were significantly lower in Alzheimer disease brains than in controls. These findings suggest that the hyperphosphorylation of τ in Alzheimer disease brain could result from a protein dephosphorylation defect in vivo. The decrease in the phosphatase activities in Alzheimer disease might also be involved in the formation of β-amyloid by augmenting the amyloidogenic pathway processing of β-amyloid precursor protein.     

The Protein Phosphatase Inhibitors Okadaic Acid and Calyculin A Induce Apoptosis in Human Osteoblastic Cells

To determine whether protein phosphorylation and dephosphorylation can affect apoptosis in osteoblastic cells, we examined the effects of okadaic acid (OA) and calyculin A (CA) on cultured human osteoblastic cells Saos-2 and MG63, and mouse osteoblastic MC3T3-E1 cells. After reaching confluence, these cells were exposed to varying concentrations of OA or CA. OA and CA induced cell death in all three cell lines in a dose- and time-dependent manner. Marked nuclear condensation and fragmentation of chromatin were also observed in these cells by using the Hoechst 33342 stain. DNA ladder formation, a hallmark of apoptosis, was detected in Saos-2 and MG63 cells, but not in MC3T3-E1 cells by treatment of OA or CA. In the Saos-2 cells, OA- and CA-induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 10 and 2 nM,respectively, and was time-dependent from 14 to 48 h. DNA ladder formation in response to OA and CA was revealed by using conventional ethidium bromide staining of electrophoresed DNA without using autoradiography. Beyond the maximal effects at the respective concentrations, however, cell death did not indicate DNA laddering, suggesting that phosphatase activity may be required for ladder formation. Our results indicate that apoptosis in the cultured osteoblastic cells is induced by moderate inhibition of PP-1 or PP-2A based on the known selectivity of okadaic acid and of calyculin A.     

Neurotoxic and Synaptic Effects of Okadaic Acid,an Inhibitor of Protein Phosphatases

Protein phosphorylation and dephosphorylation reactions, catalyzed by kinases and phosphatases, are involved in the regulation of a wide variety of physiological processes. In the nervous system, such reactions seem to modulate the function of several proteins crucial in synaptic transmission, including voltage-gated and ligand-gated channels, neurotransmitter release, and neurotransmitter transporters. On the other hand, hyperphosphorylation of certain cytoskeletal proteins or receptors may lead to neuronal death. In the present work we review the neurotoxic effect of okadaic acid (OKA), a potent and specific inhibitor of the serine/threonine protein phosphatases 1 and 2A, as well as its action on synaptic function. We analyze recent findings demonstrating that the microinjection of OKA in rat hippocampus induces neuronal stress, hyperexcitation and neurodegeneration, and discuss their possible relationships to alterations of protein phosphorylation-dephosphorylation observed in Alzheimer's disease brain. These results suggest that protein hyperphosphorylation due to inhibition of phosphatases in vivo induces neuronal stress and subsequent neurodegeneration.     

The Protein-Phosphatase Inhibitor Okadaic Acid Mimics MSH-Induced and Melatonin-Reversible Melanosome Dispersion in Xenopus laevis Melanophores

The present study describes the ability of 315 nM okadaic acid to induce melanosome dispersion in cultured Xenopus laevis melanophores. This effect of okadaic acid is similar to that of a-melanocyte stimulating hormone (MSH) and can be reversed by melatonin treatment; it indicates that a member of the protein-phosphatase 1 or 2A families must be active for maintenance of the aggregated state. Higher concentrations of okadaic acid (1 μM) attenuate the response of Xenopus melanophores to melatonin leading to the hypothesis that melatonin action is mediated by the calcium/calmodulin activated phosphatase 2B. This hypothesis seems unlikely, however, since the calcium/calmodulin inhibitors TFP and W7 do not prevent melatonin-induced pigment aggregation, but instead induce aggregation on their own.     

Farnesylated RhoB Prevents Cell Cycle Arrest and Actin Cytoskeleton Disruption Caused by the Geranylgeranyltransferase I Inhibitor GGTI-298

Here we demonstrate that the geranylgeranyltransferase-I inhibitor GGTI-298 inhibits the RhoB pathway and disrupts stress fiber and focal adhesion formation in NIH-3T3 cells. Farnesylated V14RhoB-CAIM (resistant to GGTI-298), but not geranylgeranylated V14RhoB (-CLLL), prevented inhibition of actin stress fiber and focal adhesion formation, underlining the critical role of RhoB. In contrast, farnesylated, V14RhoA (-CVLS) was unable to prevent effects of GGTI 298 on cytoskeleton organization. Furthermore, the ability of GGTI-298 to induce p21WAF and to block cells in the G0/G1 phase of the cell cycle was also prevented by farnesylated V14RhoB but not by farnesylated V14RhoA. Moreover, treatment with GGTI-298 of cells expressing farnesylated RhoB results in accumulation of these cells in the G2/M phase. Therefore, the RhoB pathway is a critical target of GGTI-298.     

Okadaic Acid Induces Selective Arrest of Protein Transport in the Rough Endoplasmic Reticulum and Prevents Export into COPII-Coated Structures

Quantitative immunoelectron microscopy and subcellular fractionation established the site of endoplasmic reticulum (ER)-Golgi transport arrest induced by the phosphatase inhibitor okadaic acid (OA). OA induced the disappearance of transitional element tubules and accumulation of the anterograde-transported Chandipura (CHP) virus G protein only in the rough ER (RER) and not at more distal sites. The block was specific to the early part of the anterograde pathway, because CHP virus G protein that accumulated in the intermediate compartment (IC) at 15°C could gain access to Golgi stack enzymes. OA also induced RER accumulation of the IC protein p53/p58 via an IC-RER recycling pathway which was resistant to OA and inhibited by the G protein activator aluminium fluoride. The role of COPII coats in OA transport block was investigated by using immunofluorescence and cell fractionation. In untreated cells the COPII coat protein sec 13p colocalized with p53/p58 in Golgi-IC structures of the juxtanuclear region and peripheral cytoplasm. During OA treatment, p53/p58 accumulated in the RER but was excluded from sec 13p-containing membrane structures. Taken together our data indicate that OA induces an early defect in RER export which acts to prevent entry into COPII-coated structures of the IC region.     

Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 n M ) and okadaic acid (50 n M ) reduced significantly ( p < 0.01) the synthesis of [³H]ACh from [³H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration-dependent manner ( p < 0.01). This depletion was not the result of a drug-induced increase of spontaneous ACh release, which was not changed significantly ( p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration-dependent manner ( p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly ( p > 0.4) in the presence of calyculin A or okadaic acid. Both high-affinity and low-affinity [³H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration-dependent manner in the presence of calyculin A or okadaic acid; these agents reduced V _max values for high- and low-affinity choline uptake ( p < 0.01) with no significant change in K _m values ( p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.     

Relationship Between Phosphatase Activity and Cytotoxic Effect of Two Protein Phosphatase Inhibitors,Okadaic Acid and Pervanadate,on Human Myeloid Leukemia Cell Line

Protein phosphatases are signalling molecules that regulate a variety of fundamental cellular processes including cell growth, metabolism and apoptosis. The aim of this work was to correlate the cytotoxicity of pervanadate and okadaic acid on HL60 cells and their effect on the phosphatase obtained from these cells. The cytotoxicity of these protein phosphatase inhibitors was evaluated on HL60 cells using phosphatase activity, protein quantification and MTT reduction as indices. The major phosphatase presents in the cellular extract showed high activity (80%) and affinity (Km=0.08?mM) to tyrosine phosphate in relation to p-nitrophenyl phosphate (pNPP)—(Km=0.51?mM). Total phosphatase (pNPP) was inhibited in the presence of 10?mM vanadate (98%), 200?μM pervanadate (95%) and 100?μM p-chloromercuribenzoate (80%) but okadaic acid caused a slight increase in enzyme activity (25%). When the HL60 cells were treated with the phosphatase inhibitors (pervanadate and okadaic acid) for 24?hours, only 20% residual activity was observed in presence of 200?μM pervanadate, whereas in the presence of okadaic acid this inhibitory effect was not observed. However, in respect to mitochondrial function, cell viability decreased about 80% in the presence of 100?nM okadaic acid. The total protein content was decreased 25% when the cells were treated with 100?nM okadaic acid in combination with 200?μM pervanadate. Our results suggest that both phosphatase inhibitors presented different mechanisms of action on HL60 cells. However, their effect on the cell redox status have to be considered.     

A Transgene-Induced Mitotic Arrest Mutation in the Mouse Allelic with Oligosyndactylism

Oligosyndactylism (Os) is a radiation-induced mutation on mouse chromosome 8 associated with early postimplantation lethality in homozygotes and abnormal development of the limbs and kidneys in heterozygotes. The recessive lethal effect of Os is due to a mitotic block of the embryonic cells that becomes apparent at the blastocyst stage, but it is not known if the heterozygous effect of Os is due to haploinsufficiency of the gene responsible for the mitotic arrest, or is due to mutation(s) of other gene(s). We have recently described a transgene-induced recessive mutation, 94-A/K, that results in early postimplantation death of the embryos, and we have mapped this mutation to the same region of chromosome 8 where Os has been assigned. On the basis of complementation tests between transgenic and Os/+ mice, in vitro growth characteristics and increased mitotic index of 94-A/K embryos, and molecular structural analysis of 94-A and 94-K transgenic and Os/+ mice, we conclude that the 94-A/K mutation represents a new allele of Os. This insertional mutation should facilitate the isolation of a mammalian gene essential for normal progression of the cell cycle beyond metaphase.     

Activation of β-Glucan Synthases by Wall-Bound Purple Acid Phosphatase in Tobacco Cells

Wall-bound purple acid phosphatases have been shown to be potentially involved in the regulation of plant cell growth. The aim of this work was to further investigate the function of one of these phosphatases in tobacco (Nicotiana tabacum), NtPAP12, using transgenic cells overexpressing the enzyme. The transgenic cells exhibited a higher level of phosphatase activity in their walls. The corresponding protoplasts regenerating a cell wall exhibited a higher rate of β-glucan synthesis and cellulose deposition was increased in the walls of the transgenic cells. A higher level of plasma membrane glucan synthase activities was also measured in detergent extracts of membrane fractions from the transgenic line, while no activation of Golgi-bound glycan synthases was detected. Enzymatic hydrolysis and methylation analysis were performed on the products synthesized in vitro by the plasma membrane enzymes from the wild-type and transgenic lines extracted with digitonin and incubated with radioactive UDP-glucose. The data showed that the glucans consisted of callose and cellulose and that the amount of each glucan synthesized by the enzyme preparation from the transgenic cells was significantly higher than in the case of the wild-type cells. The demonstration that callose and cellulose synthases are activated in cells overexpressing the wall-bound phosphatase NtPAP12 suggests a regulation of these carbohydrate synthases by a phosphorylation/dephosphorylation process, as well as a role of wall-bound phosphatases in the regulation of cell wall biosynthesis.     

Okadaic Acid-Induced Apoptosis in Neuronal Cells: Evidence for an Abortive Mitotic Attempt

Abstract: There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B₁ and cyclin D₁ markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.     

Effect of Inorganic Phosphate on the Extracellular Acid Phosphatase Activity of Tobacco Cells Cultured in vitro

Acid phosphatase activity in culture medium of tobacco cells growing in suspension increased with the age of the culture from which the medium was obtained. The increase in the activity was accelerated by omitting inorganic phosphate from nutrient medium, and it was depressed by addition of inorganic phosphate or cycloheximide. Amylase and β-galactosidase activities were not induced by the omission of inorganic phosphate. It was concluded that derepression of acid phosphatase synthesis was involved in the increase in the extracellular acid phosphatase activity upon inorganic phosphate depletion.