okadaic-acid and Leukemia--T-Cell

Protein phosphatase 4 (PP4) is a protein phosphatase 2A (PP2A)-related, okadaic acid-sensitive, serine/threonine protein phosphatase that shares 65% amino acid identity with PP2A. Numerous studies have shown that protein phosphatase is involved in the regulation of T cell signaling and activation. In this study, we investigated the effect of overexpression of PP4 on the expression of members of the MAP kinase family in Jurkat leukemia T cells, which had previously been stimulated with UV, 12-O-tetradecanoylphorbol-13-acetate (TPA), ionomycin and okadaic acid. We found that the overexpression of PP4 expressed relatively low activity in the absence of any kind of stimulation. However, TPA, UV or ionomycin treatment strongly increased the activity of PP4. In addition, Jurkat T cells, transfected with various expression plasmids and/or stimulated with TPA, UV or ionomycin strongly induced the c-Jun N-terminal kinase (JNK) and p38, whereas the extracellular signal-regulated kinase (ERK)-1/2 kinase pathway was weekly activated. Treatment of Jurkat T cells with okadaic acid, an inhibitor of PP2, also inhibited the increase of JNK and p38 activity induced by PP4. The effect of okadaic acid on the activity of PP4 was similar to that observed in Jurkat T cells treated with a dominant negative c-Jun (dn-jun). These results indicate that the activation of JNK and p38, but not ERKs, is a target for the PP4 activity in Jurkat leukemia T cells.

Topics: Calcium Ionophores; Enzyme Activation; Enzyme Inhibitors; Humans; Ionomycin; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukemia, T-Cell; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Signal Transduction; Tetradecanoylphorbol Acetate; Ultraviolet Rays

Serine/threonine phosphatase regulation of phosphorylation-mediated intracellular signaling controls a number of important processes in mammalian cells. In this study, we show that constitutively active protein phosphatase 2A (PP2A), which is a serine/threonine phosphatase, is essential for T leukemia cell survival. Jurkat and CCRF-CEM T leukemia cells treated with the PP2A-selective inhibitor okadaic acid (OA) showed a dose- and time-dependent induction of apoptosis, as indicated by loss of mitochondrial transmembrane potential (delta psi(m)), cleavage-induced activation of caspase-3, -8, and -9, and DNA fragmentation. In addition, caspase-8 or caspase-9 inhibition with z-IETD-fmk or z-LEHD-fmk, respectively, largely prevented OA-induced apoptosis. Although OA treatment did not affect constitutive Bcl-2 expression, overexpression of Bcl-2 prevented both OA-induced DNA fragmentation and dissipation of delta psi(m). Furthermore, inhibition of caspase-3, -8, or -9 partially protected against OA-induced loss of delta psi(m). In addition, caspase-9 and caspase-3 inhibition largely prevented procaspase-3 and procaspase-8 cleavage, respectively, while caspase-8 inhibition partially interfered with procaspase-9 cleavage in OA-treated T leukemia cells. Thus, PP2A inhibition triggered the intrinsic pathway of apoptosis, which was enhanced by a mitochondrial feedback amplification loop. PP2A has also been implicated in the regulation of p38 mitogen-activated protein kinase (MAPK). Co-immunoprecipitation analysis revealed a physical association between the catalytic subunit of PP2A and p38 MAPK in T leukemia cells. Moreover, OA treatment caused p38 MAPK to be phosphorylated in a dose- and time-dependent fashion, indicating that PP2A prevented p38 MAPK activation. Although p38 MAPK activation usually promotes apoptosis, pharmacologic inhibition of p38 MAPK exacerbated OA-induced DNA fragmentation and loss of delta psi(m) in T leukemia cells, suggesting that, in this instance, the p38 MAPK signaling pathway promoted cell survival. Collectively, these findings indicate that PP2A and p38 MAPK have coordinate effects on signaling pathways that regulate the survival of T leukemia cells.

Topics: Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; DNA Fragmentation; Humans; Intracellular Membranes; Leukemia, T-Cell; Membrane Potential, Mitochondrial; Okadaic Acid; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Permeability; Phosphoprotein Phosphatases; Phosphorylation; Protein Binding; Protein Phosphatase 2; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.

Topics: Androstadienes; Chromones; Down-Regulation; Enzyme Activation; Enzyme Inhibitors; Humans; Jurkat Cells; Leukemia, T-Cell; Morpholines; Okadaic Acid; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Kinase C; Protein Kinase Inhibitors; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; RNA Interference; RNA, Small Interfering; Signal Transduction; Transfection; Wortmannin

A better understanding of dysregulated signaling pathways in cancer cells may suggest novel strategies to prevent tumor development and/or progression. Here we show that Jurkat and CCRF-CEM human T-leukemia cell lines were more sensitive than normal human T cells to the cytotoxic effect of inhibiting protein phosphatase 2A (PP2A). Inhibition of PP2A by okadaic acid (OA) caused T-leukemia cells to die by apoptosis, as indicated by DNA fragmentation, caspase-3 activation, loss of mitochondrial membrane potential (DeltaPsi(m)), and changes in nuclear morphology that were consistent with apoptosis. PP2A might therefore be a useful intracellular target for the treatment of T cell-derived leukemias. We also observed that reactive oxygen species (ROS) were generated in response to PP2A inhibition in T-leukemia cells. However, loss of DeltaPsi(m) that resulted from PP2A inhibition was not prevented by exogenous antioxidants (glutathione and N-acetyl-cysteine), indicating that OA-induced changes in mitochondrial membrane permeability were not a consequence of ROS production. Moreover, exogenous antioxidants protected CCRF-CEM T-leukemia cells from apoptosis caused by PP2A inhibition but failed to prevent OA-induced apoptosis in Jurkat T-leukemia cells, indicating a differential role for ROS in apoptosis caused by PP2A inhibition in two different human T-leukemia cell lines.

Topics: Animals; Antioxidants; Apoptosis; Caspase 3; DNA Fragmentation; Enzyme Activation; Enzyme Inhibitors; Humans; Jurkat Cells; Leukemia; Leukemia, T-Cell; Membrane Potentials; Okadaic Acid; Protein Phosphatase 2; Reactive Oxygen Species

The reduced folate carrier (RFC) is the dominant influx transporter for antifolates. A major mechanism of antifolate resistance is loss of RFC (SLC19A1) gene expression due to decreased GC-box-dependent transcription. However, despite the poor GC-box binding in multiple antifolate-resistant cell lines, normal Sp1 levels were retained. Here we explored the post-translational modifications that may disrupt Sp1 function. Phospho-affinity purification of nuclear proteins revealed that resistant cells contained approximately 8-fold more phosphorylated Sp1 than parental cells; treatment of nuclear proteins from these cells with alkaline phosphatase restored GC-box binding. As protein kinase A phosphorylates Sp1, resistant cells were treated with various cAMP-reactive agents, revealing no apparent effect on GC-box binding except for the general phosphodiesterase inhibitor IBMX. As cGMP levels also may be affected by IBMX, resistant cells were treated with 8-pCPT-cGMP, resulting in the complete restoration of GC-box binding, luciferase reporter activity, and RFC mRNA levels. This restoration was abolished in the presence of the protein phosphatase 2A inhibitor (PP2A) okadaic acid. Importantly, whereas resistant cells showed multiple phosphorylated Sp1 forms barely detectable in parental cells, treatment with 8-pCPT-cGMP resulted in their elimination; this disappearance, however, was prevented by the copresence of okadaic acid. These findings provide the first evidence that loss of RFC gene expression in antifolate-resistant cells is associated with an inhibitory Sp1 phosphorylation that can be eliminated by a cGMP-dependent activation of PP2A.

Topics: 1-Methyl-3-isobutylxanthine; Alkaline Phosphatase; Binding Sites; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Down-Regulation; Drug Resistance, Neoplasm; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Folic Acid Antagonists; GC Rich Sequence; Humans; Leukemia, T-Cell; Luciferases; Membrane Transport Proteins; Nuclear Proteins; Okadaic Acid; Phosphodiesterase Inhibitors; Phosphoprotein Phosphatases; Phosphorylation; Platelet Aggregation Inhibitors; Promoter Regions, Genetic; Protein Phosphatase 2; Protein Processing, Post-Translational; Reduced Folate Carrier Protein; Sp1 Transcription Factor; Thionucleotides; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Therapeutic agents which target NF-kappa B transcription factor may be useful in the management of AIDS, cancer and inflammation. Since oxidative stress has been implicated in the signaling pathway, the use of antioxidants to inhibit NF-kappa B activation has gained attention. In the present study, we examined the effects of catechol derivatives, nitecapone and OR-1246, which have been identified to possess potent antioxidant properties, on NF-kappa B activation by monitoring its DNA binding activity. Both nitecapone and OR-1246 (10-300 microM) inhibited NF-kappa B activation induced by tumor necrosis factor-alpha in Jurkat T (acute human leukemia) cells. Nitecapone was a better inhibitor than OR-1246. The observed effects may, at least in part, be due to the ability of the two compounds to directly inhibit DNA binding activity of activated NF-kappa B. The inhibitory capability of OR-1246 on NF-kappa B DNA binding does not appear to be a sole mechanism, as it did not inhibit NF-kappa B activation induced by okadaic acid. Hence, catechol derivatives inhibit NF-kappa B transcription factor through multiple mechanisms, and nitecapone and OR-1246 may be useful as therapeutic agents targeting NF-kappa B.

Topics: Antioxidants; Base Sequence; Catechol O-Methyltransferase Inhibitors; Catechols; Ethers, Cyclic; Humans; Leukemia, T-Cell; Molecular Sequence Data; NF-kappa B; Okadaic Acid; Pentanones; Tumor Cells, Cultured