okadaic-acid and Leukemia--Promyelocytic--Acute

While all-trans retinoic acid (ATRA) treatment in acute promyelocytic leukemia (APL) has been the paradigm of targeted therapy for oncogenic transcription factors, the underlying mechanisms remain largely unknown, and a significant number of patients still relapse and become ATRA resistant. We identified the histone demethylase PHF8 as a coactivator that is specifically recruited by RARα fusions to activate expression of their downstream targets upon ATRA treatment. Forced expression of PHF8 resensitizes ATRA-resistant APL cells, whereas its downregulation confers resistance. ATRA sensitivity depends on the enzymatic activity and phosphorylation status of PHF8, which can be pharmacologically manipulated to resurrect ATRA sensitivity to resistant cells. These findings provide important molecular insights into ATRA response and a promising avenue for overcoming ATRA resistance.

Topics: Animals; Drug Resistance, Neoplasm; Histone Demethylases; Histones; Humans; Leukemia, Promyelocytic, Acute; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Proteins; Okadaic Acid; Oncogene Proteins, Fusion; Phosphorylation; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA Interference; RNA, Small Interfering; Signal Transduction; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In this study, we aimed to investigate the potential relationship between gossypol-induced cytotoxicity of human promyelocytic leukemia cell line (HL-60) leukemic cells and intracellular serine/threonine protein phosphatase (PP) dynamics and human telomerase reverse transcriptase (hTERT) activity. Gossypol was found to be cytotoxic in HL-60 cells with the IC(50) dose of 4.5 microM. The combination of gossypol and okadaic acid in IC(50) doses revealed the increased cytotoxicity in a time-dependent manner. Treatment of cells with gossypol has shown significant decrease in PP2A activity. The expression of the PP2A catalytic subunit was downregulated in gossypol-treated cells with 24 hours' intervals. hTERT mRNA levels were gradually decreased. In conclusion, during gossypol-induced cytotoxicity, intracellular activity and expression of PP2A was decreased as well as the activity of hTERT. The variation of hTERT activity in gossypol-treated HL-60 cells may be the potential reason for the phosphatase interaction during the gossypol treatment of leukemic cells resulting in cellular cytotoxicity.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Gossypol; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Okadaic Acid; Protein Phosphatase 2; Telomerase

To study the changes in expression and activity of protein phosphatases type 2A (PP2A ) during differentiation of NB4 and NB4-MR2 cells induced by all-trans retinoic acid (ATRA), and evaluate the role of PP2A in MR2 resistance to ATRA.. ATRA, okadaic acid (OKA) and ATRA + OKA at the same dosage were incubated with NB4 and MR2 cells respectively. Wright's staining and NBT reduction test were employed to evaluate the change in the cells. The CD11b expression was measured by flow cytometry. The activity of PP2A was evaluated by serine/threonine phosphatase assay system, and the level of PP2A subunits was detected by Western blot.. 1) Wright's staining, NBT reduction test and flow cytometry results showed OKA could augment the differentiation of NB4 induced by ATRA, and OKA + ATRA induced slight differentiation of MR2 cells. 2) Phosphatase assay showed a decrease in PP2A phosphatase activity [(534 +/- 43) pmol x min(-1) x microg protein(-1)] in NB4 after ATRA treatment, accompanied with that activity [(959 +/- 83) pmol x min(-1) x microg protein(-1)] in untreated NB4 cells. OKA enhanced the inhibitory effect of ATRA on the activity in NB4. When OKA + ATRA was incubated with MR2, PP2A in the cells was significantly decreased [(229 +/- 23) pmol x min(-1) x microg protein(-1)]. 3) Western blot analysis showed that the level of PP2A catalytic subunit (PP2A/C) was decreased during the course of ATRA-induced NB4 cell differentiation, whereas expressions of every subunits of PP2A in MR2 cells were somewhat unaltered.. Expression of PP2A/C and activity of PP2A is decreased during differentiation of NB4 induced by ATRA, and no repression of the PP2 activity maybe related to MR2 resistance to ATRA.

Topics: Cell Differentiation; Cell Line, Tumor; Humans; Leukemia, Promyelocytic, Acute; Okadaic Acid; Phosphoprotein Phosphatases; Protein Phosphatase 2; Tretinoin

Oxidative stress can be involved in several cellular responses, such as differentiation, apoptosis and necrosis. Dehydrocrotonin (DCTN, diterpene lactone) from Croton cajucara, Brazilian medicinal plant, slightly induced NBT-reducing activity. In presence of protein phosphatase inhibitors significant differentiation of HL60 cells was observed. Flow cytometry analysis demonstrated that apoptosis was induced when the cells were treated with okadaic acid (OKA) and plus trans-dehydrocrotonin (t-DCTN) this effect was two-fold increased. Unlike, when the cells were treated only with t-DCTN, necrosis was observed. On the other hand, the necrosis induced by t-DCTN could be due to oxidative stress, revealed by increase of GSH content. Therefore, this differentiation pathway involves the modulation of protein phosphatases and this inhibition promotes the t-DCTN action on apoptosis induction.

Topics: Annexin A5; Apoptosis; Brazil; Cell Differentiation; Croton; Diterpenes; Diterpenes, Clerodane; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Flow Cytometry; Glutathione; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Necrosis; Okadaic Acid; Oxidative Stress; Phosphoprotein Phosphatases; Plants, Medicinal; Vanadates

Treatment of HL-60 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) (1-5 nM) induced inhibition of cell growth and the appearance of an adherent monocyte-like cell type in a dose- and time-dependent manner. The extent of TPA-induced monocytic differentiation was found to be markedly reduced by okadaic acid (OA) (35 nM). OA had to be present for the early 12 h during treatment with TPA to reduce the induction of monocytic differentiation. The majority of cells (80%) were non-adherent but morphologically resembled mature myelocytes or granulocytes after treatment with TPA (5 nM) in the presence of OA (35 nM). Vanadate (VD), on the other hand, enhanced the extent of monocytic differentiation induced by low-dose of TPA (1 nM). These results indicated that dephosphorylation by tyrosine protein phosphatase and serine-threonine protein phosphatase may play an important role in the induction of monocytic and granulocytic differentiation.

Topics: Cell Differentiation; Ethers, Cyclic; Granulocytes; Hematopoietic Stem Cells; Humans; Leukemia, Promyelocytic, Acute; Monocytes; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vanadates

The protein phosphatase inhibitors okadaic acid and calyculin A at moderate concentrations induced three types of apoptotic promyelocytic leukemia cell death, distinct with respect to ultrastructure and polynucleotide fragmentation. Calyculin A at higher concentrations (> 50 nM) induced a non-apoptotic death type with high ATP and pronounced micromitochondriosis. This suggests that protein phosphorylation pathways are involved in the triggering of several death pathways. Activation of the cAMP kinase induced yet another apoptotic death type, preferentially affecting cells in S-phase. In fact, cAMP acted in two ways to stop IPC promyelocyte proliferation: (1) block in late G1 (preventing new cells from entering DNA replication); and (2) induction of apoptosis in S-phase. cAMP and phosphatase inhibitors acted via distinct pathways. The inhibitors suppressed cAMP-induced death, but only at concentrations high enough to commit the cells to alternative, less conspicuous death types. The tumor-promoting activity of okadaic acid and calyculin A may therefore not be by protection against apoptosis. DNA fragmentation correlated with the novel feature of limited 28 S rRNA cleavage, suggesting co-ordinated polynucleotide cleavage, possibly directed against illegitimate polynucleotides, in some apoptotic death types.

Topics: Animals; Apoptosis; Cell Cycle; Cell Membrane; Cell Nucleus; Chromatin; Cyclic AMP; Cytoplasm; DNA Damage; Dose-Response Relationship, Drug; Ethers, Cyclic; Leukemia, Promyelocytic, Acute; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Rats; RNA, Ribosomal, 28S; Signal Transduction; Tumor Cells, Cultured

The proteolytic modification of plasminogen activator inhibitor 2 (PAI-2) was studied during apoptosis in the human promyelocytic leukaemic NB4 cell line during treatment with the phosphatase inhibitors okadaic acid and calyculin A as well as the protein synthesis inhibitor cycloheximide. The apoptic type of cell death was ascertained by morphological and biochemical criteria. In cell homogenates PAI-2 was probed by [125I]urokinase plasminogen activator (uPA) and detected as a sodium dodecyl sulphate-stable M(r) 80,000 complex after reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. During apoptosis a smaller (M(r) 70,000) uPA-PAI-2 complex was consistently detected. The modification was in the PAI-2 moiety, as the [125I]uPA tracer could be extracted in its intact form from the complex. Thus the cleaved PAI-2 isoform is a biochemical marker of apoptosis in the promyelocytic NB4 cell line. The modified PAI-2 isoform was also detected in homogenates made from purified human mononuclear leukaemic cells aspirated from the bone marrow of patients suffering from acute and chronic myeloid leukaemia.

Topics: Apoptosis; Biomarkers, Tumor; Bone Marrow; Cell Death; Ethers, Cyclic; Humans; Intracellular Fluid; Isomerism; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Promyelocytic, Acute; Marine Toxins; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Plasminogen Activator Inhibitor 2; Tumor Cells, Cultured

To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions of PP1 and PP2A were examined. Treatment with 1 microM ATRA caused an 85% decrease in the PP2A activity in extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells induced by ATRA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (M(r) 55,000) regulatory subunits of PP2A were relatively unaltered. Expressions of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) were not significantly affected by ATRA treatment. Northern blot analysis revealed that mRNA levels of PP2A-C beta and A alpha regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-C alpha and B (M(r) 55,000) alpha regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-C beta preceded the granulocytic maturation induced by ATRA. Expressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentiation of HL-60 cells.

Topics: Blotting, Northern; Cell Differentiation; Cytosol; Down-Regulation; Ethers, Cyclic; Glyceraldehyde-3-Phosphate Dehydrogenases; Granulocytes; Humans; Immunoblotting; Leukemia, Promyelocytic, Acute; Molecular Weight; Myosins; Okadaic Acid; Phosphoprotein Phosphatases; RNA, Messenger; Tretinoin; Tumor Cells, Cultured

Electron microscopy studies demonstrate unequivocally that the observed oligonucleosome-sized secondary DNA fragmentation in human promyelocytic HL-60 cells treated with the topoisomerase inhibitors camptothecin and teniposide is correlated with the morphological changes in cell structure typical of programmed cell death (apoptosis). Since apoptosis has been associated with potential involvement of intracellular signaling linked to the Ca2+/calmodulin and protein kinase C transduction pathways, we also investigated the effects of signaling modulators on camptothecin- and teniposide-induced secondary DNA fragmentation in HL-60 cells. Neither calcium chelators, calcium/calmodulin inhibitors (calmidazolium or cyclosporine A), protein kinase C stimulation by TPA, protein phosphatase inhibition by okadaic acid, protein kinase inhibition by staurosporine, calphostin C, genistein or H7, nor cell cycle alterations by caffeine had any detectable effect. Interestingly, most of these intracellular signaling modulators were able to induce DNA fragmentation in HL-60 cells by themselves. These results may suggest that even though modulation of these signaling pathways was unable to prevent topoisomerase inhibitor-induced apoptosis, their sole deregulations could induce apoptosis in HL-60 cells. In contrast, aphidicolin blocked camptothecin-induced secondary DNA fragmentation, indicating that replication-induced DNA damage is required for camptothecin- but not teniposide-induced secondary DNA fragmentation. Zinc, 3-aminobenzamide, and spermine also modulated both camptothecin- and teniposide-induced secondary DNA fragmentation without significant alteration of topoisomerase-mediated primary DNA strand breaks. Hence, poly(ADP-ribosyl)ation and chromatin structure may be important in modulating oligonucleosome-sized DNA fragmentation associated with apoptosis in HL-60 cells treated with topoisomerase inhibitors.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Aminoquinolines; Aphidicolin; Apoptosis; Benzamides; Calcium; Calmodulin; Camptothecin; Cell Cycle; Chromatin; Cyclosporine; DNA; Egtazic Acid; Ethers, Cyclic; Humans; Isoquinolines; Leukemia, Experimental; Leukemia, Promyelocytic, Acute; Microscopy, Electron; Naphthalenes; Okadaic Acid; Phosphoprotein Phosphatases; Piperazines; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Polycyclic Compounds; Protein Kinase C; Spermine; Staurosporine; Teniposide; Tetradecanoylphorbol Acetate; Topoisomerase I Inhibitors; Topoisomerase II Inhibitors; Tumor Cells, Cultured; Zinc

Okadaic acid, a newly recognized protein phosphatase inhibitor and a non-TPA type tumor promoter, enhanced 1 alpha 25(OH)2D3(D3)-induced HL-60 cell differentiation into monocyte/macrophage lineage but did not affect dibutyryl cyclic AMP (dbcAMP)-induced differentiation into granulocytic lineage. Okadaic acid alone did not induce any differentiation. The process of D3-induced HL-60 cell differentiation on cultivation in magnesium deficient medium can be divided into two steps namely commitment and phenotypic expression as we have previously reported (J Cell Physiol 1987;131:50; Cell Growth Diff 1991;2:415), and the effect of okadaic acid on each step was studied. The results obtained indicated that okadaic acid inhibited commitment and enhanced phenotypic expression. We have previously shown that PKC has a dual action in the process of differentiation, i.e. as a positive regulatory signal in commitment and as a negative one in phenotypic expression. Thus, although okadaic acid has been reported to enhance the phosphorylation of various proteins that are also phosphorylated by PKC, we found that it mimics the role of PKC inhibitors such as H7 and staurosporine in D3-induced HL-60 cell differentiation.

Topics: Calcitriol; Cell Differentiation; Ethers, Cyclic; Humans; Leukemia, Promyelocytic, Acute; Okadaic Acid; Protein Kinase C; Tumor Cells, Cultured

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) stimulates the transcription of many eucaryotic genes by catalyzing the phosphorylation of the cAMP-regulatory element binding protein (CREB). Conversely, the attenuation or inhibition of cAMP-stimulated gene transcription would require the dephosphorylation of CREB by a nuclear protein phosphatase. In HepG2 cells treated with the protein serine/threonine (Ser/Thr) phosphatase inhibitor okadaic acid, dibutyryl-cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter was enhanced over the level of PEPCK gene transcription observed in cells treated with dibutyryl-cAMP alone. This process was mediated, at least in part, by a region of the PEPCK promoter that binds CREB. Likewise, okadaic acid prevents the dephosphorylation of PKA-phosphorylated CREB in rat liver nuclear extracts and enhances the ability of PKA to stimulate transcription from the PEPCK promoter in cell-free reactions. The ability of okadaic acid to enhance PKA-stimulated transcription in vitro was entirely dependent on the presence of CREB in the reactions. The phospho-CREB (P-CREB) phosphatase activity present in nuclear extracts coelutes with protein Ser/Thr phosphatase type 2A (PP2A) on Mono Q, amino-hexyl Sepharose, and heparin agarose columns and was chromatographically resolved from nuclear protein Ser/Thr-phosphatase type 1 (PP1). Furthermore, P-CREB phosphatase activity in nuclear extracts was unaffected by the heat-stable protein inhibitor-2, which is a potent and selective inhibitor of PP1. Nuclear PP2A dephosphorylated P-CREB 30-fold more efficiently than did nuclear PP1. Finally, when PKA-phosphorylated CREB was treated with immunopurified PP2A and PP1, the PP2A-treated CREB did not stimulate transcription from the PEPCK promoter in vitro, whereas the PP1-treated CREB retained the ability to stimulate transcription. Nuclear PP2A appears to be the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.

Topics: Amino Acid Sequence; Base Sequence; Bucladesine; Carcinoma, Hepatocellular; Cell Nucleus; Cloning, Molecular; Cyclic AMP Response Element-Binding Protein; Ethers, Cyclic; Female; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Liver Neoplasms; Macromolecular Substances; Molecular Sequence Data; Okadaic Acid; Oligodeoxyribonucleotides; Phosphoenolpyruvate Carboxykinase (GTP); Phosphoprotein Phosphatases; Phosphorylation; Placenta; Polymerase Chain Reaction; Pregnancy; Promoter Regions, Genetic; Protein Kinases; Protein Phosphatase 2; Recombinant Proteins; Transcription, Genetic; Tumor Cells, Cultured

Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.

Topics: Calcium-Calmodulin-Dependent Protein Kinases; Cell Adhesion; Cell Differentiation; Cell Nucleus; Drug Resistance; Ethers, Cyclic; Gene Expression; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Monocytes; Okadaic Acid; Phosphoprotein Phosphatases; Protein Kinase C; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogenes; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vincristine