okadaic-acid and Leukemia--Myeloid

Adenosine is an endogenous immunomodulator that has been shown to exhibit anti-inflammatory and immunosuppressive properties through a mechanism that is not fully established. Owing to the pivotal role of nuclear factor (NF)-kappaB in these responses, we tested the hypothesis that adenosine mediates its effects through suppression of NF-kappaB activation. We investigated the effects of adenosine on NF-kappaB activation induced by various inflammatory agents in human myeloid KBM-5 cells. The treatment of these cells with adenosine suppressed TNF-induced NF-kappaB activation, but had no effect on activation of another redox-sensitive transcription factor, AP-1. These effects were not restricted to myeloid cells, as NF-kappaB activation in other lymphocytic and epithelial cell types was also inhibited. The effect on TNF-induced NF-kappaB activation was selective as adenosine had minimal effect on NF-kappaB activation induced by H(2)O(2), PMA, LPS, okadaic acid, or ceramide, suggesting differences in the pathway leading to NF-kappaB activation by different agents. Adenosine also suppressed NF-kappaB-dependent reporter gene expression activated by TNF or by overexpression of TNFR1, TRAF 2, NIK, and p65 subunit of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by adenosine was found not to be because of inhibition of TNF-induced IkappaBalpha phosphorylation and degradation or IkappaBalpha kinase activation. The suppression of TNF-induced NF-kappaB activation was unique to adenosine, as neither its metabolites (inosine, AMP, and ATP) nor pyrimidines (thymidine and uridine) had any effect. Overall, our results clearly demonstrate that adenosine selectively suppresses TNF-induced NF-kappaB activation, which may contribute to its role in suppression of inflammation and of the immune system.

Topics: Adenosine; Alkaline Phosphatase; Antigens, CD; Cells, Cultured; Ceramides; Dose-Response Relationship, Drug; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Genes, Reporter; HeLa Cells; Humans; Hydrogen Peroxide; I-kappa B Kinase; I-kappa B Proteins; Jurkat Cells; Kidney; Leukemia, Myeloid; Lipopolysaccharides; Monocytes; Neoplasm Proteins; NF-kappa B; NF-KappaB Inhibitor alpha; NF-kappaB-Inducing Kinase; Okadaic Acid; Protein Serine-Threonine Kinases; Proteins; Purinergic P1 Receptor Agonists; Receptors, Purinergic P1; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Thymidine; TNF Receptor-Associated Factor 2; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uridine

To elucidate the roles of serine/threonine protein phosphatases type 1 (PP1) and type 2A (PP2A) in methylprednisolone-induced differentiation of HL60 cells into granulocytes and K562 cells into monocytes, we examined the effect of serine/threonine protein phosphatase inhibitors, okadaic acid and Cal-A on the proliferation/differentiation of HL60 and K562 cells. Okadaic acid and Cal-A augmented methylprednisolone induced granulocytic differentiation and cell death of HL60 cells and monocytic differentiation and cell death of K562 cells in different dose ranges, respectively. These data suggest an important role of PP1 and PP2A in the mechanism leading to differentiation of leukemic cells.

Topics: Cell Death; Cell Differentiation; Enzyme Inhibitors; HL-60 Cells; Humans; Leukemia, Myeloid; Marine Toxins; Methylprednisolone; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases

We have used the human leukemia cell line K562 as a model to study the role of c-myc in differentiation and apoptosis. We have generated stable transfectants of K562 constitutively expressing two c-Myc inhibitory mutants: D106-143, that carries a deletion in the transactivation domain of the protein, and In373, that carries an insertion in the DNA-interacting region. We show here that In373 is able to compete with c-Myc for Max binding and to inhibit the transformation activity of c-Myc. K562 cells can differentiate towards erythroid or myelomonocytic lineages. K562 transfected with c-myc mutants showed a higher expression of erythroid differentiation markers, without any detectable effects in the myelomonocytic differentiation. We also transfected K562 cells with a zinc-inducible max gene. Ectopic Max overexpression resulted in an increased erythroid differentiation, thus reproducing the effects of c-myc inhibitory mutants. We also studied the role of c-myc mutants and max in apoptosis of K562 induced by okadaic acid, a protein phosphatases inhibitor. The expression of D106-143 and In373 c-myc mutants and the overexpression of max reduced the apoptosis mediated by okadaic acid. The common biochemical activity of D106-143 and In373 is to bind Max and hence to titrate out c-Myc to form non-functional Myc/Max dimers. Similarly, Max overexpression would decrease the relative levels of c-Myc/Max with respect to Max/Max. The results support a model where a threshold of functional c-Myc/Max is required to maintain K562 cells in an undifferentiated state and to undergo drug-mediated apoptosis.

Topics: Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Basic-Leucine Zipper Transcription Factors; DNA-Binding Proteins; Enzyme Inhibitors; Erythrocytes; Erythropoiesis; Gene Expression; Humans; Leukemia, Myeloid; Okadaic Acid; Phosphoric Monoester Hydrolases; Protein Binding; Proto-Oncogene Proteins c-myc; Transcription Factors; Transfection; Tumor Cells, Cultured

We investigated the effect of various protease inhibitors on several tumor necrosis factor (TNF)-mediated cellular responses. Treatment of a human myelogenous leukemia cell line, ML-1a, with TNF in the presence of cycloheximide triggers endonucleolytic activity and apoptotic cell death within 90 minutes. The general serine protease inhibitor diisopropyl fluorophosphate (DFP) and the chymotrypsin-like protease inhibitor N-tosyl-L-lysyl chloromethyl ketone (TPCK) completely abrogated TNF-induced DNA fragmentation and the formation of apoptotic bodies. However, 13 other protease inhibitors, including serine protease inhibitors, did not. The addition of TPCK to cells 30 minutes after TNF treatment completely inhibited the cytokine action, indicating that TPCK-sensitive proteases are not involved in the early stages of signal transduction. TNF is cytotoxic and induces differentiation in ML-1a cells after a 3-day incubation. TPCK had no effect on the TNF-induced cytotoxicity and differentiation, indicating that TPCK-sensitive proteases are specific for DNA fragmentation. TPCK also blocked TNF-induced activation of nuclear factor (NF)-kappa B. The dose-response and the time-course of the inhibitor, however, indicated that the site of action of TPCK for NF-kappa B activation and for DNA fragmentation are quite distinct. Therefore, we conclude that TNF activates two distinct TPCK-sensitive pathways, one leading to apoptosis and the other to NF-kappa B activation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Apoptosis; Base Sequence; Cell Differentiation; Cycloheximide; Cytotoxicity, Immunologic; DNA Damage; Ethers, Cyclic; Gene Expression Regulation, Leukemic; Humans; Isoquinolines; Leukemia, Myeloid; Molecular Sequence Data; Neoplasm Proteins; NF-kappa B; Okadaic Acid; Piperazines; Protease Inhibitors; Tosylphenylalanyl Chloromethyl Ketone; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

The differentiation of a cell line of human promyelocytic leukemia, HL-60 cells, triggered by 12-O-tetradecanoyl 13-phorbol acetate (TPA), depends on the phosphorylation of some proteins, such as 17, 27, and 34 kDa proteins, by protein kinase C. For elucidation of the mechanism of ligand-induced differentiation of HL-60 cells, the effects of okadaic acid (OA), a phosphatase inhibitor, on cell differentiation and protein phosphorylation were studied. After treatment with OA, HL-60 cells differentiated into macrophage-like cells; within 16 h, 70% or more of the treated cells adhered to plastic dishes. The adherent cells did not undergo mitosis but began activities such as phagocytosis. OA increased the phosphorylation of 17, 23, 27, and 34 kDa proteins, as did TPA. The amount of annexin I (39 kDa protein) in HL-60 cells caused to differentiate with OA was 7.5-fold that without such treatment. Kinetic analysis showed that increased transcription of annexin I mRNA caused the increase in annexin I in the differentiated cells. Thus, OA and TPA increased cellular levels of annexin I and caused the differentiation of HL-60 cells into macrophage-like cells.

Topics: Annexin A1; Cell Adhesion; Cell Differentiation; Cell Division; Cell Line; Ethers, Cyclic; Humans; Leukemia, Myeloid; Okadaic Acid; Phosphorylation; RNA, Messenger; Tetradecanoylphorbol Acetate

Bafilomycin A1, a selective inhibitor of vacuolar H+-ATPase, time- and dose-dependently induced the differentiation of M1 cells, a murine myeloid leukemic cell line, into macrophage-like cells as revealed by the phagocytosis of polystyrene latex particles. This differentiation was inhibited not only by actinomycin D and cycloheximide but also by ST-638 (an inhibitor of tyrosine kinase). However, it was affected neither by K-252a (an inhibitor of C-kinase) nor by H-89 (an inhibitor of A-kinase), in contrast to the M1 cell differentiation induced by leukemia inhibitory factor (LIF). Okadaic acid inhibited both the bafilomycin A1-induced and LIF-induced differentiation of M1 cells.

Topics: Animals; Anti-Bacterial Agents; Carbazoles; Cinnamates; Ethers, Cyclic; Growth Inhibitors; Indole Alkaloids; Interleukin-6; Isoquinolines; Leukemia Inhibitory Factor; Leukemia, Myeloid; Lymphokines; Macrolides; Macrophages; Mice; Okadaic Acid; Phagocytosis; Phosphoprotein Phosphatases; Phosphorylation; Protein Biosynthesis; Protein Kinase C; Protein Kinase Inhibitors; Proton-Translocating ATPases; RNA; Sulfides; Sulfonamides; Tumor Cells, Cultured; Vacuoles

Treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), is associated with induction of monocytic differentiation. Since PKC can act immediately upstream to the cytoplasmic Raf-1 serine/threonine protein kinase, we studied activation of Raf-1 during induction of the differentiated monocytic phenotype. The results demonstrate that Raf-1 is activated during TPA-induced monocytic differentiation of HL-60 cells. In contrast, there was little effect of TPA on this kinase in an HL-60 variant, designated HL-525, which is resistant to TPA-induced differentiation. Treatment of both HL-60 and HL-525 cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with Raf-1 activation and induction of the monocytic phenotype. Since Raf-1 can activate the mitogen-activated protein (MAP) kinases, we also studied the relationship between MAP kinase activation and monocytic differentiation. Treatment of HL-60, but not HL-525, cells with TPA was associated with increased MAP kinase activity as determined by phosphorylation of myelin basic protein and the c-Jun Y peptide. Okadaic acid-induced differentiation of both HL-60 and HL-525 cells was similarly accompanied by increases in MAP kinase activity. These findings indicated that activation of Raf-1/MAP kinase signaling is associated with induction of a differentiated monocytic phenotype and that okadaic acid bypasses a defect in this cascade in TPA-treated HL-525 cells. While recent studies have shown that HL-525 cells are deficient in PKC beta, the present results demonstrate that PKC beta expression is up-regulated in the HL-525 variant by treatment with retinoic acid. The results also demonstrate that retinoic acid-treated HL-525 cells respond to TPA with activation of Raf-1 and MAP kinase, as well as induction of monocytic differentiation. Taken together, the results indicate that activation of Raf-1/MAP kinase signaling is associated with monocytic differentiation and that stimulation of serine/threonine protein phosphorylation by TPA or okadaic acid is sufficient for reversal of the leukemic HL-60 phenotype.

Topics: Amino Acid Sequence; Calcium-Calmodulin-Dependent Protein Kinases; Cell Differentiation; Enzyme Activation; Ethers, Cyclic; Gene Expression; Genes, jun; Humans; In Vitro Techniques; Leukemia, Myeloid; Molecular Sequence Data; Monocytes; Okadaic Acid; Protein Kinase C; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; RNA, Messenger; Tetradecanoylphorbol Acetate; Tretinoin; Tumor Cells, Cultured

The phosphatase inhibitor okadaic acid was found to induce cell cycle arrest of human myeloid leukemic cell lines HL-60 and U937 in a concentration- and time-dependent manner. Exposure to low concentrations of okadaic acid (2-8nM) for 24-48 hr caused greater than 70% of cells to arrest at G2/M, with up to 40% of the cells arrested in early mitosis. Cell viability decreased rapidly after 48 hr of treatment, and morphological and DNA structure analysis indicated that this was primarily due to the induction of apoptosis. The cells arrested in mitosis by 8 nM okadaic acid could be highly enriched by density gradient centrifugation and underwent apoptosis when further cultured either with or without okadaic acid, indicating that the effects of okadaic acid were irreversible. In contrast to the effects of low concentrations of okadaic acid, high concentrations (500 nM), inhibited proliferation in less than 3 hr. Remarkably, the majority of cells also entered a mitosis-like state characterized by dissolution of the nuclear membrane and condensation and partial separation of chromosomes. However, these cells had a diploid content of DNA, indicating that the cell cycle arrest occurred at G1/S with premature chromosome condensation (PCC), rather than at G2/M. If cells were first blocked at G1/S with hydroxyurea and then treated with okadaic acid, greater than 90% developed PCC in less than 3 hr without replicating their DNA. Caffeine was not able to induce PCC in these cells, either with or without prior inhibition of DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Cycle; Cell Death; Cell Differentiation; Cell Division; Centrifugation, Density Gradient; Electrophoresis, Agar Gel; Ethers, Cyclic; Humans; Kinetics; Leukemia, Myeloid; Mitosis; Nocodazole; Okadaic Acid; Phosphoprotein Phosphatases; Tumor Cells, Cultured

The growth inhibitory effect of tumour promoters on human leukaemia and lung cancer cell lines was examined using the [3-(4,5 dimethylthiazol)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. The four cell lines used were the K562 human leukaemia cell line, its adriamycin (ADM)-resistant subline (K562/ADM), which shows the mdr phenotype, PC-9 (a human lung adenocarcinoma cell line) and its cisplatin (CDDP)-resistant subline (PC-9/CDDP), which does not show the mdr phenotype. Phorbol 12-tetradecanoate-13-acetate (TPA) and the TPA-type tumour promoters, aplysiatoxin and debromoaplysiatoxin, inhibited the growth of the two parental cell lines, K562 and PC-9. The non-TPA-type tumour promoter, okadaic acid, also inhibited the growth of the two parental cell lines in a dose-dependent manner. TPA-type and okadaic acid inhibited the growth of K562/ADM more weakly than that of K562, and showed no growth inhibition in PC-9/CDDP. Anhydrodebromoaplysiatoxin, an inactive derivative of the TPA-type tumour promoter, could suppress the growth of K562 and K562/ADM only at high concentration (more than 50 pM) and it showed similar growth inhibitory effects on the two cell lines. Okadaic acid tetramethyl ether, the inactive form of the non-TPA-type tumour promoter did not inhibit the growth of any of the cell lines. The growth inhibitory effect of these compounds was well correlated with their tumour-promoting activity. A study of the accumulation of okadaic acid revealed that the amount of 3H-okadaic acid in K562/ADM and PC-9/CDDP was similar to that in their parental cells indicating that cross-resistance to this tumour promoter in the drug-resistant cell lines is not due to a difference in the amount of drug accumulated in sensitive and resistant cells. These results suggest the presence of another common mechanism for resistance to ADM and CDDP as well as to TPA- or non-TPA-type tumour promoters.

Topics: Cell Division; Cell Line; Chemical Phenomena; Chemistry; Cisplatin; Cross Reactions; Doxorubicin; Drug Resistance; Ethers, Cyclic; Humans; Leukemia, Myeloid; Lung Neoplasms; Lyngbya Toxins; Okadaic Acid; Tetradecanoylphorbol Acetate