1.Effect of okadaic acid on differentiation of NB4 and MR2 cells induced by all-trans retinoic acid.
Xi-hui XU ; Jian OU-YANG ; Jun-hao CHEN ; Pin-hao XIE ; Yong-quan XIA
Chinese Journal of Hematology 2008;29(6):379-383
OBJECTIVETo 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.
METHODSATRA, 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.
RESULTS1) 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.
CONCLUSIONExpression 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.
Cell Differentiation ; drug effects ; Cell Line, Tumor ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Okadaic Acid ; pharmacology ; Phosphoprotein Phosphatases ; metabolism ; Protein Phosphatase 2 ; antagonists & inhibitors ; metabolism ; Tretinoin ; pharmacology
2.Inhibitory effect of Hsp70 on angiotensin II-induced vascular smooth muscle cell hypertrophy.
Ying ZHENG ; Chang Nim IM ; Jeong Sun SEO
Experimental & Molecular Medicine 2006;38(5):509-518
Angiotensin II (Ang II), which is an important mediator of both vascular responsiveness and growth, has been shown to induce vascular smooth muscle cell (VSMC) hypertrophy via the activation of a complex series of intracellular signaling events. Heat shock protein 70 (Hsp70) has recently been shown to protect against Ang II-induced hypertension. In this study, we tested the hypothesis that Hsp70 can protect VSMC from Ang II-induced hypertrophy. We treated VSMCs with Ang II to induce hypertrophy and to activate MAPK signaling pathway. We observed that the augmentation of Hsp70 expression inhibited Ang II-stimulated VSMC hypertrophy. This inhibitory effect of Hsp70 appears to be partly due to extracellular signal-regulated kinase (ERK1/2) inactivation, which in turn, may possibly result from the accumulation of MAP kinase phosphatase-1 (MKP-1).
Rats, Sprague-Dawley
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Rats
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RNA, Small Interfering/pharmacology
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Protein-Tyrosine-Phosphatase/metabolism/physiology
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Phosphoprotein Phosphatase/metabolism/physiology
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Muscle, Smooth, Vascular/*cytology/*drug effects
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Mitogen-Activated Protein Kinase 3/antagonists & inhibitors
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Mitogen-Activated Protein Kinase 1/antagonists & inhibitors
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Male
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MAP Kinase Kinase 2/metabolism
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MAP Kinase Kinase 1/metabolism
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Immediate-Early Proteins/metabolism/physiology
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Hypertrophy
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HSP70 Heat-Shock Proteins/antagonists & inhibitors/*pharmacology
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Flavonoids/pharmacology
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Enzyme Stability/drug effects
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Cells, Cultured
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Cell Cycle Proteins/metabolism/physiology
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Aorta/drug effects/pathology
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Animals
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Angiotensin II/*pharmacology
3.Calyculin A modulates activation of the NADPH-oxidase in Me2SO-differentiated HL-60 cells.
Joo In PARK ; David J UHLINGER ; Byeung Seon CHUNG ; In Hoo KIM ; Jong Young KWAK
Experimental & Molecular Medicine 1998;30(4):214-220
Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.
Arachidonic Acid/metabolism
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Ca(2+)-Calmodulin Dependent Protein Kinase/metabolism
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Cell Differentiation
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Dimethyl Sulfoxide/pharmacology*
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Enzyme Inhibitors/pharmacology*
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HL-60 Cells
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Human
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N-Formylmethionine Leucyl-Phenylalanine/pharmacology
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NADPH Oxidase/metabolism*
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Neutrophils/metabolism*
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Neutrophils/drug effects
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Oxazoles/pharmacology*
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Oxygen/metabolism
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Phosphoprotein Phosphatase/antagonists & inhibitors
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Phosphoproteins/immunology
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Signal Transduction
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Tetradecanoylphorbol Acetate/pharmacology
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Time Factors