Proteome analyses of the growth inhibitory effects of NCH-51, a novel histone deacetylase inhibitor, on lymphoid malignant cells.

Recent reports showing successful inhibition of cancer and leukemia cell growth using histone deacetylase inhibitor (HDACi) compounds have highlighted the potential use of HDACi as anti-cancer agents. However, high incidence of toxicity and low stability in vivo were observed with hydroxamic acid-based HDACi such as suberoylanilide hydroxamic acid (SAHA), thus limiting its clinical applicability. In this study, we found that a novel non-hydroxamate HDACi NCH-51 could inhibit the cell growth of a variety of lymphoid malignant cells through apoptosis induction, more effectively than SAHA. Activation of caspase-3, -8 and -9, but not -7 was detected after the treatment with NCH-51. Gene expression profiles showed that NCH-51 and SAHA similarly upregulated p21 and downregulated anti-apoptotic molecules including survivin, bcl-w and c-FLIP. Proteome analysis using two-dimensional electrophoresis revealed that NCH-51 upregulated anti-oxidant molecules including peroxiredoxin 1 and 2 and glutathione S-transferase at the protein level. Interestingly, NCH-51 induced reactive oxygen species (ROS) after 8 h whereas SAHA continuously declined ROS. Pretreatment with an antioxidant, N-acetyl-L-cysteine, abolished the cytotoxicity of NCH-51. These findings suggest that NCH-51 exhibits cytotoxicity by sustaining ROS at the higher level greater than SAHA. This study indicates the therapeutic efficacy of NCH-51 and novel insights for anti-HDAC therapy.

Okadaic acid induces both augmentation and inhibition of opsonized zymosan-stimulated superoxide production by differentiated HL-60 cells. Possible involvement of dephosphorylation of a cytosolic 21K protein in respiratory burst.

We found that okadaic acid (OA), a potent tumor promoter and a phosphatase inhibitor, has a unique opposing effect on opsonized zymosan (Op.-zym.)-elicited O2.- production by differentiated HL-60 cells in a narrow range of concentrations but does not induce any O2.- production by itself. Okadaic acid magnified the O2.- production 2.5-fold at 1.0 microM, while it inhibited it at 2.0 microM or higher concentrations. This effect of OA did not correspond to the changes in the expression of surface receptors (CD11b/CD18, CR3) for Op.-zym., because they were weakly down-regulated by OA at any concentration. Two-dimensional gel electrophoresis revealed that in the absence of OA, Op.-zym. induced rapid dephosphorylation of a cytosolic 21K protein with a very slight increase in phosphorylation of membranous p47phox, which is one of the cytosolic factors required for respiratory burst. In the presence of a stimulatory concentration (1.0 microM) of OA, the Op.-zym.-caused dephosphorylation of the 21K protein was still observed and the phosphorylation of p47phox was enhanced. In the presence of an inhibitory concentration (2.0 or 5.0 microM) of OA, the Op.-zym.-induced dephosphorylation of the 21K protein was strongly inhibited while p47phox was heavily phosphorylated. Acid hydrolysis of the 21K phosphoprotein yielded only phosphoserine as a phosphoamino acid. Furthermore, at least part of the 21K protein seemed to be associated with p67phox and p47phox, because it was co-immunoprecipitated with those cytosolic factors. These results suggest that a cytosolic 21K protein plays an important role in respiratory burst through dephosphorylation by a phosphoserine phosphatase, and that the dephosphorylated 21K protein may work synergistically with the phosphorylated p47phox on the pathway for activation of the respiratory burst oxidase.

Effects of calyculin A and FK506 on the O2- generation of rat peritoneal macrophages.

To investigate the role of phosphatase in O2- generation, the effects of the potent phosphoprotein phosphatase inhibitors, Calyculin A and FK506, were analyzed during phagocytosis using rat peritoneal macrophages. O2- generation was continuously measured after addition of opsonized zymosan (op. ZY) or IgG-coated zymosan (IgG-ZY). The rate of O2- generation was directly proportional to the number of macrophages, up to 1-2 x 10(6) cells/ml. It was found that the rate and duration of O2- generation were markedly inhibited by Calyculin A. The addition of 100 nM of Calyculin A reduced O2- generation to about one-tenth of the control value. In contrast, FK506 did not inhibit O2- generation, suggesting that calcium calmodulin phosphatase is not involved in the activation of NADPH oxidase. This result indicates that the process of dephosphorylation might involve activation of NADPH oxidase as a control mechanism in phagocytosis by rat peritoneal macrophages. Furthermore, since Calyculin A is an inhibitor of phosphatase 1 and 2A, it is suggested that dephosphorylation may be evoked by these phosphatases.

Granulocyte colony-stimulating factor-induced dephosphorylation of a 45 kD cytosolic protein in HL-60 cells differentiating into neutrophils.

Granulocyte colony-stimulating factor (G-CSF)-induced alteration of phosphoprotein during differentiation of HL-60 cells was studied. From the two-dimensional gel electrophoresis analysis of phosphoproteins, a 45 kD phosphoprotein in the cytosolic fraction of DMSO-pretreated HL-60 cells was rapidly dephosphorylated by the addition of G-CSF. This 45 kD phosphoprotein migrated into four or five spots between 4.5 and 5.5 pI. The dephosphorylation of 45 kD protein was observed within at least 10 min and reached a maximum at 60 min. Phosphoamino acid analysis showed that only serine residue of 45 kD phosphoprotein was phosphorylated, suggesting that G-CSF induced an activation of serine phosphatase. Furthermore, Staurosporine and calphostin C inhibited the phosphorylation of 45 kD protein, suggesting that protein kinase C or its downstream kinase(s) is involved in the phosphorylation of 45 kD protein. These results indicate that G-CSF causes dephosphorylation of a 45 kD cytosolic phosphoprotein which may play a role in signal transduction of G-CSF.