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.

To clarify the process of endothelial differentiation, we isolated AC133(+) cells and induced the in vitro differentiation of these cells into endothelial cells. AC133(+) cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin-coated dishes in the presence of vascular endothelial growth factor. Time-course analysis of the alteration of endothelial markers on cultured AC133(+) cells revealed that the expression of CD31 (PECAM-1) on AC133(+) cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133(+) cells. CD31-bright cells, which were sorted from cultured AC133(+) cells, differentiated more efficiently into endothelial cells than had CD31-positive or CD31-negative cells, suggesting that CD31-bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31(+) cells derived from cultured AC133(+) cells that are able to differentiate to endothelial cells as precursor cells.

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.

No abstract

We previously reported that CD31 bright cells, which were sorted from cultured AC133 ؉ cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31 ؉ cells or CD31 ؊ cells, suggesting that CD31 bright cells may be endothelial precursor cells. In this study, we found that CD31 bright cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133 ؉ cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31 bright cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.