Biomedical subjects

K Sakoe

Publications and source records attributed to K Sakoe.

24 records · Page 2Linked to original sources

PubMed1995

Biosynthesis of the so-called "a" and "asialo" pathway glycosphingolipids is differentially regulated in murine myelogenous leukemia NFS60 cells.

Regulation of "a" and "asialo" series ganglioside biosynthesis was analyzed. COS-1 cells expressing only GD1a showed high synthetic activities of GM3, GM2, GM1a, and GD1a, but little activity for GA2 synthesis. However, IL-3-dependent murine NFS60-I7, which has GM1b and GD1 alpha, exhibited high synthetic activities of GM2, GM1a, and GD1a, but GM3 synthase was only 1/6 of COS-1 and GA2 synthetic activity was low. By contrast, IL-3 gene-transfected subline NFS60-H7 expressing GD1a in addition to GM1b and GD1 alpha displayed up-regulated GM3 synthase and GA2 synthase activities, while GM2, GM1a, and GD1a synthase activities were in the same levels as in NFS60-I7 cells. Since GA2 synthetic activities were not parallel with GM2 synthase in the investigated machinery of ganglioside biosynthesis, it is strongly suggested that biosynthesis of "a" and "asialo" series gangliosides is regulated differentially from each other in murine NFS60 cell lines.

Animals↗

PubMed1995

Expression of differentiation-related phenotypes and apoptosis are independently regulated during myeloid cell differentiation.

When human promyelocytic leukemia cell line HL-60 was treated with various differentiation-inducers, apoptosis always occurred after the full appearance of differentiation-related phenotypes. However, the two phenomena could be dissociated when HL-60 cells were treated with PDBu. When HL-60 cells were cultured with PDBu for more than 36 h, apoptosis was induced following differentiation. Apoptosis was not, however, observed when PDBu was removed within 24 h, even though induction of differentiation-related phenotypes, such as NBT-reducing ability and surface marker expression, was the same as that in the control. Northern blot analysis revealed that bcl-2 mRNA was rapidly down-regulated within 6 h of the treatment with PDBu. The amount of bcl-2 mRNA recovered to that of undifferentiated HL-60 cells when PDBu was washed out within 24 h. In contrast, the recovery of bcl-2 was incomplete when the cells were treated with PDBu for more than 36 h, suggesting that bcl-2 is also a critical regulator of the cell fate during myeloid differentiation. This hypothesis was confirmed by experiments using antisense oligonucleotides, i.e., blocking the recovery of bcl-2 mRNA by antisense oligonucleotides could result in the induction of apoptosis in HL-60 cells from which PDBu was removed within 24 h. Moreover, overexpression of BCL-2 in HL-60 cells could block apoptosis during differentiation without any significant effect on differentiation itself. These results strongly suggest that apoptosis is not a simple consequence of differentiation-induction, and that apoptosis and differentiation are regulated independently in myeloid cells.

Apoptosis↗

PubMed1995

Over-expression and amplification of the CDC2 gene in leukaemia cells.

The expression and structure of the cdc2 gene, one of the master regulators of the eukaryotic cell cycle, were investigated in fresh leukaemic cells from 51 cases of various types of leukaemia. Cdc2 mRNA transcripts were detectable in approximately 40% (21/51) of cases by Northern blotting. Over-expression of cdc2 mRNA as compared to normal bone marrow cells was noted in 10/21 cases with detectable cdc2 mRNA transcripts. Amplification of the cdc2 gene was found in three cases. Cdc2 mRNA was over-expressed in these three cases, suggesting that gene amplification is a direct cause of mRNA over-expression in a subset of cases. Cell proliferative capacity was well correlated with the amount of cdc2 mRNA transcripts, i.e. 3H-thymidine incorporation was highest in cases with cdc2 mRNA over-expression and was significantly higher in cdc2-positive cases than in cdc2-negative cases. These results suggest that over-expression of CDC2, which is due to the gene amplification in some cases, might play a role in altered growth of leukaemic cells.

Blotting, Northern↗

PubMed1994

The role of cellular transcription factor E2F in the regulation of cdc2 mRNA expression and cell cycle control of human hematopoietic cells.

cdc2 mRNA transcripts were detected in immature bone marrow cells and became undetectable along with differentiation. Peripheral blood resting cells did not express cdc2 mRNA, but it was induced in T-lymphocytes when the cells reentered the cell cycle in response to specific mitogens. In contrast, cdc2 mRNA could not be induced in granulocytes and monocytes even after the culture with the appropriate stimulants. In order to investigate the mechanism of the regulation of cdc2 mRNA expression in hematopoietic cells, we isolated the 5'-flanking sequence of the cdc2 gene and found the putative E2F binding site at the position of nucleotides -124 to -117. The binding of E2F at this region was detected by a gel-retardation assay and DNaseI footprinting in phytohemagglutinin-stimulated T-lymphocytes, which was coincident with the expression of cdc2 mRNA. E2F binding was not observed in both granulocytes and monocytes. Transient chloramphenicol acetyltransferase assay revealed that the region containing E2F binding site had a strong promoter activity, and introduction of the mutation at the E2F binding site resulted in a significant loss of the activity. E2F-1 and DP-1 mRNAs were not detectable in granulocytes, monocytes and resting T-lymphocytes but were induced after the mitogenic stimulation of T-lymphocytes. The induction of E2F activity preceded the appearance of cdc2 mRNA, which is consistent with the role of E2F in the regulation of cdc2 mRNA expression. These results suggest that cdc2 mRNA expression is related to the cell cycling of normal human hematopoietic cells and that E2F plays some roles in the regulation of its expression.

Base Sequence↗

PubMed1993

A monoclonal antibody reactive with terminal lactotriaosyl residue-containing oligosaccharides and its application to characterizing cell surface expression of the glyco-epitopes in COS-1 cells.

A new monoclonal antibody (MoAb), designated JF12, reactive strongly with Lc3 (GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) but only slightly with nLc5 (GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->Cer) has been prepared after immunization of Balb/c mice with Lc3. By the flowcytometrical analyses of COS-1 cells, which was strongly stained with JF12, the reactivity completely disappeared in the confluent condition upon harvesting by the protease treatments of the cell surface. In the sparse condition, however, the cells still retained JF12 reactivity in spite of the protease treatments. This strongly suggests that the expression of terminal GlcNAc-containing glycoconjugates on the surface of COS-1 cells may be dramatically modulated by protease-sensitive membranous components being dependent upon the cell density.

Animals↗

PubMed1992

Total metabolic flow of glycosphingolipid biosynthesis is regulated by UDP-GlcNAc:lactosylceramide beta 1-->3N-acetylglucosaminyltransferase and CMP-NeuAc:lactosylceramide alpha 2-->3 sialyltransferase in human hematopoietic cell line HL-60 during differentiation.

We have previously reported that ganglioside GM3 was remarkably increased during monocytoid differentiation of human myelogenous leukemia cell line HL-60 cells and that neolacto series gangliosides (NeuAc-nLc) were enriched during granulocytoid differentiation. In addition, HL-60 was differentiated into monocytic lineage by exogenous GM3 and into granulocytoid by NeuAc-nLc. In the present report, the enzymatic bases of glycosphingolipid biosynthesis in HL-60 during differentiation induced by 12-O-tetradecanoylphorbol-13-acetate and all-trans-retinoic acid were investigated. The following results were of particular interest. (i) Lactosylceramide alpha 2-->3 sialyltransferase (GM3 synthase) was remarkably up-regulated during monocyte differentiation, while the GM3 synthase level did not change in granulocytic differentiation. (ii) By contrast, lactosylceramide beta 1-->3N-acetylglucosaminyltransferase (Lc3Cer synthase) was down-regulated during monocytic differentiation, while the activity of Lc3Cer synthase was found to increase in granulocytic differentiation. (iii) The activities of four downstream glycosyltransferases (for synthesis of NeuAc-nLc) were found to increase or to remain unchanged during monocytic and granulocytic differentiation. These results strongly suggested the following. The dramatic GM3 increase and the decrease of NeuAc-nLc during monocytic differentiation are the consequences of the up-regulation of GM3 synthase and the down-regulation of Lc3Cer synthase, although the downstream enzymes are ready to catalyze their enzyme reactions. The notable increase of NeuAc-nLc and the relative decrease of GM3 during granulocytic differentiation are the results of the unchanged level of GM3 synthase and the up-regulation of Lc3Cer synthase together with the activation of the downstream glycosyltransferases. These results suggest that these two key upstream glycosyltransferases, GM3 synthase and Lc3Cer synthase, play critical roles in regulating the glycosphingolipid biosynthesis in HL-60 cells during differentiation. This switching mechanism of these two glycosyltransferases, together with our previous findings, might be one of the most important parts of the determining system of differentiation direction in human myeloid cells into monocytic or granulocytic lineages.

Antibodies, Monoclonal↗