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.     

Myeloid calcium binding proteins: expression in the differentiated HL-60 cells and detection in sera of patients with connective tissue diseases

A differentiation antigen induced in 1,25-dihydroxyvitamin D3 (VD3)-treated HL-60 cells was identified as being comprised of the myeloid calcium binding proteins CaBP-p8 and -p14 by determining its amino acid and DNA sequence. Northern blot analysis using a DNA fragment of the gene encoding p14 as a probe indicated that the gene was not expressed in undifferentiated HL-60 cells but transcribed starting on day 1 after VD3 treatment. The level of p 14 mRNA reached a peak on day 2, then declined, and little mRNA remained on day 10, indicating that the p14 gene is activated once and then inactivated during HL-60 differentiation due to VD3. In contrast, thymidylate synthase (TSase) mRNA was present in undifferentiated HL-60 cells but disappeared quickly after VD3 treatment. Both p8 and p14 of CaBP were found at elevated levels in sera of some patients with connective tissue diseases [highly elevated in rheumatoid arthritis (RA), Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE), and progressive systemic sclerosis (PSS), and moderately in polymyositis or dermatomyositis (PM/DM) and mixed connective tissue disease (MCTD)]. These results were in sharp contrast with the finding that p14 is always at a highly elevated level but little p8 is present in the sera of cystic fibrosis (CF) patients [Bruggen et al. (1988) Nature 331, 570).     

Nuclear structure and gene activity in human differentiated cells

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.     

Expression of src family genes during monocytic differentiation of HL-60 cells

It has been reported that src family protein-tyrosine kinases were expressed specifically in a certain lineage or differentiation stage of hematopoietic cells. To understand the molecular basis for differentiation and function of monocyte/macrophage, we investigated the expressions of src family genes by the HL-60 cells stimulated with differentiation-inducing agents. TPA and vitamin D3 (D3) were used as stimulants for monocytic development, since each agent has been known to induce phenotypically specific differentiation of HL-60 cells. The fyn, fgr, and lyn genes were characteristically expressed concomitantly with phenotypic changes and expressions of nuclear proto-oncogenes, whereas src, lck, hck, and yes genes were not. In TPA-induced differentiation of HL-60 cells, both fyn and lyn genes, but not fgr gene, were expressed. In contrast, both fgr and lyn genes, but not fyn gene, were expressed in D3-induced differentiation of the cells. The independent and characteristic expressions of these genes were observed in the further advanced differentiation of HL-60 cells induced by TPA plus D3 or D3 plus human transforming growth factor-beta 1. The granulocytic differentiation of the cells treated with retinoic acid was accompanied by intense expression of fgr, but weak or no expression of lyn and fyn gene. These data indicate that each protein-tyrosine kinase encoded by src family genes may play distinct roles in development and/or functions of monocyte/macrophage-lineage cells.     

Recombinant human interferon sensitizes resistant myeloid leukemic cells to induction of terminal differentiation

Recombinant human leukocyte interferon (IFN-alpha A) inhibits growth of the human promyelocytic leukemic cell line HL-60 without inducing these cells to differentiate terminally. When IFN-alpha A is combined with agents capable of inducing differentiation in HL-60 cells, such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), cis or trans retinoic acid (RA) or dimethylsulfoxide (DMSO), growth suppression and induction of differentiation are dramatically increased. By growing HL-60 cells in increasing concentrations of TPA, RA, or DMSO, a series of sublines have been developed which are resistant to the usual growth inhibition and induction of differentiation seen when wild type HL-60 cells are exposed to these agents. Treatment of these resistant HL-60 cells with the combination of IFN-alpha A and the appropriate inducer results, however, in a synergistic suppression in cell growth and a concomitant induction of terminal differentiation. The ability of interferon to interact synergistically with agents which promote leukemic cell maturation may represents a novel means of reducing resistant leukemic cell populations.     

DADS对DJ-1核定位高表达人白血病HL-60细胞的影响

最近研究表明,DJ-1在许多肿瘤中过表达,而且DJ-1的核表达与肿瘤的生物学行为有关. 本文主要研究二烯丙基二硫（DADS）对DJ-1核定位高表达人白血病HL-60细胞生物行为学的影响,阐明DJ-1在细胞核内的功能,为临床诊断及治疗过程提供一个潜在的治疗靶点. 通过基因转染技术建立核内高表达HL-60细胞株（DJ-1/HL-60）,利用软琼脂集落形成实验、 MTT法、间接免疫荧光细胞化学实验、硝基蓝四氮唑( NBT)还原比色实验评估HL-60细胞的增殖与分化,DJ-1核定位过表达促进HL-60细胞的增殖并抑制其分化. Transwell迁移侵袭小室实验表明DJ-1核定位过表达可以促进HL-60的迁移和侵袭能力. Western blot结果表明DADS具有抑制HL-60细胞中核内DJ-1蛋白表达的能力. 说明DJ-1核定位高表达具有促进HL-60细胞增殖和迁移侵袭及抑制HL-60细胞分化的作用,DADS可以诱导DJ-1核定位高表达HL-60细胞分化及抑制迁移侵袭, DJ-1核定位高表达可减弱DADS抑制HL-60细胞增殖的作用.     

Altered expression profiles of microRNAs during TPA-induced differentiation of HL-60 cells

MicroRNAs (miRNAs) are highly conserved small non-coding RNAs that regulate gene expression through translational repression by base-pairing with partially complementary mRNAs. The expression of a set of miRNAs is known to be regulated developmentally and spatially, and is involved in differentiation or cell proliferation in several organisms. However, the expression profiles of human miRNAs during cell differentiation remain largely unknown. In an effort to expand our knowledge of human miRNAs, we investigated miRNAs during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation of human leukemia cells (HL-60) into monocyte/macrophage-like cells. Several hundred RNAs ranging from 18 to 26 nucleotides were isolated from HL-60 cells with or without TPA-induction, and subsequently characterized by sequencing, database searching, and expression profiling. By removing non-miRNA sequences, we found three novel and 38 known miRNAs expressed in HL-60 cells. These miRNAs could be further classified into subsets of miRNAs that responded differently following TPA induction, either being up-regulated or down-regulated, suggesting the importance of regulated gene expression via miRNAs in the differentiation of HL-60 cells.     

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.