Decrease in CuZn-superoxide dismutase mRNA level during differentiation of human monocytic and promyelotic leukemia cells

Change in the level of CuZn-superoxide dismutase (SOD) mRNA was examined using a molecular probe during differentiation of human monocytic leukemia U937 cells or promyelotic leukemia HL-60 cells induced by either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or dimethylsulfoxide (DMSO). CuZn-SOD mRNA levels were found to decrease during the course of differentiation, and this response is specific for differentiation, since the treatment of human B cell leukemia cells or normal diploid fibroblasts with TPA failed to have any effect on the level of CuZn-SOD mRNA. The activity of CuZn-SOD in U937 cells also decreased during differentiation, but following that of the CuZn-SOD mRNA level. The expression of the CuZn-SOD gene is thus concluded to diminish during the differentiation of HL-60 and U937 cells.     

Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line

The susceptibility of four different human cell lines (HUT 102, THP-1, MOLT-4, and HL-60) to infection by human CMV (HCMV) was studied. Only HUT 102 was susceptible and only immediate-early gene products were produced. However, THP-1, a monocytic cell line, could be infected by HCMV with a full cycle of replication after treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which produced differentiation of the cell line into cells with characteristics of mature macrophages. Late (structural) Ag were demonstrated, as were infectious virions as detected by electron microscopy and infectious center assay. HL-60, a promyelocytic cell line, was not susceptible to HCMV infection after treatment with TPA despite differentiation into adherent cells with properties of macrophages, suggesting that cellular lineage was important. Treatment with TPA after infection resulted in a greatly reduced frequency of infected cells, suggesting that pretreatment was essential. Furthermore, continued presence of TPA was unnecessary after differentiation was induced. This study establishes the precedent of productive HCMV infection in human monocytic cells. The potential mechanism and relevance of enhanced replication induced by TPA are discussed.     

Control of HL-60 monocytic differentiation. Different pathways and uncoupled expression of differentiation markers

Control of expression of the terminally differentiated phenotype was studied using the human promyelocytic leukemia cell line HL-60. Three known inducers of HL-60 monocytic differentiation were compared: 1,25-dihydroxy vitamin D3, tetradecanoylphorbol acetate (TPA), and sodium butyrate. At concentrations where all three inducers resulted in similar courses of G1/0-specific growth arrest, the kinetics of appearance of certain differentiation markers typically characteristic of mature monocytic cells was determined. The markers were inducible oxidative metabolism, non-specific esterase activity, and the cell surface determinants Mo1, My4, and Mo2. The results indicate that: Regulation of the expression of these markers during induced monocytic differentiation is not controlled in common. The three monocytic inducers do not induce the same metabolic cascade leading to differentiation. Similar states of differentiation could thus be reached by different pathways apparently due to the fact that control of expression of different differentiation markers was not tightly coupled.     

Induction of HL-60 monocytic cell differentiation promoted by a perturbation of DNA synthesis: hydroxyurea promotes action of TPA

Control of terminal cell differentiation was studied using the human promyelocytic leukemia cell line, HL-60. HL-60 cells are known to undergo terminal monocytic differentiation when continuously exposed to 1.6 nM tetradecanoylphorbol acetate (TPA). The dose-response relationship between TPA concentration and induced differentiation is relatively steep. TPA (1.1 nM) induces little G1/0 specific growth inhibition or phenotypic differentiation. In contrast, pretreating the cells with a pulse exposure to hydroxyurea promotes their capability to terminally differentiate in response to TPA. Initially exponentially proliferating cells exposed for 20 h, approximately one doubling time, to 0.3 mM hydroxyurea, a subcytotoxic dose, underwent rapid G1/0 specific growth arrest and cell differentiation in response to subsequent exposure to 1.1 nM TPA. The extent of terminal differentiation was comparable to that induced by 1.6 nM TPA. The results support the hypothesis that early events in induction of terminal HL-60 cell differentiation depend on an S phase-specific process which may involve gene amplification.     

Inducers of leukemic cell differentiation cause down-regulation of RB gene expression.

Expression of the retinoblastoma (RB) tumor suppressor gene during cell differentiation induced by dimethyl sulfoxide or sodium butyrate was studied in HL-60 human promyelocytic leukemia cells. As cells progressed through the cell cycle, the amount of RB protein per cell increased with homeostasis maintained, so that the amount of RB protein relative to the total cell mass remained almost constant. Dimethyl sulfoxide was used to induce these promyelocytic leukemia cells to undergo terminal differentiation into mature myeloid cells. There was an early reduction in the RB protein expressed per cell. The reduction in expression was similar for cells in all cell cycle phases. There was also progressively reduced expression at later times as cells terminally differentiated. This was compared to the case in which sodium butyrate was used to induce the differentiation of HL-60 cells into mature monocytic cells. An early reduction in RB protein expression per cell also occurred. It occurred for cells in all cell cycle phases as well. Thus, the induced differentiation of HL-60 cells along either the myeloid or the monocytic differentiation lineage involves an early reduction in RB expression, which is common to both pathways. The reduction anteceded proliferative arrest or differentiation. In both cases, the final, resulting G0-differentiated cells had less RB protein per cell than the proliferating, immature, leukemic precursor cells.     

Differential expression of the chromosomal high mobility group proteins 14 and 17 during the onset of differentiation in mammalian osteoblasts and promyelocytic leukemia cells

The expression of chromosomal proteins HMG 14 and HMG 17 during proliferation and differentiation into the osteoblast and monocyte phenotypes was studied. Cellular levels of HMG 14 and HMG 1 7 mRNA were assayed in primary cultures of calvarial-derived rat osteoblasts under conditions that (1) support complete expression of the mature osteocytic phenotype and development of a bone tissue-like organization; and (2) where development of osteocytic phenotypic properties are both delayed and reduced in extent of expression. HMG 14 and HMG 17 are preferentially expressed in proliferating osteoblasts and decline to basal levels post-proliferatively at the onset of extracellular matrix mineralization. In contrast, under conditions that are not conducive to extracellular matrix mineralization, HMG 14 is maximally expressed following the downregulation of proliferation. Consistent with previous reports by Bustin and co-workers [Crippa et al., 1990], HMG 14 and HMG 17 are expressed in proliferating HL-60 promyelocytic leukemia cells and downregulated post-proliferatively following phorbol ester-induced monocytic differentiation. However, differentiation into the monocyte phenotype is accompanied by reinitiation of HMG 17 gene expression. The results indicate that the levels of HMG 14 and HMG 17 mRNA are selectively down-regulated during differentiation.     

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.     

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

Activation of the pp60c-src kinase during differentiation of monomyelocytic cells in vitro.

The proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60c-src tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60c-src kinase.     

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.     

Induction of platelet-derived growth factor gene expression during megakaryoblastic and monocytic differentiation of human leukemia cell lines.

Platelet-derived growth factor (PDGF) is one of the most important polypeptide growth factors in human serum. It is composed of two polypeptide chains linked by disulfide bonds. The B-chain is encoded by the c-sis proto-oncogene, which is expressed in several malignant and non-malignant cells including K562 cells differentiating towards megakaryoblasts. Expression of the A-chain has been reported to occur in human solid tumor cell lines independently of c-sis expression. We report here the non-coordinate expression of the A- and B-chains in human leukemia cell lines. The PDGF-A and B-chain (c-sis) RNA expression as well as secretion of PDGF polypeptides are induced in the K562 cell line upon induction of megakaryoblastic differentiation with 12-O-tetradecanoyl phorbol-13-acetate (TPA) whereas erythroid differentiation induced with sodium butyrate is accompanied by c-sis expression only. Simultaneously with megakaryoblastic differentiation the RNA level for another platelet protein, the transforming growth factor-beta was also increased, but in a complex manner. The promyelocytic leukemia cell line HL-60 does not express PDGF-A RNA, whereas the promonocytic cell line U937 does. Preferential induction of the A-chain RNA is obtained in both cell lines after treatment with TPA which causes monocytic differentiation. PDGF-A expression in HL-60 cells is also observed after treatment with the tumor necrosis factor-alpha but granulocytic differentiation of HL-60 cells induced with dimethyl sulfoxide or the granulocyte colony-stimulating factor is not associated with PDGF gene expression.     

Regulation of progranulin expression in myeloid cells

Progranulin (pgrn; granulin-epithelin precursor, PC-cell-derived growth factor, or acrogranin) is a multifunctional secreted glycoprotein implicated in tumorigenesis, development, inflammation, and repair. It is highly expressed in macrophage and monocyte-derived dendritic cells. Here we investigate its regulation in myeloid cells. All-trans retinoic acid (ATRA) increased pgrn mRNA levels in myelomonocytic cells (CD34(+) progenitors; monoblastic U-937; monocytic THP-1; progranulocytic HL-60; macrophage RAW 264.7) but not in nonmyeloid cells tested. Interleukin-4 impaired basal expression of pgrn in U-937. Differentiation agents DMSO, and, in U-937 only, phorbol ester [phorbol 12-myristate,13-acetate (PMA)] elevated pgrn mRNA expression late in differentiation, suggestive of roles for pgrn in more mature terminally differentiated granulocyte/monocytes rather than during growth or differentiation. The response of pgrn mRNA to ATRA differs in U-937 and HL-60 lineages. In U-937, ATRA and chemical differentiation agents greatly increased pgrn mRNA stability, whereas, in HL-60, ATRA accelerated pgrn mRNA turnover. The initial upregulation of pgrn mRNA after stimulation with ATRA was independent of de novo protein synthesis in U-937 but not HL-60. Chemical blockade of nuclear factor-kappaB (NF-kappaB) activation impaired ATRA-stimulated pgrn expression in HL-60 but not U-937, whereas in U-937 it blocked PMA-induced pgrn mRNA expression, suggestive of cell-specific roles for NF-kappaB in determining pgrn mRNA levels. We propose that: 1) ATRA regulates pgrn mRNA levels in myelomonocytic cells; 2) ATRA acts in a cell-specific manner involving the differential control of mRNA stability and differential requirement for NF-kappaB signaling; and 3) elevated pgrn mRNA expression is characteristic of more mature cells and does not stimulate differentiation.