A system for monocytic differentiation of leukemic cells HL 60 by a short exposure to 1,25-dihydroxycholecalciferol

The human promyelocytic cell line HL 60 can be induced to differentiate toward more mature myeloid or monocytic forms by a variety of agents. This process is thought to require several days of exposure to the inducer, thus making it difficult to identify the early cellular changes which are fundamental to the differentiation program, and to relate the induction to phases of the cell cycle. In order to study the kinetics of leukemic cell differentiation we have developed a system for the induction of rapid monocytic maturation in a subpopulation of HL 60 cells. The cells are exposed to 10(-7) M 1,25-dihydroxycholecalciferol for 4 hr in serum-free medium. Subsequent incubation in a complete medium results in cellular differentiation recognizable by several criteria (phagocytosis, nonspecific esterase reaction, adherence to substratum, cell morphology) beginning at 10 hr from the exposure to the inducer. Approximately 20 hr later 30-40% of the cells in culture show the differentiated phenotype and are capable of phagocytosis. The proportion of differentiated cells in culture decreases thereafter. This system has been utilized to study the expression of c-myc oncogene in relation to the kinetics of maturation, and it was found that the inhibition of the expression of this gene precedes the onset of phenotypic differentiation by approximately 8 hr, is transient, and is accompanied by a brief retardation of cell proliferation, which resumes the normal rate within 24 hr of the exposure to the inducer.     

Cathepsin B synthesis by the HL60 promyelocytic cell line: effects of stimulating agents and anti-inflammatory compounds

Cathepsin B synthesis by the human HL60 promyelocyte cell line was investigated by immunohistochemistry and by the assay of the enzyme in cell lysates using a fluorimetric substrate. HL60 cells were shown to produce cathepsin B in response to treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). Intracellular levels of cathepsin B and immunohistochemical staining of the enzyme were related to time in culture with increasing concentrations of TPA from 1 nmol/1 to 8.0 nmol/1. Synthesis of cathepsin B was associated with TPA-induced phagocytic activity of cells in culture, expression of alpha-naphthyl acetate esterase and reduced cell division. Cathepsin B production was, therefore, related to differentiation of the HL60 promyelocytes into mature macrophage-like cells. Cathepsin B activity in HL60 cell lysates was significantly increased by incubation of the cells with 10 micrograms/ml endotoxin (lipopolysaccharide) from Escherichia coli, but not carrageenan. The production of cathepsin B by TPA-induced HL60 cells was significantly reduced by 0.25 mumol/1 dexamethasone and the non-steroidal anti-inflammatory compound 4-(6-methoxy-2-naphthyl)-butan-2-one but not by indomethacin. The HL60 promyelocytic cell line is a useful model for the study of factors affecting proteinase synthesis by human mononuclear phagocytes.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G1/G0 and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G1 DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G1 to G0.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Transient down-regulation of c-myc protooncogene expression by the alkaloid nicotine in human promyelocytic HL-60 cells.

Human promyelocytic leukemia cells (HL-60) acquire monocytic biochemical characteristics when exposed to the alkaloid nicotine. We report herein that the nicotine-induced phenotype change is accompanied by the transient reduced expression of mRNA for the protooncogene c-myc. The suppressive effect of nicotine on c-myc mRNA levels is not reversed by the addition of ADP-ribosyl transferase inhibitor, 3-aminobenzamide.     

Accessory function and interleukin 1 production by human leukemic cell lines

Highly purified T lymphocytes do not proliferate in response to mitogens, unless adherent HLA-DR-positive monocytes are added to the culture. This accessory function (AF) of monocytes requires the release of interleukin 1 (IL 1). Cells from three human leukemic cell lines, K562, HL60, and U937, could very efficiently replace monocytes in a 72-hr mitogen-induced T cell proliferation assay. The AF was clearly related to precise maturational stages of these cells; the hematopoietic precursor K562 cells spontaneously exerted high AF, but lost this property when treated with differentiation inducers. On the contrary, the promyelocytic HL60 cells and the "histiocytic" U937 cells exhibited no spontaneous AF, but acquired this property when induced to differentiate along the granulocytic and/or monocytic pathway. Three leukemic cells could not only stimulate T cells to proliferate and produce IL 2 in the presence of mitogens, but also under appropriate culture conditions these cells could produce IL 1, which could not be distinguished from normal human monocyte derived IL 1 by gel filtration and isoelectric focusing. Moreover, analysis of phenotypic markers revealed that AF and production of IL 1 could be demonstrated in different cell types and therefore are not restricted to the monocytic lineage. No HLA-DR antigen could be detected on K562 and HL60 cells. Thus, the expression of the DR antigens is not required for AF and IL 1 production in response to mitogens. These three human leukemic cell lines will provide convenient sources of human IL 1.     

Inositol derivatives down-regulate c-myc inducing growth arrest without differentiation

Cyclitol derivatives that are structurally related to myo-inositol induce growth arrest without differentiation in human promyelocytic leukemia (HL60) cells. An early effect is the rapid down-regulation of c-myc mRNA levels. This was observed also in several mouse and human lines carrying either normal or rearranged myc. The mRNA levels of a constitutive mouse myc construct transfected into HL60 were not affected at the same time. Uridine and thymidine incorporation were significantly decreased by the cyclitol treatment. These effects partly resemble those of certain differentiation inducers and those of hexachlorcyclohexane, another myo-inositol analogue. This new group of agents offers a novel approach to studying control mechanisms involving c-myc.     

Independent variations of myc amplification, inducibility, maturation, and proliferation states in HL60

We have investigated the possible relationship between c-myc gene activity and other variable traits in HL60. In a panel of variant lines, a good correlation was observed between myc gene copy number and the level of myc mRNA. There was no correlation between myc amplification or expression and the resistance of the lines to induction of terminal neutrophilic or monocytic differentiation. Therefore, myc mRNA level does not appear to determine the ability of the variant HL60 lines to respond to inducers of differentiation. Flow cytometric analyses of the expression of a differentiation antigen (AGF 4.36) revealed stable negative and positive subpopulations in growing HL60. myc amplification, expression, and inducibility were identical in these subpopulations, suggesting that variation of these traits in HL60 sublines and variants is not due to maturation state differences. myc gene copy number was also identical in transferrin receptor positive (proliferating) and negative (resting) populations. These data contradict the notion that myc amplification has been important in determining the in vitro biological properties of HL60.     

Temporal relationships between induced changes in c-myc mRNA abundance, proliferation, and differentiation in HL60 cells

Changes in the relative abundances of c-myc mRNA have been related to changes in other parameters of differentiation (histochemical, clonogenic) during the course of the differentiation of HL60 cells to monocytes/macrophages or to granulocytes. Induction of differentiation to monocytes/macrophages was marked by a rapid rate of appearance of committed cells (80 to 90% in 24 hours) and a concomitant rapid loss of c-myc mRNA. Induction of granulocytic differentiation resulted in a much slower rate of appearance of committed cells (50% in 48 hours), and a much faster rate of loss of c-myc mRNA (tenfold in 1 hour). These data are consistent with there being a direct link between down-regulation of the expression of c-myc and the onset of proliferation arrest and monocytic differentiation, but show there is no such association of c-myc mRNA abundance and proliferation or differentiation during the maturation of HL60 granulocytes.     

Macrophage glycolipid receptors for human migration inhibitory factor (MIF): differentiated HL-60 cells exhibit MIF responsiveness and express surface glycolipids which both bind MIF and convert nonresponsive cells to responsiveness

The human promyelocytic leukemia line HL-60 when treated with a phorbol diester (TPA) differentiates into cells (HL60-TPA) that respond to human migration inhibitory factor (MIF). Unresponsive HL-60 cells became responsive to MIF when preincubated with a glycolipid-enriched preparation extracted from HL60-TPA cells, human monocytes, human macrophage-like (U937) cell line, or with the purified glycolipid receptor for MIF from guinea pig peritoneal macrophages. Human blood monocytes exhibited an increased response to MIF when preincubated with glycolipids from HL60-TPA and U937 cells but not from HL-60 cells. Finally, glycolipids from HL60-TPA cells but not from HL-60 cells were able to reversibly bind MIF when covalently coupled to agarose. These studies suggest that TPA induces the differentiation of HL-60 cells into MIF-responsive cells through the expression of a glycolipid receptor for MIF.     

Growth pattern of the human promyelocytic leukaemia cell line HL60

Abstract. In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 × 10⁶ cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 ± 4.1 hr (SD), and that of the cell cycle phases was: G₁, 3.8 ± 2.2 hr; S, 15.1 ± 3 hr; and G₂, 5.4 ± 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the in vitro study of substances which may affect human leukaemic myelopoietic proliferation.     

Coupled down-regulation of the RB retinoblastoma and c-myc genes antecedes cell differentiation: possible role of RB as a "status quo" gene.

The ability of the well known morphogen, retinoic acid (RA), as well as 1,25-dihydroxy-vitamin D3 (VD), whose receptor complex binds a DNA consensus sequence related to that of the retinoic acid receptor, to regulate expression of the retinoblastoma (RB) tumor suppressor gene in a context of induced cell differentiation was characterized. HL-60 human promyelocytic leukemia cells were induced to undergo myeloid or monocytic terminal cell differentiation by these agents. To investigate the potential coupling between down-regulation of RB and c-myc oncogene expression with cell differentiation, dose response relationships for the induced down-regulation of RB and c-myc expression were compared with each other and with induced cell differentiation. The total amount of RB protein per cell increased as cells advanced through the cell cycle, but the amount of RB protein relative to the total cell mass remained approximately constant. Treated with RA or VD, an early progressive decrease in cellular content of the RB protein occurred in all cell cycle phases well before any cell cycle modulation or phenotypic differentiation. For a differentiation-defective variant HL-60 cell line, failure to differentiate was preceded by a failure to down-regulate cellular levels of the RB protein. In dose response experiments, progressively increasing RA or VD concentrations caused progressively greater reductions in RB as well as c-myc expression with an increasing fraction of cells terminally differentiating. For both RA and VD, the dose response relationships for reductions in RB and c-myc expression were similar suggesting that their down-regulation may be coupled. These observations are consistent with a model whereby RB expression acts as a cellular brake to sustain a developmentally ordained state of differentiation (i.e., preserve the "status quo"); and the down-regulation of heterogeneously distributed RB protein per cell below a threshold is part of the metabolic cascade culminating in terminal cell differentiation. Thus, RB may have a role in this developmental context.