Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60 . The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and polymorphonuclear leukocytes (PMN) over 9 d of culture in 1 .3% dimethylsulfoxide (DM SO). As the HL-60 cells mature, the rate of 02 production increases 18-fold, with a progressive shortening of the lag time required for activation . Hexosemonophosphate shunt activity rises concomitantly . Ingestion of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities . Degranulation, as measured by release of R-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective . However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of 02 generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100% that of normal PMN . DMSO-induced differentiation of HL-60 cells is a promising model for myeloid development .KEY WORDS phagocytosis " superoxide granulocyte -lysosome -lactoferrin A human promyelocytic leukemia cell line, HL-60, was recently established from the peripheral blood of a patient with acute promyelocytic leukemia (9) . It has maintained continuous growth in J . CELL. BIOLOGY © The Rockefeller University Press -0021-9525/79/08/0315/08 $1 .00 Volume 82 August 1979 315-322 suspension culture in the absence of added conditioned medium or colony-stimulating factor for over 18 mo and is tumorigenic in athymic nude mice (10). The majority of HL-60 cells are promyelocytic in morphology and histochemistry, but 4-15% of them show morphologic characteristics of more mature myeloid cells: myelocytes, meta-315 on
Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan- stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.
We have used a cloned cDNA for the major human selenoprotein, glutathione peroxidase (GPx), to assess the mode of regulation of human GPx gene (GPX-1) expression by selenium. When the HL-60 human myeloid cell line is grown in a selenium-deficient medium, GPx enzymatic activity decreases 30-fold compared with selenium-replete cells. Upon return to a medium containing selenium in the form of selenite, GPx activity in the cells starts to increase within 48 hours and reaches maximal (selenium-replete) levels at 7 days. Steady-state immunoreactive protein levels correlate with enzymatic activity. Cycloheximide inhibits the rise in GPx activity that accompanies selenium replenishment, indicating that protein synthesis is required for the increase. However, GPx mRNA levels and the rate of transcription of the human GPx gene change very little and thus appear to be independent of the selenium supply. Thus the human GPx gene appears to be regulated post-transcriptionally, probably cotranslationally, in response to selenium availability.
Stimulation of granulocyte (PMN) superoxide (O2-) production by concanavalin-A (Con-A) can be monitored continuously in the spectrophotometer. Both the rate of activation and final activity of the O2--generating system is dependent on the concentration of Con-A. Alpha methylmannoside (alpha MM) can prevent Con-A, but not phorbol myristate acetate (PMA) or zymosan, induced O2- production. Alpha MM inhibits both the rate of activation and the final rate of O2- production. When alpha MM is added after the attainment of a maximal rate of O2- production with Con-A, O2- production continues for another minute before it ceases. When PMA is added to such treated cells, it restores O2- production. Although the inhibition of O2- production by alpha MM on previously activated cells requires time, most of the bound concanavalin-A is removed immediately after the addition of alpha MM. Treatment of cells with L-1-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) prevents activation of PMN by Con-A to a greater extent than it does for either PMA or zymosan. TPCK has no effect on the binding of Con-A. TPCK, when added after Con-A, will inactivate O2- production by the cells. The addition of PMA after TPCK treatment restores O2--generating activity. Membrane-enriched particles from PMN activated with Con-A, alpha MM, and PMA demonstrate that the change in O2- production seen by whole cells is due to an alteration of the activity of the NADPH oxidase. Thus, Con-A stimulation of human PMN O2- production can be prevented and reversed by the addition of either alpha MM or TPCK and that PMA can reactivate Con-A and either alpha MM- or TPCK-treated cells. The activation, inactivation, and reactivation occur as a result of changes in the plasma membrane NADPH-dependent O2-- generating enzyme.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.