1,25-DIHYDROXYVITAMIN D-3 PROMOTES PROSTAGLANDIN E(1)-INDUCED DIFFERENTIATION OF HL-60 CELLS

Human promyelocytic HL-60 cells can be induced by biochemical agents t o differentiate in vitro towards divergent types of myelomonocytic cel ls. It has been reported that prostaglandin E(1) (PGE(1)) can induce g ranulocytic differentiation and that 1,25-dihydroxyvitamin D-3 (1,25(O H)(2)D-3) can induce monocytic differentiation. We have now examined t he effects of these compounds, both alone and in combination, on HL-60 cell differentiation. PGE(1) (1 mu g/ml) or 1,25(OH)(2)D-3 (10 nM) ea ch inhibited cell proliferation over 48-96 hours of treatment, but com bined treatment with both agents was necessary to produce a strong inh ibition. The percentage of HL-60 cells that can reduce nitroblue tetra zolium (NBT) (a characteristic index of early monocytic or granulocyti c differentiation) increased 13-fold within 72 hours of PGE(1) treatme nt, and 1,25(OH)(2)D-3 produced a fivefold stimulation. However, combi ned treatment (PGE(1) plus 1,25(OH)(2)D-3) produced a dramatic 35-fold increase. HL-60 cells did not produce significant levels of nitric ox ide (NO) before 48 hours in culture, and treatment with PGE(1) or 1,25 (OH)(2)D-3 did not significantly increase cellular NO elaboration over control levels. However, combined treatment produced a striking 12-fo ld increase over control levels. Similarly, combined treatment was nec essary to obtain the maximal time-dependent stimulation of cellular la ctate dehydrogenase (LDH) activity (a marker of granulocytic different iation) as well as acid phosphatase (ACP) activity. During this same p eriod of time, PGE(1), but not 1,25(OH)(2)D-3, markedly stimulated cel lular elaboration of interleukin (IL)-1 alpha, IL-6, and tumor necrosi s factor (TNF)-alpha, and 1,25(OH)(2)D-3 cotreatment strongly augmente d these effects. Thus, combined treatment with 1,25(OH)(2)D-3 plus PGE (1) generally augmented the apparent conversion of these cells, produc ing synergistic (multiplicative) or additive effects. Furthermore, PGE (1) induced within 48 hours the more general phenotypic changes classi cally associated with the differentiation of these cells: increased ex pression of chloroacetate esterase (ChAE) (a granulocytic marker), dec reases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in mo rphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)(2)D-3 had little effect itself on most of these pa rameters, but augmented the morphological changes induced by PGE, trea tment. Within 48 hours, the ability of these cells to reduce the tetra zolium salt WST-1, a general measure of cellular metabolic activity, w as increased by PGE(1), but not by 1,25(OH)(2)D-3; however, the combin ation of 1,25(OH)(2)D-3 and PGE(1) again produced the strongest stimul ation. Similarly, only PGE(1) significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. I n summary, our findings suggest that PGE(1), not 1,25(OH)(2)D-3, is su fficient to promote rapid in vitro differentiation of HL-60 cells alon g the granulocytic pathway; however, the PGE(1)-induced conversion of these cells is markedly augmented by cotreatment with 1,25(OH)(2)D-3. In addition, these converted HL-60 cells preferentially utilize the gl ycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mat ure granulocytes.