Human promyelocytic HL-60 cells can be induced by biochemical agents to differentiate in vitro towards divergent types of myelomonocytic cells. It has been reported that prostaglandin E(1) (PGE(1)) can induce granulocytic differentiation and that 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) can induce monocytic differentiation. We have now examined the 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) each inhibited cell proliferation over 48-96 hours of treatment, but combined treatment with both agents was necessary to produce a strong inhibition. The percentage of HL-60 cells that can reduce nitroblue tetrazolium (NBT) (a characteristic index of early monocytic or granulocytic differentiation) increased 13-fold within 72 hours of PGE(1) treatment, and 1,25(OH)(2)D-3 produced a fivefold stimulation. However, combined 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 oxide (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-fold increase over control levels. Similarly, combined treatment was necessary to obtain the maximal time-dependent stimulation of cellular lactate dehydrogenase (LDH) activity (a marker of granulocytic differentiation) as well as acid phosphatase (ACP) activity. During this same period of time, PGE(1), but not 1,25(OH)(2)D-3, markedly stimulated cellular elaboration of interleukin (IL)-1 alpha, IL-6, and tumor necrosis factor (TNF)-alpha, and 1,25(OH)(2)D-3 cotreatment strongly augmented these effects. Thus, combined treatment with 1,25(OH)(2)D-3 plus PGE(1) generally augmented the apparent conversion of these cells, producing synergistic (multiplicative) or additive effects. Furthermore, PGE(1) induced within 48 hours the more general phenotypic changes classically associated with the differentiation of these cells: increased expression of chloroacetate esterase (ChAE) (a granulocytic marker), decreases in the nuclear/cytoplasmic ratio (characteristic of development beyond the promyelocyte/myelocyte stage), and major alterations in morphology from floating spherical cells to loosely adherent, elliptical polygons. 1,25(OH)(2)D-3 had little effect itself on most of these parameters, but augmented the morphological changes induced by PGE, treatment. Within 48 hours, the ability of these cells to reduce the tetrazolium salt WST-1, a general measure of cellular metabolic activity, was increased by PGE(1), but not by 1,25(OH)(2)D-3; however, the combination of 1,25(OH)(2)D-3 and PGE(1) again produced the strongest stimulation. Similarly, only PGE(1) significantly reduced intracellular ATP levels, but combined treatments produced a more pronounced decrease. In summary, our findings suggest that PGE(1), not 1,25(OH)(2)D-3, is sufficient to promote rapid in vitro differentiation of HL-60 cells along 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 glycolytic pathway, rather than the citric acid cycle, for production of ATP, a metabolic characteristic that resembles that described for mature granulocytes.
