Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).     

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1alpha,25-dihydroxyvitamin D3.

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR.     

1alpha,25-dihydroxyvitamin D(3)-26,23-lactone analogs antagonize differentiation of human leukemia cells (HL-60 cells) but not of human acute promyelocytic leukemia cells (NB4 cells).

We examined the effects of two novel 1alpha,25-dihydroxyvitamin D(3)-26,23-lactone (1alpha,25-(OH)(2)D(3)-26,23-lactone) analogs on 1alpha,25(OH)(2)D(3)-induced differentiation of human leukemia HL-60 cells thought to be mediated by the genomic action of 1alpha, 25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non-genomic actions of 1alpha,25-(OH)(2)D(3). We found that the 1alpha,25-(OH)(2)D(3)-26,23-lactone analogs, (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9648), inhibited differentiation of HL-60 cells induced by 1alpha,25-(OH)(2)D(3). However, 1beta-hydroxyl diastereomers of these analogs, i.e. (23S)-25-dehydro-1beta-hydroxyvitamin D(3)-26, 23-lactone (1beta-TEI-9647) and (23R)-25-dehydro-1beta-hydroxyvitamin D(3)-26,23-lactone (1beta-TEI-9648), did not inhibit differentiation of HL-60 cells caused by 1alpha,25-(OH)(2)D(3). A separate study showed that the nuclear vitamin D receptor (VDR) binding affinities of the 1-hydroxyl diastereomers were about 200 and 90 times weaker than that of 1alpha-hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1alpha,25-(OH)(2)D(3). In contrast, 1beta,25-dihydroxyvitamin D(3) (1beta,25-(OH)(2)D(3)) and 1beta,24R-dihydroxyvitamin D(3) (1beta,24R-(OH)(2)D(3)) inhibited NB4 cell differentiation but not HL-60 cell differentiation. Collectively, the results suggested that 1-hydroxyl lactone analogs, i.e. TEI-9647 and TEI-9648, are antagonists of 1alpha,25-(OH)(2)D(3), specifically for the nuclear VDR-mediated genomic actions, but not for non-genomic actions.     

Antagonistic action of novel 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs on 25-hydroxyvitamin-D(3)-24-hydroxylase gene expression induced by 1alpha,25-dihydroxy-vitamin D(3) in human promyelocytic leukemia (HL-60) cells

We have demonstrated that 1alpha,25-dihydroxyvitamin D(3)-26, 23-lactone analogs, (23S)- and (23R)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647, TEI-9648, respectively), inhibit HL-60 cell differentiation induced by 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], but not differentiation caused by all-trans retinoic acid (D. Miura et al., 1999, J. Biol. Chem. 274, 16392). To assess whether the antagonistic actions of TEI-9647 and TEI-9648 in HL-60 cells are related to 1alpha,25(OH)(2)D(3) breakdown, we investigated their effects on catabolism of 1alpha,25(OH)(2)D(3). In HL-60 cells, the C-24 but not the C-23 side-chain oxidation pathway of 1alpha,25(OH)(2)D(3) has been reported. Here we demonstrate that 1alpha,25(OH)(2)D(3) was metabolized both to 24,25,26,27-tetranor-1alpha,23-(OH)(2)D(3) and 1alpha,25(OH)(2)D(3)-26,23-lactone; thus HL-60 cells constitutively possess both the 24- and the 23-hydroxylases. Metabolism of 1alpha, 25(OH)(2)D(3) was strongly suppressed by 10(-7) M TEI-9647 or 10(-6) M TEI-9648. 1alpha,25(OH)(2)D(3) alone slightly induced 24-hydroxylase gene expression by 8 h with full enhancement by 24-48 h; this induction was inhibited by 10(-6) M TEI-9647 and 10(-6) M TEI-9648 (86.2 and 31.9%, respectively) 24 h after treatment. However, analogs of TEI-9647 and TEI-9648 without the 25-dehydro functionality induced 24-hydroxylase gene expression. These results indicate that TEI-9647 and TEI-9648 clearly mediate their stereoselective antagonistic actions independent of their actions to block the catabolism of 1alpha,25(OH)(2)D(3). Therefore, TEI-9647 and TEI-9648 appear to be the first antagonists specific for the nuclear 1alpha,25(OH)(2)D(3) receptor-mediated genomic actions of 1alpha,25(OH)(2)D(3) in HL-60 cells.     

Regulation of 25-hydroxyvitamin D3 metabolism in a human promyelocytic leukemia cell line (HL-60): 1,25-dihydroxyvitamin D3 stimulates the synthesis of 24,25-dihydroxyvitamin D3

The human promyelocytic leukemia cell line HL-60 undergoes macrophage-like differentiation after exposure to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3. In the current study, we demonstrate that 1,25(OH)2D3 also regulates 25-hydroxyvitamin D3 [25(OH)D3] metabolism in HL-60 cells. The presence of 1,25(OH)2D3 in the culture medium of HL-60 cells stimulated the conversion of 7-10% of the substrate [25(OH)D3] to a more polar metabolite, which was identified as 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] from the elution positions on sequential HPLC systems and the sensitivity to periodate treatment. The HL-60 subclone HL-60 blast, which is unresponsive to 1,25(OH)2D3 in terms of differentiation, also responded to 1,25(OH)2D3 treatment with the production of 24,25(OH)2D3. Maximal stimulation of 24,25(OH)2D3-synthesis (approximately 7 pmol/5 X 10(6) cells) in HL-60 cells was noted with a 12-h exposure to 10(-9) M 1,25(OH)2D3. The ability of vitamin D3 metabolites other than 1,25(OH)2D3 to induce the synthesis of 24,25(OH)2D3 in HL-60 cells was, with the exception of 1 alpha-hydroxyvitamin D3, in correlation with their reported affinities for the specific 1,25(OH)2D3 receptor which is present in HL-60 cells. Treatment of HL-60 cells with phorbol diesters abolished the 1,25(OH)2D3 responsiveness, while treatment with dimethylsulfoxide and interferon gamma did not markedly alter the 25(OH)D3 metabolism of HL-60 cells. Small amounts (approximately 1% of substrate) of two 25(OH)D3 metabolites, which comigrated with 5(E)- and 5(Z)-19-nor-10-keto-25-hydroxyvitamin D3 on two HPLC solvent systems, were synthesized by HL-60 cells, independently from 1,25(OH)2D3 treatment or stage of cell differentiation. Our results indicate that 1,25(OH)2D3 influences 25(OH)D3 metabolism of HL-60 cells independently from its effects on cell differentiation.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Inhibition of COX activity by NSAIDs or ascorbate increases cAMP levels and enhances differentiation in 1alpha,25-dihydroxyvitamin D3-induced HL-60 cells

Arachidonic acid metabolism is modulated during differentiation induced by 1alpha,25(OH)(2)D(3) in HL-60 cells. Antioxidants that affect arachidonic acid metabolism enhance this differentiation program. Ascorbate also enhances differentiation in 1alpha,25(OH)(2)D(3)-induced cells depending on the induction of cAMP. The aim of this work was to study if this cAMP rise depends on modulation of arachidonic acid metabolism by ascorbate. Cyclooxygenase inhibitors, indomethacin and aspirin, increased cAMP levels and also enhanced 1alpha,25(OH)(2)D(3)-induced differentiation in HL-60 cells. Ascorbate did not affect the release of arachidonic acid-derived metabolites but decreased the levels of TXB(2) and PGE(2), suggesting the inhibition of cyclooxygenase. On the other hand, free arachidonic acid increased both cAMP levels and differentiation in the absence or presence of 1alpha,25(OH)(2)D(3). Neither cyclooxygenase inhibitors nor ascorbate modified AA effect. Then, inhibition of cyclooxygenase activity by ascorbate could accumulate free arachidonic acid or other metabolites that increase cAMP levels and enhance differentiation in 1alpha,25(OH)(2)D(3)-induced HL-60 cells.     

Biological activity of fluorinated vitamin D analogs at C-26 and C-27 on human promyelocytic leukemia cells, HL-60

Vitamin D compounds added to the culture medium induce HL-60 cells to differentiate into macrophage/monocytes via a receptor mechanism. This system provides a biologically relevant assay for the study of biopotency of vitamin D analogs. Using this system, the biological activity of various fluorinated derivatives of vitamin D3 was compared with that of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). As assessed by cell morphology, nitroblue tetrazolium reduction and nonspecific esterase activity, 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3 (26,27-F6-1,25-(OH)2D3) and 26,26,26,27,27,27-hexafluoro-1,24-dihydroxyvitamin D3 (26,27-F6-1,24-(OH)2D3) were about 10 times as potent as 1,25-(OH)2D3 in suppressing HL-60 cell proliferation and inducing cell differentiation. The biological activity of 26,26,26,27,27,27-hexafluoro-1-hydroxyvitamin D3 (26,27-F6-1-OH-D3) was equal to that of 1,25-(OH)2D3 in this system. 1,25-(OH)2D3 and its fluorinated analogs exerted their effects on HL-60 cells in a dose-dependent manner. HL-60 cells have a specific receptor for 1,25-(OH)2D3 with an apparent Kd of 0.25 nM, identical with that of chick intestinal receptor. While the binding affinities of 26,27-F6-1,25-(OH)2D3 and 26,27-F6-1,24-(OH)2D3 for chick intestinal receptor were lower than that of 1,25-(OH)2D3 by factors of 3 and 1.5, respectively, they were as competent as 1,25-(OH)2D3 in binding to HL-60 cell receptor. The ability of 26,27-F6-1-OH-D3 to compete for receptor protein from HL-60 cells and chick intestine was about 1/70 that of 1,25-(OH)2D3. These results indicate that trifluorination of carbons 26 and 27 of vitamin D3 can markedly enhance the effect on HL-60 cells.     

Appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells during monocytic differentiation: enhancement of thromboxane synthesis by 1 alpha,25-dihydroxyvitamin D-3

The appearance of the arachidonic acid metabolic pathway in human promyelocytic leukemia (HL-60) cells was investigated during 1 alpha,25-dihydroxyvitamin D-3-induced monocytic differentiation. 1 alpha,25-Dihydroxyvitamin D-3-treated HL-60 cells acquired the ability to convert [1-14C]arachidonic acid to thromboxane B2 and prostaglandin E2 during monocytic differentiation. The major cyclooxygenase product synthesized by the HL-60 cells after 3-4 days exposure to 1 alpha,25- dihydroxyvitamin D-3 (48 nM) was thromboxane B2 and its production was about 19-25-times higher than that of untreated HL-60 cells. The percent conversion of thromboxane B2 from [1-14C]arachidonic acid in the 1 alpha,25-dihydroxyvitamin D-3 (48 nM, 3 day exposure)-treated HL-60 cells was about 4.4% as compared to that (about 0.3%) of the untreated cells, whereas the percent conversion of thromboxane B2 from [1-14C]prostaglandin H2 in the 1 alpha,25-dihydroxyvitamin D-3-treated cell homogenate was about 22.4% as compared to that (about 13.6%) of the untreated cell homogenate. The stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on thromboxane B2 production from [1-14C]arachidonic acid and from [1-14C]prostaglandin H2 in HL-60 cells was inhibited by the addition of cycloheximide (1 microgram/ml). However, 1 alpha,25-dihydroxyvitamin D-3 (48 nM) did not significantly stimulate the arachidonic acid release either in HL-60 cells or in 1 alpha,25-dihydroxyvitamin D-3-induced cells. These results suggest that the stimulatory effect of 1 alpha,25-dihydroxyvitamin D-3 on the thromboxane production in HL-60 cells was not due to the activation of phospholipase A2 but due to the induction of fatty acid cyclooxygenase and thromboxane synthetase activities. Thromboxane A2 actively produced during the monocytic differentiation of HL-60 cells could influence the cell adhesiveness of the monocyte-macrophage-differentiated cells.     

Differentiation-inducing activity of retinoic acid isomers and their oxidized analogs on human promyelocytic leukemia HL-60 cells.

Retinoidal activity of retinoic acid isomers [all-trans-retinoic acid (ATRA), 9-cis-retinoic acid (9CRA) and 13-cis-retinoic acid (13CRA)] and their oxidized derivatives [19-hydroxy and 19-oxo derivatives of ATRA (19-hydroxy-ATRA and 19-oxo-ATRA), 19-oxo derivative of 9CRA (19-oxo-9CRA), and 19-hydroxy derivative of 13CRA (19-hydroxy-13CRA)] was evaluated by means of a human promyelocytic leukemia HL-60 cell differentiation induction assay. All the compounds examined showed this activity with ED50 values of 2-30 nM, which are in accordance with their binding activity to nuclear retinoic acid receptors (RARs).     

Isolation of apoptosis- and differentiation-inducing substances toward human promyelocytic leukemia HL-60 cells from leaves of Juniperus taxifolia

A chloroform extract of the leaves of Juniperas taxifolia exhibited a marked antiproliferative effect on human promyelocytic leukemia HL-60 cells at a concentration of 2.5 microg/ml. Deoxypodophyllotoxin (4) was identified in the extract as an outstanding antiproliferative compound, and five diterpenes (1-3, 5, and 6) were isolated as known compounds with weak or no cytotoxicity. These compounds were examined for their respective apoptosis- and differentiation-inducing activities toward HL-60 cells by DNA fragmentation and NBT-reducing assays, respectively. Among them, 7alpha-hydroxysandaracopimaric acid (6) was found to have a potent differentiation-inducing activity in a dose-dependent manner at 0.125-2 microg/ml (0.39-6.29 microM), together with apoptosis-inducing activity at concentrations of more than 2.5 microg/ml (7.86 microM). Deoxypodophyllotoxin (4) that exerted cytotoxic and apoptosis-inducing activities at 2 ng/ml (5 nM) did not induce differentiation at the same concentration, and the other diterpenes (1-3 and 5) showed no effect on cell differentiation, even at 5 microg/ml. It was thus demonstrated for the first time that 7alpha-hydroxysandaracopimaric acid was an effective differentiation-inducing compound toward HL-60 cells.     

Immunological identification of 1,25-dihydroxyvitamin D3 receptors in human promyelocytic leukemic cells (HL-60) during homologous regulation

Immunological techniques were utilized to detect 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) receptor levels and to characterize physical/chemical changes in receptors in human promyelocytic leukemic cells (HL-60) during continuous exposure to hormone. The monoclonal antibody (IVG8C11) raised against the porcine intestinal 1,25-(OH)2D3 receptor immunoprecipitated quantitatively 1,25-(OH)2D3 receptors in nuclear extracts from HL-60 cells. The highly enriched immunoprecipitated receptors were electrophoresed on sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes, which were probed with 125I-labeled IVG8C11. The basal receptor from the cells treated with 1,25-(OH)2D3 for 2 h was detected as a single form at 53 kDa. Moreover, receptors were shown to be up-regulated at 12 h and down-regulated at 48 and 72 h in the continuous presence of hormone as evidenced by the ratio of density of the bands, 1.0 (2 h):4.2 (12 h):1.2 (48 h):0.9 (72 h), as measured by laser scanning densitometry. The up- and down-regulated receptors were also detected as single forms and had the same molecular mass as the basal receptor. Therefore, the data presented here strongly support the hypothesis of homologous regulation of 1,25-(OH)2D3 receptors in intact human target cells.     

Etoposide stimulates 1,25-dihydroxyvitamin D3 differentiation activity,hormone binding and hormone receptor expression in HL-60 human promyelocytic cells

The simultaneous administration of the DNA topoisomerase II inhibitor etoposide (0.15 mM) and 1,25-dihydroxyvitamin D₃ (VD₃) (10 nM) synergistically induced the differentiation of HL-60 human promyelocytic leukemia cells. Similar results were obtained using U-937 human promonocytic cells, or the topoisomerase II inhibitors doxorubicin (15 nM) and mitoxantrone (2.5 nM). When sequential treatments were used, pre-incubation with VD₃ had little effect on the subsequent action of etoposide, while pre-incubation with etoposide greatly potentiated the subsequent action of VD₃. In addition, etoposide treatment stimulated VD₃ binding activity and increased VD₃ receptor mRNA and protein levels. The increase in hormone receptor expression may explain, at least in part, the capacity of topoisomerase inhibitors to potentiate the differentiation inducing activity of VD₃.     

Barbiturates enhance retinoic acid or 1,25-dihydroxyvitamin D3-induced differentiation of leukemia HL-60 cells.

In the presence of 1 nM retinoic acid (RA), pentobarbital markedly enhanced differentiation of HL-60 cells to granulocytic cells. In the absence of RA, pentobarbital by itself did not induce cell differentiation. Similarly, pentobarbital enhanced the action of 1,25-dihydroxyvitamin D3 to induce differentiation of HL-60 cells into monocyte/macrophage lineage. The potency of various barbiturates to enhance cell differentiation was closely correlated with their activity to inhibit protein kinase C of HL-60 cells. In contrast to staurosporine, however, barbiturates did not affect the action of differentiation inducers of other types such as dimethyl sulfoxide, dibutyryl cyclic AMP or actinomycin D.     

Detection of a novel cyclooxygenase metabolite produced by human promyelocytic leukemia (HL-60) cells

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.     

Side-chain-modified analogs of calcitriol cause resistance of human HL-60 promyelocytic leukemia cells to drug-induced apoptosis

Calcitriol and some of its analogs have antiproliferative activity, but at the same time, can cause resistance to apoptosis induced by known cytostatic drugs. In this paper, we examined the effects of treatment with calcitriol or its side-chain-modified analogs, analog of Vitamin D2, coded PRI-1906, with monohomologated and unsaturated side-chain and the analog of Vitamin D3, coded PRI-2191, with (24R) hydroxyl group, and those of known cytostatics (genistein, etoposide, doxorubicin, cisplatin, and taxol) on the apoptosis of HL-60 promyelocytic leukemia cells. HL-60 cells were incubated in three different sequences: (1) pre-treatment with calcitriol or its analogs and then treatment with cytostatics; (2) pre-treatment with cytostatics and then treatment with calcitriol; (3) simultaneous treatment with calcitriol and cytostatics. Apoptosis was examined either by DNA fragmentation in agarose gel electrophoresis or by cell-cycle analysis in a FACS Calibur flow cytometer. We showed that pre-treatment with calcitriol or one of its side-chain-modified analogs PRI-1906 or PRI-2191 caused resistance of HL-60 promyelocytic leukemia cells to genistein-, doxorubicin-, cisplatin-, and taxol-induced apoptosis. Simultaneous exposure of HL-60 cells to calcitriol and drug caused a significant decrease in the apoptotic level of HL-60 cells compared with cells treated with drug alone. The pre-treatment of HL-60 cells with drug and then treatment with calcitriol did not increase the level of apoptosis compared with the drug effect alone. These results indicate the potential limitations of calcitriol analogs for treatment of leukemia.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

Induction of monocytic differentiation of HL-60 cells by 1,25-dihydroxyvitamin D analogs

1,25-Dihydroxyvitamin D3, the hormonal form of vitamin D, induces differentiation of HL-60 human promyelocytes into monocyte-like cells in vitro. We assessed the relative activity of 30 analogs of 1,25-dihydroxyvitamin D3 in inducing development of monocytic markers in HL-60 cells. The three differentiation markers assayed were nonspecific acid esterase activity, nitro blue tetrazolium reducing activity, and phagocytic capacity. Of the known metabolites of vitamin D, 1,25-dihydroxyvitamin D3 is the most active; 50% of the cells exhibit the mature phenotype following a 4-day treatment with 10(-8) M 1,25-dihydroxyvitamin D3. Removal of either the C-1 or C-25-hydroxyl group reduces activity by 2 orders of magnitude, while epimerization of the 1 alpha- to 1 beta-hydroxyl group virtually abolishes activity. Elongation of the steroidal side chain of 1,25-dihydroxyvitamin D3 by addition of one carbon at C-24 or C-26 improves the potency by an order of magnitude. Truncation of the steroidal side chain leads to a 10-fold reduction in activity for each carbon removed. Elimination of the C-26 and C-27 methyl groups reduces activity 100-fold. Analogs with short aliphatic side chains as 1 alpha-hydroxyhomo- and bishomopregnacholecalciferol have surprisingly high activity, being only 20-fold less potent than the natural hormone. The activity of most analogs in the HL-60 system parallels their known relative affinities for the well characterized 1,25-dihydroxyvitamin D3 receptor in chick intestine, providing further evidence that this function of 1,25-dihydroxyvitamin D3 is receptor mediated.