Antagonistic Action of Novel 1α,25-Dihydroxyvitamin D3-26,23-lactone Analogs on Differentiation of Human Leukemia Cells (HL-60) Induced by 1α,25-Dihydroxyvitamin D3 *

We examined the effects of two novel 1α,25-dihydroxyvitamin D3-26,23-lactone (1α,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 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3) and its analogues. We found that the 1α,25-lactone analogues (23S)-25-dehydro-1α-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1α-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S,25R)-1α,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 1α,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 1α,25(OH)2D3(10−8 m) mediated induction of p21WAF1,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 1α,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1α,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.

It is widely accepted that the fundamental biological activities of the hormonal form of vitamin D 3 , 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ), 1 are to stimulate intestinal calcium absorption and to increase bone calcium mobilization (1,2). In recent years, however, many new biological functions different from those mentioned above have been reported (3); these include inhibition of cell proliferation and induction of cell differentiation (4), modulation of immunological responses (5), stimulation of insulin secretion (6,7), and neurobiological functions (8,9). 1␣,25(OH) 2 D 3 is believed to mediate biological responses as a consequence of its interaction with both a nuclear receptor (VDR) to regulate gene transcription (10,11) and with a putative cell membrane receptor to generate rapid nongenomic effects (12), including the opening of voltage-gated calcium and chloride channels (13), and activation of mitogenactivated protein kinase (14).
To better understand the interactions of the ligand/VDR interacting with a vitamin D-responsive element (DRE) located on the promoter of regulated genes, it would be helpful to identify analogues of 1␣,25(OH) 2 D 3 that can modulate or antagonize these interactions. However, to date the only known antagonist of 1␣,25(OH) 2 D 3 is the analogue 1␤,25(OH) 2 D 3 , which blocks rapid nongenomic responses but is without effect on the classical nuclear VDR (15).
Normally proliferating human promyelocytic leukemia cells (HL-60) show promyelocytic features and no differentiated functions (for example, nitro blue tetrazolium (NBT)-reducing activity, monocyte-specific esterase activity, and cell surface * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Binding Affinity to VDR and to Vitamin D-binding Protein (DBP)-A competitive receptor binding assay for 1␣,25(OH) 2 D 3 and 1␣,25-lactone analogues was performed using VDR from HL-60 cells as described previously (31,32). Exponentially proliferating HL-60 cells were disrupted by sonication in TEDK buffer (50 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, 5 mM dithiothreitol, 300 mM KCl). After ultracentrifugation at 105,000 ϫ g for 60 min, supernatant was collected and used as VDR fraction. [26,27-methyl-3 H]1␣,25(OH) 2 D 3 (specific activity, 179 Ci/ mmol, 15,000 dpm, 15.7 pg) and various amounts of 1␣,25-lactone analogues to be tested were dissolved in 50 ml of absolute ethanol in 12 ϫ 75-mm polypropylene tubes (Sarstedt, Nü mbrecht, Germany). 1 ml of the HL-60 cell VDR fraction and 1 mg of gelatin were added to each tube in an ice bath. The assay tubes were incubated in a shaking water bath for 1 h at 25°C and then chilled in an ice bath. 1 ml of 40% polyethylene glycol 6000 in distilled water was added to each tube, which was then mixed vigorously and centrifuged at 2,260 ϫ g for 60 min at 4°C. After the supernatant was decanted, the bottom of the tube containing the pellet was cut off into a scintillation vial containing 10 ml of dioxane-based scintillation fluid consisting of 10% naphthalene and 0.5% Omnifluor (DuPont) in 1,4-dioxane. The radioactivity was measured with Beckman liquid scintillation counter (model LS6500) using an external standard. In the assay, using chick intestinal VDR, 0.2 mg of protein/ml of chick VDR was used instead of HL-60 cell VDR fraction. A competitive binding assay of DBP in FBS for 25(OH)D 3 and 1␣,25-lactone analogues was performed as described previously (33).
Cytohistochemical Assay-Cell morphology, NBT-reducing activity and monocytic cell-specific esterase (␣-naphthylbutyrate (␣-NB) used as a substrate) activity was used as cell differentiation markers. HL-60 cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Exponentially proliferating cells were collected, suspended in fresh medium, and seeded in culture vessels. 24-well culture plates (Falcon, Becton Dickinson and Co., Franklin Lakes, NJ) were used. Cell concentration at seeding was adjusted to 2 ϫ 10 4 cells/ml and seeding volume was 1 ml/well. 1␣,25(OH) 2 D 3 and 1␣,25-lactone analogues dissolved in ethanol were added to the culture medium at 0.1% volume and cultured with cells for 4 days at 37°C in a humidified atmosphere of 5% CO 2 /air without medium change. The same amount of vehicle was added to the control culture. NBT reduction assay was performed according to the method of Collins et al. (34). Briefly, cells were collected and washed with PBS. After washing, cells were suspended in serumfree medium, and NBT/TPA solution (dissolved in PBS) was added. Final concentrations of NBT and TPA were 0.1% and 100 ng/ml, respectively. Then, cell suspensions were incubated at 37°C for 25 min. After incubation, cells were collected by centrifugation and resuspended in FBS. Cytospin smears were prepared, and the counterstaining of nucleus was done with Kernechrot solution. At least 500 cells per preparation were observed.
␣-NB esterase activity was measured as follows: cell seeding, treatment, and collection were performed according to the method described above. Cells were resuspended in FBS and then cytospin smears were prepared. Esterase activity of cells was examined after staining with an esterase staining kit. For cell morphology examination, cytospin smears were stained with May-Grü nwald-Giemsa solution.
Cell Surface Marker Expression-Cells were treated with compounds and collected according to the same methods described above. Collected cells were suspended in PBS, and antibodies were added. After incubation on ice for 30 min, cells were collected and washed with PBS. Cells were resuspended in PBS, and the cell surface marker expression was measured with fluorescent-activated cell sorter (FACS) (Becton Dickinson and Co.).
Reverse Transcription PCR of p21 WAF1,CIP1 and ␤-Actin-RNA of HL-60 cells was extracted and purified using CLONsep total RNA isolation kit (CLONTECH Laboratories, Inc., Palo Alto, CA). 2 mg of total RNA were reverse-transcribed with 50 units of murine leukemia virus reverse transcriptase (Takara Biomedicals, Shiga, Japan) in 20 ml containing 1 mM deoxyribonucleoside triphosphates, 5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 20 units of RNase inhibitor (RNasin, Promega Corp., Madison, WI), 2.5 mM oligo(dT) primer. Samples were diluted to 100 ml with buffer containing 2 mM MgCl 2 , 10 mM Tris-HCl, pH 8.3, 50 mM KCl. 100 pmol of each primer and 2.5 units Taq DNA polymerase (Takara Biomedicals, Shiga, Japan) were added, and samples were covered with mineral oil and then subjected to PCR amplification in a programmed thermal cycler. PCR primer was selected with OLIGO TM (National Bioscience), referring to the mRNA sequence registered in GenBank TM . For p21 WAF1,CIP1 amplification, the PCR primers were 5Ј to 3Ј AGGAGGCCCGTGAGCGATGGAAC and ACAAGTGGG-GAGGAGGAAGTAGC. PCR cycles were as follows: 1 min at 94°C for denaturation, 1 min at 59°C for annealing, 1 min at 72°C for polymerization, 26 cycles. For ␤-actin amplification, the PCR primers were 5Ј to 3Ј GATATCGCCGCGCTCGTCGTCGAG and CAGGAAGGAAGGCT-GGAAGAGTGC. PCR cycles were as follows: 1 min at 94°C for denaturation, 1 min at 61°C for annealing, 1 min at 72°C for polymerization, 20 cycles. PCR products were analyzed by 2% agarose gel electrophoresis (about 400-base pair product was obtained in p21 WAF1,CIP1 PCR and about 800-base pair product was obtained in ␤-actin PCR).
Luciferase Reporter Gene Assay-The promoter region of the rat 24-hydroxylase gene (Ϫ291/ϩ9), which also contains two DREs (a gift from Dr. Y. Ohyama, Hiroshima University, Japan) (35) were cloned into a luciferase reporter vector pGV-B2 (Toyo Ink Co. Ltd., Tokyo, Japan). The DNA sequences of these plasmids were confirmed using an ABI 373A DNA sequencer (PE Applied Biosystems, Tokyo, Japan). The luciferase activities of the cell lysates were measured with a luciferase assay kit (Toyo Ink Co. Ltd.) according to the manufacturer's manual. Transactivation measured by luciferase activities was standardized by the galactosidase activities of the same cells determined by a ␤-galactosidase enzyme assay system (Promega). The receptor binding affinities of 1␣,25-lactone and its analogues to VDR prepared from HL-60 cells are shown in Fig. 2 and summarized in Table I. The VDR binding affinities of TEI-9647 and TEI-9648 were 10 and 8%, respectively, as compared with 1␣,25(OH) 2 D 3 . Their binding affinities to VDR of HL-60 cells were 120 -140 times stronger than that of the naturally occurring (23S,25R)-1␣,25(OH) 2 D 3 -26,23-lactone. In contrast, the binding affinities of TEI-9616 and the naturally occurring (23S,25R)-1␣,25(OH) 2 D 3 -26,23-lactone to the VDR of HL-60 cells were about 237 (0.48%) and 1,400 (0.07%) times weaker than that of 1␣,25(OH) 2 D 3 . Similar results were obtained using chick intestinal VDR.
ATRA and 9-cis-RA are also known to promote cell differentiation of HL-60 cells but into granulocytes. TPA can also induce HL-60 cell differentiation into macrophage-like cells. We examined whether TEI-9647 and TEI-9648 could inhibit HL-60 cell differentiation induced by these compounds. In data not presented, we found that neither TEI-9647 nor TEI-9648 could cause inhibition even after treatment at 10 Ϫ6 M.
Collectively, Figs TEI-9647 and TEI-9648 to inhibit the ability of both 1␣,25(OH) 2 D 3 and KH-1060 to mediate the complex process of HL-60 cell differentiation via the VDR. To assess the potential antagonistic action of these two lactone analogues on specific 1␣,25(OH) 2 D 3 /VDR-activated genes, two separate assays were conducted. The results presented in Figs. 7 and 8 suggest that TEI-9647 is a specific antagonist for VDR/DRE activation of gene expression. However, an alternative interpretation for these results is that the TEI-9647 enhances the catabolism of 1␣,25(OH) 2 D 3 in the cell culture system over 24 h (Fig. 7) to 48 h. (Fig. 8). Thus, the apparent inhibition of the 1␣,25(OH) 2 D 3 agonist effect could have been due to a reduction in the effective concentration of the secosteroid. However, in light of the results pre- sented in Fig. 9, this seems not to be a valid concern.  a This is a naturally occurring vitamin D metabolite (18). The structures of the lactone-metabolite and its three analogs are given in Fig. 1. b The summary is presented from data similar to that shown in Figs
The antagonist activity of TEI-9647 and TEI-9648 has been confirmed in two other systems. In COS-7 cells (which are devoid of both the VDR and the 25(OH)D 3 -24-hydroxylase), after co-transfection with the cDNA for both the VDR and the promoter of the 24-hydroxylase, TEI-9647 was found to antagonize 1␣,25(OH) 2 D 3 action (Fig. 8). Secondly, TEI-9647 antag-onized gene expression of p21 WAF1,CIP1 regulated by 1␣,25(OH) 2 D 3 (Fig. 7). Collectively these results described the first example of a stereospecific vitamin D secosteroid, which functions as an antagonist of the nuclear receptor for 1␣,25(OH) 2 D 3 .
The biological significance of the natural (23S,25R)-1␣,25(OH) 2 D 3 -26,23-lactone is not yet fully understood, though it is a major metabolite of 1␣,25(OH) 2 D 3 under physiological conditions (50). The metabolic pathways leading to 1␣,25-lac- tone production from 1␣,25(OH) 2 D 3 are well investigated (19), whereas the further metabolism of 1␣,25-lactone is not entirely known. It has been previously reported that the 25-dehydration reaction of 1␣,25(OH) 2 D 3 can occur in vivo, resulting in the production of both 24-dehydro-1␣-hydroxyvitamin D 3 and 25dehydro-1␣-hydroxyvitamin D 3 (51). In the case of the naturally occurring 1␣,25-lactone, a similar 25-dehydration reaction may possibly take place resulting in the production of TEI-9647. If our hypothesis is true, TEI-9647 should be present under physiological conditions and could possibly act as a negative regulator of hormonal action of 1␣,25(OH) 2 D 3 in vivo. We are now trying to identify further metabolites of the natural 1␣,25-lactone in vivo and determining whether they include TEI-9647.
In conclusion, our data strongly suggest that the novel 1␣,25lactone analogues, TEI-9647 and TEI-9648, may be antagonists of VDR/DRE-mediated genomic actions. They are the first antagonists that possess such properties, and they may be useful compounds for basic research on vitamin D 3 action. Also, it is clear that the 26,23-lactone ring functionality can change the biological properties of 1␣,25(OH) 2 D 3 in an unexpected fashion.