Daishiro Miura

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.

The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

The Pig‐a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N‐acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig‐a is the only gene involved in GPI anchor synthesis that is on the X‐chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI‐anchored cell surface proteins, almost always have mutations in the Pig‐a gene. These properties of the Pig‐a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig‐a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10−6) and rats treated with multiple doses of the potent mutagen, N‐ethyl‐N‐nitrosourea, display Pig‐a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat‐dose toxicology studies. Environ. Mol. Mutagen., 2010. Published 2010 Wiley‐Liss, Inc.

Accumulation and persistence of Pig-A mutant peripheral red blood cells following treatment of rats with single and split doses of N-ethyl-N-nitrosourea.

We previously reported the development of an in vivo gene mutation assay using the phosphatidylinositol glycan complementation group A gene (Pig-A) as an endogenous reporter. The assay quantifies mutation in rat peripheral red blood cells (RBCs) by flow cytometric detection of cells negative for glycosylphosphatidyl inositol (GPI)-anchored protein surface markers. In this study, we examined the accumulation and persistence of Pig-A mutant RBCs in rats treated with N-ethyl-N-nitrosourea (ENU) using two dosing schedules. Male F344 rats were given single i.p. injections of 8.9, 35.6, or 142.4 mg/kg ENU or four equal weekly doses totaling 35.6 or 142.4 mg/kg ENU (8.9 mg/kgx4 or 35.6 mg/kgx4; split-dose groups). Before the treatment and through 26 weeks after the single dose or beginning the split-dose regimen, peripheral RBCs were collected and Pig-A mutant frequencies measured as RBCs negative for the GPI-anchored protein, CD59. Mean CD59-negative RBC frequencies in negative control rats ranged from 3.9 x 10(-6) to 28.7 x 10(-6) and displayed no time-related trend. With single ENU doses, CD59-negative RBC frequencies increased in a time- and dose-related manner. Maximum responses were observed beginning at 6 weeks post-treatment (57.3 x 10(-6) in the 8.9 mg/kg group; 186.9 x 10(-6) in the 35.6 mg/kg group; 759.2 x 10(-6) in the 142.4 mg/kg group), and these elevated mutant frequencies persisted to the last sampling time. In addition, splitting the dose of ENU into four weekly doses produced nearly the same mutant frequency as when given as a single dose: the maximum responses after four weekly doses of 8.9 or 35.6 mg/kg were 176.8 x 10(-6) and 683.3 x 10(-6), respectively. These results indicate that ENU-induced Pig-A mutant RBCs accumulate in a near additive fashion in rats, and once present in the peripheral blood, persist for at least 6 months. These characteristics of Pig-A mutation could be important for detecting weak mutagens by repeated or subchronic/chronic dosing protocols.

Analysis of the Molecular Mechanism for the Antagonistic Action of a Novel 1α,25-Dihydroxyvitamin D3 Analogue toward Vitamin D Receptor Function

We have recently reported that 23(S)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone (TEI-9647) efficiently blocks the differentiation of HL-60 cells induced by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) (Miura, D., Manabe, K., Ozono, K., Saito, M., Gao, Q., Norman, A. W., and Ishizuka, S. (1999) J. Biol. Chem. 274, 16392–16399). To clarify the molecular mechanisms of this antagonism, we examined whether TEI-9647 antagonizes the genomic effects of 1α,25(OH)2D3. 10−7 to 10−9 m TEI-9647 inhibited the transactivation effect of 10−8 m1α,25(OH)2D3 in a dose-dependent manner, while TEI-9647 alone did not activate the reporter activity driven by SV40 promoter containing two vitamin D response elements in Saos-2 cells. The antagonistic effect of TEI-9647 was also observed using the rat 24-hydroxylase gene promoter, but the effect was weaker in HeLa and COS-7 cells than in Saos-2 cells. TEI-9647 also exhibited antagonism in an assay system where the VDR fused to the GAL4 DNA-binding domain and the reporter plasmid containing the GAL4 binding site were used in Saos-2 cells, but did not in HeLa cells. TEI-9647 reduced the interaction between VDR and RXRα according to the results obtained from the mammalian two-hybrid system in Saos-2 cells, but did not in HeLa cells. The two-hybrid system also revealed that the interaction between VDR and SRC-1 was reduced by TEI-9647 in Saos-2 cells. These results demonstrate that the novel 1α,25(OH)2D3 analogue, TEI-9647, is the first synthetic ligand for the VDR that efficiently antagonizes the action of 1α,25(OH)2D3, although the extent of its antagonism depends on cell type.

A novel and practical route to A-ring enyne synthon for 1α,25-dihydroxyvitamin D3 analogs: Synthesis of A-ring diastereomers of 1α,25-dihydroxyvitamin D3 and 2-methyl-1,25-dihydroxyvitamin D3

A novel and practical route to the A-ring enyne synthon (2), which can be versatile for a variety of A-ring analogs of 1α,25-dihydroxyvitamin D3 (1), was developed. This novel method led to an improved synthesis of the A-ring diastereomers of 1, the compounds 13–15, and synthesis of the new analogs, 2-methyl-1,25-dihydroxyvitamin D3 (4) with its all possible diastereomers. The biological evaluation of the 2-methyl analogs showed the ααβ-isomer to be more potent than 1.

Further development of the rat Pig-a mutation assay: measuring rat Pig-a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes.

Recent studies indicate that the Pig‐a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig‐a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid‐specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 × 106 CD71‐positive RETs could be evaluated by flow cytometry for Pig‐a mutant frequency within 5 to 8 min. CD59‐deficient RET and BME frequencies of more than 100 × 10−6 and 80 × 10−6 were detected 1 week after treating rats with 40 mg/kg N‐ethyl‐N‐nitrosourea; by comparison, the frequency of CD59‐deficient total RBCs in these rats was 13.2 × 10−6. The frequency of spontaneous Pig‐a mutant RETs and BMEs was less than 5 × 10−6 and 15 × 10−6, respectively. Since ∼98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59‐deficient Pig‐a mutations in cells of erythroid lineage. Conducting concurrent Pig‐a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.

Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression.

Our previous rat studies indicate that the endogenous Pig-a gene is a promising reporter of in vivo mutation and potentially useful as the basis for an in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100mg/kgN-ethyl-N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM Pig-a mutants transit from the BM and accumulate in PB.

1α,25‐Dihydroxyvitamin D3‐26,23‐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 1α,25‐dihydroxyvitamin D3‐26,23‐lactone (1α,25‐(OH)2D3‐26,23‐lactone) analogs on 1α,25(OH)2D3‐induced differentiation of human leukemia HL‐60 cells thought to be mediated by the genomic action of 1α,25‐dihydroxyvitamin D3 (1α,25‐(OH)2D3) and of acute promyelocytic leukemia NB4 cells thought to be mediated by non‐genomic actions of 1α,25‐(OH)2D3. We found that the 1α,25‐(OH)2D3‐26,23‐lactone analogs, (23S)‐25‐dehydro‐1α‐hydroxyvitamin D3‐26,23‐lactone (TEI‐9647) and (23R)‐25‐dehydro‐1α‐hydroxyvitamin D3‐26,23‐lactone (TEI‐9648), inhibited differentiation of HL‐60 cells induced by 1α,25‐(OH)2D3. However, 1β‐hydroxyl diastereomers of these analogs, i.e. (23S)‐25‐dehydro‐1β‐hydroxyvitamin D3‐26,23‐lactone (1β‐TEI‐9647) and (23R)‐25‐dehydro‐1β‐hydroxyvitamin D3‐26,23‐lactone (1β‐TEI‐9648), did not inhibit differentiation of HL‐60 cells caused by 1α,25‐(OH)2D3. 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 1α‐hydroxyl diastereomers, respectively. Moreover, none of these lactone analogs inhibited NB4 cell differentiation induced by 1α,25‐(OH)2D3. In contrast, 1β,25‐dihydroxyvitamin D3 (1β,25‐(OH)2D3) and 1β,24R‐dihydroxyvitamin D3 (1β,24R‐(OH)2D3) 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 1α,25‐(OH)2D3, specifically for the nuclear VDR‐mediated genomic actions, but not for non‐genomic actions.

Flow cytometric detection of Pig-A mutant red blood cells using an erythroid-specific antibody: Application of the method for evaluating the in vivo genotoxicity of methylphenidate in adolescent rats†

A modified flow cytometry assay for Pig‐A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague‐Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 × /day on postnatal days (PNDs) 29–50. Eight additional rats (4/sex) were injected i.p. with N‐ethyl‐N‐nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/μl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614–629), the HIS49 Pig‐A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU‐treated rats, the modification increased the overall sensitivity of the assay 2–3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig‐A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El‐Zein et al. [2005]: Cancer Lett 230: 284–291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans. Environ. Mol. Mutagen. 2010. Published 2009 by Wiley‐Liss, Inc.

Synthesis and biological activity of 2-methyl-20-epi analogues of 1α,25-dihydroxyvitamin D3

Abstract Synthesis and biological evaluation of all eight possible A-ring diastereomers of 2-methyl-20-epi-1,25-dihydroxyvitamin D3 are described. Among the analogues synthesized, 2α-methyl-20-epi-1α,25-dihydroxyvitamin D3 exhibited exceptionally high potency. The double modification of 2-methyl substitution and 20-epimerization yielded analogues with unique activity profiles.