Synergistic enhancement by 12-O-tetradecanoylphorbol-13-acetate and dibutyryl cAMP of 1alpha,25-dihydroxyvitamin D3 action in human promyelocytic leukemic HL-60 cells.

We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3)-induced 24-hydroxylation activity in HL-60 cells by increasing 1,25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1,25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1,25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1 h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1,25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1,25-(OH)2D3, besides up-regulation of VDR.     

Effect of antimetabolites and thymidine blockage on the induction of differentiation of HL-60 cells by retinoic acid or 1 alpha,25-dihydroxyvitamin D3

Induction of differentiation of the HL-60 human promyelocytic leukemia cell line by retinoic acid or 1 alpha,25(OH)2D3 was analyzed under the condition in which cellular DNA synthesis was inhibited by several antimetabolites or blocked by thymidine. The results demonstrate that differentiation occurs in the absence of DNA synthesis and that some inhibitors of DNA synthesis may enhance the differentiation of HL-60 cells by the above inducers. Among the antimetabolites used, the enhancement of induction of differentiation by hydroxyurea was shown to be more effective than that of Ara-C or aphidicolin. The effect of thymidine blockage was similar to that of hydroxyurea. These different effects may be due to the different points at which the cell cycle is blocked by these agents. These results seem to be common in both the differentiation of the granulocytic line induced by retinoic acid and of the macrophage line induced by 1 alpha,25(OH)2D3. The present study also suggests that combination treatment with the inhibitor of DNA synthesis and the inducer of differentiation could be beneficial in the clinical therapy of leukemia. The mode of action of clinical low-dose Ara-C treatment is also discussed.     

Functional defect of variant clones of a human myeloid leukemia cell line (HL-60) resistant to 1 alpha,25-dihydroxyvitamin D3

Two variant clones of a human myeloid leukemia cell line (HL-60) resistant to the active metabolite of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], were isolated from a 1 alpha,25(OH)2D3-sensitive parent clone, and the mechanism of the resistance was examined. When the parent clone was incubated with 120 nM 1 alpha,25(OH)2D3, cell growth was suppressed to half of the control and about half of the cells exhibited phagocytic activity and C3 rosette formation on day 3. The variant clones, however, were resistant to 120 nM 1 alpha,25(OH)2D3. One of the variant clones was also insensitive to other potential inducers such as 12-O-tetradecanoyl-phorbol-13-acetate, actinomycin D, and dimethyl sulfoxide. When the variant clones were incubated with 1 alpha,25(OH)2[3H]D3, they took up much less radioactivity than the parent clone into the whole cells, and into the cytosol protein-bound and chromatin-bound fractions. The variant clones were found to possess reduced amounts of the cytosol receptor protein to which 1 alpha,25(OH)2D3 was specifically bound; but the hormone-receptor complex could be transferred to the chromatin acceptor sites similarly both in the wild type clone and its variant clones, indicating that one of the major defects in the 1 alpha,25(OH)2D3-resistant clones is the reduced amounts of the specific cytosol receptor. These results support the concept that 1 alpha,25(OH)2D3 induces differentiation of human myeloid leukemia cells by a receptor-mediated mechanism.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 and its sensitivity to ketoconazole in 1 alpha,25-dihydroxyvitamin D3-differentiated HL60 cells

Regulation of the metabolism of [3H]25-hydroxyvitamin D3 ([ 3H]25-(OH)D3) in vitro to material with the characteristics of [3H]24,25-dihydroxyvitamin D3 ([3H]24,25-(OH)2D3) has been studied in the human promyelocytic cell line HL60. Synthesis of 24,25-(OH)2D3 was induced in a dose-dependent manner in cells pretreated with 0.1-100 nM 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) for 4 days. This treatment also inhibited cell proliferation and stimulated differentiation to a macrophage phenotype that was characterized by staining for non-specific esterase (NSE) activity. The ability to synthesize [3H]24,25-(OH)2D3 from [3H]25-(OH)D3 and the expression of NSE activity both responded to changes in concentration of 1 alpha,25-(OH)2D3 in the culture medium in a parallel manner. Synthesis of [3H]24,25-(OH)2D3 was linear when the incubation time was between 1 and 8 h and the cell number between 1 and 12 x 10(6) cells/incubation. The optimum substrate concentration for its synthesis was 125 nM, giving an apparent Michaelis constant of 360 nM. The identity of the [3H]24,25-(OH)2D3 synthesized by these cells was confirmed by co-chromatography with authentic 24,25-(OH)2D3 on normal-phase and reverse-phase high-performance liquid chromatography systems and by its reaction to sodium-m-periodate. Cells that had been exposed to 100 nM 1 alpha,25-(OH)2D3 for 4 days synthesized 2.17 +/- 0.07 (S.E.M.) pmol 24,25-(OH)2D3/10(6) cells per h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of terminal differentiation of cultured mouse epidermal cells by 1 alpha,25-dihydroxyvitamin D3

Terminal differentiation of mouse epidermal cells in primary culture was found to be regulated by 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), the hormonal form of vitamin D3 produced by sequential hydroxylations in the liver and kidney. Epidermal differentiation was stimulated dose dependently by 1 alpha,25(OH)2D3 at concentrations of 0.12 nM or more. In the presence of the vitamin, stratified foci increased in number and size and contiguous foci coalesced. Basal cells in the treated cultures decreased sharply and underwent differentiation into squamous and enucleated cells which sloughed off into the medium during cultivation. The size and density of the cells became larger and lighter during the course of differentiation. 1 alpha,25(OH)2D3 markedly stimulated formation of a cornified envelope, a structure with chemically stable cross-links formed beneath the plasma membrane. Of several derivatives of vitamin D3 examined, 1 alpha,25(OH)2D3 was the most potent in inducing epidermal differentiation. Stimulation of epidermal differentiation was also observed in low calcium medium. DNA synthesis was inhibited dose dependently by 1 alpha,25(OH)2D3. A specific receptor for 1 alpha,25(OH)2D3 was found in the cytosol fraction of the epidermal cells. Scatchard plot analysis revealed that the receptor has an apparent dissociation constant (Kd value) of 54 pM and maximum binding value (Nmax) of 43 fmol/mg protein. The specificity of the receptor was demonstrated by analog competition in the following order: 1 alpha,25(OH)2D3 much greater than 25-hydroxyvitamin D3 greater than 1 alpha-hydroxyvitamin D3 greater than 24R,25-dihydroxyvitamin D3.     

Metabolism of 1alpha,25(OH)2D3 and its 20-epi analog integrates clonal expansion, maturation and apoptosis during HL-60 cell differentiation.

Induction of growth arrest and monocyte differentiation of HL-60 leukemia cells by 1alpha,25 dihydroxyvitamin D3 (1alpha,25(OH)2D3) is well established. By contrast, we have observed, that 1alpha,25(OH)2D3 and its metabolites play separate roles in clonal expansion and survival of differentiating HL-60 cells. Cells that had differentiated by 48 h (CD14 positive) grew slower than control cells, whereas CD14 negative cells were growing faster at this time point. Inhibiting 1alpha,25(OH)2D3 or 1alpha,25(OH)2-20-epi-D3 metabolism, by the 25(OH)D3-24-hydroxylase inhibitor ketoconazole, abolished hyperproliferation of CD14 negative cells. Instead, both the onset of differentiation and subsequent apoptosis were enhanced. These events were associated with immediate up-regulation of the cyclin-dependent kinase inhibitor p21(waf1) and a lack of sustained expression, respectively. Stimulation and inhibition of growth by vitamin D3-related compounds was observed to be concentration and metabolite specific. Low amounts of 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated HL-60 cell growth. At higher concentrations, 1alpha,25(OH)2-20-epi-D3 was a more potent inducer than 1alpha,24,25(OH)3-20-epi-D3 of HL-60 differentiation; 1alpha,25(OH)2-20-epi-24-oxo-D3 was exclusively pro-differentiative at all concentrations. 1alpha,25(OH)2-20-epi-D3 and 1alpha,24,25(OH)3-20-epi-D3 stimulated proliferation of KG-1a leukemia cells, but neither of these compounds nor 1alpha,25(OH)3-20-epi-24-oxo-D3 exerted pro-differentiative effects on these cells. These findings shed new light on the pro- and anti-proliferative effects of 1alpha,25(OH)2D3 and lead to the postulate that metabolism of 1alpha,25(OH)2D3 and its 20-epi analog regulates different subsets of genes so as to co-ordinate population expansion and the differentiation process. Furthermore, 1alpha,25(OH)2D3 metabolism and/or sensitivity to the effects of metabolites may be altered in transformed cells to derive a clonal advantage.     

Polyamines in 1 alpha, 25-dihydroxycholecalciferol-induced differentiation of human promyelocytic leukemia cells, HL-60

The human promyelocytic leukemia cell line, HL-60, differentiated into macrophage/monocytes in the presence of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3], as assessed by the percentage of morphologically mature cells and their ability to reduce nitroblue tetrazolium. In this study of the mechanism involved, the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase (SAT), the rate-limiting enzymes of polyamine metabolism, as well as the cellular levels of polyamine were measured. ODC activity reached a peak 24 h after the addition of 1 alpha,25(OH)2D3 and then decreased, while SAT activity gradually increased as differentiation commenced. An increase in putrescine and decreases in spermidine and spermine were also observed. Addition of alpha-difluoromethylornithine, an irreversible inhibitor of ODC, with or without methylglyoxalbis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, caused no effect on 1 alpha,25(OH)2D3-induced cell differentiation, although the cellular levels of putrescine and spermidine decreased markedly. Addition of alpha-difluoromethylornithine markedly suppressed cell proliferation; this effect was reversed by the addition of exogenous putrescine. Addition of exogenous spermidine or spermine to overcome activation of SAT also had no effect on 1 alpha,25(OH)2D3-induced cell differentiation. These results suggest both that polyamine metabolism is not important in 1 alpha,25(OH)2D3-induced differentiation of HL-60 cells, but that it is intimately involved in the proliferation of these cells.     

Cooperative effects of gamma interferon and 1-alpha,25-dihydroxyvitamin D3 in inducing differentiation of human promyelocytic leukemia (HL-60) cells

Various agents induce differentiation of human leukemia cells in vitro. Most of these agents cause myeloid differentiation, but phorbol diesters, 1-alpha,25-dihydroxyvitamin D3 (1,25[OH2]D3), and certain lymphokines cause differentiation to monocyte-like cells. The purpose of this study was to determine the cooperative effects of 1,25(OH2)D3 and the lymphokine gamma interferon (IFN-gamma) on HL-60 cell differentiation. The recombinant human IFN-gamma or 1,25(OH2)D3 caused a slight reduction in the proliferation of the HL-60 cells (30%-40% reduction at doses of 100-200 U/ml [0.25-0.50 nM] IFN-gamma, or 5-25 nM 1,25[OH2]D3). HL-60 cells treated with 100 U/ml IFN-G had an eightfold increase in expression of nonspecific esterase (NSE) and a twofold increase in H2O2 production in response to phorbol myristate acetate (PMA). 1,25(OH2)D3 enhanced NSE expression eight- to 30-fold and H2O2 secretion twofold in response to PMA. There was also enhanced expression of HLA-DR and the receptor for C3bi. The 1,25(OH2)D3- and IFN-gamma-differentiating effects appeared to be additive or synergistic. Populations of IFN-gamma-treated HL-60 cells (but not the 1,25[OH2]D3-treated cells) had multinucleated giant cells. The polykaryons had NSE activity and had some properties of macrophage polykaryons or osteoclasts. 1,25(OH2)D3 did not augment the IFN-gamma-induced polykaryon formation.     

1 alpha,25-dihydroxyvitamin D3 corrects osteomalacia in hypoparathyroidism and pseudohypoparathyroidism

Deficiency of circulating 1 alpha-25-dihydroxyvitamin D3 (1 alpha,25(OH)2D) regularly occurs in hypoparathyroidism (HP) and pseudohypoparathyroidism (PHP). Osteomalacia is occasionally found in the two diseases. Two patients, one with HP and the other with PHP, both with symptomatic and biopsy-proven osteomalacia, were studied before and after treatment with 1 alpha,25(OH)2D3. Laboratory values before treatment were as follows: serum immunoreactive parathyroid hormone was undetectable in the patient with HP and was elevated in the patient with PHP. Serum 25-hydroxyvitamin D, measured by binding assay, was 131.5 and 61.9 nmol/l (normal: 69.1 +/- 15.9 nmol/l); serum 24,25-dihydroxyvitamin D, measured by binding assay, was 13.9 and 3.8 nmol/l (normal: 3.4 +/- 1.4 nmol/l); serum 1 alpha,25(OH)2D, measured by bioassay, was 28.6 and 29.0 pmol/l (normal: 77.3 +/- 22.8 pmol/l) and, measured by receptor assay, was 36.2 and 41.0 pmol/l (normal: 71.8 +/- 35.8 pmol/l) in the HP and PHP patients, respectively. Serum calcium was low and serum inorganic phosphate was high in both cases. Treatment with 1 alpha,25(OH)2D3 (3-5 micrograms per day for 10-12 months) restored serum calcium and inorganic phosphate to normal, alleviated bone pain and healed the osteomalacia as shown on repeat bone biopsy. Our results provide further evidence that isolated deficiency of 1 alpha,25(OH)2D may cause osteomalacia or rickets.     

Effect of 1alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 on metalloproteinase activity and cell maturation in growth plate cartilage in vivo.

Recent studies indicate that 1alpha,25-dihydroxyvitamin D3 (1alpha,25[OH]2D3) and 24R,25-dihydroxyvitamim D3 (24R,25[OH]2D3) differentially regulate proliferation, differentiation, and matrix synthesis of growth plate chondrocytes. To determine whether both metabolites play the same or different roles in vivo, we used the vitamin D-deficient rat as a model. Rickets was induced and then reversed by administering a single dose of ergocalciferol, 1alpha,25(OH)2D3, or 24R,25(OH)2D3 and euthanizing the animals after 4, 24, 48, or 72 h. Growth plates were either processed for histology and histomorphometry or extracted with buffered guanidine-HCl. Neutral metalloproteinase activity in the extracts was measured by use of aggrecan-containing beads, and collagenase activity was determined by use of radioactive type I collagen. The levels of tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator were also determined. The morphology of the growth plate varied as a function of treatment. While 24R,25(OH)2D3 appeared to affect cell maturation and 1alpha,25(OH)2D3 appeared to affect terminal differentiation and calcification, response to ergocalciferol was indicative of the combined responses to the individual metabolites. Enzyme activity was regulated in a differential manner. Treatment with ergocalciferol produced a rapid decline in both neutral metalloproteinase and collagenase activities that was statistically significant by 4 h. By contrast, 1alpha,25(OH)2D3 had no effect on neutral metalloproteinase activity but caused a significant decrease in both active and total collagenase activity by 4 h, while 24R,25(OH)2D3 decreased neutral metalloproteinase activity by 48 h and had no effect on collagenase activity. Ergocalciferol had no effect on TIMP levels at any time examined, whereas 1alpha,25(OH)2D3 caused an increase at 48 and 72 h and 24R,25(OH)2D3 completely blocked TIMP production at 4 and 24 h. By contrast, plasminogen activator activity by ergocalciferol was decreased at 4 h, increased by 1alpha,25(OH)2D3 at 4 and 24 h, and decreased by 24R,25(OH)2D3 at all time points examined. These in vivo results confirm our previous cell culture observations showing that growth plate chondrocytes are differentially regulated by 1alpha,25(OH)2D3 and 24R,25(OH)2D3. Moreover, they show definitively that these two vitamin D metabolites play distinct roles not only in regulating neutral metalloproteinase and collagenase activities in growth plate cartilage but in cell maturation and calcification of this tissue in vivo.     

1alpha,25-dihydroxyvitamin D3 inhibits prostate cancer cell growth by androgen-dependent and androgen-independent mechanisms

We recently reported that 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibits the growth of the LNCaP human prostate cancer cell line by an androgen-dependent mechanism. In the present study we examined the actions and interactions of 1,25-(OH)2D3 and the androgen 5alpha-dihydrotestosterone (DHT) on two new human prostate cancer cell lines (MDA), MDA PCa 2a and MDA PCa 2b. Scatchard analyses revealed that both cell lines express high affinity vitamin D receptors (VDRs) with a binding affinity (Kd) for [3H]1,25-(OH)2D3 of 0.1 nM. However, the MDA cell lines contain low affinity androgen receptors (ARs) with a Kd of 25 nM for [3H]DHT binding. This is 50-fold lower than the AR in LNCaP cells (Kd = 0.5 nM). Their response to DHT is greatly reduced; 2a cells do not respond to 100 nM DHT, and 2b cells show a modest response at that high concentration. 1,25-(OH)2D3 causes significant growth inhibition in both MDA cell lines, greater (for 2b cells) or lesser (for 2a cells) than that in the LNCaP cell line. Moreover, 1,25-(OH)2D3 significantly up-regulates AR messenger RNA in all three cell lines, as shown by Northern blot analysis. The growth inhibitory effect of 1,25-(OH)2D3 on LNCaP cells is blocked by the pure antiandrogen, Casodex, as we previously reported. However, Casodex (at 1 microM) did not block the antiproliferative activity of 1,25-(OH)2D3 in MDA cells. In conclusion, the growth inhibitory action of 1,25-(OH)2D3 in the MDA cell lines appears to be androgen independent, whereas the actions of 1,25-(OH)2D3 in LNCaP cells are androgen dependent. Most importantly, the MDA cell lines, derived from a bone metastasis of human prostate carcinoma, remain sensitive to 1,25-(OH)2D3, a finding relevant to the therapeutic application of vitamin D and its low calcemic analogs in the treatment of advanced prostate cancer.     

1alpha,25-Dihydroxy-3-epi-vitamin D3 a physiological metabolite of 1alpha,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes

Recent studies of metabolism using pharmacological substrate concentrations of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)(2)D3] in several tissues including primary cultures of human keratinocytes, bovine parathyroid cells and bone cells led to the identification of 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)(2)-3-epi-D3] as a major natural metabolite of 1alpha,25(OH)(2)D3. In the present study, we demonstrate that human keratinocytes incubated with 25-hydroxy[26,27-(3)H] vitamin D3 produce 1alpha,25(OH)(2)-3-epi-D3 along with 1alpha,25(OH)(2)D3. The production of 1alpha,25(OH)(2)-3-epi-D3 is also identified in human keratinocytes incubated with physiological substrate concentrations of 1alpha,25(OH)(2)D3. Unlike 24-hydroxylase, the major enzyme involved in the further metabolism of 1alpha,25(OH)(2)D3 in human keratinocytes, the enzyme(s) responsible for the production of 1alpha,25(OH)(2)-3-epi-D3 is constitutive and is not inhibited by ketoconazole. It is also noted that 1alpha,25(OH)(2)-3-epi-D3 is further metabolised in human keratinocytes into several as yet unidentified metabolites, the production of which is inhibited to a great extent by SDZ 89-443, an inhibitor of 24-hydroxylase. This finding indicates that the 24-hydroxylase like in the case of 1alpha,25(OH)(2)D3, also plays a major role in the metabolism of 1alpha,25(OH)(2)-3-epi-D3. The results obtained from the metabolism studies performed in parallel among 25OHD3, 1alpha,25(OH)(2)D3 and 1alpha,25(OH)(2)-3-epi-D3 indicate that 1alpha,25(OH)(2)-3-epi-D3 and its metabolites exhibit higher metabolic stability. In summary, we demonstrate for the first time that 1alpha,25(OH)(2)-3-epi-D3 is a physiological metabolite of 1alpha,25(OH)(2)D3 in human keratinocytes. Also, 1alpha,25(OH)(2)-3-epi-D(3) is further metabolised in human keratinocytes mainly through the activity of 24-hydroxylase. Furthermore, our finding of the relative metabolic stability of 1alpha,25(OH)(2)-3-epi-D3 and especially its metabolites when compared to 1alpha,25(OH)(2)D3 and its metabolites provides an important explanation for its previously observed potent inhibitory effect on keratinocyte growth in spite of its low affinity to vitamin D receptor.     

Regulation of 25-hydroxyvitamin D3-1 alpha-hydroxylase and production of 1 alpha,25-dihydroxyvitamin D3 by human dendritic cells

25-Hydroxyvitamin D3-1 alpha-hydroxylase (25(OH)D3-1 alpha-hydroxylase), the key enzyme of 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) production, is expressed in monocyte-derived macrophages (MACs). Here we show for the first time constitutive expression of 25(OH)D3-1 alpha-hydroxylase in monocyte-derived dendritic cells (DCs), which was increased after stimulation with lipopolysaccharide (LPS). Accordingly, DCs showed low constitutive production of 1,25(OH)2D3, but activation by LPS increased 1,25(OH)2D3 synthesis. In addition, 25(OH)D3-1 alpha-hydroxylase expression was found in blood DCs but not in CD34+-derived DCs. Next we analyzed the functional consequences of these results. Addition of 1,25(OH)2D3 at concentrations comparable with those produced by DCs inhibited the allostimulatory potential of DCs during the early phase of DC differentiation. However, terminal differentiation decreased the responsiveness of DCs to 1,25(OH)2D3. In conclusion, DCs are able to produce 1,25(OH)2D3 especially following stimulation with LPS. Terminal maturation renders DCs unresponsive to the effects of 1,25(OH)2D3, but those cells are able to suppress the differentiation of their own precursor cells in a paracrine way through the production of 1,25(OH)2D3.     

A potent analog of 1alpha,25-dihydroxyvitamin D3 selectively induces bone formation

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] is a principal regulator of calcium and phosphorus homeostasis through actions on intestine, kidney, and bone. 1,25(OH)(2)D(3) is not considered to play a significant role in bone formation, except for its role in supporting mineralization. We report here on the properties of 2-methylene-19-nor-(20S)-1alpha,25(OH)(2)D(3) (2MD), a highly potent analog of 1,25(OH)(2)D(3) that induces bone formation both in vitro and in vivo. Selectivity for bone was first demonstrated through the observation that 2MD is at least 30-fold more effective than 1,25(OH)(2)D(3) in stimulating osteoblast-mediated bone calcium mobilization while being only slightly more potent in supporting intestinal calcium transport. 2MD is also highly potent in promoting osteoblast-mediated osteoclast formation in vitro, a process essential to both bone resorption and formation. Most significantly, 2MD at concentrations as low as 10(-12) M causes primary cultures of osteoblasts to produce bone in vitro. This effect is not found with 1,25(OH)(2)D(3) even at 10(-8) M, suggesting that 2MD might be osteogenic in vivo. Indeed, 2MD (7 pmol/day) causes a substantial increase (9%) in total body bone mass in ovariectomized rats over a 23-week period. 1,25(OH)(2)D(3) (500 pmol three times a week) only prevented the bone loss associated with ovariectomy and did not increase bone mass. These results indicate that 2MD is a potent bone-selective analog of 1,25(OH)(2)D(3) potentially effective in treating bone loss diseases.     

Differentiation of mouse myeloid leukemia cells induced by 1 alpha,25-dihydroxyvitamin D3.

Mouse myeloid leukemia cells can be induced to differentiate into macrophages in vitro by 1 alpha,25-dihydroxyvitamin D3, the active form of vitamin D3. The minimal concentration of 1 alpha,25-dihydroxyvitamin D3 to induce the cell differentiation was 0.12 nM. The degree of cell differentiation in various markers induced by 12 nM 1 alpha,25-dihydroxyvitamin D3 was nearly equivalent to that induced by 1 microM dexamethasone, the most potent known stimulator. Among several markers of the differentiation by 1 alpha,25-dihydroxyvitamin D3, phagocytic activity was induced within 24 hr, and this was followed by induction of lysozyme and locomotive activities. Similar changes were also induced by 0.01-1 microM 1 alpha-hydroxyvitamin D3. 25-Hydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3 showed only weak inducing activity. These results suggest the possibility that, in addition to its wellknown biological activities in enhancing intestinal calcium transport and bone mineral mobilization, 1 alpha, 25-dihydroxyvitamin D3 is involved in the differentiation of bone marrow cells.