Interleukin-4 inhibits the differentiation of mouse myeloid leukemia M1 cells induced by dexamethasone, D-factor/leukemia inhibitory factor and interleukin-6, but not by 1 alpha,25-dihydroxyvitamin D3

The effects of interleukin-4(IL-4) on the growth and differentiation of mouse myeloid leukemia M1 cells induced by various differentiation inducers were investigated. IL-4 alone did not have any significant effect on the growth or differentiation of M1 cells, but inhibited their differentiation induced by dexamethasone, D-factor/leukemia inhibitory factor, or interleukin 6. IL-4 also restored the proliferation of M1 cells after growth inhibition during their induction of differentiation by inducers. In contrast, IL-4 enhanced inhibition of growth and induction of differentiation of M1 cells by 1 alpha,25-dihydroxyvitamin D3. These results indicate that modulation of differentiation of M1 cells by IL-4 depends on the differentiation inducer.     

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.     

Fusion of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 involves extracellular, but not intracellular, calcium

We have reported that the active form of vitamin D3, 1 alpha, 25-dihydroxy-vitamin D3 [1 alpha, 25(OH)2D3], directly induces the fusion of mouse alveolar macrophages (Abe et al: Proc. Natl. Acad. Sci. USA 80:5583-5587, 1983). The fusion process can be divided into two phases: the 1 alpha,25(OH)2D3-dependent priming phase (0-18 hr) and the calcium-dependent progression phase (18-72 hr) (Jin et al: J. Cell. Physiol. 137:110-116, 1988). In the present study, we examined the role of calcium in the progression phase of macrophage fusion induced by 1 alpha,25(OH)2D3. Macrophages pretreated with 1 alpha,25(OH)2D3 for 48 hr in a low-calcium (0.13 mM) medium began to fuse quickly 30 min after the culture medium was switched to a normal calcium (1.85 mM) medium. Of various cations tested, calcium was the most effective in inducing fusion, followed by strontium and manganese. Magnesium, potassium, and sodium had no effect. Calcium ionophores such as A23187 and ionomycin did not induce fusion in the low-calcium medium, nor did they potentiate fusion in the media containing higher concentrations of calcium. The intracellular concentration of free Ca2+, measured by a fluorescent method using fura-2 AM, was 116 +/- 1 nM in the macrophages pretreated with 1 alpha,25(OH)2D3 for 48 hr in the low-calcium medium. When calcium chloride was added to the assay system at a final concentration of 1.85 mM, the cytosolic free Ca2+ concentration did not increase appreciably (from 116 to 144 nM). But the macrophages began to fuse quickly when CaCl2 was added. In contrast, adding ionomycin increased cytosolic free Ca2+ from 116 to 440 nM, but no fusion occurred. These results clearly indicate that the extracellular, but not the intracellular, calcium is involved in the progression phase of the fusion of mouse alveolar macrophages primed by 1 alpha,25(OH)2D3.     

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 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.     

Inhibition by 1 alpha,25-dihydroxyvitamin D3 of dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Regulation of erythroid differentiation by vitamin D3 derivatives was examined in Friend erythroleukemia cells. After Friend cells were cultured for 5 days with 1.5% dimethyl sulfoxide (DMSO), as much as 70% of the cells became benzidine-positive and the hemoglobin content increased in parallel with the increase of benzidine-positive cells. The DMSO-induced erythroid differentiation was markedly inhibited by concurrent addition of the active form of vitamin D3, 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Of the vitamin D3 derivatives tested, 1 alpha,25(OH)2D3 was the most potent in inhibiting DMSO-induced erythroid differentiation. 1 alpha,25(OH)2D3 alone was totally ineffective in both cell growth and erythroid differentiation. These results together with our previous reports indicate that 1 alpha,25(OH)2D3 is somehow involved not only in myeloid differentiation, but also in erythroid differentiation.     

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.     

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.     

Oncoprotein Cot1 represses kinase suppressors of Ras1/2 and 1,25-dihydroxyvitamin D3-induced differentiation of human acute myeloid leukemia cells

Metabolites and derivatives of vitamin D are well-known inducers of monocytic differentiation, but the mechanistic basis for their action is not fully elucidated. Here we show that the product of protooncogene Cot1 represses the monocytic phenotype in human acute myeloid leukemia (AML) cells induced to differentiate by 1,25-dihydroxyvitamin D(3) (1,25D), even though the expression of cellular Cot1 increases early in the process of 1,25D-induced differentiation. Interestingly, the expression of the two members of the Kinase Suppressor of Ras (KSR) family of molecular scaffolds, known to be positive regulators of Ras signaling and of 1,25D-induced differentiation, increases in parallel with Cot1 in 1,25D-treated cells. However, KSR1/2 are negatively regulated by Cot1, as determined by transfection of siCot1, and confirmed by a reverse effect of ectopic expression of Cot1. The effect of Cot1 in AML cells appears to be cell-type specific, as previous reports in other cell types found KSR-2 to be a negative regulator of Cot1, a reverse relationship. Also in contrast to findings in other cells, in AML cells Cot1 exerts negative control on the MAP kinase pathways, since siCot1 increases the levels of activated Raf1, p90RSK, JNK1, c-jun, and p38, though not of MEK/ERK. These findings have implications for therapy of AML, since in AML cells active MAPKs hasten cell differentiation, and specific pharmacological inhibitors of Cot1 kinase activity have recently became available, thus making Cot1 a "druggable" target.     

Possible involvement of 1 alpha,25-dihydroxyvitamin D3 in proliferation and differentiation of 3T3-L1 cells

Growth of 3T3-L1 cells was inhibited by 10(-10)-10(-7)M of 1 alpha,25-dihydroxy vitamin D3 [1 alpha,25(OH)2D3] in a dose- and time-dependent manner. The potency of 1 alpha,25(OH)2D3 in inducing differentiation was low, since 3T3-L1 cells cultured with 1 alpha,25(OH)2D3 did not become mature adipocyte-like cells but were changed to slightly rounded cells containing small droplet-like substances in the cytoplasm and glycerophosphate dehydrogenase (sn-glycerol-3-phosphate: NAD+2-oxidoreductase, EC 1.1.1.8), the marker enzyme of differentiation to adipocyte, did not increase. These results together with the natural occurrence of this vitamin indicate that 1 alpha,25(OH)2D3 may play an important role in the cell growth and differentiation besides such known action as intestinal calcium transport and bone mineral mobilization.     

Raf-1 signaling is required for the later stages of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells but is not mediated by the MEK/ERK module

We are interested in determining the signaling pathways for 1,25-dihydroxyvitamin D3 (1,25D)-induced differentiation of HL60 leukemic cells. One possible candidate is Raf-1, which is known to signal cell proliferation and neoplastic transformation through MEK, ERK, and downstream targets. It can also participate in the regulation of cell survival and various forms of cell differentiation, though the precise pathways are less well delineated. Here we report that Raf-1 has a role in monocytic differentiation of human myeloid leukemia HL60, which is not mediated by MEK and ERK, but likely by direct interaction with p90RSK. Specifically, we show that Raf-1 and p90RSK are increasingly activated in the later stages of differentiation of HL60 cells, at the same time as activation of MEK and ERK is decreasing. Transfection of a wild-type Raf-1 construct enhances 1,25D-induced differentiation, while antisense Raf-1 or short interfering (si) Raf-1 reduces 1,25D-induced differentiation. In contrast, antisense oligodeoxynucleotides (ODN) and siRNAs to MEK or ERK have no detectable effect on differentiation. In late stage differentiating cells Raf-1 and p90RSK are found as a complex, and inhibition of Raf-1, but not MEK or ERK expression reduces the levels of phosphorylated p90 RSK. These findings support the thesis that Raf-1 signals cell proliferation and cell differentiation through different intermediary proteins.     

The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1alpha,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1alpha,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1alpha,25(OH)2D3 or its 6-s-cis-conformer analog, 1alpha,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1beta-hydroxyl diastereomers, including 2beta-methyl-1beta,25(OH)2D3 and 2beta-methyl-3-epi-1beta,25(OH)2D3. Very interestingly, 2beta-methyl-3-epi-1alpha,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1alpha-hydroxyl group. Other 1alpha-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the alpha- and beta-chair conformers. The 2beta-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the beta-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.     

Increased expression of N-acetylglucosaminyltransferase-V in human hepatoma cells by retinoic acid and 1alpha,25-dihydroxyvitamin D3

UDP-N-acetylglucosamine: alpha-6-D-mannoside beta-1,6N-acetylglucosaminyltransferase-V activities were determined in human hepatoma cell lines of Hep3B and HepG2, and also compared with those of normal liver tissues and primary hepatocytes. When GlcNAcbeta1-2Manalpha1-3(GlcNAcbeta1-2Manalpha1-4)(Manbeta1-4GlcNAc-2-amino pyridine (GlcN,GlcN-biant-PA) and UDP-GlcNAc were used as substrates, the enzymes displayed optimum temperatures of 50 degrees C, optimum pHs of 6.5 in each case, K(m) values for UDP-GlcNAc to be 5.8 (Hep3B) and 4.5 mM (HepG2) and K(m) values for GlcN,GlcN-biant-PA (mM) to be 1.28 (Hep3B) and 2.4 (HepG2). This indicates that values of Hep3B GlcNAc-transferase-V were distinguishable with HepG2 enzyme. Furthermore, Hep3B enzyme in membrane fraction showed about 1.5-fold higher specific activity (1.423 pmol/(h mg) than that (1.066 pmol/(h mg)) of HepG2. Normal hepatocytes are characterized by very low level of GlcNAc-transferase-V activity whereas hepatoma cells contained high activities. Treatment of hepatoma cells with retinoic acid and 1alpha,2,5-dihydroxyvitamin D(3) (Vit-D(3)) resulted in an increase in GlcNAc-transferase-V activity, while treatment with dimethyl sulfoxide and cytosine-arabinoside resulted in decrease in the enzyme activity. Although retinoic acid (RA) treated cells shows a changed GlcNAc-transferase-V mRNA expression, expression of marker proteins such as alpha-fetoprotein and albumin was not changed. This is the first demonstration of GlcNAc-transferase-V activity in RA and Vit-D(3)-treated hepatoma cell lines.     

Induction of differentiation of human leukemia cells by inhibitors of myosin light chain kinase

Inhibitors of myosin light chain kinase, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9) and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7), induced Nitroblue tetrazolium reducing activity, lysozyme activity and morphological maturation of human monoblastic U937, THP-1 and promyelocytic HL-60 cells, but not of erythroblastic K562 cells. However, three analogs of ML-9, which are an inhibitor and an activator of protein kinase C, and a calmodulin antagonist, respectively, did not induce differentiation of the cells.     

Targeting 1alpha,25-dihydroxyvitamin D3 antiproliferative insensitivity in breast cancer cells by co-treatment with histone deacetylation inhibitors

Proliferation of the non-malignant breast epithelial cell line, MCF-12A, is sensitively and completely inhibited by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (ED90 = 70 nM), We used real time RT-PCR to demonstrate that the relative resistance to 1alpha,25(OH)(2)D(3) of MDA-MB-231 cells (ED50 > 100 nM) correlated with significantly reduced Vitamin D receptor (VDR) and increased NCoR1 nuclear receptor co-repressor mRNA (0.1-fold reduction in VDR and 1.7-fold increase in NCoR1 relative to MCF-12A (P < 0.05)). This molecular lesion can be targeted by co-treating cells with 1alpha,25(OH)(2)D(3) or potent analogs and the histone deacetylation inhibitor trichostatin A (TSA). For example, the co-treatment of 1,25-dihydroxy-16,23,Z-diene-26,27-hexafluoro-19-nor Vitamin D(3) (RO-26-2198) (100 nM) plus TSA results in strong additive antiproliferative effects in MDA-MB-231 cells. This may represent novel chemotherapeutic regime for hormone insensitive breast cancer.     

Parathyroid hormone inhibits 25-hydroxyvitamin D3-24-hydroxylase mRNA expression stimulated by 1 alpha,25-dihydroxyvitamin D3 in rat kidney but not in intestine.

Using a cDNA probe for rat renal 24-hydroxylase, expression of its mRNA was compared in the rat kidney and intestine. Vitamin D-deficient rats received a single injection of 1 alpha,25-dihydroxyvitamin D3. Expression of 24-hydroxylase mRNA was first detected in the kidney at 3-h post-injection and increased thereafter. Similarly, 24-hydroxylase mRNA was expressed in the intestine after 1 alpha,25-dihydroxyvitamin D3 injection. However, the dose level of 1 alpha,25-dihydroxyvitamin D3 required to induce the intestinal 24-hydroxylase mRNA expression was only 1/100 the amount required to induce renal 24-hydroxylase mRNA. Induction of intestinal 24-hydroxylase mRNA expression by 1 alpha,25-dihydroxyvitamin D3 was far more rapid than that of renal 24-hydroxylase mRNA. Thyroparathyroidectomy shortened the time required to induce expression of renal, but not intestinal, 24-hydroxylase mRNA. Administration of either parathyroid hormone or cAMP to vitamin D-deficient rats greatly reduced the expression of 24-hydroxylase mRNA in the kidney but not in the intestine. When rats were fed a vitamin D-repleted diet containing 0.7% (adequate) or 0.03% (low) calcium for 2 weeks, intestinal expression of 24-hydroxylase mRNA could be induced only in the low calcium group. In contrast, renal mRNA expression was preferentially stimulated in the adequate calcium group. These results clearly demonstrate that the expression of 24-hydroxylase mRNA is down-regulated by parathyroid hormone in the kidney but not in the intestine.