Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation

We examined the expression of HLA-DR and HLA-DQ antigens and mRNA from myeloid and lymphoid cells obtained from normal volunteers and established cell lines. Cytofluorometric analysis and immunoprecipitation were performed using murine monoclonal antibodies specific for HLA-DR (L-243) and HLA-DQ (Leu 10). The expression of mRNA for HLA-DR and HLA-DQ chains was determined by Northern blot and RNA dot-blot analysis. Lymphoid cell lines expressed both HLA-DR and HLA-DQ antigens, with consistently higher levels of expression of DR. Myeloid cell lines of early myeloblast or bipotent (myeloid-erythroid) phenotype (KG-1, KG-1a, HEL) expressed HLA-DR at high levels, whereas cell lines manifesting a greater degree of myeloid maturation (ML-3, HL- 60, U937) expressed DR at low or undetectable levels. The HLA-DQ antigen was expressed at low levels on the surface of KG-1 and KG-1a cells but was not detectable on other myeloid cell lines. The expression of mRNA for HLA-DR and HLA-DQ chains paralleled the pattern of expression of the respective antigens. The HL-60 and U-937 cells stimulated to differentiate in vitro to macrophages with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] were induced to express detectable levels of HLA-DR antigens. Exposure to gamma-interferon (gamma-IFN) increased the expression of HLA-DR antigens by all myeloid cell lines. Induction of differentiation in vitro with either 1,25(OH)2D3 or dimethyl sulfoxide potentiated this effect of gamma-IFN. Expression of the HLA-DQ antigens was increased on KG-1 myeloblasts after exposure to gamma-IFN. HLA-DQ expression could not be detected on other myeloid cell lines after exposure to gamma-IFN, nor was HLA-DQ expression stimulated by gamma-IFN after HL-60 and U-937 cells were induced to differentiate to macrophagelike cells in vitro. These results provide additional evidence that expression of the HLA-DR and HLA-DQ genes may be independently regulated in human myeloid cells.     

Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism.

We describe several novel analogs of the seco-steroid 1,25(OH)2-vitamin D3[1,25(OH)2D3] and their effects on differentiation and proliferation of HL-60 human myeloid leukemic cells in vitro as well as their effects on calcium metabolism in vivo. The 1 alpha-25(OH)2-16ene-23yne-26,27F6-vitamin D3 is the most potent analog reported to date, having about 80-fold more activity than the reference 1,25(OH)2D3 for inhibition of proliferation and induction of differentiation of HL-60 cells. Also, this analog decreased RNA expression of MYC oncogene in HL-60 by 90% at 5 x 10(-10) mol/L. Intriguingly, intestinal calcium absorption and bone calcium mobilization mediated in vivo by 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3 was found to be markedly (15-fold) less than that of 1,25(OH)2D3. In addition, 1 alpha-25(OH)2D3 bound to 1,25(OH)2D3 receptors of both HL-60 and intestine more avidly than did 1 alpha-25(OH)2-16ene-23yne-26,27F6-D3. This novel analog may open up new therapeutic strategies for several hematopoietic, skin, and bone abnormalities and may provide a new tool to understand how vitamin D3 seco-steroids induce cellular differentiation.     

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.     

Novel vitamin D analogs that modulate leukemic cell growth and differentiation with little effect on either intestinal calcium absorption or bone mobilization

Induction of terminal differentiation of leukemic and preleukemic cells is a therapeutic approach to leukemia and preleukemia. The 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active form of vitamin D3, can induce differentiation and inhibit proliferation of leukemia cells, but concentrations required to achieve these effects cause life-threatening hypercalcemia. Seven new analogs of 1,25(OH)2D3 were discovered to be either equivalent or more potent than 1,25(OH)2D3 as assessed by: (a) inhibition of clonal proliferation of HL-60, EM-2, U937, and patients' myeloid leukemic cells: and (b) induction of differentiation of HL-60 promyelocytes. Furthermore, these analogs stimulated clonal growth of normal human myeloid stem cells. The most potent analog, 1,25-dihydroxy-16ene-23yne-vitamin D3, was about fourfold more potent than 1,25(OH)2D3. This analog decreased clonal growth and expression of c-myc oncogene in HL-60 cells by 50% within ten hours of exposure. Effects on calcium metabolism of these novel analogs in vivo was assessed by intestinal calcium absorption (ICA) and bone calcium mobilization (BCM). Each of the analogs mediated markedly less (10 to 200-fold) ICA and BCM as compared with 1,25(OH)2D3. To gain insight into the possible mechanism of action of these new analogs, receptor binding studies were done with 1,25(OH)2-16ene-23yne-D3 and showed that it competed only about 60% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in HL-60 cells and 98% as effective as 1,25(OH)2D3 for receptors present in chick intestinal cells. In summary, we have discovered seven novel vitamin D analogs that are more potent than the physiologic 1,25(OH)2D3 as measured by a variety of hematopoietic assays. In contrast, these compounds appear to have the potential to be markedly less toxic (induction of hypercalcemia). These novel vitamin D compounds may be superior to 1,25(OH)2D3 in a number of clinical situations including leukemia/preleukemia; they will provide a tool to dissect the mechanism of action of vitamin D seco-steroids in promoting cellular differentiation.     

Differentiating agents facilitate infection of myeloid leukemia cell lines by monocytotropic HIV-1 strains [published erratum appears in Blood 1991 Apr 1;77(7):1624]

Monocytotropic human immunodeficiency virus type 1 (HIV-1) isolates from patients with acquired immunodeficiency syndrome (AIDS) infect mononuclear phagocytes as well as activated T cells, but do not usually infect immature human myeloid cell lines in vitro. The HL-60 promyelocytic/myeloblastic cell line and the promonocytic line, U937, were susceptible to productive infection by monocytotropic HIV-1 isolates (HIV-1JR-FL and HTLV-IIIBa-L) after treatment with retinoic acid, dimethyl sulfoxide, dibutyryl cAMP, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), or 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Virus production was only detected when these compounds were added before virus infection. Virus replication did not correlate with CD4 receptor expression because undifferentiated HL-60 cells express CD4 and the level of CD4 expression did not increase after differentiation in the presence of retinoic acid, 1,25(OH)2D3, or TPA. A mature monocytic cell line (THP-1) was capable of infection without pretreatment, and treatment with differentiating agents enhanced virus production. A chronically infected cell line (J-HL-60) was isolated after HIV-1JR-FL infection of HL-60 cells treated with retinoic acid. Virus production in this cell line was enhanced more than 10-fold after differentiation in the presence of 1,25(OH)2D3 or TPA. The majority of virus production by 1,25(OH)2D3-treated J-HL-60 cells was associated with the mature, adherent population. Molecular analysis of a cloned line of J-HL-60 showed integration of a single DNA provirus. These results suggest that cellular factors associated with precursor cell differentiation along the myelomonocytic pathway are required for optimal replication of monocytotropic HIV-1 strains in vitro.     

Association between the expression of the c-myc oncogene mRNA and the expression of the receptor protein for 1,25-dihydroxyvitamin D3

Studies in lymphocytes have indicated similarities in the state of activation, the time kinetics, and the pathologic states associated with the expression of the c-myc oncogene, and the expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor protein. Here, we have sought evidence for an association between c-myc and the 1,25-(OH)2D3 receptor protein in mammalian cells other than lymphocytes. Comparing two rat osteogenic sarcoma cell lines, one that produces constitutively relatively high levels of the 1,25-(OH)2D3 receptor protein (ROS 17/2.8) and one in which the 1,25-(OH)2D3 receptor protein is practically undetectable (ROS 2/3), we found that the 1,25-(OH)2D3 receptor-expressing cell line also expressed c-myc mRNA. In contrast, the cell line in which the 1,25-(OH)2D3 receptor was undetectable did not express c-myc mRNA. Furthermore, we transfected mouse skin fibroblasts (NIH 3T3) with a recombinant plasmid carrying the human c-myc oncogene. We found a dramatic increase in the 1,25-(OH)2D3 receptor concentration in five separate clonal lines of NIH 3T3 cells transfected with the c-myc-carrying plasmid compared to their nontransfected counterparts or to NIH 3T3 fibroblasts transfected with the vector plasmid alone. The receptor protein of the transfected cells exhibited biochemical characteristics indistinguishable from those of classical receptors for 1,25-(OH)2D3. The increased expression in the transfected cells appeared specific for the receptor for 1,25-(OH)2D3; receptors for sex steroids were not detected in the nontransfected NIH 3T3 cells and remained undetectable after transfection with c-myc. Moreover, the level of the glucocorticoid receptor protein, which was expressed in the nontransfected cells, did not change upon transfection with c-myc.     

Novel vitamin D(3) analog, 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3), that modulates cell growth, differentiation, apoptosis, cell cycle, and induction of PTEN in leukemic cells

The active form of vitamin D(3), 1,25(OH)(2)D(3), inhibits proliferation and induces differentiation of a variety of malignant cells. A new class of vitamin D(3) analogs, having 2 identical side chains attached to carbon-20, was synthesized and the anticancer effects evaluated. Four analogs were evaluated for their ability to inhibit growth of myeloid leukemia (NB4, HL-60), breast (MCF-7), and prostate (LNCaP) cancer cells. All 4 analogs inhibited growth in a dose-dependent manner. Most effective was 21-(3-methyl-3-hydroxy-butyl)-19-nor D(3) (Gemini-19-nor), which has 2 side chains and removal of the C-19. Gemini-19-nor was approximately 40 625-, 70-, 23-, and 380-fold more potent than 1,25(OH)(2)D(3) in inhibiting 50% clonal growth (ED(50)) of NB4, HL-60, MCF-7, and LNCaP cells, respectively. Gemini-19-nor (10(-8) M) strongly induced expression of CD11b and CD14 on HL-60 cells (90%); in contrast, 1,25(OH)(2)D(3) (10(-8) M) stimulated only 50% expression. Annexin V assay showed that Gemini-19-nor and 1,25(OH)(2)D(3) induced apoptosis in a dose-dependent fashion. Gemini-19-nor (10(-8) M, 4 days) caused apoptosis in approximately 20% of cells, whereas 1,25(OH)(2)D(3) at the same concentration did not induce apoptosis. Gemini-19-nor increased in HL-60 both the proportion of cells in the G(1)/G(0) phase and expression level of p27(kip1). Moreover, Gemini-19-nor stimulated expression of the potential tumor suppressor, PTEN. Furthermore, other inducers of differentiation, all-trans-retinoic acid and 12-O-tetradecanoylphorbol 13-acetate, increased PTEN expression in HL-60. In summary, Gemini-19-nor strongly inhibited clonal proliferation in various types of cancer cells, especially NB4 cells, suggesting that further studies to explore its anticancer potential are warranted. In addition, PTEN expression appears to parallel terminal differentiation of myeloid cells.     

1 alpha,25-dihydroxyvitamin D3-induced upregulation of calcineurin during leukemic HL-60 cell differentiation

Cyclosporin A and FK506, at concentrations that inhibited phosphatase activity of calcineurin in HL-60 cellular lysates, augmented the proliferation of leukemic HL-60 cells. These immunosuppressants did not affect 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]-induced monocytic differentiation of HL-60 cells, but did abrogate the 1,25(OH)2D3- induced inhibition of HL-60 cell growth. Treatment with 20 nmol/L 1,25(OH)2D3 led to a progressive increase in calcineurin phosphatase activity in subcellular fractions from HL-60 cell extracts, the increase in this activity appeared to parallel the phenotypic and functional changes of HL-60 cells during monocytic differentiation induced by 1,25(OH)2D3. Immunoblot analysis indicated that increase in calcineurin activity was concordant with the increased expressions of calcineurin catalytic subunit isozymes, calcineurin A alpha (CNA alpha), and calcineurin A beta(CNA beta), and a regulatory calcineurin B subunit (CNB) proteins, which were preceded by a coordinate increase in the levels of CNA alpha, CNA beta and CNB mRNAs. The expression of calmodulin remained unaltered throughout 1,25(OH)2D3-induced monocytic differentiation. These results suggest that calcineurin activation has a net negative effect on HL-60 cell proliferation, and that the increased expression of calcineurin may be involved in 1,25(OH)2D3- induced inhibition of HL-60 cell proliferation.     

Avian and mammalian receptors for 1,25-dihydroxyvitamin D3: in vitro translation to characterize size and hormone-dependent regulation.

In vitro translation of cellular poly(A)+ RNA coupled with immunoprecipitation was developed as a technique for characterizing 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptors and assessing receptor mRNA activity. Cell-free translation of poly(A)+ RNA isolated from chicken intestine revealed two immunoprecipitable forms of avian receptor at 60 kDa and 58 kDa. These two species were identical in electrophoretic mobility to those detected directly in intestinal cytosol by immunoblot analysis. Liver, a tissue devoid of 1,25-(OH)2D3 binding activity, contained no apparent translatable receptor mRNA. 1,25-(OH)2D3 receptors were also synthesized in vitro employing poly(A)+ RNA obtained from several cultured mammalian cell lines. Selective immunoprecipitation revealed a single form of receptor at 54 kDa in mouse fibroblasts (3T6) and pig kidney cells (LLC-PK1) and a 52-kDa species in human breast carcinoma (T47D). Each of these in vitro translated mammalian 1,25-(OH)2D3 receptors migrated identically with its cellular counterpart that was synthesized in vivo employing metabolic labeling of cell protein with [35S]methionine. In vitro translation of poly(A)+ RNA derived from mouse 3T6 cells treated with 1,25-(OH)2D3 for 24-48 hr disclosed a 5-fold increase in receptor mRNA activity over untreated control cells. These results are consistent with the conclusions that 1,25-(OH)2D3 receptors are protein species ranging from 52 to 60 kDa and that, though their functional and immunological domains have been evolutionarily conserved, an inverse relationship apparently exists between phylogenetic status and receptor mass. The data also support the hypothesis that the presence of 1,25-(OH)2D3 leads to a significant increase in receptor mRNA activity in 3T6 cells, indicative of receptor autoregulation.     

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.     

1,25-Dihydroxycholecalciferol and macrophage differentiation with aging

Aging is attended by both decreased levels of circulating 1,25-dihydroxy-vitamin D (1,25(OH)2D) and alterations of immune function. We have explored the relationship of these events via the effects of the steroid hormone on macrophage differentiation, using both the human leukemic cell line HL-60, which has the capacity to differentiate along a monocytic or granulocytic pathway, and authentic bone-marrow-derived macrophage precursors. When treated with 1,25(OH)2D, HL-60 cells undergo monocytic differentiation, as documented by the appearance of macrophage-specific membrane antigens and esterase activity. Also, 1,25(OH)2D increases [Ca2+]i in a slow tonic manner, an event that parallels f-Met-Leu-Phe (fMLP) receptor expression. The rise of [Ca2+]i is derived from influx of extracellular Ca2+ and is associated with increased inositol trisphosphate (IP3)-stimulated Ca2+ release from intracellular stores. On the other hand, while prevention of the 1,25(OH)2D-generated increase in [Ca2+]i leads to reduced superoxide generation, it does not block monocytic differentiation. 1,25(OH)2D also targets to authentic bone-marrow-derived macrophage precursors at all stages of differentiation. In CSF-1-dependent cells, the steroid produces doubling of expression of the mannose receptor, a macrophage-specific membrane protein, which is also expressed by differentiated osteoclasts. The macrophage-maturing effect of 1,25(OH)2D was further explored by analyzing its effect on fMLP signal transduction in HL-60 cells. While virgin HL-60 cells are unresponsive to fMLP, cells incubated for 24 h with 1,25(OH)2D respond to fMLP stimulation with a 60% increase in [Ca2+]i, and possess greater IP3-sensitive calcium stores than virgin cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

1 Alpha,25-dihydroxyvitamin D3 receptor distribution and effects in subpopulations of normal human T lymphocytes

Receptors for 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are expressed upon activation of human lymphocytes, and the hormone inhibits in vitro the proliferation of mitogen-activated lymphocytes. In this study we examined the distribution of the 1,25-(OH)2D3 receptor protein in the two major subsets of T lymphocytes (T helper and T suppressor cells) and the effect of the hormone on their respective rates of proliferation. We activated normal lymphocytes in the presence of monocytes with phytohemagglutinin and subsequently isolated the T helper (T4-positive) and the T suppressor (T8-positive) subsets using monoclonal antibodies and complement-mediated lysis. In parallel experiments, we first isolated monocyte-depleted T4 and T8 cells and then activated them using phytohemagglutinin and a phorbol ester. Using either approach we found that both T4 and T8 lymphocytes expressed the 1,25-(OH)2D3 receptor protein upon activation. The concentration of this protein, its affinity for the ligand (Kd, approximately 10(-10) mol/L), and its sedimentation characteristics (S = 3.3) were indistinguishable in the two T cell subsets. Furthermore, the time kinetics of expression of the receptor after activation were very similar in the two subsets. Nevertheless, 1,25-(OH)2D3 inhibited in a dose-dependent fashion the rate of proliferation of the helper subset, but had no effect on the proliferation of suppressor cells. The finding of a dissimilar effect of 1,25-(OH)2D3 on the proliferation of the T helper and T suppressor cells despite their indistinguishable receptor status suggests that the 1,25-(OH)2D3 receptors of the T cells might not be involved in the effects of the hormone on T-cell proliferation, and that the 1,25-(OH)2D3-induced inhibition of mitogen-activated T4 cell proliferation could be mediated indirectly.     

Induction of bone-type alkaline phosphatase in human vascular smooth muscle cells: roles of tumor necrosis factor-alpha and oncostatin M derived from macrophages

Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-gamma (IFN-gamma) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-gamma and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-gamma and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of beta-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-alpha (TNF-alpha) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-alpha and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-gamma and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-alpha and OSM.     

The involvement of calcium ions in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) was found to suppress growth of human leukemic cells (HL-60), and to induce the differentiation of these cells to monocyte-like cells. The purpose of the present study was to examine the role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells. Incubation of the HL-60 cells with 1,25(OH)2D3 (10(-7) M) for 4 days caused a significant inhibition of 50% of cell growth. The number of differentiated cells increased simultaneously from 24 x 10(3) +/- 2 x 10(3) in the controls to 658 x 10(3) +/- 32 x 10(3) in the 1,25(OH)2D3 (10(-7) M)-treated cells. The role of calcium ions in the effects of 1,25(OH)2D3 on HL-60 cells was first studied by changing the available calcium in the medium and by measuring the effect of 1,25(OH)2D3 on intracellular Ca2+ levels. Limitation of the available Ca2+ by means of ethyleneglycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or verapamil enhanced the inhibitory effect on proliferation and decreased the number of differentiated cells obtained by 1,25(OH)2D3 alone. These effects could be abolished by restoring the Ca2+ levels. The role of the intracellular free Ca2+ ions in the effect of 1,25(OH)2D3 was further illustrated by measuring the intracellular Ca2+ levels. The intracellular free Ca2+ concentration in 1,25(OH)2D3 (10(-7) M)-treated HL-60 cells rose significantly from 117.0 +/- 6.3 nM in the untreated HL-60 cells to 145.0 +/- 7.5 nM in the treated cells (p less than 0.02). Addition of verapamil moderated the increase in intracellular free Ca2+ (125.0 +/- 5.2 nM) obtained by 1,25(OH)2D3 alone. Thus the elevation of intracellular free Ca2+ caused by 1,25(OH)2D3 treatment may be involved in the effect of the hormone on the HL-60 cells.     

1,25-Dihydroxyvitamin D3 enhances cytosolic free calcium in HL-60 cells

1,25-Dihydroxyvitamin D3 (1,25[OH]2D3) caused a rise in the concentration of intracellular free calcium ions ([Ca2+]i) in HL-60 cells. This effect of 1,25(OH)2D3 parallels its suppression of cell proliferation and its induction of cell differentiation into monocyte-like cells. The changes in [Ca2+]i are dose and time dependent. The concentration of 1,25(OH)2D3 (10(-7) M) that induced maximal differentiation also caused the maximal increase in intracellular Ca2+. The rise in cytoplasmic free Ca2+ concentration was not immediate and reached statistical significance only after 24 h. The [Ca2+]i reached its peak at 48 h (134 +/- 4 nM vs 101 +/- 3 nM in controls) and remained stable at this level. The increase in intracellular Ca2+ was found to be related to new protein synthesis, because it was inhibited in the presence of specific RNA and protein synthesis inhibitors. The rise in [Ca2+]i was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3 (24,25[OH]2D3), a vitamin D metabolite that does not induce the differentiation of HL-60 cells. In contrast, 25-hydroxyvitamin D3 (25-OH-D3) and phorbol 12-myristate 13-acetate (TPA), both of which induce differentiation in this cell line, also increase [Ca2+]i. In conclusion, the present study emphasizes that a significant increase in intracellular free Ca2+ occurs in the effect of 1,25(OH)2D3 on HL-60 cells.     

Effect of 1,25-(OH)2-vitamin D3 on growth, homologous receptor and c-myc regulation in C3H/10T1/2 cells

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor concentration, cell proliferation, and the steady-state level of c-myc mRNA were examined in the C3H/10T1/2 mouse embryo fibroblasts, before and after exposing the cells to 1,25-(OH)2D3. The non-transformed, logarithmically growing C3H/10T1/2 Cl 8 cells contained a high concentration of 1,25-(OH)2D3 receptor (164 fmol/mg of protein). An up-regulation of the 1,25-(OH)2D3 receptor and a potent inhibition of cell growth were observed by exposing the cells to 10 nM 1,25-(OH)2D3. The concentration of 1,25-(OH)2D3 receptor in the two chemically transformed, tumorigenic cell lines. C3H/10T1/2 Cl 16 and C3H/10T1/2 TPA 482, was 218 and 63 fmol/mg of protein, respectively. In the two transformed cell lines, 10 nM 1,25-(OH)2D3 had only negligible effect on cell growth. In the Cl 16 cells, an up-regulation of the 1,25-(OH)2D3 receptor was demonstrated, but only a weak up-regulation was found in the TPA 482 cells by the 1,25-(OH)2D3 treatment. No major changes were found in c-myc mRNA levels by the 1,25-(OH)2D3 treatment. Despite inhibition of cell growth, the steady-state level of c-myc mRNA was slightly induced (35%, mean) in the Cl 8 cells compared to control cells. In the transformed cells, no consistent change of the c-myc level was found. In contrast to earlier reports, we did not find any correlation between the 1,25-(OH)2D3 receptor and c-myc level, nor did we find any decrease of c-myc mRNA by 1,25-(OH)2D3 treatment in the C3H/10T1/2 fibroblasts.     

Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn- SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.     

1,25-Dihydroxyvitamin D3 modulates colony-stimulating factor-1 receptor binding by murine bone marrow macrophage precursors.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been shown to be a potent agent for monocyte/macrophage differentiation in leukemic cell lines. This study examines the differentiational effects of 1,25-(OH)2D3 in authentic murine bone marrow immature uncommitted precursor cells collected by pretreatment of murine bone marrow with 5-fluorouracil (5-FU). Early precursor cells, collected 1 day after 5-FU treatment, are characterized by low expression of the colony-stimulating factor-1. (CSF-1) receptor and dependence on both CSF-1 and interleukin-1 for growth. Intermediate precursors were collected 5 days after 5-FU treatment and required either CSF-1 or interleukin-3 for growth. Intermediate precursors expressed relatively higher levels of the CSF-1 receptor. Addition of 1,25-(OH)2D3 to these populations inhibited proliferation, as measured by [3H]thymidine incorporation and soft agar colony assay. Furthermore, 1,25-(OH)2D3 caused a more rapid appearance of the CSF-1 receptor in early precursor cells in a dose-dependent metabolite-specific manner. Conversely, CSF-1 receptor expression was decreased in intermediate precursors treated with the steroid. This decrease in receptor expression was also dose dependent and metabolite specific. These findings demonstrate that 1,25-(OH)2D3 1) targets to authentic bone marrow macrophages at various stages of differentiation and 2) modulates expression of the CSF-1 receptor, a protein which, in turn, regulates monocytic maturation.