Cooperative regulation of c-myc expression in differentiation of human promyelocytic leukemia induced by recombinant gamma-interferon and 1,25-dihydroxyvitamin D3

The biological activity of recombinant human gamma-interferon (IFN-gamma) to induce the phenotypic differentiation of human promyelocytic leukemia cell line HL-60 and to regulate c-myc expression was evaluated. Treatment with IFN-gamma increased monocyte-associated cell surface antigens detected by monocyte-specific monoclonal antibodies in a dose- and time-dependent manner. These antigenic changes were accompanied by a functional differentiation, determined by the increase of phagocytic capability and superoxide generation. IFN-gamma was also found to suppress the growth of HL-60 cells and reduce expression of a c-myc oncogene. These phenotypic and morphological changes to macrophage-like cells induced by IFN-gamma were similar to those by 1,25-dihydroxyvitamin D3, whereas the plasma membrane antigenic changes were different. Moreover, the combination of IFN-gamma and suboptimal doses of 1,25-dihydroxyvitamin D3 have synergistic effects in augmenting mature monocyte specific antigens (Mo2, 63D3, OKM1, and OKM5). In the reduction of c-myc expression by these drugs, a cooperative effect was observed with the inhibition of transferrin receptor expression and cell growth. These results indicate that human recombinant IFN-gamma induces a monocyte phenotype in the HL-60 cells via a mechanism different from the action of 1,25-dihydroxyvitamin D3.     

Requirement of differentiative signals of both interferon-gamma and 1,25-dihydroxyvitamin D3 for induction and secretion of interleukin-1 by HL-60 cells

HL-60, a promyelocytic cell line, was treated with both recombinant interferon-gamma (IFN-gamma) and 1,25-(OH)2 vitamin D3 (D3), and the effect on monocyte-specific markers was assessed. IFN-gamma and D3 modulated different stages in the monocytic differentiation with respect to interleukin-1 (IL-1) production and secretion. D3 induced production of an intracellular IL-1 activity that was not secreted after lipopolysaccharide stimulation. IFN-gamma did not induce intracellular IL-1 but differentiated HL-60 cells, which had been treated with D3, so that lipopolysaccharide stimulated IL-1 release. Both D3 and IFN-gamma individually enhanced expression of nonspecific esterase; in combination the two agents potentiated each other. Expression of cell surface Leu-M3 antigen was also enhanced by the combination of these two agents. Thus, IFN-gamma not only potentiated expression of D3-induced markers but also conferred phenotypic properties characteristic of monocytes. IFN-gamma may play a role in the differentiation of bone marrow cells to mature monocytes.     

Potentiation by 1-alpha,25-dihydroxyvitamin D3 of cytotoxicity to HL-60 cells produced by cytarabine and hydroxyurea

A short (4-hr) exposure to 1-4 X 10(-7) M 1,25-dihydroxyvitamin D3 [(1,25(OH)2D3); 1-alpha,25-dihydroxycholecalciferol] induced transient differentiation in a clone (R2AB2) of human promyelocytic leukemia cells (HL-60) but caused no permanent growth impairment and no detectable cytotoxicity. This treatment with 1,25(OH)2D3 also produced an inhibition of DNA synthesis that was promptly reversed when 1,25(OH)2D3 was removed. When such treatment with 1,25(OH)2D3 immediately followed a sublethal exposure to drugs that inhibit DNA synthesis, including the cancer chemotherapeutic agents cytarabine and hydroxyurea, the proportion of HL-60 cells lethally damaged was increased. This finding was demonstrated by morphologic evidence of cell damage and disintegration, an increased permeability to trypan blue, loss of cells from culture, and a reduced clonogenic potential of the treated cells. Exposure to 1,25(OH)2D3 before treatment with a cytotoxic agent had a slightly protective rather than a damaging effect. These observations suggest that the presence of 1,25(OH)2D3 markedly reduces the capacity of HL-60 cells to repair DNA damage or to reduce the intracellular concentration of cytotoxic agents.     

Effects of calmodulin antagonists and cytochalasins on proliferation and differentiation of human promyelocytic leukemia cell line HL-60

To identify the possible roles of Ca2+-related proteins, calmodulin and microfilaments in leukemic cells, we tested the effect of calmodulin antagonists and cytochalasins on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. The growth of HL-60 was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and trifluoperazine dihydrochloride. In contrast, the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced differentiation of HL-60, as judged by plasma-membrane antigenic changes detected by monoclonal antibodies (OKM1, OKT9), nitroblue tetrazolium reduction, and induction of phagocytotic capacity, was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, although phagocytosis was depressed by N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide or N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide. Trifluoperazine dihydrochloride also failed to inhibit the antigenic change induced by 1,25-(OH)2D3. Cytochalasins B and D, microfilament-disrupting agents, inhibited the cytoplasmic division and the growth of HL-60 but did not inhibit the 1,25-(OH)2D3-induced differentiation. These findings suggest that the calmodulin- and microfilament-dependent process may be involved in the proliferation of HL-60, but not in the differentiation induced by 1,25-(OH)2D3.     

Recombinant gamma-interferon and lipopolysaccharide enhance 1,25-dihydroxyvitamin D3-induced cell differentiation in human promyelocytic leukemia (HL-60) cells

The induction of cell differentiation by a combination of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], recombinant gamma-interferon (rec gamma-IFN), and a lipopolysaccharide from E. coli (LPS) was studied in a clonal population (clone-9) of human promyelocytic HL-60 leukemia cells in vitro. Treatment of clone-9 cells with 10(-9) to 10(-7)M 1,25-(OH)2D3 yielded a macrophage cell differentiation. The addition of 10 or 100 U/ml of gamma-IFN and 2 or 10 micrograms/ml LPS caused a further increase in expression of the different differentiation markers. The most pronounced effects involved increases in cell attachment to the surface of tissue-culture Petri dishes and in lysozyme, nonspecific esterase, and cytolytic activities. The combined treatment with 1,25-(OH)2D3 and rec gamma-IFN and LPS also caused an increase in the percent of multinucleated giant cells. These results indicate the effectiveness of combining different agents in inducing cell differentiation in HL-60 cells. A similar approach may be useful in controlling myeloid leukemias in vivo.     

Differentiated HL-60 promyelocytic leukaemia cells produce a factor inducing differentiation

The bipotential human promyelocytic leukaemia cell line HL-60 can be induced to differentiate into monocytic or granulocytic cells by treatment with 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) or dimethylsulphoxide (DMSO) respectively. Conditioned media (CM) from 1,25(OH)2D3- or DMSO-treated cells were able to induce monocytic differentiation in fresh HL-60 cells as measured by induction of non-specific esterase and macrophage surface markers. CM from 1,25(OH)2D3-treated cells also led to a dose dependent loss of proliferative capacity in soft agar colony assays. These effects were not due to a toxic effect of the CM or to residual inducer present in the CM. gamma-interferon and GM-CSF were apparently not responsible for these effects. CM from the human histiocytic lymphoma cell line U937 led to only a low level of induction of macrophage differentiation in fresh HL-60 cells. The defect in HL-60 leukaemic cells may therefore be at the level of induction of an autonomously-produced differentiation factor.     

Growth inhibition and differentiation induction in human monoblastic leukaemia cells by 1alpha-hydroxyvitamin D derivatives and their enhancement by combination with hydroxyurea.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent inducer of differentiation in myeloid leukaemia cells, but its clinical use is limited because of its hypercalcaemic activity. We examined the ability of 1,25(OH)2D3 in combination with several anti-cancer drugs to inhibit the proliferation of, and induce differentiation in, human monoblastic leukaemia U937 cells. Hydroxyurea (HU), cytarabine and camptothecin showed effective synergism with 1,25(OH)2D3 with regard to growth inhibition, while daunorubicin and etoposide had only modest synergistic effects. HU and cytarabine effectively enhanced nitroblue tetrazolium-reducing activity induced by 1,25(OH)2D3. HU also enhanced the morphological maturation and expression of CD11b and CD14 in cells treated with 1,25(OH)2D3. Among the anti-cancer drugs examined, HU had the greatest synergistic effects with 1,25(OH)2D3 with regard to growth inhibition and differentiation induction in U937 cells. HU also enhanced the differentiation of other myeloid leukaemia HL-60, ML-1, THP-1, P39/TSU, P31/FUJ and NB4 cells induced by 1,25(OH)2D3 and that of U937 cells induced by 24-epi-1,25(OH)2D2 and 1,25(OH)2D7. Interestingly, 1alpha(OH)D derivatives (1alpha-hydroxyvitamin D3, D2, D4 and D7) effectively induced the differentiation of monoblastic leukaemia U937, P39/TSU and P31/FUJ cells. HU also enhanced the growth inhibition and differentiation of U937 cells induced by 1alpha(OH)D derivatives. As 1alpha(OH)D derivatives preferentially act on monocytic cells, they may be useful in the treatment of acute monocytic leukaemia, both alone and in combination with HU.     

Cell cycle sensitivity of HL-60 cells to the differentiation-inducing effects of 1-alpha,25-dihydroxyvitamin D3

A recently described system for monocyte-like differentiation of HL-60 cells was utilized to determine if the initiation of this pathway can be linked to a set of replicative cellular events. The standard induction system consisted of a 4-h exposure to 100 nM 1-alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] followed by determination of nonspecific esterase and phagocytic activity 24 h later. The cell cycle status was ascertained by the incorporation of [3H]thymidine and autoradiography. Studies in which cell cycle block in the G1/S phase boundary region was produced by a partial inhibition of DNA synthesis with thymidine, or sodium butyrate, showed that the exposure of such semisynchronous cultures to 1,25(OH)2D3 resulted in an increased proportion of differentiated cells. Conversely, blocking the cell cycle with vinblastine (G2/M block) or theobromine (mid-G1 block) inhibited the initiation of differentiation by 1,25(OH)2D3. Experiments in which the differentiated cells were examined for the cell cycle position at the time of the exposure to 1,25(OH)2D3 by [3H]thymidine labeling and autoradiography confirmed that the late G1 and early S phase cells are those which predominate in the differentiated fraction of 1,25(OH)2D3-treated HL-60 cultures. These results link pre- and early replicative cellular events to the induction of monocytic differentiation by 1,25(OH)2D3.     

Phenotypic differentiation-linked growth inhibition in human leukemia cells by active vitamin D3 analogues

Active vitamin D₃ induced phenotypic differentiation of the human promyelocytic leukemia cell line, HL-60, cultured in serum-free medium. Upon exposure to 10^?1-10^?7 M 1,25-(OH)₂D₃ 1,24S-(OH)₂D₃ or 1,24R(OH)₂D₃, monocyte-granulocyte-associated plasma membrane antigens of HL-60 cells detected by monoclonal antibodies, OKM1, 63D3 and Mo2, quantitated by fluorescence-activated cell sorter analysis, were increased time- and dose-dependently. After expose to 1,25-(OH)₂D₃, promotion of this antigenic expression was detected within 16 h, and the induction of differentiation continued until 96 h. The number of cells bearing transferrin receptors recognized by the monoclonal antibody, OKT9, and its density on the surface of HL-60 cells were decreased symmetrically. These effects appeared in parallel with the inhibition of cell growth, poly(ADP-ribose) content and de novo DNA-RNA synthesis. These findings indicate that 1,24S-(OH)₂D₃ stimulates differentiating induction of HL-60 like 1,25-(OH)₂D₃ in vitro and that the decrease of transferrin receptor is apparently correlated with the inhibition of proliferation.     

Structure-function studies on analogues of 1 alpha,25-dihydroxyvitamin D3: differential effects on leukemic cell growth, differentiation, and intestinal calcium absorption

The hormonally active form of vitamin D, 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], is an efficient stimulator of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM) in humans and experimental animals and, as well, has been shown to be effective in inducing differentiation and inhibiting proliferation of leukemia cells. Thus, it has been proposed that analogues of 1,25(OH)2D3 could be synthesized which might allow for separation of biological functions, i.e., promote a differentiation of leukemia cells without a significant stimulation of ICA or BCM, both biological effects which can cause hypercalcemia in humans. Here we report the results of an evaluation of four analogues of the previously studied (Zhou et al., Blood, 74:82-92, 1989) 1 alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 [1,25(OH)2-16-ene-23-yne-D3]; these analogues allowed evaluation of the consequences of (a) the presence or absence of six deuterium atoms on carbons 26 and 27 of the side chain and (b) the deletion or substitution by a fluorine atom of the 1 alpha-hydroxyl group on the A-ring. The 1,25(OH)2-16-ene-23-yne-D3 analogue was found to be 7-fold more potent than the parent 1,25(OH)2D3 with respect to (a) inhibition of clonal proliferation of HL-60 cells as well as (b) induction of differentiation of HL-60 promyelocytes. Variants of this analogue which possessed the six deuterium atoms on carbons 26 and 27 were slightly less active than the 1,25(OH)2-16-ene-yne-D3. However, replacement of the 1 alpha-hydroxyl group by a 1-fluoro group, or the absence of the 1-hydroxyl group, resulted in analogues that were somewhat less effective than the parent 1,25(OH)2D3 in achieving these biological responses but more potent as inhibitors of the renal mitochondrial 25-OH-D3-1 alpha-hydroxylase, the site of endogenous production of 1,25(OH)2D3. ICA and BCM were assessed in vivo in vitamin D-deficient chickens, and each of the analogues was markedly less potent than the standard 1,25(OH)2D3. The analogue 1,25(OH)2-16-ene-23-yne-D3 had 2% of the ICA and 3% of the BCM activity of the parent 1,25(OH)2D3. Absence of the 1 alpha-hydroxyl group or substitution of the 1-fluoro group for the 1-hydroxyl group significantly diminished both the ICA and BCM activity in comparison to 1,25(OH)2-16-ene-23-yne-D3. Receptor binding studies indicated that 1,25(OH)2-16-ene-23-yne-D3 competed about 75% as effectively as 1,25(OH)2D3 for 1,25(OH)2D3 receptors present in both chick intestinal cells and HL-60 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

24-Oxo metabolites of vitamin D3 analogues: disassociation of their prominent antileukemic effects from their lack of calcium modulation

The seco-steroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] inhibits proliferation and induces differentiation of malignant cells including those of the hematopoietic system. The 24-oxo metabolite of 1,25(OH)(2)D(3) also has prominent antiproliferative activities against various cancer cells. We chemically synthesized five novel 24-oxo vitamin D(3) analogues and evaluated their abilities both to inhibit clonal growth and induce differentiation of myeloid leukemia cells and to cause hypercalcemia. The 1alpha,25-dihydroxy-16-ene-D(3) [1,25(OH)(2)-16-ene-D(3)] and 1alpha,25-dihydroxy-16-ene-19-nor-D(3) [1,25(OH)(2)-16-ene-19-nor-D(3)] and their 24-oxo metabolites showed greater potency than 1,25(OH)(2)D(3) in their abilities to inhibit clonal proliferation of HL-60, NB4, and U937 leukemic cell lines as measured by methylcellulose soft-gel assay. Their inhibition of clonal growth was irreversible as analyzed by pulse exposure studies. The synthetic analogues also had greater potency than 1,25(OH)(2)D(3) to induce differentiation of HL-60 and NB4 cells as measured by generation of superoxide, nonspecific esterase production, and induction of CD11b and CD14 cell surface antigens and to increase the proportion of these cells in the G(0)-G(1) phase of the cell cycle. For most assays, the 24-oxo metabolite was slightly more potent than the unmodified analogue, and 50% activity was usually found in the nanomolar range. These analogues and their 24-oxo metabolites also inhibited fresh leukemic cell clonal proliferation. Expression of p27(KIP1), a cyclin-dependent kinase inhibitor that plays an important role in blocking the cell cycle, was found by Western blot analysis to be induced by the analogues and their 24-oxo metabolites in both HL-60 and U937 cells, suggesting a possible mechanism by which these analogues inhibit leukemic growth. Notably, the calcemic activity tested by injections of 1alpha,25-dihydroxy-16-ene-24-oxo-19-nor-D(3) in mice was at least 12-fold less than 1alpha,25(OH)(2)-16-ene-19-nor-D(3). Taken together, chemically synthesized 24-oxo metabolites of 1alpha,25(OH)(2)-16-ene-D(3) and 1alpha,25(OH)(2)-16-ene-19-nor-D(3) irreversibly inhibited proliferation and induced differentiation of acute myeloid leukemia cells with minimal toxicity; these compounds may have a role in the maintenance phase of therapy for acute myeloid leukemia.     

Dephosphorylation of specific proteins in HL-60 cells by 1 alpha, 25-dihydroxyvitamin D3: possible involvement of cAMP-dependent protein kinase.

Changes of phosphoprotein patterns in HL-60 cells were studied during short exposures to 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3]. One hundred nanometers 1,25(OH)2D3 dephosphorylated at least three proteins in 6 h: phosphoproteins with molecular weights of 82 kD (pp82), 33 kD (pp33), and 31 kD (pp31). Phosphorylation of pp33 and pp31 was also suppressed by 1 mM dbcAMP, and dephosphorylation of the two protein by 1,25(OH)2D3 was inhibited by 8 microM H-8, an inhibitor of cAMP-dependent protein kinase (PKA). Furthermore, 8 microM H-8 inhibited dephosphorylation of the two proteins when it was added with 1,25(OH)2D3. On the other hand, 10 nM TPA gave no significant change to these two phosphoproteins. These results suggest the possibility that PKA is involved in the early stages of 1,25(OH)2D3-induced HL-60 cell differentiation through specific protein dephosphorylation.     

1,25-Dihydroxyvitamin D3-regulated expression of genes involved in human T-lymphocyte proliferation and differentiation

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited the secretion of gamma-interferon from human T-lymphocytes activated by the calcium ionophore, A23187, or phytohemagglutinin with or without 12-O-tetradecanoylphorbol-13-acetate. The agent also inhibited cell proliferation and interleukin 2 secretion by these cells. The inhibition of gamma-interferon secretion was time and dose dependent and partially abolished by the addition of exogenous human recombinant interleukin 2. To elucidate the molecular events by which 1,25-(OH)2D3 inhibits cell proliferation and lymphokine secretion, complementary DNA probes were used to follow the expression of genes involved in human T-lymphocyte proliferation and differentiation. 1,25-(OH)2D3 inhibited the expression of interleukin 2 and gamma-interferon messenger RNA in human lymphocytes activated by phytohemagglutinin and 12-O-tetradecanoylphorbol-13-acetate. It also inhibited the accumulation of c-myc protooncogene messenger RNA and, to a lesser extent, interleukin 2 receptor messenger RNA in these cells. However, it did not affect the expression of the HLA-DR gene. These results suggest that 1,25-(OH)2D3 selectively regulates T-lymphocyte activation-related genes at the level of messenger RNA.     

Potentiation of 1-beta-D-arabinofuranosylcytosine cytotoxicity to HL-60 cells by 1,25-dihydroxyvitamin D3 correlates with reduced rate of maturation of DNA replication intermediates

S-phase-active cytotoxic drugs selectively damage leukemic cells, but the mechanisms of this action are not clear. We have investigated the previously reported potentiation of toxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) to HL-60 cells by the differentiation-inducing steroid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and compared the results with the effects of other drugs which inhibit DNA synthesis. Determination of the intracellular content of the active metabolite of ara-C, ara-CTP, excluded more prolonged retention of the drug as the basis for potentiation of cytotoxicity. Alkaline elution of replicating DNA showed that 1,25(OH)2D3 added with or immediately after ara-C or hydroxyurea reduced the rate maturation of the replicating DNA and resulted in an increased proportion of smaller DNA fragments. However, pretreatment of the cells with 1,25(OH)2D3 inhibited this effect of the drugs on replication of DNA. No direct effect of 1,25(OH)2De on replicating DNA could be detected. The results suggest that the early events which initiate cell differentiation may protect an intact DNA replicative machinery from S-phase-active drugs but reduce the rate of DNA maturation once DNA integrity has been compromised by inhibitors of DNA synthesis.     

Alternative differentiation of human promyelocytic leukemia cells (HL-60) induced selectively by retinoic acid and 1 alpha,25-dihydroxyvitamin D3

Induction of hematopoietic differentiation was investigated in human promyelocytic leukemia cells [HL-60] using two lipophilic vitamins, retinoic acid and 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Both vitamins suppressed proliferation and induced differentiation of HL-60 cells, but 1 alpha,25(OH)2D3 was 70- to 100-fold more potent than was retinoic acid on a molar basis. Simultaneous treatment with suboptimal concentrations of 1 alpha,25(OH)2D3 (0.12 to 1.2 nM) and retinoic acid (10 to 100 nM) showed additive effects in reducing nitroblue tetrazolium, a common marker for monocyte-macrophage and granulocyte differentiation. For the study of alternative differentiation of the cells by the two vitamins, we used monoclonal antibodies specific for either human monocyte-macrophages or granulocytes and other markers specific for macrophage differentiation such as alpha-naphthyl acetate esterase activity and adherence to the dish surface. HL-60 cells were induced to differentiate alternatively into macrophages by 1 alpha,25(OH)2D3 or into granulocytes by retinoic acid. When HL-60 cells were treated with various concentrations of 1 alpha,25(OH)2D3 (1.2 to 120 nM) in the presence of 1000 nM retinoic acid which is a concentration sufficient to induce maximal granulocyte differentiation, the appearance of the markers for monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 was not at all affected by the retinoic acid. These results indicate that 1 alpha,25(OH)2D3 and retinoic acid have additive effects in inducing differentiation of HL-60 cells, but monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 occurs much more readily than does granulocyte differentiation by retinoic acid.     

The role of granulocyte-macrophage colony-stimulating factor in induction of monocytic differentiation of HL-60 cells: synergistic interaction with 1 alpha, 25-dihydroxyvitamin D3 and interferon-gamma in inducing interleukin-1 beta.

1 alpha, 25-Dihydroxyvitamin D3 (D3) (100 nM) and interferon-gamma (IFN-gamma) (100 U/ml) cooperatively inhibited the proliferation of HL-60 cells, and synergistically induced their monocytic differentiation. The growth-promoting effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/ml) was inhibited appreciably by D3 and slightly by IFN-gamma. Despite the clear difference in their effects on growth of HL-60 cells, both IFN-gamma and GM-CSF in combination with D3 induced cell cycle changes, decreasing the number of cells in the S phase and increasing their percentage in the G1/0 phase. GM-CSF alone had no effect on differentiation, but enhanced differentiation induced by D3 distinctly though to a limited extent, and also enhanced monocytic differentiation, including morphological changes of HL-60 cells in the presence of D3 and IFN-gamma. GM-CSF as well as D3 and IFN-gamma induced interleukin-1 beta (IL-1 beta) production by the HL-60 cells, clearly indicating their importance in differentiation of these cells. IFN-gamma and GM-CSF had mutually potentiating effects and induced maximum IL-1 beta production in response to lipopolysaccharide (LPS) in the presence of D3. Thus despite its growth-promoting effect, GM-CSF is a potential inducer of monocytic differentiation of human myeloid leukemia cells, because in cooperation with IFN-gamma it induced monocyte-macrophage differentiation of HL-60 cells in the presence of D3.     

Inhibition of phorbol ester-induced phenotypic differentiation of HL-60 cells by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor

To identify the possible role of calcium ions in cell differentiation, we studied the extracellular Ca2+ requirement and the effect of Ca2+/phospholipid-dependent protein kinase (protein kinase C) inhibitor on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. HL-60 cells grew equally well in 0.1 and 1.0 mM Ca2+ media. The addition of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 1,25-dihydroxyvitamin D3, and all-trans-beta-retinoic acid inhibited the cell growth and induced mature macrophage and granulocyte phenotypes in 1.0 mM Ca2+ medium. 1,25-Dihydroxyvitamin D3 and all-trans-beta-retinoic acid induced HL-60 differentiation to the same degree in 0.1 mM Ca2+ and 1.0 mM Ca2+ media. However, TPA failed to induce HL-60 differentiation or to inhibit proliferation in a 0.1 mM Ca2+ medium. The decrease of extracellular Ca2+ from 1.0 to 0.1 mM caused a significant drop in the intracellular Ca2+ level in undifferentiated and TPA-treated HL-60 cells, although no rapid change in cytosolic Ca2+ was detected in response to TPA addition. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor, inhibited proliferation of HL-60 cells in a dose-dependent manner. In contrast, H-7 selectively restored the proliferation of TPA-treated HL-60 cells and inhibited TPA-induced phenotypic differentiation. However, the same concentrations of 1-(5-isoquinolinylsulfonyl)-2,3-dimethylpiperazin and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, analogues of H-7 that inhibit protein kinase C more weakly, had no effect on the proliferation or differentiation induction. H-7 also suppressed 1,25-dihydroxyvitamin D3- and all-trans-beta-retinoic acid-induced phenotypic changes of HL-60 cells but did not eliminate the growth inhibition by these inducers. These results demonstrate the Ca2+ requirement and the protein kinase C involvement in phorbol ester-induced phenotypic differentiation of HL-60 cells.     

Involvement of intracellular pH elevation in the effect of 1,25-dihydroxyvitamin D3 on HL-60 cells

Besides its effect in inhibiting proliferation and inducing differentiation of HL-60 to monocyte-like cells, 1,25-dihydroxyvitamin D3 also causes a rise in intracellular pH (pHi) from 7.17 +/- 0.02 to 7.3 +/- 0.05, as measured by the fluorescence of 2',7'-bis(carboxyethyl)-5-(6)-carboxy-fluorescein. The effect of 1,25-dihydroxyvitamin D3 on pHi is dose dependent in a parallel manner to its effect on the proliferation and differentiation processes. The elevation of pHi by 1,25-dihydroxyvitamin D3 is gradual but precedes the differentiation process. A significant increase in pHi was obtained after 16 h of incubation with the hormone and reached its maximum level after 48 h. pHi then dropped back to its initial level, which is also similar to that of peripheral normal blood monocytes. A rise in pHi was not observed during incubation of HL-60 cells with 24,25-dihydroxyvitamin D3, a metabolite which does not promote their differentiation. In contrast, other agents such as phorbol 12-myristate 13-acetate known to induce differentiation in this cell line do cause an increase in pHi. Moreover, alkalinization of HL-60 cells by NH4Cl causes induction of differentiation to monocytes. The results suggest that a rise in pHi plays a role in regulating the molecular mechanism of the inhibition of proliferation and the induction of differentiation in HL-60 cells.