Comparative assessment of the activity of beta-carotene, retinoyl-beta-D-glucuronide and retinoic acid on growth and differentiation of a human promyelocytic leukemia cell line HL-60

We compared the ability of all-transretinoic acid (RA), all-trans-retinoyl-beta-D-glucuronide (RAGL), and all-trans-beta-carotene (BC) to inhibit growth and to induce differentiation of the human promyelocytic leukemia cell line HL-60 into morphologically mature granulocytes. BC was made water-soluble by the solutol-solvent-system. RA (1 microM) could induce differentiation of 85% of the HL-60 cells after a total incubation time of 180 hours, RAGL (5 microM) induced 64% of the cells, whereas 33% of the HL-60 cells were differentiated after incubation with BC (10 microM), which was determined by assessing cell functional capacity to reduce nitroblue tetrazolium dye in response to phorbolesters. The absence of RA in RAG and BC treated cells gives strong evidence that RAG and BC exert intrinsic biological effects.     

Chemistry of loteprednol etabonate and related steroids. II. Reactions at ring C and NMR structural studies of the resulting compounds

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.     

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.     

Cell-water transfer and stability of biological structures (resonance of biological structures)

We demonstrated that adrenomedullin (AM) is produced and secreted from human leukemia cell lines (THP-1 and HL-60) as well as peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages. Immunoreactive AM accumulated in the culture media of THP-1 and HL-60 cells increased according to their differentiation into macrophage-like cells. Retinoic acid exerted synergistic effects on AM secretion from THP-1 and HL-60 cells when administered with tumor necrosis factor-alpha, lipopolysaccharide or 12-O-tetradecanoyl phorbol-13-acetate. AM was shown to increase the scavenger receptor activity on THP-1 cells. Thus, monocytes/macrophages should be recognized as sources of AM, and the secreted AM may modulate the function of macrophages.     

Tandem-in-time mass spectrometry method for the sub-parts-per-trillion determination of 2,3,7,8-chlorine-substituted dibenzo-p-dioxins and -furans in high-fat foods

Phosphorylation of the nucleosome core histone H3 (H3) on Ser-10 is thought to be a prerequisite for chromatin condensation at mitosis. Although during interphase, cell differentiation, or mitogenic activation of quiescent cells, changes in chromatin structure that involve local chromatin condensation/decondensation also occur, little is known about H3 phosphorylation during these transitions. Using the recently developed sensitive marker to monitor H3 phosphorylation, namely, the mAb that recognizes the phosphorylated epitope of H3 (anti-H3-P mAb), the status of H3 phosphorylation was assayed in individual human lymphocytes after their mitogenic stimulation (G0 to G1 transition) and in human leukemic HL-60 cells induced to differentiate by all-trans-retinoic acid (RA), 1,25-dihydroxyvitamin D3 (vit D3), dimethyl sulfoxide (DMSO), or phorbol myristate acetate (PMA). Multiparameter flow cytometry was used to correlate H3 phosphorylation with cell cycle position. The specificity of the anti-H3-P mAb was confirmed by the loss of its binding following cell treatment with alkaline phosphatase. The presence of phosphorylated H3 was detected during interphase in HL-60 cells and in normal lymphocytes at a level severalfold lower than during mitosis. No significant changes in H3 phosphorylation were observed during lymphocyte stimulation. Unexpectedly, the level of H3 phosphorylation was over fourfold higher in monocytes than in lymphocytes or granulocytes from peripheral blood. The punctate pattern of labeling with anti-H3-P mAb in monocyte nuclei suggests that H3 is phosphorylated in small clusters of adjacent nucleosomes. Differentiation of HL-60 cells was accompanied by a rise in H3 phosphorylation, which was higher after induction by RA, vit D3, and PMA (approx. threefold) than after DMSO (approximately 20%). The data indicate that in addition to being a critical event during chromatin condensation at mitosis, H3 phosphorylation plays a role during chromatin changes accompanying differentiation of HL-60 cells, in particular, along the monocytic lineage. The high level of H3 phosphorylation in monocytes may serve as a marker of these cells and is being explored as a possible diagnostic and prognostic tool in monocytic leukemias.     

Induction of the c-ski proto-oncogene by phorbol ester correlates with induction of megakaryocyte differentiation

Overexpression of v-ski blocks the terminal differentiation of chicken erythroblasts, and in cooperation with v-sea causes transformation of these cells, indicating that c-ski may play a role in regulating either proliferation or differentiation in hematopoietic cells. We examined c-ski expression in four different myeloid cell lines which can be induced to differentiate by exposure to phorbol 12-myristate 13-acetate (PMA). Two of the cell lines are multipotent and have the ability to differentiate into either erythrocytes or megakaryocytes (K562 and HEL cells), one cell line differentiates exclusively into megakaryocytes (CHRF-288-11), and the fourth cell line differentiates into either monocytes or granulocytes (HL-60). Our findings indicate that c-ski mRNA is up regulated by PMA only in those cell lines which respond by differentiating along the megakaryocyte lineage. The extent of differentiation and the observed increase in c-ski mRNA levels are positively correlated with the PMA concentration used to induce differentiation. Experiments in which CHRF-288-11 cells were treated with the protein kinase C (PKC) activator bryostatin 1 indicate that c-ski mRNA induction is not a general effect of PKC activation. The results strongly suggest that c-ski expression is correlated with megakaryocyte maturation.     

Fibronectin-mediated cell adhesion is required for induction of 92-kDa type IV collagenase/gelatinase (MMP-9) gene expression during macrophage differentiation. The signaling role of protein kinase C-beta

Induction of the 92-kDa gelatinase (MMP-9) gene expression is associated with macrophage differentiation. In this study, we explored the regulatory mechanisms underlying this differentiation-associated MMP-9 gene expression in human HL-60 myeloid leukemia cells and human peripheral blood monocytes. Phorbol 12-myristate 13-acetate (PMA) markedly induced MMP-9 gene expression in HL-60 cells; the induction closely paralleled the timing and extent of PMA-induced cell adhesion and spreading, a hallmark of macrophage differentiation. Similarly, treatment with PMA or macrophage-colony stimulating factor stimulated adherence and spreading of blood monocytes with a concurrent 7- or 5-fold increase in MMP-9 production, respectively. In protein kinase C (PKC)-beta-deficient HL-60 variant cells (HL-525), PMA failed to induce cell adhesion and MMP-9 gene expression. Transfecting HL-525 cells with a PKC-beta expression plasmid restored PKC-beta levels and PMA inducibility of cell adhesion and spreading as well as MMP-9 gene expression. Induction of cell adhesion and MMP-9 gene expression in HL-60 cells and blood monocytes was strongly inhibited by neutralizing monoclonal antibodies to fibronectin (FN) and its receptor alpha5 beta1 integrin. HL-525 cells, which constitutively display high levels of surface alpha5 beta1 integrin, adhered and spread on immobilized FN with concomitant induction of MMP-9 gene expression. Cytochalasins B and D were each a potent inhibitor of MMP-9 production. Our results suggest that alpha5 beta1 integrin-mediated interaction of immature hematopoietic cells with FN plays a critical role in modulating matrix-degrading activities during macrophage differentiation.     

Potentiation of retinoic acid-induced differentiation of HL-60 cells by prostaglandin EP2 receptor

Human promyeloid HL-60 cells are differentiated by all-trans retinoic acid (RA) to granulocytes, and prostaglandin (PG) E2 potentiates the RA-induced differentiation. Here we examined which subtype of PGE receptors was involved in this potentiating activity of PGE2. Northern blot analysis demonstrated that HL-60 cells expressed three subtypes of PGE receptor, EP2, EP3, and EP4. Among various EP agonists, and EP2-selective agonist, butaprost, preferentially potentiated the RA-induced differentiation of HL-60 cells. Butaprost not only decreased the half-maximal concentration of RA but also increased the maximal level of the differentiation. Butaprost concentration-dependently stimulated the cAMP formation, and 8-Br-cAMP strongly potentiated the RA-induced differentiation. These results demonstrate that the EP2 receptor enhances the RA-induced differentiation of HL-60 cells via stimulation of adenylate cyclase.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

Ethical concerns in modern medical practice

Differentiation of HL-60 cells along the granulocytic lineage by DMSO in the presence of transforming growth factor-beta and low concentrations of 1,25-dihydroxyvitamin D3 leads to the upregulation of 5-lipoxygenase activity in 100,000 g supernatants and intact cells to levels which are comparable to normal granulocytes. Similarly, differentiation of the human monocytic cell line Mono Mac 6 by 1,25-dihydroxyvitamin D3 and transforming growth factor-beta strongly upregulates the 5-lipoxygenase pathway. Here, we describe an assay system for leukotriene biosynthesis inhibitors which is based on the in-vitro differentiation of HL-60 and Mono Mac 6 cells. Different leukotriene biosynthesis inhibitors like the nonredox type inhibitor ZM 230487, the redox type inhibitor BW A4C and the FLAP inhibitor MK886 were tested and the results were compared with an assay system based on normal human granulocytes. ZM 230487, BWA4C and MK886 showed similar potencies in these cell lines as compared to normal leukocytes. Thus, the in-vitro differentiation of HL-60 and Mono Mac 6 cells provides an excellent model for the screening of drugs affecting the 5-lipoxygenase pathway.     

New developments in hematopoietic stem cell expansion

In the present study we investigated the effects of various doses of gamma-irradiation, followed by induction of granulocytic differentiation with all-trans-retinoic acid (ATRA), on proliferative rate, differentiation capability and oxidative metabolism of leukaemic cells from two different myeloid leukaemia cell lines, HL-60 and PLB-985. Regarding the effects of such combined treatment on the proliferative capabilities of HL-60 and PLB-985 cell lines, we showed that their growth kinetics were similar after 2 Gy gamma-irradiation combined with ATRA. However, with doses >2 Gy, the behaviour of the cell lines differed largely. Indeed, HL-60 appeared to be more radiosensitive than PLB-985 regarding cell viability and proliferation. Besides, whatever dose of irradiation (2, 5 or 10 Gy) was applied, ATRA was still able to induce differentiation of HL-60 and PLB-985 into granulocytes that retained the capacity to produce superoxide anion. The results of these in vitro studies suggest that leukaemia cell lines retain their ability to respond to ATRA, a granulocytic-differentiating inducer following high doses of irradiation. This may have implications for the use of radiation therapy in combination with ATRA for the treatment of extramedullary infiltrations of myeloid leukaemias in humans.     

Stereoselective chiral inversion of pantoprazole enantiomers after separate doses to rats

Serglycin is the major proteoglycan in most hematopoietic cells, including monocytes and macrophages. The monoblastic cell line U937-1 was used to study the expression of serglycin during proliferation and differentiation. In unstimulated proliferating U937-1 cells serglycin mRNA is nonconstitutively expressed. The level of serglycin mRNA was found to correlate with the synthesis of chondroitin sulfate proteoglycan (CSPG). The U937-1 cells were induced to differentiate into different types of macrophage-like cells by exposing the cells to PMA, RA, or VitD3. These inducers of differentiation affected the expression of serglycin mRNA in three different ways. The initial upregulation seen in the normally proliferating cells was not observed in PMA treated cells. In contrast, RA increased the initial upregulation, giving a reproducible six times increase in serglycin mRNA level from 4 to 24 h of incubation, compared to a four times increase in the control cells. VitD3 had no effect on the expression of serglycin mRNA. The incorporation of (35S)sulfate into CSPG decreased approximately 50% in all three differentiated cell types. Further, the (35S)CSPGs expressed were of larger size in PMA treated cells than controls, but smaller after RA treatment. This was due to the expression of CSPGs, with CS-chains of 25 and 5 kDa in PMA and RA treated cells, respectively, compared to 11 kDa in the controls. VitD3 had no significant effect on the size of CSPG produced. PMA treated cells secreted 75% of the (35S)PGs expressed, but the major portion was retained in cells treated with VitD3 or RA. The differences seen in serglycin mRNA levels, the macromolecular properties of serglycin and in the PG secretion patterns, suggest that serglycin may have different functions in different types of macrophages.     

Simplified analysis of pathogenic leptospiral serovars by random amplified polymorphic DNA fingerprinting

Recent studies have suggested that platelet activating factor (PAF) plays an important role in various reproductive functions, including ovulation, implantation and parturition, and that the local concentration of PAF is modulated by PAF-acetylhydrolase (PAF-AH), a potent PAF inactivator. In this study, we investigated the possible effects of various bioactive substances, which are present at high concentrations in the human pregnant uterus, on PAF-AH secretion from decidual macrophages using a monocyte-macrophage model system, human myelocytic leukaemia cells (HL-60). By treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), HL-60 cells were transformed to macrophage-like cells, which secreted PAF-AH into the culture medium time- and dose-dependently. After treatment with 10(-8) M TPA, the effects of various substances on the secretion of PAF-AH were examined. Among the substances examined, cortisol and TGF-beta suppressed PAF-AH secretion from TPA-stimulated HL-60 cells in a significant and dose-dependent way. Endothelin, epidermal growth factor, and brain natriuretic peptide had no significant effect on PAF-AH secretion from TPA-stimulated HL-60 cells. These results suggest that local PAF concentrations in the pregnant uterus might be regulated, at least partly, by cortisol and TGF-beta; thus these substances may play a role in the initiation of parturition via regulation of local PAF concentrations.     

Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.     

Retinoic acid induced mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase-dependent MAP kinase activation needed to elicit HL-60 cell differentiation and growth arrest

Retinoic acid (RA) activated the extracellular signal-regulated kinase (ERK) 2 mitogen-activated protein kinase (MAPK) of HL-60 human myeloblastic leukemia cells before causing myeloid differentiation and cell cycle arrest associated with hypophosphorylation of the retinoblastoma (RB) tumor suppressor protein. ERK2 activation by mitogen-activated protein/ERK kinase (MEK) was necessary for RA-induced differentiation in studies using PD98059 to block MEK phosphorylation. G0 growth arrest and RB tumor suppressor protein hypophosphorylation (which is typically associated with induced differentiation and G0 arrest), two putatively RB-regulated processes, also depended on ERK2 activation by MEK. Activation of ERK2 by RA occurred within hours and persisted until the onset of RB hypophosphorylation, differentiation, and arrest. ERK2 activation was probably needed early, because delaying the addition of PD98059 relative to that of RA restored most of the RA-induced cellular response. In contrast to RA (which activates RA receptors (RARs) and retinoid X receptors in HL-60 cells with its metabolite retinoids), a retinoid that selectively binds RAR-gamma, which is not expressed in HL-60 cells, was relatively ineffective in causing ERK2 activation. This is consistent with the need for a nuclear retinoid receptor function in RA-induced ERK2 activation. RA reduced the amount of unphosphorylated RAR-alpha, whose activation is necessary for RA-induced differentiation and arrest. This shifted the ratio of phosphorylated:unphosphorylated RAR-alpha to predominantly the phosphorylated form. Unlike other steroid thyroid hormone receptors susceptible to phosphorylation and activation by MAPKs, RAR-alpha was not phosphorylated by the activated ERK2 MAPK. The results thus show that RA augments MEK-dependent ERK2 activation that is needed for subsequent RB hypophosphorylation, cell differentiation, and G0 arrest. The process seems to be nuclear receptor dependent and an early seminal component of RA signaling causing differentiation and growth arrest.