Prosomes (proteasomes) changes during differentiation are related to the type of inducer

The core of the 26S proteasome, the 20S prosome, is a highly organized multi-protein complex found in large amount in malignant cells. Differentiation of several cell lines, including the monoblastic U937 and the lymphoblastoid CCRF-CEM, is accompanied by a general decrease in the prosome concentration when phorbol-myrirtic-acetate (PMA) and retinoic acid plus dihydroxyvitamine D3 (RA+VD) are used. Incubation of U937 cells for three days with PMA or RA+VD causes differentiation, but the resulting patterns of prosome labeling in the cell and on the plasma membrane are not the same. In contrast, the same kind of prosome changes occur in U937 and CCRF-CEM cells when PMA is used as inducer. The intracellular distribution of prosomes is also linked to malignancy and differentiation. Prosomes are found in the nucleus and the cytoplasm of cancer cells; and treatment with RA+VD decreases the prosomes in the nucleus whereas PMA causes various prosome proteins changes. These results indicate that prosomes are important in cell regulation and in the expression of malignancy.     

Regulation of the growth and differentiation of a human monocytic cell line by lymphokines. I. Induction of superoxide anion production and chemiluminescence

The U937 human monocytic cell line was studied to determine its ability to generate a respiratory burst after stimulation with phorbol myristate acetate (PMA) or opsonized zymosan. U937 cells cultured in normal medium produced virtually no superoxide anion or chemiluminescence in response to either stimulus. In contrast, U937 cells cultured in medium containing soluble factors from activated lymphocytes produced significant O2- and chemiluminescence when stimulated with PMA or opsonized zymosan. The chemiluminescence in response to PMA was maximal in U937 cells precultured with these soluble factors for 3 days, whereas maximal responsiveness to opsonized zymosan was not observed until 5 to 6 days of lymphokine exposure. Although this ability to generate a respiratory burst persisted for a number of days in U937 cells that were subsequently recultured in normal medium, this responsiveness was gradually lost in the continued absence of these factors. The data indicate that the U937 monocytic cell line can be activated or induced to differentiate by soluble factors released by activated lymphocytes. In the process, these cells acquire the ability to generate a respiratory burst. The U937 cell line may serve as a useful model for the study of the ontogeny and regulation of the respiratory burst during human monocytic differentiation.     

Granulocyte-macrophage colony stimulating factor modulates the production of TNF alpha by differentiated U937 cells infected with Leishmania major

In this work, the production of tumor necrosis factor alpha (TNF alpha) during interaction of human phagocytes with the intracellular parasite Leishmania major was further investigated. The human monocytic cell line U937, differentiated with a combination of 1 alpha, 25 dihydroxyvitamin D3 (VD) and retinoic acid (RA), or with granulocyte macrophage colony stimulating factor (GM-CSF) was used. Differentiated U937 cells were infected with Leishmania major promastigotes, and TNF alpha was assayed in cell culture supernatants. It was found that the cytokine was produced only by U937 cells differentiated with VD/RA and further incubated with GM-CSF and LPS or interferon gamma (IFN gamma). L. major induced TNF alpha production only in the presence of GM-CSF. No direct relationship was found, however, between production of TNF alpha and resistance of differentiated U937 cells to infection with L. major.     

Serglycin expression during monocytic differentiation of U937-1 cells

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.      

Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.     

The cellular location and specificity of bacterial cytochrome c peroxidases.

We have found that an anti-CD11c monoclonal antibody (MAb) inhibits the respiratory burst induced in phorbol 12-myristate 13-acetate (PMA)-differentiated U937 cells as well as in human peripheral blood monocytes and neutrophils upon cell stimulation with concanavalin A. The MAb had no effect, however, when the added stimulus was fMet-Leu-Phe or PMA. Flow cytometry analyses indicated that concanavalin A was able to interact with CD11c. The anti-CD11c MAb inhibited significantly concanavalin A binding to differentiated U937 cells, and concanavalin A blocked binding of anti-CD11c MAb to the cells. Binding of labelled concanavalin A to membrane proteins which were separated by PAGE and transferred to nitrocellulose paper indicated that proteins with apparent molecular masses similar to those of CD11c (150 kDa) and CD18 (95 kDa) molecules were the main concanavalin A-binding proteins in differentiated U937 cells as well as in mature neutrophils. Similar experiments carried out in the presence of the anti-CD11c MAb showed a specific and significant inhibition of concanavalin A binding to the CD11c molecule. These results indicate that concanavalin A binds to the CD11c molecule and this binding is responsible for the concanavalin A-induced respiratory burst in PMA-differentiated U937 cells as well as in human mature monocytes and neutrophils.     

Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2•− excretion versus H2O2 diffusion

Abstract The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques. Differentiated U937 cells essentially produced extracellular superoxide anion (O₂^•−), whatever the stimulus used. Monocytes, however, responded to Salmonella typhimurium , phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively. Furthermore, H₂O₂ but not O₂^•− was detected in the extracellular oxidative response of monocytes. Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O₂^•−, providing sufficient concentrations of extracellular horseradish peroxidase were present. However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen differentiated U937 cells. Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O₂^•− in the extracellular medium while, within monocytes, O₂^•− is rapidly dismutated in H₂O₂ which can eventually diffuse outside the cell. Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells.     

Induction of NF-KB during monocyte differentiation by HIV type 1 infection

The production of human immunodeficiency virus type 1 (HIV-1) progeny was followed in the U937 promonocytic cell line after stimulation either with retinoic acid or PMA, and in purified human monocytes and macrophages. Electrophoretic mobility shift assays and Southwestern blotting experiments were used to detect the binding of cellular transactivation factor NF-KB to the double repeat-KB enhancer sequence located in the long terminal repeat. PMA treatment, and not retinoic acid treatment of the U937 cells acts in inducing NF-KB expression in the nuclei. In nuclear extracts from monocytes or macrophages, induction of NF-KB occurred only if the cells were previously infected with HIV-1. When U937 cells were infected with HIV-1, no induction of NF-KB factor was detected, whereas high level of progeny virions was produced, suggesting that this factor was not required for viral replication. These results indicate that in monocytic cell lineage, HIV-1 could mimic some differentiation/activation stimuli allowing nuclear NF-KB expression.     

The oxidative burst triggered by Salmonella typhimurium in differentiated U937 cells requires complement and a complete bacterial lipopolysaccharide

The bacterial and serum factors involved in the oxidative response triggered by Salmonella typhimurium in differentiated U937 cells were investigated. Complement activation was shown to be required, using sera deficient in complement factors. An original dot-blot technique was developed to study the activation of complement by either bacteria or purified lipopolysaccharide (LPS). Both O-specific and lipid A segments of LPS were found to play a role in the triggering of the oxidative response. Lipid A was responsible for bacterial C3-derived opsonization by inducing an antibody-independent activation of complement classical pathway, whereas O-specific polysaccharide chains (O-Ag) were involved in cellular activation. Inhibition experiments using anti-cell surface marker monoclonal antibodies showed the involvement of the α chain of CR3 (CD11b) in the oxidative response developed by differentiated U937 cells in response to S. typhimurium infection. Whether both iC3b and O-Ag interact with different domains of CR3 or whether the binding of O-Ag occurs via a not yet identified receptor remains to be determined.     

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.     

Induction of the oxidative response and of concanavalin A-binding capacity in maturing human U937 cells

Differentiation of U937 cells with phorbol myristate acetate (PMA) induces high stimulation by concanavalin A of the respiratory burst as well as an increase in concanavalin A-binding cell capacity. New concanavalin A-binding proteins are detected as differentiated U937 cells acquire their capacity to be activated by concanavalin A. We identified several concanavalin A-binding proteins, of molecular mass 30-200 kDa, in PMA-differentiated cells, but only some of them seem to be directly related to the concanavalin A effect on the respiratory burst. One of these candidates could be a glycoprotein with an apparent molecular mass of 140 kDa which behaved as a major concanavalin A-binding protein and is expressed on differentiated cells at the time these cells respond maximally to concanavalin A.     

Changes in the amount and distribution of prosomal subunits during the differentiation of U937 myeloid cells: high expression of p23K

Abstract. Prosomes (Proteasomes/Multicatalytic proteinase (MCP)-complexes) are protein particles built of 28 subunits in variable composition, having proteinase activity. We have studied the changes in prosomal subunits p29K, p31K and the highly expressed p23K during the differentiation of U937 cells. Control cells had little prosomal subunit p31K in the cytoplasm, while p29K antigen was detected in both the nucleus and cytoplasm; more p23K antigen was found in the cytoplasm than in the nucleus. Flow cytometry demonstrated a biphasic intracellular decrease in prosomes during differentiation induced by phorbol-myristic-acetate (PMA) and retinoic acid plus 1,25-dihydroxycholecalciferol (RA + VD). p23K and p29K decreased both in the cytoplasm and the nucleus of differentiated cells, though the p23K antigen was concentrated near vesicles and the plasma membrane in PMA-induced cells. The p31K antigens disappeared from RA + VD-induced cells, while in PMA-induced cells, cytoplasmic labelling was unchanged and nuclear labelling was increased. Small amounts of prosomal proteins p23K and p29K were found on the outer membrane of un-induced cells. While there was no labelling on the outer membrane of RA + VD-induced cells, p23K protein increased on the plasma membrane of PMA-induced cells. The prosome-like particle protein p21K was not present to any significant extent in the intracellular compartment of control or induced cells; however, p21K was detected on the outer surface of control cells and was increased only in PMA-induced cells. The culture medium of control and induced cells contained no p21K, p23K, p29K or p31K. RA + VD seemed to induce a general decrease of prosomal subunits within the cells and at the outer surface, whereas PMA caused a migration toward the plasma membrane and an increase at the outer surface. These changes in the distribution and type of prosomes in RA + VD- and PMA-induced cells indicate that prosomes may play a part in differentiation, especially p23K which is the most highly expressed protein among those studied and presents the more important changes.     

U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Modulation of CD157 expression in multi-lineage myeloid differentiation of promyelocytic cell lines

CD157/BST-1 is expressed on mature myeloid cells but not on their precursors in vivo. Also CD38, a homologous gene to CD157, is upregulated in promyelocytic HL-60 cells by the monocyte and granulocyte differentiation-inducing 1alpha,25dihydroxyvitamin D3 (VD3) and all-trans retinoic acid (ATRA), respectively. We have examined whether CD157 expression is upregulated when the promyeloid HL-60 and/or U937 cells are induced to differentiate into mature phenotypes in vitro. VD3 treatment irreversibly upregulated the expression of CD157 in HL-60 cells but not in U937 cells in a time- and concentration-dependent manner when analyzed by flow cytometry, immunoblotting and/or RT-PCR. Different monocyte and granulocyte lineage inducers induced CD157 expression to varying extents while the macrophage differentiation-inducing phorbol 12-myristate 13-acetate (PMA) induced its down-regulation. Time-kinetics of VD3 treatment of HL-60 cells showed that the appearance of CD157 and CD11b (a differentiation marker) antigens were not substantial up to 24 hours but increased subsequently although the appearance of CD38 became significant within 6 hours. Two-color staining of VD3-treated HL-60 cells displayed an apparently linear correlation between CD157 and CD11b expression. Dibutyryl cAMP (cAMP agonist) and forskolin (cAMP-increasing agent) augmented the VD3-dependent induction of CD157 and CD11b expression while PGE1 (cAMP-decreasing agent) inhibited it, suggesting the involvement of a cAMP-dependent mechanism in VD3-induced CD157 upregulation. Co-treatment of HL-60 cells with VD3 plus TNF-alpha or ara-C produced an additive effect on CD157 upregulation. The upregulated CD157 in the VD3-differentiated HL-60 cells was able to activate CD157-dependent tyrosine kinase signal when cross-linked with anti-CD157 antibody.     

Intracellular Ca2+ mobilization in immature and more mature U937 induced to differentiate by dimethyl sulfoxide or phorbol myristate acetate

Intracellular Ca2+ mobilization in U937 cells was studied. Stimulation of immature U937 cells with leukotriene B4 (LTB4) increased intracellular Ca2+ levels, whereas stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) failed to increase intracellular Ca2+ levels. U937 cells cultured with 1.5% dimethyl sulfoxide (DMSO) for 4 days (DMSO-U937 cells) responded to LTB4 and possessed the ability to respond to fMLP. U937 cells cultured with 1 ng/ml phorbol myristate acetate (PMA) for 4 days (PMA-U937 cells) lost the ability to respond to LTB4, although they responded to fMLP. Treatment of DMSO-U937 cells with 100 ng/ml PMA for 3 min suppressed intracellular Ca2+ increase induced by LTB4 and fMLP. The fMLP-induced Ca2+ rise in PMA-U937 cells was not suppressed by a further treatment with 100 ng/ml PMA. DMSO-U937 cells responded to inositol 1,4,5-trisphosphate (IP3), indicating that IP3 functions as a messenger of intracellular Ca2+ mobilization from endoplasmic reticulum in U937. The magnitude and duration of the rise in Ca2+ induced by IP3 in DMSO-U937 cells treated with 100 ng/ml PMA for 3 min were similar to those of the controls. When DMSO-U937 cells were Ca2+-depleted, addition of Ca2+ resulted in a transient overshoot of Ca2+ influx. However, the transient overshoot was not observed, when PMA-U937 cells were tested. These results indicate that Ca2+ efflux in PMA-U937 cells is increased by an activated exit pump, which may be directly or indirectly related to the functional state of PMA-U937 cells.     

CCL229细胞经诱导后蛋白激酶C及抑制剂活性的变化

检测以维甲酸(RA), 1, 25－二羟基维生素D₃(1,25(OH)₂VD₃)诱导2d、佛波酯（PMA）诱导6h的人大肠癌细胞CCL₂₂₉的蛋白激酶C（PKC）及其抑制剂活性．结果显示：诱导后PKC总活性升高(P<0.05); RA, 1, 25(OH)₂VD诱导引起细胞质PKC活性增加,PMA诱导后细胞膜PKC比率（细胞膜活性／总活性）显著升高(P<0.01);诱导后PKC抑制剂活性均降低,其中1, 25 (OH)₂VD₃组与对照组有显著差异(P<0.05);提示PMA引起PKC从细胞质向细胞膜转移,不同药物诱导后PKC及其抑制剂活性出现不同的相对均衡关系．     

The THP-1 cell line is a urokinase-secreting mononuclear phagocyte with a novel defect in the production of plasminogen activator inhibitor-2

Mononuclear phagocytes regulate the generation of plasmin by secreting urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-2 (PAI-2). We investigated the production of plasminogen activator (PA) and PA inhibitor by the human monocytic leukemia cell line, THP-1. Similar to U937 monoblast-like cells and peripheral blood monocytes (PBM), THP-1 cells produce a PA that is specifically neutralized by anti-uPA antibody and comigrates with human high molecular mass uPA (54 kDa) on casein-plasminogen zymogaphy. PA activity could be dissociated from intact THP-1 cells by brief treatment with a weak acid-glycine buffer, indicating that the uPA is secreted and bound to receptors on the plasma membrane. Regulation of uPA proceeds normally in THP-1 cells, with cell-associated PA activity increasing from 77 +/- 20 to 163 +/- 26 and 325 +/- 30 mPU/10(6) cells in response to PMA and LPS, respectively; parallel increases in steady state levels of uPA mRNA were observed. In contrast to normal expression of uPA activity, functional PAI-2 could not be demonstrated in either the conditioned media or cell lysates of THP-1 under basal or stimulated conditions. Both U937 and PBM secrete low levels of PA inhibitor activity that increase substantially in response to stimulation with PMA and LPS. Immunoreactive PAI-2, measured by ELISA, was undetectable in THP-1 lysates or conditioned medium, but was consistently present in U937 and PBM, paralleling the presence of PA inhibitor activity. THP-1 cells express low levels of an abnormally sized mRNA for PAI-2 and demonstrate a regulatory defect whereby steady state levels of PAI-2 mRNA are markedly reduced upon stimulation with PMA or LPS. By contrast, U937 and PBM respond to identical stimulation with increases in PAI-2 mRNA. We conclude that THP-1 cells express a structurally abnormal species of PAI-2 mRNA, with complete loss of inhibitory activity as well as altered function of PMA- and LPS-responsive regulatory elements.     

Enhancement of interferon-induced 2–5 oligoadenylate synthetase activity by retinoic acid in human histiocytic lymphoma U937 cells and WISH cells

The effect of retinoic acid (RA) on the level of interferon (IFN)-induced 2-5 oligoadenylate (2-5A) synthetase activity was examined in human histiocytic lymphoma U937 cells and WISH cells** in order to ascertain the role of this polymerase in interaction between IFNs and RA. Cultures containing both IFNs (1-100 U/ml) and RA (0.1-10 microM) consistently had higher levels of enzyme activity than corresponding cells treated with IFN alone and this was true for all three types of IFNs in both cell lines. The potentiating effect of RA was dose- and time-dependent and under optimal conditions, the induction of the synthetase was synergistic between IFN-beta (10-100 U/ml) and RA (0.1-10 microM). Furthermore, pretreatment (but not posttreatment) with RA followed by subsequent treatment with IFNs preferentially induced higher levels of enzyme activity in U937 cells but not in WISH cells. In addition, our results indicated that the modulating effect of RA on IFNs did not involve interaction at the receptor level and the level of enhancement of 2-5A synthetase activity was not in parallel with either cell-growth arrest or promotion of differentiation. Lastly, the present study raises the possibility that interactions between IFNs and RA, in either a synergistic or antagonistic manner, may be mediated through amplification of the 2-5A system.     

Role of CD14 expression in the differentiation-apoptosis switch in human monocytic leukemia cells treated with 1alpha,25-dihydroxyvitamin D3 or dexamethasone in the presence of transforming growth factor beta1.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.