Atromentin-induced apoptosis in human leukemia U937 cells.

In the course of screening for apoptotic substances that induce apoptosis in human leukemia U937 cells, a fungal strain, F000487, which exhibits potent inducible activity, was selected. The active compound was purified from an ethyl acetate extract of the microorganism by Sep-pak C18 column chromatography and HPLC, and was identified as atromentin by spectroscopic methods. This compound induced caspase-3 processing in human leukemia U937 cells. The caspase-3 and poly (ADP-ribose) polymerase (PARP) were induced by atromentin in a dose-dependent manner. Furthermore, DNA fragmentation was also induced by this compound in a dose-dependent manner. These results show that atromentin potently induces apoptosis in U937 cells and that atromentin-induced apoptosis is related to the selective activation of caspases.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

The broad-spectrum caspase inhibitor Boc-Asp-CMK induces cell death in human leukaemia cells.

Synthetic caspase inhibitors and particularly broad-spectrum caspase inhibitors can prevent cells from death or at least slow down cell death process and abrogate some apoptotic hallmarks [Kitanaka, C., Kuchino, Y., 1999. Caspase-independent programmed cell death with necrotic morphology. Cell Death and Differentiation 6, 508-515]. However, not all synthetic caspase inhibitors diminish cell death. We have found that the broad-spectrum caspase inhibitor Boc-Asp-CMK induced cell death at micromolar concentrations in human leukaemia cells. Interestingly, low concentrations of Boc-Asp-CMK induced cell death with apoptotic hallmarks. Increasing concentrations of Boc-Asp-CMK led to necrotic cell death. The switch between apoptosis and necrosis seemed to depend upon the degree of inhibition of executioner caspases, including caspase-3/7 with Boc-Asp-CMK. Interestingly, caspase-3 processing was not inhibited even for the highest concentration of Boc-Asp-CMK used. We assume, that toxic properties of Boc-Asp-CMK can be attributed to the chloromethylketone residuum in its molecule, as its analogue Boc-Asp-FMK with fluoromethylketone residuum was more than 13 times less toxic. Our results further indicated that toxicity of Boc-Asp-CMK might arise from its interference with mitochondrial metabolism.

Purification, characterization and biological activities of a garlic oligosaccharide.

A novel oligosaccharide was purified from garlic (Allium sativum L.) bulbs via hot water extraction, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The molecular weight of the oligosaccharide was determined to be 1800. A nuclear magnetic resonance (NMR) study showed that ten fructose molecules were connected by beta1-2 linkage to a terminal glucose. The oligosaccharide had cytotoxic activities against human malignant lymphoma cells (U937) and colon adenocarcinoma cells (WiDr) in vitro. Furthermore, this oligosaccharide significantly suppressed the growth of murine colon adenocarcinoma cells (colon 26) in vivo. The oligosaccharide also stimulated interferon-gamma production by human peripheral blood lymphocyte in vitro, indicating that it may activate the immunological pathways and suppress the growth of tumors in vivo.

Involvement of p38 mitogen-activated protein kinase in E. coli-induced U937 apoptosis.

OBJECTIVE: To investigate whether the effect of E. coli on U937 cell lines apoptosis is mediated via p38 mitogen-activated protein kinase (MAPK) activation. METHODS: The U937 cell lines were treated with E. coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry. p38 activities were detected by Western blotting. RESULTS: E. coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E. coli induced apoptosis in U937 cells. CONCLUSION: The activation of the p38 MAPK in U937 cell lines by E. coli is a major pathway to mediate the apoptosis.

Different fates of intracellular glutathione determine different modalities of apoptotic nuclear vesiculation.

U937 monocytic cells show two main apoptotic nuclear morphologies, budding and cleavage, that are the result of two independent morphological routes, since they never interconvert one into the other, and are differently modulated by stressing or physiological apoptogenic agents [Exp Cell Res 1996; 223:340-347]. With the aim of understanding which biochemical alterations are at the basis of these alternative apoptotic morphologies, we performed an in situ analysis that showed that in U937 cells intracellular glutathione (GSH) is lost in cells undergoing apoptosis by cleavage, whereas it is maintained in apoptotic budding cells. Lymphoma cells BL41 lose GSH in apoptosis, and show the cleavage nuclear morphology; the same cells latently infected with Epstein Barr Virus (E2r line) undergo apoptosis without GSH depletion and show the budding nuclear morphology. GSH depletion is not only concomitant to, but is the determinant of the cleavage route, since the inhibition of apoptotic GSH efflux with cystathionine or methionine shifts the apoptotic morphology from cleavage to budding. Accordingly, cystathionine or methionine antagonizes apoptosis in the all-cleavage BL41, without affecting the all-budding E2r.

Modulation of the p38 MAPK (mitogen-activated protein kinase) pathway through Bcr/Abl: implications in the cellular response to Ara-C.

The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.

Pharmacologic mitogen-activated protein/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase inhibitors interact synergistically with STI571 to induce apoptosis in Bcr/Abl-expressing human leukemia cells.

Interactions between the kinase inhibitor STI571 and pharmacological antagonists of the mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) cascade have been examined in human myeloid leukemia cells (K562 and LAMA 84) that express the Bcr-Abl kinase. Exposure of K562 cells to concentrations of STI571 that minimally induced apoptosis (e.g., approximately 200 nM) resulted in early suppression (i.e., at 6 h) of p42/44 MAPK phosphorylation followed at later intervals (i.e., > or =24 h) by a marked increase in p42/44 MAPK phosphorylation/activation. Coadministration of a nontoxic concentration of the MEK1/2 inhibitor PD184352 (5 microM) prevented STI571-mediated activation of p42/44 MAPK. Cells exposed to STI571 in combination with PD184352 for 48 h demonstrated a very dramatic increase in mitochondrial dysfunction (e.g., loss of DeltaPsim and cytosolic cytochrome c release) associated with procaspase-3 activation, poly(ADP-ribose) polymerase cleavage, and the appearance of the characteristic morphological features of apoptosis. Similar results were obtained using other pharmacological MEK1/2 inhibitors (e.g., PD 98059 and U0126) as well as another leukemic cell line that expresses Bcr-Abl (e.g., LAMA 84). However, synergistic induction of apoptosis by STI571 and PD184352 was not observed in human myeloid leukemia cells that do not express the Bcr-Abl kinase (e.g., HL-60 and U937) nor in normal human peripheral blood mononuclear cells. Synergistic potentiation of STI571-mediated lethality by PD184352 was associated with multiple perturbations in signaling and apoptotic regulatory pathways, including caspase-dependent down-regulation of Bcr-Abl and Bcl-2; caspase-independent down-regulation of Bcl-x(L) and Mcl-1; activation of JNK, p38 MAPK, and p34(cdc2); and diminished phosphorylation of Stat5 and CREB. Significantly, coexposure to PD184352 strikingly increased the lethality of a pharmacologically achievable concentration of STI571 (i.e., 1-2 microM) in resistant K562 cells expressing marked increases in Bcr-Abl protein levels. Together, these findings raise the possibility that treatment of Bcr-Abl-expressing cells with STI571 elicits a cytoprotective MAPK activation response and that interruption of the latter pathway (e.g., by pharmacological MEK1/2 inhibitors) is associated with a highly synergistic induction of mitochondrial damage and apoptosis. They also indicate that in the case of Bcr-Abl-positive cells, simultaneous interruption of two signal transduction pathways may represent an effective antileukemic strategy.

Block of granulocytic differentiation of 32Dcl3 cells by AML1/ETO(MTG8) but not by highly expressed Bcl-2.

The chimeric gene, AML1/ETO (MTG8), generated in t(8;21) acute myeloid leukemia enhances the expression of Bcl-2. To evaluate whether this enhancement is the primary role of AML1/ETO in leukemogenesis, effects of over-expression of Bcl-2 in the murine myeloid precursor cell line, 32Dcl3, were examined. When 32Dcl3 cells expressing exogenous Bcl-2 were induced to differentiate, the onset of morphological differentiation was delayed. However, even the cells expressing very high levels of exogenous Bcl-2 eventually underwent differentiation without a significant decrease in the synthesis of Bcl-2. On the contrary, 32Dcl3 cells stably expressing AML1/ETO were completely resistant to differentiation and continued to grow in the presence of G-CSF. These results are consistent with the interpretation that stimulation of Bcl-2 expression is not the primary target of AML1/ETO.

Induction of apoptosis in human hematopoietic U937 cells by granulocyte-macrophage colony-stimulating factor: possible existence of caspase 3-like pathway.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induced apoptosis in human hematopoietic U937 cells by itself and in a synergistic manner with tumor necrosis factor (TNF). GM-CSF-induced apoptosis was not inhibited by caspase inhibitors YVAD-CMK, DEVD-CHO and z-VAD-FMK, under the condition that these inhibitors potently suppressed TNF-induced apoptosis. Both GM-CSF and TNF induced caspase 3-like activity in this cell line though the time course was distinct between two cytokines, and combined stimulation of cells with GM-CSF plus TNF induced additive or synergistic activation of caspase 3-like activity. Amount of immunoreactive cleaved forms of caspase 3 recognized by specific antibody was completely dissociated with its enzymatic activity when the cells were stimulated with GM-CSF, but not with TNF. These results indicate that GM-CSF induces apoptosis of U937 cells via unknown pathway, which seems to be mediated by caspase 3-like activity, yet not caspase 3 itself, resistant to the caspase inhibitors, and synergistically interacts with conventional caspase 3 pathway of TNF. Possible involvement of caspases 1 and 8 (-like activity) but not caspase 7 in this pathway was also suggested.

Mechanisms of resistance to apoptosis in human AML blasts: the role of differentiation-induced perturbations of cell-cycle checkpoints.

Alterations in the response of leukaemic cells to apoptosis-inducing stimuli may account for resistance to chemotherapy and treatment failure, either by disruption of the apoptotic pathway itself or by altered DNA repair; quiescent cells and those with disrupted cell-cycle checkpoints may also display decreased apoptosis. Quiescence can be induced by the differentiation of myeloid cells, and this led us to investigate whether the modulation of drug-induced apoptosis associated with differentiation might be a model for quiescence-associated resistance generally. We have demonstrated that resistance to idarubicin-induced apoptosis increased with greater duration of incubation of HL60 and U937 cells with ATRA and 1,25(OH)2 D3 and that this protective effect correlated with the degree of G0/G1 accumulation. In addition, the cytoprotective effects held for other classes of cytotoxic drugs with different mechanisms of action to idarubicin. Prolonged exposure to idarubicin or vinblastine was associated with diminution of the protective effect and re-entry of cells into cycle. The full cytoprotective effect was restored by resupplementation with ATRA or 1,25(OH)2 D3 during exposure to idarubicin, with concomitant persistence of G0/G1 accumulation. Differentiating agents prevented the accumulation of leukaemic cells at the G2/M checkpoint in response to low concentrations of idarubicin. Understanding how differentiating agents modulate these cell-cycle checkpoints, and how quiescent cells evade apoptosis, may allow the development of therapeutic strategies to limit such apoptosis-inhibiting effects and maximise cell kill from chemotherapy.

Purified recombinant human prosaposin forms oligomers that bind procathepsin D and affect its autoactivation.

Before delivery to endosomes, portions of proCD (procathepsin D) and proSAP (prosaposin) are assembled into complexes. We demonstrate that such complexes are also present in secretions of cultured cells. To study the formation and properties of the complexes, we purified proCD and proSAP from culture media of Spodoptera frugiperda cells that were infected with baculoviruses bearing the respective cDNAs. The biological activity of proCD was demonstrated by its pH-dependent autoactivation to pseudocathepsin D and that of proSAP was demonstrated by feeding to saposin-deficient cultured cells that corrected the storage of radioactive glycolipids. In gel filtration, proSAP behaved as an oligomer and proCD as a monomer. ProSAP altered the elution of proCD such that the latter was shifted into proSAP-containing fractions. ProSAP did not change the elution of mature cathepsin D. Using surface plasmon resonance and an immobilized biotinylated proCD, binding of proSAP was demonstrated under neutral and weakly acidic conditions. At pH 6.8, specific binding appeared to involve more than one binding site on a proSAP oligomer. The dissociation of the first site was characterized by a K(D1) of 5.8+/-2.9x10(-8) M(-1) (calculated for the monomer). ProSAP stimulated the autoactivation of proCD and also the activity of pseudocathepsin D. Concomitant with the activation, proSAP behaved as a substrate yielding tri- and disaposins and smaller fragments. Our results demonstrate that proSAP forms oligomers that are capable of binding proCD spontaneously and independent of the mammalian type N-glycosylation but not capable of binding mature cathepsin D. In addition to binding proSAP, proCD behaves as an autoactivable and processing enzyme and its binding partner as an activator and substrate.

Induction of differentiation of human myeloid leukemia cells by immunosuppressant macrolides (rapamycin and FK506) and calcium/calmodulin-dependent kinase inhibitors.

OBJECTIVE: Potent immunosuppressants, such as rapamycin, FK506, and ascomycin, are known to regulate the phosphorylation of proteins. The purpose of this study was to investigate the effects of these immunosuppressants on differentiation of several human myeloid leukemic cell lines. MATERIALS AND METHODS: Human myeloid leukemic cell lines were cultured with each immunosuppressant, and several differentiation markers were assayed. RESULTS: Rapamycin effectively induced granulocytic differentiation of human myeloid leukemic HL-60 and ML-1 cells. In addition to morphologic differentiation, it also induced nitroblue tetrazolium reduction, lysozyme activity, and expression of CD11b in HL-60 cells. The commitment to differentiation was observed after treatment with rapamycin for 1 day, indicating that the effect of rapamycin was irreversible. FK506 and ascomycin induced differentiation of HL-60 cells, but at higher concentrations than rapamycin. A calcium/calmodulin-dependent kinase (CaMK) was copurified with FKBP52 immunophilin, a binding protein of immunosuppressants. We also found that the CaMK inhibitors KN62 and KN93 induced differentiation of HL-60 cells. Rapamycin and CaMK inhibitors induced differentiation of human myeloid leukemia ML-1 and K562, but not of other cell lines such as NB4, U937, or HEL. CONCLUSION: Immunosuppressants and CaMK inhibitors induced differentiation of HL-60, ML-1, and K562 cells.

The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic endothelial cells.

Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease (CVD). One proposed mechanism for the beneficial effect of carotenoids is through functional modulation of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell (HAEC) cultures (passages 4-8) for 24 h with each of the five most prevalent carotenoids in human plasma, which are alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 micromol/l. Carotenoids were solubilized in 0.7% (v/v) tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate solubilities in aqueous medium, final concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 micromol/l and monolayers accumulated 647, 158, 7, 113, and 9 pmol/mg protein, respectively. Monolayers were then stimulated with IL-1beta (5 ng/ml) for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay (ELISA). To assess endothelial cell adhesion to monocytes, IL-1beta-stimulated monolayers were incubated for 10 min with 51Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with beta-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13%, respectively. Pre-incubation with beta-carotene and lutein significantly reduced E-selectin expression by 38 and 34%, respectively. Pre-treatment with beta-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18%, respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1beta-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25%, respectively. Thus, among the carotenoids, lycopene appears to be most effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface.

Parvovirus H-1-induced cell death: influence of intracellular NAD consumption on the regulation of necrosis and apoptosis.

The autonomous parvovirus H-1 exerts tumor-suppressive effects in living organisms and has been shown to specifically interfere with the survival of transformed cells in culture. The mechanism(s) by which H-1 virus induces death of transformed cells is not yet well understood. It has recently been reported that H-1 virus induces apoptotic cell death in the human monocytic U937 cell line, as assessed by biochemical and morphological changes of infected cells (Rayet, B., Lopez-Guerrero, J.-A., Rommelaere, J., Dinsart, C., 1998. Induction of programmed cell death by parvovirus H-1 in U937 cells: connection with the TNFalpha signalling pathway. J. Virol. 72, 8893-8903). Here we show that parvovirus H-1 infection induced early biochemical changes pointing to apoptotic events also in the transformed human keratinocyte cell line, HeLa, and the transformed rat fibroblast cell line, P1. Morphologic changes, however, and in particular the early breakdown of plasma membrane integrity, suggested that apoptosis did not go to completion, leading to necrotic cell death as the major result of parvovirus infection of HeLa and P1 cells. Parvovirus infection of these, and to a significantly lesser extent of U937 cells, was accompanied by rapid depletion of intracellular NAD stores. Inhibition of NAD-consuming enzymes interfered with parvovirus-induced NAD depletion and increased the proportion of H-1 virus-infected cells displaying apoptotic features of cell death. In contrast, a similar prevention of NAD depletion through stimulation of NAD production had little influence on the cell death pathway, suggesting that NAD-consuming enzymes may promote necrosis in a direct way rather than through inducing the overall drop of intracellular NAD.

Induction of type 1 programmed cell death in U937 cells by the antioxidant, butylated hydroxy-toluene or the free radical spin trap, NTBN.

Oxidative stress can initiate programmed cell death and contributes to the patho-physiology of a number of diseases. Low micromolar to millimolar concentrations of various antioxidants or free radical scavengers promote cell growth and reduce cellular suicide induced by several functionally distinct agents, including some known to produce oxidative stress. Severe anoxia or inhibitors of oxidative phosphorylation also initiate programmed cell death. These results seem paradoxical. In order to compare the response of U937 monoblastoid cells to higher concentrations of an antioxidant or a free radical-spin trap, cells were cultured with 20-80 microM concentrations of butylated hydroxy-toluene or with 5 to 60 mM concentrations of the free radical spin trap, N-tertiary butyl phenyl-nitrone. At these concentrations, both agents inhibited cellular proliferation and induced oligosomic DNA, detected by its 'laddering' after electrophoresis on agarose, confirmed by TUNEL (BHT) or flow cytometric (NTBN) evidence of hypodiploid DNA and ultrastructural evidence of a type 1 programmed cell death. The ability of hydroxy-toluenes to oxidize DNA and promote carcinogenesis and whether free radical spin traps could augment or interfere with the response of malignantly transformed cells to chemotherapy or ionizing radiation provide the raison d'etre of these studies.

A G-quadruplex-interactive agent, telomestatin (SOT-095), induces telomere shortening with apoptosis and enhances chemosensitivity in acute myeloid leukemia.

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is up-regulated in the vast majority of human neoplasias but not in normal somatic tissues. Therefore, the telomerase complex represents a promising universal therapeutic target in cancer. Telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to inhibit telomerase activity. We examined G-quadruplex interactive agent, telomestatin (SOT-095), for its ability to inhibit the proliferation of human leukemia cells, including freshly obtained leukemia cells. Telomere length was determined by either the terminal restriction fragment method or flow-FISH, and apoptosis was assessed by flow cytometry. Moreover, chemosensitivity was examined in telomestatin-treated U937 cells before ultimate telomere shortening. Treatment with telomestatin reproducibly inhibited telomerase activity in U937 and NB4 cells followed by telomere shortening. Enhanced chemosensitivity toward daunorubicin and cytosine-arabinoside was observed in telomestatin-treated U937 cells, before ultimate telomere shortening. Telomere shortening associated with apoptosis by telomestatin was evident in some freshly obtained leukemia cells from acute myeloid leukemia patients, regardless of sub-types of AML and post-myelodysplasia AML. These results suggest that disruption of telomere maintenance by telomestatin limits the cellular lifespan of AML cells, as well. However, in a minority of AML patients apoptosis was not evident, thus indicating that resistant mechanism might exist in some freshly obtained AML cells. Therefore, further investigation of telomestatin as a therapeutic agent is warranted.

The leukocyte integrins are regulated by transcriptional and post-transcriptional mechanisms in a leukemic cell that overexpresses protein kinase C-zeta.

Overexpression of protein kinase C-zeta (PKC-zeta) in the leukemic myeloid cell line U937 (U937-PKC-zeta cells), previously shown to induce leukemic cell differentiation, resulted in nearly complete downregulation of leukocyte integrins CD11a, CD11b, CD11d, and CD18, but not CD11c from the cell surface. The steady-state level of mRNAs for the downregulated leukocyte integrins was not detectable by Northern analysis. Nuclear run-on analysis revealed that transcription of all the leukocyte integrin genes except CD11c was reduced 70-90% as compared to control U937-Vector cells [U937 cells transfected with the empty vector pSV2M(2)6]. Transfection analysis of CD11-promoter-luciferase constructs confirmed that transcription of the leukocyte integrin genes was drastically downregulated in U937-PKC-zeta cells. The two c-jun binding sites in the CD11c promoter were essential for continued expression of CD11c in U937-PKC-zeta cells. Additionally, the 3' untranslated region (3' UTR) from CD11b, when fused to the luciferase gene, lead to the destabilization of this chimeric mRNA in U937-PKC-zeta cells. This indicates that downregulation of CD11b expression in U937-PKC-zeta cells is also the result of reduced stability of CD11b mRNA. Thus, overexpression of PKC-zeta in U937 cells leads not only to leukemic cell differentiation, but also to differential regulation of the leukocyte integrins.

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells.

To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0 degrees C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca2+ concentration ([Ca2+]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The effects of hyperthermia on LPO and [Ca2+]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (delta psi m) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca2+ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamin E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca2+]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca2+]i arising from increased expression of IP3R1 and LPO. Additional increase in [Ca2+]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.

Differential effects of retinoids on nitric oxide production by promonocytic U937 cells and ZR-75-1 human breast cancer cells.

We demonstrated the presence of inducible and endothelial nitric oxide synthases in histiocytic lymphoma U937 cells by staining with anti-iNOS and anti-eNOS antibodies. We also investigated the effects of retinol and retinoic acid on nitric oxide production by both U937 cells and ZR-75-1 human breast cancer cells. U937 cells which had been treated with either retinol or retinoic acid (10-10-10-6 M) exhibited no significant difference in nitric oxide secretion into conditioned medium. Conversely, for ZR-75-1 cells, both retinol and retinoic acid (10-10-10-6 M) caused a significant (p<0.001) increase in the amount of nitrite secreted. Our results indicate that retinoid induced growth inhibition of breast cancer cells is associated with an increase in NO production, however, an increase in NO synthesis does not mediate retinoid induced differentiation of U937 cells.