HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination.

An ATM-dependent cellular signal, a DNA-damage response, has been shown to be involved during infection of human immunodeficiency virus type-1 (HIV-1), and a high incidence of malignant tumor development has been observed in HIV-1-positive patients. Vpr, an accessory gene product of HIV-1, delays the progression of the cell cycle at the G2/M phase, and ATR-Chk1-Wee-1, another DNA-damage signal, is a proposed cellular pathway responsible for the Vpr-induced cell cycle arrest. In this study, we present evidence that Vpr also activates ATM, and induces expression of gamma-H2AX and phosphorylation of Chk2. Strikingly, Vpr was found to stimulate the focus formation of Rad51 and BRCA1, which are involved in repair of DNA double-strand breaks (DSBs) by homologous recombination (HR), and biochemical analysis revealed that Vpr dissociates the interaction of p53 and Rad51 in the chromatin fraction, as observed under irradiation-induced DSBs. Vpr was consistently found to increase the rate of HR in the locus of I-SceI, a rare cutting-enzyme site that had been introduced into the genome. An increase of the HR rate enhanced by Vpr was attenuated by an ATM inhibitor, KU55933, suggesting that Vpr-induced DSBs activate ATM-dependent cellular signal that enhances the intracellular recombination potential. In context with a recent report that KU55933 attenuated the integration of HIV-1 into host genomes, we discuss the possible role of Vpr-induced DSBs in viral integration and also in HIV-1 associated malignancy.

Tyrosine phosphorylation and intracellular alkalinization are early events in human neutrophils stimulated by tumor necrosis factor, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor.

Tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte CSF (G-CSF) primed human neutrophils for enhanced release of superoxide in time- and dose-dependent manners. The priming effects of these cytokines were detected at 3 min and maximal at 10 min of preincubation. The potency of the maximal effect was TNF > GM-CSF > G-CSF. Exposure of human neutrophils to TNF, GM-CSF and G-CSF resulted in tyrosine phosphorylation of a 42-kDa protein and intracellular alkalinization in a dose-dependent manner. The dose-response curves for triggering of tyrosine phosphorylation and intracellular alkalinization by each cytokine were similar to those for priming the cells. The potency of the maximal effect on tyrosine phosphorylation was TNF > GM-CSF > G-CSF, whereas that on intracellular alkalinization was GM-CSF > TNF > G-CSF. Tyrosine phosphorylation was detected at 3 min and maximal at 5-10 min after stimulation with each cytokine. Tyrosine phosphorylation induced by TNF declined at 20-40 min, whereas that induced by GM-CSF or G-CSF was maintained for at least 40 min. Intracellular alkalinization induced by each cytokine required a lag time of 3-5 min and was sustained for at least 40 min. Tyrosine phosphorylation preceded or occurred concomitantly with intracellular alkalinization and priming of the cells. These findings indicate that tyrosine phosphorylation and intracellular alkalinization are early events in human neutrophils stimulated by TNF, GM-CSF and G-CSF, and that these early events may, at least in part, mediate activation or priming of human neutrophils by these cytokines.

Activation of the respiratory burst and tyrosine phosphorylation of proteins in human neutrophils: no direct relationship and involvement of protein kinase C-dependent and -independent signaling pathways.

Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.

Bcl-2 down-regulation causes autophagy in a caspase-independent manner in human leukemic HL60 cells.

To understand the roles of bcl-2 for the survival of leukemic cells, we constructed human leukemic HL60 transformant lines in which full length bcl-2 antisense message was conditionally expressed by a tetracycline-regulatable expression system. Cell growth was completely inhibited after antisense message induction and massive cell death was induced. Electron microscopic examinations show that cells died by autophagy, but not by apoptosis. The morphology and the function of mitochondria remained intact: neither the reduction in mitochondrial membrane potential nor the nuclear translocation of AIF, a mitochondrial protein that translocates to nuclei in cases of apoptosis, was observed. Caspase inhibitors did not rescue bcl-2-antisense-mediated autophagy. Thus, bcl-2 is essential for leukemic cell survival and its down-regulation results in autophagy. Cell Death and Differentiation (2000) 7, 1263 - 1269.

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.

CREB antisense oligonucleotides induce non-apoptotic cell death in proliferating leukemia cells, but not normal hematopoietic cells, by a bizarre non-antisense mechanism.

We report that antisense phosphorothioate oligodeoxyribonucleotides (PS-ODNs) against cyclic AMP response element-binding protein (CREB) induce the death of human leukemia cell lines including HL-60, Kasumi-1 and K562, OCI-AML1a and also primary leukemia cells isolated from patients with acute myelocytic leukemia and chronic myelocytic leukemia in blastic crisis. In contrast, normal human bone marrow CD34+ cells and normal peripheral blood lymphocytes were resistant to the antisense-mediated cell death. We found that antisense-treated HL-60 cells had prominent nuclear fragmentations but lacked apoptotic features including internucleosomal DNA cleavage and TUNEL positivity. Cell cycle analysis demonstrated a remarkable reduction in G1 phase population along with a mild accumulation of S phase and good preservation of G2/M phase, indicating cells died at G2/M without cycling into G1 phase. None of the sense-sequenced PS-ODNs induced cell death. Further, neither the expression nor the message of CREB protein was reduced by antisense treatment, indicating that cell death was mediated by a non-antisense mechanism. On the other hand, no consensus oligonucleotide sequence for cell death induction was detected. Rather, we found a good correlation between the melting temperatures and the anti-proliferative activities of the oligonucleotides. Thus, CREB antisense PS-ODNs selectively induce a non-apoptotic cell death in leukemic cells by an unknown hybridization-dependent mechanism.

Apoptosis/differentiation-inducing effects of vitamin K2 on HL-60 cells: dichotomous nature of vitamin K2 in leukemia cells.

We originally reported that vitamin K2 (VK2) analogs, including menaquinone 4 (MK4) but not vitamin K1, effectively induce apoptosis in various types of primary cultured leukemia cells and leukemia cell lines in vitro. It has also been reported by others that VK2 showed the differentiation-inducing activity in leukemia cell lines. To investigate the discrepancy between apoptosis- and differentiation-inductions of leukemia cells by VK2 treatment, we used bcl-2 gene transfected HL-60 cells (HL-60-bcl-2) which resulted in five-fold over-expression of BCL-2 protein, and then compared the effects of MK4 to the control HL-60-neo cells. Seventy-two hours of exposure to various concentrations of MK4 resulted in growth inhibition of these cells in a dose-dependent manner (0.1-50 microM), however, HL-60-bcl-2 was less sensitive against MK4. MK4 potently induced apoptosis of HL-60-neo cells along with the depolarization of mitochondrial membrane potential and caspase-3 activation. Notably, HL-60-bcl-2 was almost completely resistant to apoptosis induction in response to MK4, although cell growth inhibition was still observed. In spite of the abrogation of apoptosis induction, about 90% of HL-60-bcl-2 cells were arrested in the G0/G1 phase within 48 h of exposure to 10 microM of MK4 accompanied by up-modulation of p27KIP1 expression. Concomitantly, HL-60-bcl-2 cells underwent monocytic differentiation. These data suggest that VK2 also shows the differentiation inducing effects on leukemia cells which are resistant against VK2-inducing apoptosis. The dichotomous nature of VK2 against leukemia cells appears to have clinical benefits for the treatment of patients with leukemias and myelodysplastic syndromes.

Activation of extracellular signal-regulated kinases and CREB/ATF-1 mediate the expression of CCAAT/enhancer binding proteins beta and -delta in preadipocytes.

The essential role of CCAAT/enhancer binding proteins (C/EBPs) beta and delta for adipocyte differentiation has been clearly established. In preadipocytes, their expression is up-regulated by the activation of leukemia inhibitory factor receptor (LIF-R) and prostacyclin receptor (IP-R) via the extracellular signal-regulated kinase (ERK) pathway and cAMP production, respectively. However, the molecular mechanisms by which LIF and prostacyclin-induced signals are propagated to the nucleus and the transcription factors mediating ERK and cAMP-induced C/EBP gene expression were unknown. Here we report that both pathways share cAMP responsive element binding protein/activation transcription factor 1 (CREB/ATF-1) as common downstream effectors. LIF-R and IP-R activation induced binding of CREB and/or ATF-1 to C/EBP promoters and CREB-dependent transcription. Expression of dominant negative forms of CREB dramatically reduced the LIF- and prostacyclin-stimulated C/EBP beta and C/EBP delta expression. Upon stimulation of the IP-R, the ERK pathway was activated in a PKA-dependent manner. ERK activation by the PKA pathway was not required for CREB/ATF-1 phosphorylation but rather was necessary for CREB-dependent up-regulation of C/EBPs expression. Our findings suggest that ERK activation is required for CREB transcriptional activity, possibly by recruitment of a coactivator.

Oxidative stress as a necessary factor in room temperature-induced apoptosis of HL-60 cells.

HL-60 cells undergo apoptosis when placed at room temperature (RT) [Shimura et al. (1997) FEBS Lett. 417, 379-384]. We report that superoxide anion radical, one of the reactive oxygen species (ROS), was produced after RT treatment. Affinity blot analysis with a biotinylated YVAD-CHO detected the generation of processed peptides with molecular masses of 15-25 kDa. Activation of such an ICE-like protease was completely abolished by N-acetylcysteine and exogenously expressed Bcl-2, known as antioxidants. We concluded that oxidative stress was a critical factor in the signal cascade of the apoptosis. Western blot analysis and experiments using tetrapeptide inhibitors suggested that caspases-1, -3, -4, -6, and -9 did not have an essential role in the apoptotic cascade. It is interesting that cyclosporin A (CsA) blocked RT-induced apoptosis with an inhibition of cytochrome c release from mitochondria. CsA, however, generated a significant amount of ROS with considerable reduction of mitochondrial membrane potential, implying that oxidative stress was one necessary factor for RT-induced apoptosis. It is also likely that mitochondrial membrane potential and the release of apoptotic factors from cytoplasm are differently regulated. Taken together with the reports that some Burkitt lymphoma cells showed apoptosis when exposed at low temperature followed by rewarming, and that hepatocytes or liver endothelial cells are susceptible to cold-induced apoptosis through the ROS function, we propose that studying the mechanism of RT-induced apoptosis of HL-60 cells may provide a therapeutic strategy for pathological conditions involving ROS, such as neurodegenerative diseases and ischemia.

Stimulation and priming of human neutrophils by IL-1 alpha and IL-1 beta: complete inhibition by IL-1 receptor antagonist and no interaction with other cytokines.

Together, interleukin-1 alpha (IL-1 alpha) and IL-1 beta primed human neutrophils for enhanced release of superoxide (O2-) stimulated by chemotactic peptide, chemokine, and plant lectin, and alone, each triggered O2- release in a dose-dependent manner. The maximal priming and triggering effect was obtained by high concentrations (50 to 500 ng/mL) of IL-1 alpha or IL-1 beta, though IL-1 beta was more effective than IL-1 alpha at suboptimal concentrations. Priming effect of IL-1 was very rapid and maximal within 10 minutes, whereas O2- release triggered by IL-1 was gradual and continued for 90 to 120 minutes. Combined stimulation of human neutrophils with IL-1 plus granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage CSF (GM-CSF) resulted in additive priming effect, and combined stimulation of neutrophils with IL-1 plus G-CSF, GM-CSF, or tumor necrosis factor (TNF) resulted in additive triggering effect, even when the maximal concentration of each cytokine was used. These priming and triggering effects of IL-1 alpha and IL-1 beta on the respiratory burst in human neutrophils were completely inhibited by IL-1 receptor antagonist (IL-1ra). Furthermore, only the net effect of IL-1 was inhibited by IL-1ra, even when human neutrophil was stimulated with IL-1 plus other cytokines to release O2-. Present results indicate that IL-1 does stimulate the respiratory burst activity in human neutrophils via receptor-mediated mechanism and suggest that the post-IL-1-receptor signaling pathways linked to the activation system of the respiratory burst are independent from those utilized by other cytokines in human neutrophils.

Increased respiratory burst activity of neutrophils in patients with aplastic anemia: effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor.

The superoxide (O2-)-releasing capacity in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) and the priming effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on FMLP-induced O2-release were investigated in neutrophils from 13 patients with aplastic anemia (AA). The O2(-)-releasing capacity of AA neutrophils (0.85 +/- 0.36 nmol/5 min/1 x 10(5) cells, n = 13) was significantly (p < 0.01) increased as compared with that of normal neutrophils (0.24 +/- 0.12 nmol/5 min/1 x 10(5) cells, n = 17). There was no close relationship between the O2(-)-releasing capacity and the peripheral blood neutrophil count or the plasma concentration of C-reactive protein. The plasma concentrations of G-CSF and GM-CSF were not elevated to the detectable levels (< 0.1 ng/ml and < 0.2 ng/ml, respectively) in all patients tested. FMLP-induced O2(-)-release was further enhanced by pretreatment of cells with rhG-CSF or rhGM-CSF for 10 min at 37 degrees C, except that no significant priming by rhG-CSF was observed in five patients. The priming effect of rhGM-CSF was consistently greater than that of rhG-CSF in all patients. The i.v. administration of rhGM-CSF (6 micrograms/kg body weight/day) to one patient resulted in an increase in neutrophil O2(-)-release stimulated by FMLP. These findings indicate that neutrophils from AA patients are already primed in vivo for enhanced release of O2- and that these neutrophil functions are further potentiated by rhG-CSF or rhGM-CSF.

Increased neutrophil respiratory burst in myeloproliferative disorders: selective enhancement of superoxide release triggered by receptor-mediated agonists and low responsiveness to in vitro cytokine stimulation.

The neutrophil superoxide (O2-)-producing capacity in 57 patients with chronic myeloproliferative disorders (MPDs) and eight patients with chronic myelomonocytic leukemia (CMML) was investigated. O2- release in neutrophils stimulated by chemotactic peptide was markedly increased in all types of chronic MPD, including chronic myelogenous leukemia in both chronic phase and blastic crisis, polycythemia vera, and essential thrombocythemia, but was normal in CMML, which is thought to be a myelodysplastic disorder rather than MPD. Increase in O2(-)-producing capacity in MPD was also observed when other receptor-mediated agonists such as interleukin-8 and concanavalin A were used, but not when phorbol ester, a direct activator of protein kinase C, was used as the triggering agonist of O2- release. Priming effects of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and tumor necrosis factor (TNF) on chemotactic peptide-induced O2- release was observed in all patients with MPD and CMML, though fold enhancement of priming effects was much less in MPD compared with normal subjects. In addition, the priming effects of TNF were less than those of GM-CSF in 10 cases, whereas the priming effects of TNF were consistently and markedly greater than those of GM-CSF in normal subjects. Tyrosine phosphorylation of 42-kDa protein stimulated by G-CSF, GM-CSF, and TNF was observed in CML neutrophils to be identical to that in normal neutrophils. Present results indicate specific potentiation of the receptor-mediated route of signaling that is linked to the respiratory burst and downregulated responsiveness to cytokines in neutrophils in patients with all types of chronic MPD, suggesting in vivo priming of patient neutrophils via certain mechanism by cytokines or related stimuli in these hematological disorders.

Signal transduction pathways in normal human monocytes stimulated by cytokines and mediators: comparative study with normal human neutrophils or transformed cells and the putative roles in functionality and cell biology.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) -3 induced tyrosine phosphorylation of 92-kDa protein in normal human monocytes. We identified this 92-kDa protein as STAT5, but not as STATs1, 3, and 6 nor c-fes and vav protooncogene products, and demonstrated its translocation to the nucleus, enhancement of specific DNA binding capacity, and potentiation of trancriptional activity by GM-CSF. N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA) induced tyrosine phosphorylation of 42- and 44-kDa proteins, which were identified as extracellular signal-regulated kinase (ERK), in human monocytes. In marked contrast to neutrophils and MO7e cells, GM-CSF did not induce tyrosine phosphorylation and activation of ERK in monocytes. Among upstream signaling molecules of ERK, Shc was constitutively associated with Grb2 and was not tyrosine-phosphorylated by GM-CSF and FMLP, and Sos1 and c-Raf-1 were not phosphorylated by GM-CSF, IL-3, TNF, and FMLP in monocytes, whereas all these signaling molecules were affected and/or utilized by GM-CSF in MO7e cells. In contrast to neutrophils, p38 was constitutively phosphorylated and agonist-dependent phosphorylation and activation was not detected in human monocytes. Superoxide release stimulated by FMLP was inhibited partially by PD98059 or SB203580, a specific inhibitor of ERK or p38 pathway, and was almost completely inhibited by the combination of both inhibitors, whereas PMA-induced superoxide release was resistant to these two inhibitors in monocytes. PD98059 inhibited GM-CSF-dependent proliferation of MO7e cells. Present results indicate trancriptional roles of STAT5 and functional roles of ERK and/or p38 in normal human monocytes stimulated by physiological receptor-mediated agonists GM-CSF and FMLP. Possible roles of ERK in proliferation of transformed cells were also suggested.

Activation of human monocyte functions by tumor necrosis factor: rapid priming for enhanced release of superoxide and erythrophagocytosis, but no direct triggering of superoxide release.

Tumor necrosis factor (TNF), like granulocyte-macrophage colony-stimul ating factor (GM-CSF), rapidly primed human monocytes for enhanced release of superoxide (O-2) stimulated by receptor-mediated agonists, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate (PMA), which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of suspended monocytes with 10 U/mL TNF for 10 minutes at 37 degrees C. The potency of the maximal priming effect was TNF> GM-CSF, and the combined effect of TNF and GM-CSF was greater than that of each cytokine alone. GM-CSF induced an increase in cytoplasmic pH but TNF did not. These findings suggest that TNF and GM-CSF activate monocytes through different mechanisms. TNF and GM-CSF by themselves never triggered O-2 release in suspended monocytes or monocytes adherent to endothelial cells, although both cytokines triggered massive release of O-2 in human neutrophils. In additions, TNF and GM-CSF induced tyrosine phosphorylation of a 42-kD protein in neutrophils but not in monocytes. These findings suggest that the TNF-receptor- or GM-CSF-receptor-mediated signaling pathways for triggering O-(2) release is active in neutrophils but inactive or defective in monocytes. TNF also enhanced phagocytosis of sialidase-treated autologous erythrocytes by monocytes, and this effect was further potentiated in the presence of autologous fresh serum. The significant enhancement of erythrophagocytosis was obtained at 1 U/mL TNF. At this concentration of TNF, the expression of C3bi-receptor (CD11b/CD18) was upregulated. These findings show that TNF rapidly primes human monocytes for enhanced release of O-(2) and erythrophagocytosis and suggest that TNF activates monocytes through autocrine or paracrine mechanisms at the inflammatory sites inasmuch as TNF is primarily produced by activated monocytes/macrophages.

Activation and priming of human monocytes by monocyte chemotactic activating factor: cooperation with other inflammatory cytokines and close association between an increase in cytoplasmic free Ca2+ and intracellular acidification.

Both monocyte chemotactic and activating factor (MCAF) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) stimulated an increase in cytoplasmic free Ca2+ ([Ca2+]i) and changes in intracellular pH (pHi) in human monocytes in parallel at lower concentrations and stimulated superoxide (O2-) release and changes in transmembrane potential in parallel at higher concentrations. The changes in pHi were characterized by initial rapid acidification followed by sustained alkalinization, and the changes in transmembrane potential were characterized by initial depolarization followed by partial repolarization. The time courses of all responses stimulated by MCAF and FMLP were similar to each other, although the magnitude of all responses was less in MCAF-stimulated cells. MCAF by itself was a very weak stimulus for inducing O2- release. However, MCAF primed monocytes and enhanced O2- release stimulated by FMLP. The priming effect of MCAF was maximal within 5 minutes of preincubation, and the dose-response curves for priming were identical to those for triggering of an increase in [Ca2+]i. Treatment of monocytes with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), abolished not only the increase in [Ca2+]i but also the changes in pHi (both acidification and alkalinization) induced by MCAF or FMLP. MCAF further potentiated FMLP-induced O2- release in tumor necrosis factor (TNF)-, granulocyte-macrophage colony-stimulating factor (GM-CSF)-, or IL-3-primed monocytes. These findings suggest that MCAF, alone or in concert with other cytokines, primes monocytes for enhanced release of 02-, and that MCAF- or FMLP-induced intracellular acidification and alkalinization are closely associated with an increase in [Ca2+]i, but not O2- release.

Characterization of room temperature induced apoptosis in HL-60.

We found that exposure to room temperature (RT/21 degrees C) causes apoptosis in HL-60 cells. Here we characterized RT-induced apoptosis in HL-60. After exposure to RT, apoptosis starts within 6 h and more than 80% of the cells underwent apoptosis within 20 h. All cells, however, were committed to apoptosis after 16 h and no viable cells could be recovered. The caspase-1 inhibitor (YVAD-CHO) effectively blocked apoptosis, whereas the caspase-3 inhibitor (DEVD-CHO) did not. About 20% of newly obtained early passage HL-60 cells (passage 10) also underwent apoptosis by RT treatment. These data suggest that some population in HL-60 which responds to RT with apoptosis became dominant during passaging.

Involvement of apoptosis-inducing factor during dolichyl monophosphate-induced apoptosis in U937 cells.

Dolichyl monophosphate (Dol-P) has been found to induce apoptosis in human leukemia U937 cells. During this apoptotic execution, the increase of plasma membrane fluidity (5-20 min), caspase-3-like protease activation (2-4 h), chromatin condensation and DNA ladder formation (3-4 h) were observed successively. Here, we report that reduction in mitochondrial transmembrane potential and translocation of apoptosis-inducing factor (AIF) are early events (1-3 h) in the apoptotic process induced by Dol-P in U937 cells. The AIF was concentrated around nuclei and partly translocated to the nuclei, which was confirmed by immunocytochemistry using specific anti-AIF antibody. Both caspase-8 and caspase-3 inhibitors blocked only DNA fragmentation but not mitochondrial processes, AIF migration and chromatin condensation. These results indicate that mitochondrial changes are an early step in the apoptosis induced by Dol-P and AIF is one of the important factors which induce chromatin condensation in nuclei.

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60.

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.

Room temperature-induced apoptosis of Jurkat cells sensitive to both caspase-1 and caspase-3 inhibitors.

We recently reported that HL-60 cells underwent apoptosis when exposed to room temperature (RT) (21 degrees C). RT-induced apoptosis of HL-60 cells is inhibited by the caspase-1 inhibitor (YVAD-CMK), but not by the caspase-3 inhibitor (DEVD-CHO). In this study, we studied RT-induced apoptosis in 15 human cell lines of hematopoietic lineage and found that the Jurkat cell line also responded to RT by a different apoptotic process. RT-induced apoptosis of Jurkat cells was attenuated by YVAD-CMK as well as DEVD-CHO. Increased caspase activity on DEVD-AMC, which was inhibited by both YVAD-CMK and DEVD-CHO added to the cell culture, was also detected. The involvement of caspase-3 itself, however, was not recognized by Western blot analysis. In contrast, the processing of caspase-3 was observed in the apoptotic HL-60 cells. These data implicate the presence of the redundant processes of apoptosis induced by RT treatment.

Diverse effects of cytochalasin B on priming and triggering the respiratory burst activity in human neutrophils and monocytes.

Cytochalasin B, despite its potent enhancing effect on superoxide (O2-) release triggered by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and many other agonists, significantly inhibited O2- release triggered by interleukin 8 (IL-8) and platelet-activating factor in human neutrophils. Cytochalasin B also enhanced changes in membrane potential stimulated by FMLP but inhibited those stimulated by IL-8. Using IL-8 as a triggering agonist, we found that the priming effect of tumor necrosis factor (TNF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on O2- release was slightly but significantly potentiated by cytochalasin B. O2- release triggered by TNF and GM-CSF was completely abolished by cytochalasin B. In contrast to these diverse effects of cytochalasin B on O2- release, changes in cytoplasmic pH stimulated by FMLP, IL-8, TNF, and GM-CSF were not or were only minimally affected by cytochalasin B. Unlike human neutrophils, human monocytes stimulated by FMLP showed inhibition of O2- release and changes in membrane potential in response to cytochalasin B, and the priming effect of TNF and GM-CSF on O2- release in human monocytes was completely abolished by cytochalasin B. These findings indicate the diverse effects of cytochalasin B on phagocytes and suggest distinct regulatory mechanisms according to the functions, agonists, and cell types.