Upregulation of thrombomodulin antigen levels in U937 cells by combined stimulation with estradiol-17beta and vitamin K2 (menaquinone 4)

The expression of thrombomodulin (TM) and tissue factor (TF) antigens by estradiol and vitamin K2 were studied in human leukemic cell lines including U937 (monoblastic leukemia), NB4 (acute promyelocytic leukemia), and HL60 (acute myeloblastic leukemia). Combined stimulation with estradiol-17beta and menaquinone 4 (MK4), homologue of vitamin K2, showed a remarkable increase of total TM antigen level only in U937 cells among these leukemic cell lines, whereas a single treatment of each agent showed a modest or a moderate increase. A synergistic effect of cotreatment with estradiol-17beta and MK4 was observed in an optimum concentration of 1.0 micromol of estradiol-17beta and 1.0 micromol of MK4. Estrogen receptors were detected only in U937 cells among these cell lines, and the competitive assay with an antiestrogenic agent showed a suppression on TM expression in a dose-dependent manner. In the mean time, concerning expression of TF antigens, if at all, only a very slight decrease was observed by costimulation with estradiol-17beta and MK4 in U937 and NB4 cells, whereas all-trans-retinoic acid (ATRA) showed a remarkable decrease in surface TF antigen levels in NB4 cells and also a modest decrease in U937 cells. These findings suggest that estradiol-17beta would up-regulate TM antigen expression via estrogen receptors and in cooperation with MK4, showing a different mechanism from ATRA.     

Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines

Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal-induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal-induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia.     

Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death

Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.     

Effects of human bone marrow stromal cell line (HFCL) on the proliferation,differentiation and apoptosis of acute myeloid leukemia cell lines U937, HL-60 and HL-60/VCR

This study investigated the effects of human bone marrow fibroblastoid stromal cell line (HFCL) on the proliferation, differentiation and chemosensitivity of acute myeloid leukemia cells (AML) in vitro coculture. By setting up coculture system of sensitive U937, HL-60 cell line and multidrug-resistant (MDR) HL-60/VCR cells in direct contact with human bone marrow fibroblastoid stromal cell line HFCL, or separated by transwell, the proliferation of AML cells cocultured with HFCL cells was inhibited, compared with AML cells alone. And NBT positive cells increased slightly. The percentage of G1 phase cells of AML cells cocultured with HFCL cells was higher than that without HFCL cells, and that of S phase cells was lower. The expression of CD11b and CD14 increased. Meanwhile HL-60 and HL-60/VCR cells treated by TPT were observed to have apoptosis characteristic morphological changes. The proportion of G0/G1 HL-60 and HL-60/VCR cells treated with TPT increased and the sub-G1 increased. The percentage of Annexin V-positive cells and apoptotic cells increased with expression of activated Caspase-3 and the reduced expression of Bcl-2. But when they were cocultured with HFCL cells, the percentage of Annexin V-positive cells and apoptotic cells decreased and sub-G1 reduced. After indirect contact with HFCL cells the expression of activated Caspase-3 decreased and the expression of Bcl-2 increased. After direct contact with HFCL cells for 96 h, the expression levels of 582 genes in HL-60 cells were up-regulated, and 1,323 genes were down-regulated at least twofold by Affymetrix GeneChip Human Genome U133 set A. The expression change in some genes, such as HL14, was confirmed by RT-PCR and northern blot. In a word, HFCL cells could inhibit the proliferation, induce the monocytic differentiation of U937, HL-60 and HL-60/VCR cells, and prevent TPT-induced apoptosis in HL-60 and HL-60/VCR cells via modulation of Bcl-2 and active Caspase-3. Many genes might take part in the influence of HFCL cells on AML cells, which may give important insights into the interaction of bone marrow stromal cells and leukemic cells.     

Analysis of myelomonocytic leukemic differentiation by a cell surface marker panel including a fucose-binding lectin from Lotus tetragonolobus

The fucose-binding lectin from Lotus tetragonolobus ( FBL -L) has been previously shown to bind specifically to normal cells of the myeloid and monocytic lineages. The purpose of this study was to explore the utility of fluoresceinated FBL -L as a leukemia differentiation marker in conjunction with a panel of other frequently used surface markers (Fc receptor, HLA-DR, OKM1, and antimonocyte antibody). FBL -L reacted with leukemic cells in 8/9 cases of clinically recognized acute myeloid leukemia, including myeloid blast crisis of chronic granulocytic leukemia, 3/3 cases of chronic phase chronic myelogenous leukemia, and in 2/7 cases of clinically undifferentiated acute leukemia. Correlations were noted between reactivity with FBL -L, and DR and Fc receptor expression. Among continuous cell lines, FBL -L bound with high intensity to a majority of HL-60 and U937 cells. The less well differentiated myeloblast cell lines, KG-1, KG1a , and HL-60 blast II, exhibited less FBL -L binding than HL-60 and U937. A moderate proportion of K562 cells exhibited low level binding of FBL -L. Several lymphoblastic cell lines exhibited a pattern of low intensity binding that was distinguishable from the high intensity binding pattern of the myeloblastic lines. FBL -L reactivity of U937 was enhanced by induction of differentiation with leukocyte conditioned medium, but not dimethylsulfoxide. Such treatments induced contrasting patterns of change of HL-60 and U937 when labeled with OKM1, alpha-Mono, and HLA- DR. These studies demonstrate the application of FBL -L to analysis and quantitation of myelomonocytic leukemic differentiation.     

Resveratrol,a Natural Product Derived from Grapes,Is a New Inducer of Differentiation in Human Myeloid Leukemias

A natural product, resveratrol (3,4,40-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other food products, is known as a cancer chemopreventive agent. We studied the in vitro biological activity of this compound by examining its effect on proliferation and differentiation in myeloid leukemia cell lines (HL-60, NB4, U937,THP-1, ML-1, Kasumi-1) and fresh samples from 17 patients with acute myeloid leukemia. Resveratrol (20 microM, 4 days) alone inhibited the growth in liquid culture of each of the 6 cell lines. Resveratrol (10 microM) enhanced the expression of adhesion molecules (CD11a, CD11b, CD18, CD54) in each of the cell lines except for Kasumi-1. Moreover, resveratrol (25 microM, 4 days) induced 37% of U937 cells to produce superoxide as measured by the ability to reduce nitroblue tetrazolium (NBT). The combination of resveratrol (10 microM) and all-trans-retinoic acid (ATRA) (50 nM, 4 days) induced 95% of the NB4 cells to become NBT-positive, whereas <1% and 12% of the cells became positive for NBT after a similar exposure to either resveratrol or ATRA alone, respectively. In U937 cells exposed to resveratrol (25 microM, 3 days), the binding activity of nuclear factor-kappaB (NFkappaB) protein was suppressed. Eight of 19 samples of fresh acute leukemia cells reduced NBT after exposure to resveratrol (20 microM, 4 days). Taken together, these findings show that resveratrol inhibits proliferation and induces differentiation of myeloid leukemia cells.     

A CD33-specific single-chain immunotoxin mediates potent apoptosis of cultured human myeloid leukaemia cells

A novel single-chain immunotoxin was constructed by combining a CD33-specific single chain Fv (scFv) antibody fragment with an engineered variant of Pseudomonas exotoxin A (ETA). The variant toxin carries the KDEL peptide at its C-terminus, a cellular peptide mediating improved retrograde transport to the endoplasmic reticulum. The purified recombinant fusion protein induced potent apoptosis of the human myeloid cell lines U937, HL-60 and THP-1. Up to 98% of U937 cells were eliminated after treatment for 72 h with a single dose of 500 ng/ml (c. 7 nmol/l). Killing was antigen-specific and occurred by apoptosis. A control protein, consisting of a CD19-specific scFv antibody fragment fused to the ETA-KDEL toxin, failed to induce death of the CD19-negative cell lines U937, HL-60 and THP-1. The CD33-ETA toxin also mediated apoptosis of fresh patient-derived acute myeloid leukaemia cells from bone marrow and peripheral blood. The pronounced antigen-restricted cytotoxicity of the novel fusion protein makes it a candidate for further evaluation of its therapeutic potential.     

CD36介导氧化型低密度脂蛋白诱导U937细胞泡沫化和凋亡

为探讨清道夫受体ＣＤ３６在氧化型低密度脂蛋白诱导Ｕ９３７细胞泡沫化和凋亡中的作用,用氧化型低密度脂蛋白温育Ｕ９３７细胞,观察Ｕ９３７细胞泡沫化过程中ＣＤ３６的表达时序和泡沫细胞的凋亡;用ＣＤ３６单克隆抗体阻断Ｕ９３７细胞的吞噬作用,观察Ｕ９３７细胞吞噬蓄积胆固醇和细胞凋亡的改变。细胞胆固醇以修饰的酶荧光法测定;用流式细胞术检测异硫氰酸荧光素－抗ＣＤ３６单克隆抗体特异标记的ＣＤ３６和细胞的凋亡情况;用逆转录聚合酶链反应检测ＣＤ３６ｍＲＮＡ的表达。结果发现,８０ｍｇ／Ｌ氧化型低密度脂蛋白与Ｕ９３７细胞温育２４ｈ可增加细胞内总胆固醇,４８ｈ时可形成典型的泡沫细胞;ＣＤ３６表达呈现时序性改变,６ｈ即可检出ＣＤ３６表达增高,２４ｈ达到最高值,４８ｈ表达略有降低,ＣＤ３６ｍＲＮＡ的转录与ＣＤ３６的表达一致。用２００ｍｇ／Ｌ     

Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF

DT(388)-GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT(388)-GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT(388)-GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT(388)-GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C(6)-ceramide inhibited protein synthesis (EC(50) approximately 5 microM) and decreased viability (EC(50) approximately 6 microM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT(388)-GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.     

Monoclonal antibodies (series Bra-) of broad (non-lineage) or restricted (myelomonocytic) reactivities elicited with granulocytes or K 562 cells

A series of monoclonal antibodies was obtained by hybridoma technology after immunization with granulocytes from healthy donors or K 562 immature erythroid-myeloid leukemia cells. Three different types of reactivities with examined hematopoietic-, nonhematopoietic cells and cell lines were observed by microscopic immunofluorescence, immunocytofluorometry and enzyme-linked immunoassay (ELISA), as follows: (i) broad, non-lineage type of two monoclonal antibodies (Bra10G and Bra7F1) with examined hematopoietic and nonhematopoietic human neoplastic cell lines, (ii) non-lineage type of reactivity restricted to hematopoietic cell lines, and (iii) restricted (myelomonocytic) pattern of binding to myeloid cell lines and healthy donors' granulocytes (monoclonal antibodies Bra4F1, BraC8 and Bra1F2). Monoclonal antibodies Bra10G and BraC6 were shown to immunoprecipitate specifically a heterodimeric two-chain cell surface protein p200,95 from cell lysates of lactoperoxidase radioiodinated U 937 cells with recognized epitope localized on the heavy chain (as shown by immunoblotting experiments). Antibodies with restricted myelomonocytic type of reactivity exhibited minor quantitative differences in their microscopic immunofluorescence and immunocytofluorometric patterns of reactivities with examined myeloid leukemia cell lines (K 562, HL 60, U 937) and healthy donors' granulocytes. The monoclonal antibody Bra4F1 was defined by the 4th International Workshop on Leukocyte Differentiation Antigens as CD15 (with typical selective reactivity towards myelomonocytic leukemia cells and cell lines as well as healthy donors' granulocytes) recognizing the X-hapten carbohydrate antigenic determinant.     

Expression of CD9 (p24) antigen on hematopoietic cells following treatment with phorbol ester

Expression of the leukemia-associated cell surface antigen p24 (CD9) on human hematopoietic cell lines and B-cell chronic lymphocytic leukemia (B-CLL) cells was analyzed before and after treatment with the phorbol ester 12-o-tetradecanoyl-phorbol 13-acetate (TPA). Little or no expression of CD9 was detected in any of the cell lines used or in B-CLLs before treatment with TPA. After exposure to TPA, HL-60, Epstein-Barr virus-immortalized B-cell lines, Molt-3, MT-2 and B-CLLs showed markedly augmented CD9 expression. U937 and K562 showed slight increases of CD9 expression. However, no expression of CD9 was induced in CCRF-CEM or HUT-102. Although CD9 is known to be one of the most useful markers of pre-B-cell common acute lymphoblastic leukemia, the expression of CD9 does not seem to be restricted to any specific cell lineage and can be induced in various hematopoietic cell lineages by treatment with TPA.     

Lineage infidelity of a human myelogenous leukemia cell line

We have analyzed the organization and expression of the immunoglobulin heavy and light chain gene in the human myeloblastic leukemic sublines, ML1, ML2, and ML3, and in the human myeloid leukemic cell lines, HL-60, U937, THP1, and K562. ML1, ML2, and ML3 cells, despite a predominant granulocytic phenotype, express a rearrangement of the immunoglobulin heavy chain gene that typically occurs during the early stages of the B cell differentiation pathway. No rearrangement was found in any of the other cell lines tested. These findings strongly support the notion that, at least in some cases, acute myeloid leukemia (AML) cells represent highly atypical cells with profoundly altered gene expression, rather than cells arrested at a well-defined stage of the myeloid lineage.     

19-Nor-1α,25-dihydroxyvitamin D<Subscript>2</Subscript> (paricalcitol): effects on clonal proliferation,differentiation, and apoptosis in human leukemic cell lines

PURPOSE: 19-Nor-1alpha,25-dihydroxyvitamin D(2) (paricalcitol) is an analogue of 1,25(OH)(2)D(3) with reduced calcemic effects that is approved for the suppression of parathyroid hormone in chronic renal failure. Paricalcitol has recently been reported to have anticancer activity in prostate cancer. In order to explore paricalcitol as a potential agent against leukemia, we tested its effects on HL-60 and U937 leukemia cell lines. METHODS: We studied cellular differentiation via expression of CD11b and CD14 surface antigens using flow cytometry, and via the nitroblue tetrazolium (NBT) assay. Cell cycle was analyzed using propidium iodide staining. Apoptosis was assessed with the annexin V assay. Cellular proliferation was determined via colony inhibition on semisolid medium. RESULTS: Paricalcitol induced the maturation of HL-60 and U937 cells, as shown by increased expression of CD11b differentiation surface antigen. CD14 showed increased expression in HL-60 but not in U937 cells. After exposure to paricalcitol at 10(-8) M for 72 h, the ability of HL-60 cells to reduce NBT was markedly increased. Conversely, U937 cells were unchanged. Paricalcitol inhibited colony formation of both HL-60 and U937 cell lines in semisolid medium after a 10-day incubation (estimated IC(50) of 3x10(-8) M in HL-60 cells and 4x10(-8) M in U937 cells). Paricalcitol at 10(-8) M and 10(-7) M caused a significant dose- and time-dependent increase of apoptosis in HL-60 cells ( P<0.05). In both HL-60 and U937 cells, exposure to 10(-7) M paricalcitol for 72 h increased the number of cells in G(0)/G(1) phase, and decreased the number of cells in S phase. CONCLUSIONS: Paricalcitol inhibits colony formation, induces maturation and causes cell cycle arrest in HL-60 and U937 cells. Additionally, paricalcitol induces apoptosis in HL-60 cells. These findings support the further evaluation of paricalcitol as an antileukemia agent.     

Icaritin induces AML cell apoptosis via the MAPK/ERK and PI3K/AKT signal pathways

Icaritin, a hydrolytic product of icaritin, is isolated from the traditional Chinese medicinal herb epimedium. Icaritin inhibits the proliferation of several tumor cell lines, but its effect on acute myeloid leukemia (AML) and underlying mechanisms remain to be identified. In the present study, we demonstrated that icaritin inhibits the proliferation of human AML cell lines NB4, HL60, and U937, in a dose- and time-dependent manner. Importantly, icaritin showed anti-leukemia activity on bone marrow mononuclear cells from 15 newly diagnosed AML patients. Flow cytometry analyses indicated that icaritin induces AML cells apoptosis. Icaritin induced activation of caspase-9, -3, -7 and the cleavage of PARP as measured by Western blotting. Icaritin downregulates p-ERK and p-AKT and inhibits the expression of c-myc. These results suggest that icaritin is a promising candidate drug for the treatment of AML. The underlying mechanisms of icaritin anti-AML activity are associated with inhibition of the MAPK/ERK and PI3K/AKT signals and downregulation of c-myc.     

The tyrphostin adaphostin interacts synergistically with proteasome inhibitors to induce apoptosis in human leukemia cells through a reactive oxygen species (ROS)-dependent mechanism

Interactions between the tyrphostin adaphostin and proteasome inhibitors (eg, MG-132 and bortezomib) were examined in multiple human leukemia cell lines and primary acute myeloid leukemia (AML) specimens. Cotreatment of Jurkat cells with marginally toxic concentrations of adaphostin and proteasome inhibitors synergistically potentiated mitochondrial damage (eg, cytochrome c release), caspase activation, and apoptosis. Similar interactions occurred in other human leukemia cell types (eg, U937, HL-60, Raji). These interactions were associated with a marked increase in oxidative damage (eg, ROS generation), down-regulation of the Raf/MEK/ERK pathway, and JNK activation. Adaphostin/MG-132 lethality as well as mitochondrial damage, down-regulation of Raf/MEK/ERK, and activation of JNK were attenuated by the free-radical scavenger NAC, suggesting that oxidative damage plays a functional role in antileukemic effects. Ectopic expression of Raf-1 or constitutively active MEK/ERK or genetic interruption of the JNK pathway significantly diminished adaphostin/MG-132-mediated lethality. Interestingly, enforced Raf or MEK/ERK activation partially diminished adaphostin/MG-132-mediated ROS generation, suggesting the existence of an amplification loop. Finally, the adaphostin/MG-132 regimen displayed similar toxicity toward 5 primary AML samples but not normal hematopoietic progenitors (eg, bone marrow CD34+ cells). Collectively, these findings suggest that potentiating oxidative damage by combining adaphostin with proteasome inhibitors warrants attention as an antileukemic strategy.     

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.