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The dynamic rearrangement of the actin cytoskeleton is an essential component of many mechanotransduction and cellular force generation pathways. Here we use periodic surface topographies with feature sizes comparable to those of in vivo collagen fibers to measure and compare actin dynamics for two representative cell types that have markedly different migratory modes and physiological purposes: slowly migrating epithelial MCF10A cells and polarizing, fast-migrating, neutrophil-like HL60 cells. Both cell types exhibit reproducible guidance of actin waves (esotaxis) on these topographies, enabling quantitative comparisons of actin dynamics. We adapt a computer-vision algorithm, optical flow, to measure the directions of actin waves at the submicron scale. Clustering the optical flow into regions that move in similar directions enables micron-scale measurements of actin-wave speed and direction. Although the speed and morphology of actin waves differ between MCF10A and HL60 cells, the underlying actin guidance by nanotopography is similar in both cell types at the micron and submicron scales.

Background: HL-60 cells have been used in in vitro experiments of neutrophils rolling. They lose uniform spherical appearance and enhance deformability by differentiation to neutrophil-like cells, which would affect their rolling characteristics., Objective: We investigate the influence of differentiation and coating of target substrate on the fundamental rolling characteristics of the cells under a constant pressing force which mimics the pressing force to the vessel wall by erythrocytes in vivo., Methods: Motions of undifferentiated and differentiated HL-60 cells on plain or MPC-polymer-coated flat glass substrate were compared using a homemade inclined centrifuge microscope system., Results: Most of the cells alternated between stop and go during the motion. The differentiation resulted in a high temporal ratio of the non-moving state and low mean velocity during the moving state, together with a high suppression performance of cell adhesion by the polymer. It was also suggested that the cells were mostly rolling but that the coating probably induced an infrequent slip on the substrate to stabilize the cells motion., Conclusions: Differentiation strongly affects adhesivity of HL-60 cells but less affects the mean velocity. Our findings also demonstrate the importance of the pressing force and advantage of the present system with respect to classical flow chambers.

Tryptanthrin is a natural product which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signaling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK which led to the activation of intrinsic and extrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of tyrosine 705 and serine 727 p-STAT3. As IL-6 is the main driver of the STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, PD98059 significantly reduced the apoptotic effects of TBr, thus showing the direct involvement of p-ERK in TBr mediated cell death. It was further shown that apoptotic protein Bax silencing in HL-60 cells resists TBr mediated ERK dependent apoptosis. In summary, for the first time we report the mechanism of TBr mediated cell death in human leukemia cell lines by targeting STAT3 and ERK pathways.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine secreted by stromal cells and plays a role in the differentiation of bone marrow stem cells and proliferation of neutrophils. Therefore, G-CSF is widely used to reduce the risk of serious infection in immunocompromised patients; however, its use in such patients is limited because of its non-persistent biological activity. We created an N-linked glycosylated form of this cytokine, hG-CSF (Phe140Asn), to assess its biological activity in the promyelocyte cell line HL60. Enhanced biological effects were identified by analyzing the JAK2/STAT3/survivin pathway in HL60 cells. In addition, mutant hG-CSF (Phe140Asn) was observed to have enhanced chemoattractant effects and improved differentiation efficiency in HL60 cells. These results suggest that the addition of N-linked glycosylation was successful in improving the biological activity of hG-CSF. Furthermore, the mutated product appears to be a feasible therapy for patients with neutropenia.

Benzene is a well-known environmental pollutant that can induce hematotoxicity, aplastic anemia, acute myelogenous leukemia, and lymphoma. Benzene toxicity is likely mediated through metabolites induced by means of multiple pathways. Although benzene metabolites are known to induce oxidative stress and disrupt the cell cycle, the mechanism underlying leukemogenesis is not fully understood. The aim of this study was to analyze the genome-wide expression profiles of human promyelocytic leukemia HL-60 cells that had been exposed to benzene and its metabolites. This was carried out using whole human genome oligonucleotide microarrays to ascertain potential biomarkers. Genes that were differentially expressed (>1.5-fold and p-values <0.05) after exposure to benzene (BZ), hydroquinone (HQ), and 1,4-benzoquinone (BQ) were then classified with GO, KEGG and GSEA pathway annotation. All genes that were identified were then functionally categorized as being involved in the cell cycle, the p53 signaling pathway, apoptosis, the MAPK signaling pathway, or the T cell receptor signaling pathway. Functionally important genes were further validated by means of real-time RT-PCR. The results showed that EGR1, PMAIP1, AR, CCL2, CD69, HSPA8, SLC7A11, HERPUD1, ELK1, and MKI57 genes altered their expression profiles. Similar expression profiles were also found in human erythromyeloblastoid leukemia K562 cells and in human leukemic monocyte lymphoma U937 cells. In conclusion, gene expression profiles along with GO, KEGG and GSEA pathway annotation analysis have provided an insight into the leukemogenesis as well as highlighted potential gene-based biomarkers of human leukemia cell lines when they are exposed to benzene and its metabolites., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Leukocyte antigen CD38 expression is an early marker of all-trans retinoic acid (ATRA) stimulated differentiation in the leukemic cell line HL-60. It promotes induced myeloid maturation when overexpressed, whereas knocking it down is inhibitory. It is a type II membrane protein with an extracellular C-terminal enzymatic domain with NADase/NADPase and ADPR cyclase activity and a short cytoplasmic N-terminal tail. Here we determined whether CD38 enzymatic activity or the cytoplasmic tail is required for ATRA-induced differentiation. Neither a specific CD38 ectoenzyme inhibitor nor a point mutation that cripples enzymatic activity (CD38 E226Q) diminishes ATRA-induced differentiation or G1/0 arrest. In contrast a cytosolic deletion mutation (CD38 Δ11-20) prevents membrane expression and inhibits differentiation and G1/0 arrest. These results may be consistent with disrupting the function of critical molecules necessary for membrane-expressed CD38 signal transduction. One candidate molecule is the Src family kinase Fgr, which failed to undergo ATRA-induced upregulation in CD38 Δ11-20 expressing cells. Another is Vav1, which also showed only basal expression after ATRA treatment in CD38 Δ11-20 expressing cells. Therefore, the ability of CD38 to propel ATRA-induced myeloid differentiation and G1/0 arrest is unimpaired by loss of its ectoenzyme activity. However a cytosolic tail deletion mutation disrupted membrane localization and inhibited differentiation. ATRA-induced differentiation thus does not require the CD38 ectoenzyme function, but is dependent on a membrane receptor function., (Copyright © 2010. Published by Elsevier Inc.)

Phospholipase D (PLD), a major source of lipid second messengers (phosphatidic acid, diglycerides) in many cell types, is tightly regulated by protein kinases, but only a few of them have been identified. We show here that protein kinase B (AKT) is a novel major signaling effector of PLD activity induced by the formylpeptide f-Met-Leu-Phe (fMLP) in human neutrophil-like HL-60 cells (dHL-60 cells). AKT inhibition with the selective antagonist AKTib1/2 almost completely prevented fMLP-mediated activity of PLD, its upstream effector ERK1/2, but not p38 MAPK. Immunoprecipitation studies show that phosphorylated AKT, ERK, and PLD2 form a complex induced by fMLP, which can be prevented by AKTib1/2. In cell-free systems, AKT1 stimulated PLD activity via activation of ERK. AKT1 actually phosphorylated ERK2 as a substrate (K(m) 1 μm). Blocking AKT activation with AKTib1/2 also prevented fMLP- but not phorbol 12-myristate 13-acetate-mediated NADPH oxidase activation (respiratory burst, RB) of dHL-60 cells. Impaired RB was associated with defective membrane translocation of NADPH oxidase components p67(phox) and p47(phox), ERK, AKT1, AKT2, but not AKT3. Depletion of AKT1 or AKT2 with antisense oligonucleotides further indicates a partial contribution of both isoforms in fMLP-induced activation of ERK, PLD, and RB, with a predominant role of AKT1. Thus, formylpeptides induce sequential activation of AKT, ERK1/2, and PLD, which represents a novel signaling pathway. A major primarily role of this AKT signaling pathway also emerges in membrane recruitment of NOX2 components p47(phox), p67(phox), and ERK, which may contribute to assembly and activation of the RB motor system, NADPH oxidase.

This study explored the effects of nuclear factor-kappa B (NF-κB) inhibitor Bay 11-7082 on Fas/FasL system and Fas-mediated apoptosis in cell line HL-60 cells. The mRNA and protein levels of Fas, FasL, and X-linked inhibitor of apoptosis protein (XIAP) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM); the level of sFasL was evaluated by enzyme-linked immunosorbent assay (ELISA); and apoptosis was determined by FCM. After treatment with Bay 11-7082, the mRNA and protein levels of FasL and XIAP in HL-60 cells were significantly lower than in the controls (P<0.05), but the mRNA and protein levels of Fas and sFasL did not change significantly (P>0.05). Apoptotic rate of HL-60 cells treated with Bay 11-7082 was significantly higher than in the controls (P<0.05). Therefore, we conclude that Bay 11-7082 can enhance Fas-mediated apoptosis in HL-60 cells by downregulating FasL and XIAP levels.

Tescalcin is a 25-kDa EF-hand Ca(2+)-binding protein that is differentially expressed in several mammalian tissues. Previous studies demonstrated that expression of this protein is essential for differentiation of hematopoietic precursor cell lines and primary stem cells into megakaryocytes. Here we show that tescalcin is expressed in primary human granulocytes and is upregulated in human promyelocytic leukemia HL-60 cells that have been induced to differentiate along the granulocytic lineage. However, during induced macrophage-like differentiation of HL-60 cells the expression of tescalcin is downregulated. The decrease in expression is associated with a rapid drop in tescalcin mRNA level, whereas upregulation occurs via a post-transcriptional mechanism. Tescalcin is necessary for HL-60 differentiation into granulocytes as its knockdown by shRNA impairs the ability of HL-60 cells to acquire the characteristic phenotypes such as phagocytic activity and generation of reactive oxygen species measured by respiratory burst assay. Both up- and downregulation of tescalcin require activation of the MEK/ERK cascade. It appears that commitment of HL-60 cells toward granulocytic versus macrophage-like lineage correlates with expression of tescalcin and kinetics of ERK activation. In retinoic acid-induced granulocytic differentiation, the activation of ERK and upregulation of tescalcin occurs slowly (16-48 h). In contrast, in PMA-induced macrophage-like differentiation the activation of ERK is rapid (15-30 min) and tescalcin is downregulated. These studies indicate that tescalcin is one of the key gene products that is involved in switching differentiation program in some cell types., (Copyright 2010 Elsevier Inc. All rights reserved.)

The leukemia cell line HL60 is widely used in studies of the cell cycle, apoptosis, and adhesion mechanisms in cancer cells. We conducted a focused cytogenetic study in an HL60 cell line, by analyzing GTG-banded chromosomes before and after treatment with pisosterol (at 0.5, 1.0, and 1.8 microg/ml), a triterpene isolated from Pisolithus tinctorius, a fungus collected in the Northeast of Brazil. Before treatment, 99% of the cells showed the homogeneously staining region (HSR) 8q24 aberration. After treatment with 1.8 microg/ml pisosterol, 90% of the analyzed cells lacked this aberration. We further performed a pulse test, in which the cells treated with pisosterol (0.5, 1.0, and 1.8 microg/ml) were washed and re-incubated in the absence of pisosterol. Only 30% of the analyzed cells lacked the HSR 8q24 aberration, suggesting that pisosterol probably blocks the cells with HSRs at interphase. No effects were detected at lower concentrations. At the highest concentration examined (1.8 microg/ml), pisosterol also inhibited cell growth, but this effect was not observed in the pulse test, reinforcing our hypothesis that, at the concentrations tested, pisosterol probably does not induce cell death in the HL60 line. The results found for pisosterol were compared with those for doxorubicin. Cells that do not show a high degree of gene amplification (HSRs and double-minute chromosomes) have a less aggressive and invasive behavior and are easy targets for chemotherapy. Therefore, further studies are needed to examine the use of pisosterol in combination with conventional anti-cancer therapy.

Kaempferia parviflora Wall. ex. Baker is a Thai herb containing many flavonoids that have anti-inflammatory, anti-allergic and antioxidant activities. The objective of this study was to demonstrate apoptotic effects of Kaempferia parviflora Wall. ex. Baker rhizome ethanolic extract on HL-60 cells in vitro. The extract suppressed HL-60 cell growth and decreased cell viability in a dose- and time-dependent manner. Apoptotic cell death was demonstrated by changes in cell morphology, externalization of phosphatidylserine on the cell surface, loss in mitochondrial transmembrane potential and activation of caspase 3. Apoptosis induced by K. parviflora Wall. ex. Baker rhizome ethanolic extract was enhanced by treatment with paclitaxel or doxorubicin, and inhibitors of Akt, PI3-K and MEK.

Background: High density oligonucleotide tiling arrays are an effective and powerful platform for conducting unbiased genome-wide studies. The ab initio probe selection method employed in tiling arrays is unbiased, and thus ensures consistent sampling across coding and non-coding regions of the genome. These arrays are being increasingly used to study the associated processes of transcription, transcription factor binding, chromatin structure and their association. Studies of differential expression and/or regulation provide critical insight into the mechanics of transcription and regulation that occurs during the developmental program of a cell. The time-course experiment, which comprises an in-vivo system and the proposed analyses, is used to determine if annotated and un-annotated portions of genome manifest coordinated differential response to the induced developmental program., Results: We have proposed a novel approach, based on a piece-wise function - to analyze genome-wide differential response. This enables segmentation of the response based on protein-coding and non-coding regions; for genes the methodology also partitions differential response with a 5' versus 3' versus intra-genic bias., Conclusion: The algorithm built upon the framework of Significance Analysis of Microarrays, uses a generalized logic to define regions/patterns of coordinated differential change. By not adhering to the gene-centric paradigm, discordant differential expression patterns between exons and introns have been identified at a FDR of less than 12 percent. A co-localization of differential binding between RNA Polymerase II and tetra-acetylated histone has been quantified at a p-value < 0.003; it is most significant at the 5' end of genes, at a p-value < 10-13. The prototype R code has been made available as supplementary material [see Additional file 1].

Induction of terminal differentiation of neoplastic cells offers potential for a novel approach to cancer therapy. One of the agents being investigated for this purpose in preclinical studies is 1,25-dihydroxyvitamin D(3) (1,25D), which can convert myeloid leukemia cells into normal monocyte-like cells, but the molecular mechanisms underlying this process are not fully understood. Here, we report that 1,25D upregulates the expression of hKSR-2, a new member of a small family of proteins that exhibit evolutionarily conserved function of potentiating ras signaling. The upregulation of hKSR-2 is direct, as it occurs in the presence of cycloheximide, and occurs primarily at the transcriptional level, via activation of vitamin D receptor, which acts as a ligand-activated transcription factor. Two VDRE-type motifs identified in the hKSR-2 gene bind VDR-RXR alpha heterodimers present in nuclear extracts of 1,25D-treated HL60 cells, and chromatin immunoprecipitation assays show that these VDRE motifs bind VDR in 1,25D-dependent manner in intact cells, coincident with the recruitment of RNA polymerase II to these motifs. Treatment of the cells with siRNA to hKSR-2 reduced the proportion of the most highly differentiated cells in 1,25D-treated cultures. These results demonstrate that hKSR-2 is a direct target of 1,25D in HL60 cells, and is required for optimal monocytic differentiation.

Iron is required for the differentiation of HL-60 cells along the monocyte lineage in vitro, reflecting a requirement for iron in the transcriptional induction of the p21(WAF1/CIP1) gene. To determine if the same requirement holds true for differentiation in other cell lineages and for primary human CD34(+) bone marrow precursor cells, we induced granulocyte differentiation by treating HL-60 cells with dimethyl sulphoxide, and erythroid or megakaryocytic differentiation by treating K562 cells with butyrate or phorbol myristate acetate, respectively. Nitro blue tetrazolium reduction, expression of haem, or expression of CD41 was used to assess granulocytic, erythroid, or megakaryocytic differentiation respectively. Purified CD34(+) cells were cultured with granulocyte/macrophage-colony stimulating factor and stem cell factor to induce myelomonocytic differentiation. Iron deprivation was induced by desferrioxamine. p21(WAF1/CIP1) antisense oligonucleotides were used to inhibit p21 expression. Iron deprivation blocked p21 induction as judged by real-time polymerase chain reaction assays. In addition, both iron deprivation and p21 antisense blocked CD34(+) cell differentiation. These observations were not explained by induction of widespread apoptosis under conditions of iron deprivation. We concluded that both iron and functional p21(WAF1/CIP1) are required for in vitro differentiation of human haematopoietic precursors along multiple cell lineages.

The induction of apoptosis by 66 alkaloids of the quinoline, quinolizidine, pyrrolizidine, isoquinoline, indole, terpene, tropane, steroid, purine, and piperidine type, of 9 cardiac glycosides, 11 non-protein amino acids and 10 further secondary metabolites was assayed in HL-60 cell cultures and measured by quantification of the subdiploid DNA content by flow cytometry, detection of DNA fragmentation by gel electrophoresis, and cell morphology. Several alkaloids of the isoquinoline, quinoline, and indole type were active, whereas quinol-izidine, tropane, pyrrolizidine, terpene and piperdine alkaloids were mostly inactive. The proapoptotic alkaloids can be characterized by their property to inhibit protein biosynthesis and their intercalation into DNA at the same time, or by their inhibition of microtubule formation. All cardiac glycosides, which are both membrane detergents and Na+,K+-ATPase inhibitors, are potent apoptosis inducers. Also proapoptotic were a few non-protein amino acids, podophyllotoxin and the flavonoid quercetin.

To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODNs) on apoptosis, we designed four ASODNs targeting different regions of caspase-3 mRNA and transfected them into human leukemia HL-60 cells. The transfected cells were given 10 Gy gamma-irradiation followed by incubation for 18 h and measurement of apoptosis and caspase-3 expression. Our results showed that ASODN-2 targeting the 5' non-coding region of sites -62 to -46, and ASODN-3 targeting the 5' coding region of sites -1 to 16, both reduced apoptosis measured by gel electrophoresis and flow cytometry. Hoechst 33258 staining and TUNEL assay revealed that apoptotic indexes in the ASODN-2 and ASODN-3 groups were significantly lower than those in the untransfected and mismatched oligodeoxynucleotide (MODN) groups. Immunocytochemistry, Western blotting and RT-PCR showed that expression levels of caspase-3 protein and mRNA in both ASODN-2 and ASODN-3 groups were decreased compared with those in the untransfected and MODN groups. In conclusion, caspase-3 mRNA ASODNs can inhibit gamma-radiation-induced apoptosis of HL-60 cells and reduce expression of caspase-3 protein and mRNA. The results suggest that antisense approach may be useful for therapeutic treatment of certain neurodegenerative diseases in which apoptosis is involved.

Background: Chemo- and radiotherapeutic responses of leukemia cells are modified by integrin-mediated adhesion to extracellular matrix. To further characterize the molecular mechanisms by which beta1 integrins confer radiation and chemoresistance, HL60 human acute promyelocytic leukemia cells stably transfected with beta1 integrin and A3 Jurkat T-lymphoma cells deficient for Fas-associated death domain protein or procaspase-8 were examined., Methodology/principal Findings: Upon exposure to X-rays, Ara-C or FasL, suspension and adhesion (fibronectin (FN), laminin, collagen-1; 5-100 microg/cm(2) coating concentration) cultures were processed for measurement of apoptosis, mitochondrial transmembrane potential (MTP), caspase activation, and protein analysis. Overexpression of beta1 integrins enhanced the cellular sensitivity to X-rays and Ara-C, which was counteracted by increasing concentrations of matrix proteins in association with reduced caspase-3 and -8 activation and MTP breakdown. Usage of stimulatory or inhibitory anti beta1 integrin antibodies, pharmacological caspase or phosphatidylinositol-3 kinase (PI3K) inhibitors, coprecipitation experiments and siRNA-mediated beta1 integrin silencing provided further data showing an interaction between FN-ligated beta1 integrin and PI3K/Akt for inhibiting procaspase-8 cleavage., Conclusions/significance: The presented data suggest that the ligand status of beta1 integrins is critical for their antiapoptotic effect in leukemia cells treated with Ara-C, FasL or ionizing radiation. The antiapoptotic actions involve formation of a beta1 integrin/Akt complex, which signals to prevent procaspase-8-mediated induction of apoptosis in a PI3K-dependent manner. Antagonizing agents targeting beta1 integrin and PI3K/Akt signaling in conjunction with conventional therapies might effectively reduce radiation- and drug-resistant tumor populations and treatment failure in hematological malignancies.

Aim: To study the effects of ferulic acid (FA) on E-selectin expression in human umbilical vein endothelial cells (HUVECs) activated by lipopolysaccharide and leukocyte-endothelial cell adhesion., Methods: The effects of FA on E-selectin and E-selectin mRNA expression were determined by flow cytometry and reverse transcription polymerase chain reaction. The effect of FA on HL60-HUVEC adhesion was evaluated with the method of staining the cells by Rose Bengal., Results: The expression of E-selectin and E-selectin mRNA were down regulated by FA (0.62 and 0.41 mmol x L(-1), respectively). HL60 cells adhered to activated HUVECs were also reduced by FA (0.62 and 0.41 mmol x L(-1), respectively)., Conclusion: FA can inhibit the expression of E-selectin and E-selectin mRNA and HL60-HUVEC adhesion. This may contribute to its protective effect against ischemia-reperfusion injury.

We examined the behavior of polymorphonuclear leukocytes (PMNs) and monocytes during subcutaneous plasma extravasation in guinea-pigs. Plasma extravasation was induced by intradermal injection of zymosan-activated plasma (ZAP). The degree of extravasation correlated logarithmically with the concentration of injected ZAP, and was composed of PMN-dependent and -independent components. The latter was mediated primarily by histamine. The former accounted for 40-50% of the total plasma extravasation, peaked within 15 min, and then rectilinearly decreased with a half-life between 30 and 40 min. Histological examination of skin at 15 min after ZAP injection demonstrated PMN attachment to the luminal surface of venule endothelial cells, without evidence of PMN extravasation. We next examined whether monocyte infiltration of subcutaneous tissue played a causal role in plasma extravasation. Monocyte-predominant infiltration was initially caused by an intradermal injection of a monocyte-specific chemotactic factor, the S19 ribosomal protein (RP S19) dimer. Monocyte infiltration did not induce plasma extravasation even in guinea-pigs with elevated peripheral blood monocyte levels following administration of a macrophage-colony stimulating factor. A simultaneous injection of prostaglandin E2, a vasodilating agent, with RP S19 dimer also did not induce plasma extravasation. In contrast, a simultaneous injection of RP S19 dimer with ZAP changed the leukocyte infiltration pattern from PMN-predominant to monocyte-predominant, and almost completely suppressed the PMN-dependent component of the ZAP-induced plasma extravasation. The lack of plasma extravasation in the monocyte-predominant pattern was reproduced when a strong monocyte infiltration was induced by an intradermal injection of apoptotic cells. We conclude that leukocyte-induced plasma extravasation is specific for PMN, and is not due to a physical leakage of plasma during leukocyte emigration. Rather, plasma extravasation is probably caused by a cognate interaction between PMNs and postcapillary venule endothelial cells.

To evaluate the mediation of cell adhesion by Dendrostellera lessertii, an anti-cancer medicinal plant in Iranian folk medicine, the adhesion of thrombin activated human platelets to the cultured monocytes or HL-60 cells was investigated under the effect of the plant extract (0.4 mg/ml) and one of its purified components, 3-hydrgenkwadaphnin (0.2 nM). Treatment of the platelets with the plant extract or/3-hydrgenkwadaphnin, for various time intervals, followed by their activation by thrombin resulted in 80-90% reduction in the number of monocytes with more than 10 attached platelets. Similarly, under almost all identical conditions, the adhesion of the activated platelets to HL-60 cells was decreased by 90%. The adhesion of thrombin activated platelets to the plant extract treated HL-60 cells was also reduced significantly (95%). These data clearly indicates that Dendrostellera lessertii is capable of mediating tumor metastasis through affecting the adhesion properties of the cells., (Copyright 2004 Elsevier Inc.)

We compared immunohistochemical distribution of glycosphingolipids globotriosylceramide (GbOse(3)Cer) and globotetraosylceramide (GbOse(4)Cer) with that of E-selectin on human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor (TNF)-alpha. HUVECs activated by TNF-alpha were characterized by the highest expression of E-selectin and greatest adhesion of HL-60 cells as well compared to stimulation with interleukin-1beta or lipopolysaccharide. HUVECs activated by TNF-alpha also stained intensely with globoside antibodies, especially with the GbOse(3)Cer-directed one, staining being redistributed in a concentration-dependent manner. These results indicate the possible role of GbOse(3)Cer and GbOse(4)Cer in immune effector mechanisms of endothelium such as adhesion.

The effect of all-trans retinoid acid (ATRA) on the expression of Notch1 gene by real-time reverse transcriptase polymerase chain reaction (RT-PCR) in acute promyelocytic leukemia cells (APL), NB4, and HL-60 lacking t(15;17) was studied. The cells were treated with ATRA 0.5 microM for up to 96 hours. The increased transcript level of Notch1 was in concert with that of CD11b in NB4 cells, but not in HL-60 cells. The expression of Notch1 gene might be specific for APL cells. As Notch1 gene is involved in the differentiation and leukemogenesis in lymphoid neoplasm, observations suggest that Notch1 is involved in ATRA-modulated differentiation process in APL.

Rhein is an anthraquinone compound enriched in the rhizome of rhubarb, a traditional Chinese medicine herb showing anti-tumor promotion function. In this study, we first reported that rhein could induce apoptosis in human promyelocytic leukemia cells (HL-60), characterized by caspase activation, poly(ADP)ribose polymerase (PARP) cleavage, and DNA fragmentation. The efficacious induction of apoptosis was observed at 100 microM for 6h. Mechanistic analysis demonstrated that rhein induced the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondrion to cytosol, and cleavage of Bid protein. Rhein also induced generation of reactive oxygen species (ROS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. However, these actions seem not to be associated with the apoptosis induction because antioxidants including N-acetyl cysteine (NAC), Tiron, and catalase did not block rhein-induced apoptosis, although they could block the generation of ROS and the phosphorylation of JNK and p38 kinase. Our data demonstrate that rhein induces apoptosis in HL-60 cells via a ROS-independent mitochondrial death pathway.

Background and Aim of the Study: Histopathological studies of rejected orthotopic heart transplants suggest that cardiac valve endothelium is spared the inflammatory cell infiltration and tissue damage that occurs in the myocardium. To test whether this apparent protection from leukocyte invasion might be an inherent feature of the valve endothelium, leukocyte adhesion molecule expression and function were analyzed in human pulmonary valve endothelial cells (HPVEC). Use of cultured HPVEC allowed delineation of the potential contribution of functional adhesion molecules from the contribution of hemodynamic forces exerted on the leaflet surface in vivo, Methods and Results: HPVEC express E-selectin, ICAM-1, and VCAM-1 in response to the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) similarly to other types of cultured human endothelial cells. In a static cell adhesion assay, E-selectin-mediated adhesion of HL-60 cells, a human promyelocytic leukemia cell line, and U937 cells, a human monocytic cell line, was determined in cells treated with TNF-alpha for 5 h. After 24 h of TNF-alpha, adhesion of U937 cells to HPVEC was mediated primarily by VCAM-1, consistent with the high expression of VCAM-1 and diminished expression of E-selectin at 24 h., Conclusion: These results demonstrate that HPVEC express functional leukocyte adhesion molecules in vitro and suggest that cardiac valve endothelium is competent to initiate leukocyte adhesion. Thus, other factors, such as the hemodynamic forces exerted on the valve, may contribute to the apparent protection from inflammatory cell infiltration in vivo.

It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage. In this study, we compared the expression profiles of cancer-related genes by cDNA microarray analysis during the differentiation of human promyelocytic leukemia HL-60 cells into either monocytes or granulocytes. RNA was isolated at times 0, 6, 12, 24, 36, 48, and 72 h following stimulation of differentiation with all-trans retinoic acid (all-trans RA) or 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and hybridized to the microarray gene chips containing 872 genes related to cell-cycles, oncogenes and leukemias. Several genes were commonly or differentially regulated during cell differentiation into either lineage, as demonstrated by both hierarchical and self-organizing map clustering analysis. At 72 h the expression levels of 45 genes were commonly up- or down-regulated at least a twofold in both lineages. Most importantly, 32 genes including alpha-L-fucosidase gene and adducin gamma subunit gene were up- or down-regulated only in all-trans RA-treated HL-60 cells, while 12 genes including interleukin 1beta and hypoxia-inducible factor 1alpha were up- or down-regulated only in 1,25-(OH)(2)D(3)-treated HL-60 cells. The expression of selected genes was confirmed by Northern blot analysis. As expected, some genes identified have not been examined during HL-60 cell differentiation into either lineage. The identification of genes associated with a specific differentiation lineage may give important insights into functional and phenotypic differences between two lineages of HL-60 cell differentiation.

Alterations in gene expression during apoptosis in HL-60 cells were identified by a cDNA based array analysis. Apoptosis was induced in the human promyelocytic leukemia cell line, HL-60, by incubation with 30 microM etoposide for 5 hours. Changes in gene expression occurring during apoptosis in these cells were detected using the "ATLAS cDNA Expression Array" technique. 40 genes were identified as differentially expressed in the apoptotic cells by at least a factor of two. 30 of these genes were down-regulated during apoptosis. Many of the down-regulated genes reflected decreased proliferative activity in the cells as well as decreased activity of survival pathways. Most of the genes, which were up-regulated during apoptosis, were genes involved in pathways leading to cell death and suppression of proliferation. Based on the up-regulations observed at the mRNA level, it is speculated that etoposide-induced apoptosis in the HL-60 cells proceeds via pathways involving factors such as TNFalpha, IGFBP3, SAPK1, AP-1 and GADD153/CHOP10. Four genes, which showed changes at the mRNA level, were also analyzed by Western blotting in order to confirm the observed differences at the protein level.

We studied the mechanism of the cytotoxic effects of 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT; induction with 1 mM ALA for 4 h followed by a blue light dose of 18 J/cm(2)) on the human promyelocytic leukemia cell line HL60 using biochemical and electron microscopy methods. The disruption of mitochondrial membrane potential, deltapsi(m), was paralleled by a decrease in ATP level, unmasking of the mitochondrial antigen 7A6, release of cytochrome c into the cytoplasm, activation of caspases 9 and 3 and cleavage of poly(ADP-ribose) polymerase (PARP). This was followed by DNA fragmentation. These data suggest that ALA-PDT activates the mitochondrial apoptotic pathway. The level of endoplasmic reticulum Ca(2+)-binding chaperones ERp57 and ERp72 and of anti-apoptotic proteins Bcl-2 and Bcl-x(L) was decreased whereas that of Ca(2+)-binding protein calmodulin and the stress protein HSP60 was elevated following ALA-PDT. Inhibition of the initiator caspase 9, execution caspase 3 and Ca(2+)-dependent protease m-calpain, did not prevent DNA fragmentation. We conclude that, in our in vitro model, ALA-based photodynamic treatment initiates several signaling processes in HL60 cells that lead to rapidly progressing apoptosis, which is followed by slow necrosis. Two apoptotic processes proceed in parallel, one representing the mitochondrial pathway, the other involving disruption of calcium homeostasis and activation of the endoplasmic reticulum stress-mediated pathway., (Copyright 2002 Elsevier Science B.V.)

K-562 cells were cultured in HL-60 cell growth-conditioned medium (GCM) for up to 96h. Myeloperoxidase (MPO) mRNA was transiently detected by reverse transcription-polymerase chain reaction (RT-PCR) techniques at 12, 24, and 48h. The de novo expression of MPO protein was subsequently detectable by intracellular flow cytometry at 24, 48, 72 and 96h. Immunogold staining and cytochemical analysis demonstrated granularly-sequestered MPO in approximately 40% of HL-60 GCM-cultured cells after 48h of culture. The sequential detection of MPO mRNA and MPO biosynthesis is considered an indicator of serial maturation evocative of myeloblastic cells, and suggest that K-562 cells maintain the ability to differentiate along this lineage.

Enteral nutrition with eicosapentaenoic acid (EPA; 20:5 n-3) and gamma-linolenic acid (GLA; 18:3 n-6) decreased pulmonary inflammation by reducing neutrophil counts and chemotactic factors in bronchoalveolar lavage fluid during acute respiratory distress syndrome (ARDS). We hypothesize that the anti-inflammatory effects of EPA and GLA may be due, in part, to induction of neutrophil apoptosis. The purpose of this study was to determine whether EPA and GLA, alone or in combination, trigger apoptotic cell death in the human promyelocytic leukemia HL-60 cell line. HL-60 cells were incubated with 10, 20, 50, and 100 micromol/L EPA, GLA, or various combinations of EPA and GLA for 2, 4, 8, 12, and 24 hs. Oleic acid (18:1 n-9) was used as a fatty acid control. Flow cytometry using dual staining with propidium iodide and annexin V-FITC assessed apoptosis, necrosis, and viability. Apoptosis was verified by DNA fragmentation as assessed by agarose gel electrophoresis. EPA, GLA, and various combinations of EPA and GLA significantly induced apoptosis and reduced cell viability in HL-60 cells. Viability was significantly reduced to the same extent with the combination of 50 micromol/L EPA\20 micromol/L GLA compared with 100 micromol/L EPA. These data indicate that EPA and GLA, alone or in combination, reduce cell survival by induction of apoptosis. Thus, induction of apoptosis by select dietary n-3 (EPA) and n-6 (GLA) polyunsaturated fatty acids may be the mechanism of the resolution of pulmonary inflammation in ARDS.

We have carried out a detailed comparison of the motile properties of differentiated HL-60 cells and human peripheral blood neutrophils. We compared the effects of chemotactic stimuli and of inhibitors of signalling proteins on morphology, chemokinesis and chemotaxis of neutrophils and differentiated HL-60 cells using videomicroscopy and a filter assay for chemotaxis. We also assessed expression of signalling and cytoskeletal proteins using Western blotting. Chemotactic peptide induced a front-tail polarity in HL-60 cells comparable to that of neutrophils. Chemokinetic and chemotactic responses to chemotactic peptide were also very similar for both cell types, concerning mean speed of migration, the fraction of migrated cells and the concentration of stimulus optimal for activation. The cytokine interleukin-8 was in contrast clearly less effective in activating motile responses of differentiated HL-60 cells as compared to neutrophils. An important functional role of Rho-activated kinases and phosphatidylinositol 3-kinase in motile responses of HL-60 cells, consistent with their upregulation during differentiation, could be confirmed using inhibitors with specificity for the corresponding enzymes. The only difference observed here between HL-60 cells and neutrophils concerned the differential effects of a protein kinase C inhibitor.In summary, the results presented here show that differentiated HL-60 cells, stimulated with chemotactic peptide, are a valid model system to study molecular mechanisms of neutrophil emigration.

We frequently use competitive PCR in the plateau phase in quantifying DNA species with a small number of cells. However, the basic issues of this method are poorly understood. Here, first we analyze this method theoretically under a generalized condition that competitor and target DNA products accumulate with different amplification efficiencies. We show a theoretical reason that competitive PCR might quantify DNA more accurately during the plateau phase than during the exponential phase. Second, we demonstrate that the theoretical predictions are supported by the experimental results of beta-globin gene amplification using the lysates of human diploid fibroblast WS1 cells. We also demonstrate that we can correctly quantify target DNA by keeping the starting concentration of target DNA close to a constant preset value while using a constant number of PCR cycles and by using WS1 cells as control. Finally, we show the experimental errors in routine measurements of c-myc copy number/cell in human leukemia HL-60 cells with various levels of c-myc multiplication. The number of c-myc copies/cell was determined with an error rate of less than 10%, where agarose gel bands were stained with ethidium bromide for the product quantitation., ((c)2002 Elsevier Science (USA).)

Human promyelocytic leukaemia cells (HL-60) differentiate into neutrophil-like cells that die spontaneously by apoptosis when treated with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to and inhibit caspases 3, 7, and 9 activity and the induction of apoptosis. In this study, we demonstrate that undifferentiated HL-60 cells express IAP. During their differentiation, IAP expression is decreased at the mRNA and protein levels. In addition, we show that there is a corresponding increase in the expression and functional activity of active caspases 3 and 9. This activity was associated with the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we demonstrate that blocking caspase activity does not alter the decrease in IAP protein expression during differentiation but prevents caspase activation, IAP cleavage, and the induction of apoptosis. This result shows that the loss of IAP expression is independent of the induction of apoptosis and is solely related to the differentiation process. However, IAP cleavage is caspase-dependent. Terminal differentiation results in an altered apoptotic phenotype that is associated with the induction of HL-60 cell apoptosis.

It has been reported that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase suppress cell proliferation and induce apoptosis. One inhibitor which induces apoptosis is mevastatin. However, the molecular mechanism of apoptosis induction is not well understood so the effects of mevastatin on various functions of HL-60 cells were investigated. We confirmed that mevastatin activated caspase-3 by release of cytochrome c (Cyt. c) from mitochondria through a membrane permeability transition mechanism and also induced typical fragmentation and ladder formation of DNA in HL-60 cells. These effects were inhibited by mevalonate, a metabolic intermediate of cholesterol biosynthesis. Mevalonate and geranylgeraniol (GGOH) inhibited DNA fragmentation whereas farnesol (FOH) did not. Mevastatin also induced cell differentiation to monocytic cells via a mevalonate inhibitable mechanism. Furthermore, mevastatin decreased the amount of an isoprenylated membrane bound Rap1 small GTPase concomitant with an increase in cytosolic Rap1 which occurred before apoptosis and differentiation. On the contrary, both mevastatin and geranylgeranylacetone (GGA), which competes with geranylgeranyl pyrophosphate, induced membrane depolarization of isolated mitochondria without swelling and Cyt. c release. These results suggest that mevastatin-induced apoptosis of HL-60 cells might be caused indirectly by activation of the caspase cascade through the modulation of mitochondrial functions and that some relationship between a certain small GTPase molecule, such as Rap1, and mevastatin-induced apoptosis may exist.

In the HL-60 sublines that were isolated after a long-term continuous culture, abnormally stained or abnormally banded regions on chromosomes replaced extrachromosomal double minutes. The c-MYC gene is amplified in these structures. We followed the c-MYC gene loci during a consecutive passage by using FISH, and have found a large extrachromosomal element (LEE) that preexisted at the earliest passage in a very small fraction of cells. No chromosomal integration of c-MYC sequences was observed in up to 225 passages. The LEEs persistently evolved during culture and were not excluded from the nucleus. In the LEE-positive cells, the spontaneous differentiation was blocked and the granulocytic differentiation that was induced by treatment with dimethyl sulfoxide was reversed by withdrawal of the drug. The c-MYC gene integration into LEEs is unlikely to lead to these phenotypes. The reversibility might be related to the reversible c-MYC down-regulation during the early phase of the drug treatment of HL-60 cells at early cell passages., (Copyright 2001 Academic Press.)

DNA oxidative damage was measured in human promyelocytic leukemia HL-60 cells, in the same cells committed to granulocytic differentiation with dimethyl sulfoxide (DMSO) or all-trans-retinoic acid (RA) and in mature human peripheral granulocytes (HPG). DNA damage was evaluated as single strand breaks and 8-OHdG adducts, measured by single cell electrophoresis or by monoclonal antibodies, respectively. The basal levels of either marker of DNA damage were higher in undifferentiated HL-60 cells than in HPG and DMSO- or RA-differentiated cells. Treatment with H(2)O(2) increased 8-OHdG formation in all cells, but the levels of DNA damage remained higher in undifferentiated cells as compared to the differentiated ones. Three lines of evidence suggested that the higher levels of DNA damage observed in undifferentiated cells were at least in part attributable to a reduced detoxification of reactive oxygen species (ROS). First, undifferentiated cells were shown to accumulate higher levels of dichlorodihydrofluorescein-detectable ROS than HPG and DMSO- or RA-differentiated cells. Second, undifferentiated HL-60 cells were characterized by reduced levels of GSH and lower GSH/GSSG ratios as compared to the differentiated cells. Third, pretreatment of undifferentiated HL-60 cells with antioxidants such as alpha-tocopherol or beta-carotene suppressed the elevation of ROS and the formation of 8-OHdG induced by H(2)O(2). Further evidence for the importance of the oxidant/antioxidant balance was obtained by modulating the iron-catalyzed decomposition of H(2)O(2) to hydroxyl radicals in undifferentiated HL-60 cells. In fact, pretreatment with FeSO(4) increased the formation of 8-OHdG induced by H(2)O(2), whereas pretreatment with the iron chelator deferoxamine produced the opposite effect. These results illustrate correlations between the oxidant/antioxidant balance and DNA damage and suggest that the capability of a cell population to withstand oxidative stress and DNA damage may depend on its degree of differentiation.

Reactive oxygen species play a critical role in the onset of apoptosis induced by various extracellular stimuli, including ionizing radiation. Therefore active regulation of reactive oxygen species-metabolizing enzymes may be one response to an apoptotic stimulus. In this regard, HP100 cells, H(2)O(2)-resistant variants derived from human leukemia HL60 cells, display an interesting phenotype in which the activity of catalase is constitutively high, whereas its mRNA is reduced after X-ray irradiation. In the present study, we investigated the molecular mechanisms underlying this phenomenon. By combining analyses from nuclear run-on, reporter gene transient transfection, genomic footprinting, site-directed mutagenesis, electrophoretic mobility shift analysis, and Western blotting experiments, we found that constitutively elevated catalase expression is strongly regulated at the transcriptional level by both Sp1 and CCAAT-recognizing factors and that much higher levels of nuclear Sp1 and NF-Y are present in HP100 nuclei as compared with HL60 nuclei. In addition, we demonstrated an X-ray-inducible association of a WT1/Egr-related factor with an overlapping Sp1/Egr-1 recognition sequence located within the core promoter of the catalase gene. This association may lead to inactivation of the promoter by disturbing or competing with the transactivating ability of Sp1.

This report is a comparative study of the in vitro cell-growth inhibitory activity of six new trifluoromethylquinoline derivatives on human promyelocytic leukemic cells and the evaluation of their DNA binding characteristics. 2,8-Bis(trifluoromethyl)quinoline-4-(N(4)-ethyl-5-nitroimidazolo)methylene ketone shows the highest cell-growth inhibitory activity with IC(50) of 10+/-2.5 microM, causes extensive changes in the morphology of the HL-60 cells and appears to induce apoptosis. The other compounds are only moderately growth-inhibitory, while 8-trifluoromethylquinoline-4-(N(4)-ethyl-5-nitroimidazolo)methylene ketone is actually stimulatory to cell growth. Except for the latter, the cell growth inhibitory activity of these compounds appears to be due their ability to bind to and intercalate DNA.

Human promyelocytic leukemia (HL-60) cells were irradiated with 0.5-100 Gy of gamma radiation and studied for 48 h post irradiation to determine the mode of death and progression of cells through the phases of the cell cycle. HL-60 cells are much more sensitive to radiation-induced loss of clonogenicity (D0 = 2.2 Gy) than to induction of apoptosis at 6 h (D0 for nonapoptotic cells = 32.6 Gy). After doses 20-50 Gy, the onset of massive apoptosis occurred and nonapoptotic cells were in G1/G0 phase of the cell cycle. In contrast, 6 h after irradiation with doses 2.5-10 Gy maximum cells were in S-phase and 16-24 h after irradiation were arrested in G2-phase. Maximum apoptosis occurred 48 h after irradiation with doses 3.5-10 Gy, and cells that died by necrosis were found in 9-44%.

The mechanism of biological effects of extremely-low-frequency electromagnetic field involve induced changes of Ca2+ transport through plasma membrane ion channels. In this paper we discusses the effects of externally applied Vi = 30 sin 100 pi t(mV) on the induced Ca2+ flux of HL-60 leukemia cells. After signal excitation or sham excitation the response of the intracellular calcium levels changes not only with the different concentrations of the activator but also with the activated states of cells.

Although there is evidence that changes in cellular ionic concentrations are important early events in apoptosis, the regulation of ion fluxes across the plasma membrane during this process is poorly understood. We report here that Bcl-2 overexpression results in up-regulation of capacitative Ca2+ entry (CCE) and that SKF-96365, an inhibitor of CCE, is a potent inducer of apoptosis. Cells that overexpress Bcl-2 are resistant to SKF-96365-mediated apoptosis and to its inhibition of CCE. Enhanced CCE can be reversed with ouabain, suggesting that Bcl-2-associated plasma membrane hyperpolarization plays a role in up-regulating CCE and may partially explain the antiapoptotic effect of Bcl-2.

Sphingosine induces a biphasic increase in cytosolic-free Ca(2+)([Ca(2+)](i)) with an initial peak followed by a sustained increase in HL-60 cells differentiated into neutrophil-like cells. The initial peak is not affected by the presence of ethylene glycol bis (beta-aminoethyl ether) N, N, N', N-tetraacetic acid (EGTA) in the buffer and appears to be dependent on conversion of sphingosine to sphingosine -1-phosphate (S1P) by sphingosine kinase, since it is blocked by the presence of N, N-dimethylsphingosine (DMS), which, like sphingosine, causes a sustained increase itself. The sustained increase that is elicited by sphingosine or DMS is abolished by the presence of EGTA in the buffer. The sustained sphingosine-induced Ca(2+)influx does not appear due to Ca(2+)influx through store-operated Ca(2+)(SOC) channels, since the influx is not inhibited by SKF 96365, nor is it augmented by loperamide. In addition, sphingosine and DMS attenuate the Ca(2+)influx through SOC channels that occurs after depletion of intracellular stores by ATP or thapsigargin. Both the initial peak and the sustained increase in [Ca(2+)](i)elicited by sphingosine can be blocked by phorbol 12-myristate 13-acetate (PMA)-elicited activation of protein kinase C. Thus, in HL-60 cells sphingosine causes a mobilization of Ca(2+)from intracellular Ca(2+)stores, which requires conversion to S1P, while both sphingosine and DMS elicit a Ca(2+)influx through an undefined Ca(2+)channel and cause a blockade of SOC channels., (Copyright 2000 Harcourt Publishers Ltd.)

CD11d encodes the latest alpha-subunit of the leukocyte integrin family to be discovered, and it is expressed predominantly in myelomonocytic cells. We have isolated a genomic clone that contains CD11d and showed this gene to be 11,461 bp downstream and oriented in the same direction as the related CD11c gene. CD11d transcription begins 69-79 nucleotides upstream of the ATG codon. Transfection analysis of CD11d-luc reporter constructs revealed that the -173 to +74 region is sufficient to confer leukocyte-specific expression of luciferase in myelomonocytic cells (THP1 and HL60), B-cells (IM9), and T-cells (Jurkat). Transfection analysis showed that down-regulation of CD11d expression by phorbol ester was myelomonocyte-specific and is mediated by one or more cis-elements within the -173 to +74 region. In vitro DNase I footprint analysis and electrophoretic mobility shift analysis showed that Sp1 and Sp3 bind at -63 to -40. Deletion of the Sp-binding site significantly reduced CD11d promoter activity. Overexpression of either Sp1 or Sp3 in THP1 cells led to activation of the CD11d promoter even in the presence of phorbol ester, whereas down-regulation of either factor by antisense oligonucleotides decreased CD11d promoter activity. In contrast, overexpression of Sp3 in IM9 and Jurkat cells down-regulated CD11d promoter expression. In vivo genomic footprinting revealed that the -63 to -40 region is bound by a Sp protein in unstimulated HL60 cells but not in phorbol ester-stimulated HL60 cells. In contrast, this site is bound in both unstimulated and phorbol ester-stimulated IM9 and Jurkat cells. Together, these results show that myelomonocyte-specific phorbol ester down-regulation of CD11d is mediated through both Sp1 and Sp3.

Interstitial deletions of the q arm of chromosome 5 have been associated with acute myelogenous leukemia (AML); therefore, accurate identification of rearrangements of this chromosome in a model cell line, HL-60, is important for understanding the critical genes involved in this disease. In this study, we employed a newly developed technology termed spectral karyotyping to delineate chromosomal rearrangements in this cell line. Our study revealed a derivative of chromosome 7 that resulted from translocations of chromosome arms 5q and 16q to 7q; that is, der(7)t(5;7)(?;q?)t(5;16)(?;q?). Interestingly, both chromosomes 5 and 7 were also involved in translocations with chromosome 16 in der(16) t(5;16)(q?;q?22-24) and der(16)t(7;16)(?;q?22-24), respectively. Other notable chromosomal abnormalities that were not previously reported in the HL-60 included an insertion of chromosome 8 in the q arm of chromosome 11, a translocation between chromosomes 9 and 14, and a translocation between chromosomes 14 and 15. In an attempt to define the loss of the 5q31.1 region in HL-60, we performed fluorescence in situ hybridization analysis by utilizing bacterial artificial chromosomes BAC1 and BAC2 that spanned the IL9 and EGR1 gene interval, which was previously shown to be a critical region of loss in AML. We showed that a copy of both BAC1 (spanning the D5S399 locus) and BAC2 (spanning the D5S393 locus centromeric to BAC1) were present in the normal chromosome 5, but a second copy of BAC1 was lost and a second copy of BAC2 was inserted in the der(16)t(7;16) chromosome. Thus, not only was this study the first to use the new 24-color karyotyping technique to identify several novel chromosomal rearrangements in HL-60, but it also narrowed the 5q31.1 critical region of deletion to the region represented by BAC1.

An exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2-6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth.

Oxidative phosphorylation within the inner mitochondrial membrane generates the majority of cellular adenosine triphosphate (ATP) required for normal physiological functions (including regulation of cell volume and solute concentration, maintenance of cellular architecture, and synthesis of essential macromolecules). Its efficient functioning depends on the maintenance of an electrochemical gradient and is tightly coupled to the energetic demands of the cell and/or tissue. Commitment to and completion of the cell division cycle are sensitive to changes in the availability of mitochondrially derived ATP, although the relationship between cell cycle and mitochondrial physiology is poorly understood. Using vital, mitochondrial-specific fluorochromes to differentiate between mitochondrial mass (10-N-nonyl acridine orange) and mitochondrial membrane potential (Rhodamine 123), together with a quantification of total cellular ATP levels, it was possible to generate profiles of these mitochondrial characteristics in HL-60 cells at different stages of their cell cycle. The data suggest that the availability of ATP changes in a cell cycle-specific manner and cannot be predicted by changes in mitochondrial mass or membrane potential. Furthermore, transition points in the cell cycle where ATP availability is low with respect to the amount of functional inner mitochondrial membrane have been observed. We suggest that these cell cycle phase transitions are sensitive to inhibition of mitochondrial activity because the basal levels of available ATP at these points are nearer to a theoretical "minimal threshold" below which cell cycle progression is inhibited.

The steady-state level of nucleophosmin/B23 mRNA decreased during berberine-induced (25 microg/ml, 24 to 96 hr) apoptosis of human leukemia HL-60 cells. A decline in telomerase activity was also observed in HL-60 cells treated with berberine. A stable clone of nucleophosmin/B23 overexpressed in HL-60 cells was selected and found to be less responsive to berberine-induced apoptosis. About 35% to 63% of control vector-transfected cells (pCR3) exhibited morphological characteristics of apoptosis, while about 8% to 45% of nucleophosmin/B23-over-expressed cells (pCR3-B23) became apoptotic after incubation with 15 microg/ml berberine for 48 to 96 hr. DNA extracted from pCR3 cells contained more fragmented DNA than pCR3-B23 cells during treatment with 15 microg/ml berberine for 24 to 48 hr. Our results indicate that berberine-induced apoptosis is associated with down-regulation of nucleophosmin/B23 and telomerase activity. We also suggest that nucleophosmin/B23 may play an important role in the control of the cellular response to apoptosis induction.

Jaspamide, a naturally occurring cyclic peptide isolated from the marine sponge Hemiastrella minor, has fungicidal and growth-inhibiting activities. Exposure of promyelocytic HL-60 cells and human monocytes to jaspamide induces a dramatic reorganization of actin from a typical fibrous network to focal aggregates. HL-60 cells exposed to 5 x 10(-8) mol/L or 10(-7) mol/L jaspamide exhibited a reduced proliferation rate. In addition, 10(-7) mol/L jaspamide induced maturation of HL-60 cells as indicated by the appearance of a lobulated nucleus in 55% +/- 5% of the cells and immunophenotypic maturation of the leukemia cells (upregulation of CD16 and CD14 B antigens). Further characterization has shown that F-actin is aggregated both in HL-60 cells and in human monocytes exposed to 10(-7) mol/L jaspamide. Well-spread cultured human monocytes contracted and adopted round shapes after treatment with jaspamide. Moreover, a dose-dependent increase in both total actin and de novo synthesized portions of the soluble actin was observed in jaspamide-treated HL-60 cells. Jaspamide treatment inhibits ruffling and intracellular movement in HL-60 cells and monocytes, but does not affect phagocytic activity or respiratory burst activity. The consequential effects of jaspamide-induced actin reorganization on ruffling, versus its negligible effect on phagocytosis and oxidative burst, may shed light on molecular mechanisms of actin involvement in these processes. Jaspamide disrupts the actin cytoskeleton of normal and malignant mammalian cells with no significant effect on phagocytic activity and may, therefore, be considered as a novel therapeutic agent.

We have previously shown that in myeloid leukemic cells, daunorubicin (DNR) induces apoptosis via the activation of the sphingomyelin-ceramide pathway. We have now investigated sphingomyelin (SM) hydrolysis, ceramide generation, and apoptosis in vincristine-selected multidrug resistant (MDR) HL-60 cells (HL-60/Vinc), compared with their parental counterparts. We show that DNR triggers the SM cycle (stimulation of neutral sphingomyelinase, SM hydrolysis, and ceramide generation) and apoptosis in both parental and MDR cells, when used at isotoxic doses (ie., 1 and 100 microM for HL-60 and HL-60/Vinc, respectively). However, in MDR cells treated with either 10 microM DNR or 1 microM DNR in association with the P-glycoprotein (P-gp) blocker verapamil (treatment conditions which yield an intracellular DNR concentration similar to that achieved with 1 microM in the parental cells), we were unable to detect SM hydrolysis, ceramide generation and apoptosis. This implies that inhibition of the DNR-induced SM cycle in MDR cells is not directly related to P-gp. We have also investigated the influence of intracellular drug localization on the DNR-induced SM-cycle in HL-60/Vinc cells. In these cells, DNR at 10 microM is mainly localized in cytoplasmic vesicles, while the drug is diffusely distributed when used at 100 microM. A diffuse distribution pattern was also observed when MDR cells were treated with 1 microM DNR in association with the cyclosporine derivative PSC-833, but not with verapamil. In parallel, PSC-833, but not verapamil, restored the induction of the SM cycle and the apoptotic potential of DNR, and markedly increased drug cytotoxicity in MDR cells. Our results suggest that altered intracellular drug transport plays an important role in limiting ceramide generation and cell death in MDR cells.