Response Of HL-60 Cells Research Articles

Quantitative Monitoring of GPCR-Mediated Spatiotemporal IP3 Dynamics Using Confocal Fluorescence Microscopy.

Activation of G protein-coupled receptors upon chemoattractant stimulation induces activation of multiple signaling pathways. To fully understand how these signaling pathway coordinates to achieve directional migration of neutrophils, it is essential to determine the dynamics of the spatiotemporal activation profile of signaling components at the level of single living cells. Here, we describe a detailed methodology for monitoring and quantitatively analyzing the spatiotemporal dynamics of 1,4,5-inositol trisphosphate (IP3) in neutrophil-like HL60 cells in response to various chemoattractant fields by applying Förster resonance energy transfer (FRET) fluorescence microscopy.

Abstract 4678: The role of EGR1 and AP1 in acute myeloid leukemia cell reprogramming toward cell cycle arrest and apoptosis

Abstract EGR and AP1 transcription factors are rapidly upregulated in acute myeloid leukemia cells responding to treatment with phorbol esters. Phorbol ester induced transcriptome changes are accompanied by phenotypic changes including cell cycle arrest, myeloid differentiation, and ultimately apoptosis. Here we present results documenting the transcriptome alterations of the phorbol ester response in HL-60 cells by DNA microarray and RNA-seq analyses, and explore the specific roles of EGR and AP1 in determining the evolving transcriptional program. Overexpression of egr1 or c-fos recapitulates many of the phorbol ester-induced transcriptome alterations and affects cell proliferation, differentiation and survival. Identification of changes in EGR1 and FOS/AP1 target gene expression supports the hypothesis that EGR and AP1 are involved in a genetic reprogramming of AML cells leading to loss of the leukemic phenotype and recovery of apoptotic competency. We also provide a comprehensive bioinformatic analysis of AML patient sample data provided by The Cancer Genome Atlas and the published study from Tyner et al., Nature 562:526-531 (2018) accessible through the Beat AML data viewer (Vizome) regarding expression of egr1. Egr1 high expression correlates with improved overall survival and a particular mutational signature distinct from patients with low egr1 expression. We define a mutational and transcriptional signature for AML patients based on egr1 expression levels that correlates with clinical outcomes. Citation Format: Michael Roberts, Lauren Kageler, Kayla Bendinelli, Shannon Bonner, Ashir Borah, Jeffrey Forrester. The role of EGR1 and AP1 in acute myeloid leukemia cell reprogramming toward cell cycle arrest and apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4678.

Frontline Science: A flexible kink in the transmembrane domain impairs β2 integrin extension and cell arrest from rolling.

β2 integrins are the main adhesion molecules in neutrophils and other leukocytes and are rapidly activated by inside-out signaling, which results in conformational changes that are transmitted through the transmembrane domain (TMD). Here, we investigated the biologic effect of introducing a proline mutation in the β2 integrin TMD to create a flexible kink that uncouples the topology of the inner half of the TMD from the outer half and impairs integrin activation. The β2 integrin alpha chains, αL, αM, αX, and αD, all contain an inserted (I) domain with homology to von Willebrand factor A domain. β2 activation was monitored in a homogenous binding assay of 2reporter monoclonal antibodies: KIM127 reporting extension (E+ ) and mAb24 reporting the high-affinity (H+ ) conformation of the β2 I-like domain. The proline mutation partially diminished chemokine-induced extension, but not the high-affinity conformation. The proline mutation in the TMD of β2 completely inhibited arrest of rolling HL-60 cells in response to the chemokine IL-8. TMD mutant HL-60 cells rolling on P-selectin and ICAM-1 were unable to reduce their rolling velocity in response to IL-8. Quantitative dynamic footprinting live-cell imaging showed that blocking TMD topology transmission impaired the chemokine-induced activation of β2, limiting the appearance of extended high-affinity (E+ H+ ) β2. This also resulted in a defect in early spreading (3min after arrest), which could be overcome by forced integrin activation using Mn2+ . We conclude that the TMD proline mutation severely impairs β2 integrin extension, cell arrest, and early spreading.

P0200 Use of polyamine transport system by polyamine anthracene conjugates in human leukaemic cells

Background It is well known that cancer cells have increased polyamine concentrations compared with normal cells. This relation is due to a combination of increased polyamine uptake via the polyamine transport system (PTS) as well as increased biosynthesis. Thus, we may be able to exploit the PTS for selective delivery of cytotoxic agents to cancer cells by attaching them to polyamine vectors. Such an approach should reduce non-specific toxicities and therefore decrease the side-effects associated with chemotherapy. This concept is also supported by the nature of PTS, which can accommodate a wide range of substrates. Three compounds combining the DNA intercalator, anthracene, with a polyamine side chain have been synthesised: Ant 4, Ant 44, and Ant 444.The aim of this study was to investigate the mode of cell death induced by anthracene conjugates in HL-60 cell lines. Methods We used a variety of techniques to study the response of HL-60 cells to Ant 4, Ant 44, and Ant 444. Findings Deconvolution microscopy analysis revealed that the conjugates entered the cells. Quantitative analysis using high-performance liquid chromatography showed that the amount of Ant 4 in cells increased 30% from 6 h to 12 h. Ant 44 showed the highest level in cells after 6 h of exposure. Ant 444 showed a gradual increase between 0 h to 48 h. The indirect evidence using pre-treated cells with 5 mmol difluromethylornithine (DFMO) showed a significant decrease of IC 50 compared with no DFMO pretreatment for Ant 4, Ant 44, and Ant 444. The uptake study using radiolabelled polyamines showed there is a competition uptake between radiolabelled polyamines [ 14 C] putrescine, spermidine, or spermine with Ant 4, Ant 44, and Ant 444. Interpretation Ant 4, Ant 44, and Ant 444 gain entry into HL-60 cells via the PTS. The idea of selectively targeting cancer cells via the PTS shows promise. In the future, the concept of selective targeting of cancer cells via the PTS could lead to the development of novel and effective anticancer therapies.

Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway.

The toxicities of conventional chemotherapeutic agents to normal cells restrict their dosage and clinical efficacy in acute leukemia; therefore, it is important to develop novel chemotherapeutics, including natural products, which selectively target cancer-specific pathways. The present study aimed to explore the effect of the chemopreventive agent asiatic acid (AA) on the proliferation and apoptotic rate of the leukemia cell line HL-60 and investigated the mechanisms underlying its anti-tumor activity. The effect of AA on the proliferation of HL-60 cells was evaluated using the MTT assay. Annexin V-fluorescein isothiocyanate/propidium iodide double staining followed by flow cytometric analysis as well as Hoechst 33258 staining were used to analyze the apoptotic rate of the cells. Furthermore, changes of survivin, B-cell lymphoma 2 (Bcl-2), myeloid cell leukemia 1 (Mcl-1), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 expressions were detected by western blot analysis. AA blocked the growth of HL-60 cells in a dose- and time-dependent manner. The IC50-value of AA on HL-60 cells was 46.67 ± 5.08 µmol/l for 24 h. AA induced apoptosis in a dose-dependent manner, which was inhibited in the presence of Z-DEVD-FMK, a specific inhibitor of caspase. The anti-apoptotic proteins Bcl-2, Mcl-1 and survivin were downregulated by AA in a dose-dependent manner. Concurrently, AA inhibited ERK and p38 phosphorylation in a dose-dependent manner, while JNK phosphorylation was not affected. In conclusion, the present study indicated that the p38 and ERK pathways, as well as modulation of Bcl-2 family and survivin proteins were key regulators of apoptosis induced in HL-60 cells in response to AA.

Artificial Food Colorants Inhibit Superoxide Production in Differentiated HL-60 Cells

We tested synthetic food colorants for their antioxidative potential by the in vitro superoxide generation assay in differentiated HL-60 cells in response to phorbol ester. Among the 12 colorants tested, such fluorescein-type red colorants as rose bengal showed potent inhibitory activity without any cytotoxicity under dark conditions. The intracellular accumulation and superoxide anion scavenging effect of rose bengal were at least partly involved in the inhibitory activity.

Abstract 4080: Global changes in gene expression during phorbol ester-induced differentiation of human acute myeloid leukemia cells

Abstract This study seeks to elucidate the molecular mechanisms by which the acute myeloid leukemia cell line, HL-60, is induced to differentiate into macrophage-like cells following exposure to phorbol 12-myristate 13-acetate (PMA). Changes in gene expression over the time course of PMA treatment were measured by DNA microarray analysis and select genes shown to be significantly up- and down-regulated were validated by quantitative, real-time PCR. Functional studies of transcription factor binding activities were performed by the electrophoretic mobility shift assay (EMSA). Novel findings include the rapid and sustained induction of mRNA levels for a group of CC chemokine genes (CCL2, CCL3, CCL3L1, CCL4, CCL4L1 and CCL4L2). These genes displayed 10 to 50-fold expression level increases that were observed within 3 hours of treatment and were maintained through 6 hours post-induction. The anti-proliferative effects of these peptides are being examined on a variety of leukemia cell lines. Among the most highly induced early genes were others known to encode inflammatory response proteins (TNF, IL8, C3AR1). In addition, the transcription factor profile is dramatically altered early in the differentiation time course, with the predicted activation of AP-1 family members (JUNB, FOSL1) and the concomitant inhibition of NFKB (TNFAIP3, NFKBIA, NFKBID). Other transcription factor genes were transiently up regulated (MAFB, GATA2, ETS2) and c-MYC expression was significantly down regulated and remained low during the differentiation course. Pathway analysis was used to predict gene expression changes occurring later in the differentiation process and was tested by microarray analysis. Early events in the genetic re-programming of HL-60 cells in response to the differentiation inducer PMA includes a massive up-regulation of chemokine genes and alteration of the transcription factor profile. The implication of these gene expression changes will be discussed in the context of differentiation therapies for acute myeloid leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4080.

Neutrophilic differentiation modulates the apoptotic response of HL-60 cells to sodium butyrate and sodium valproate

Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.

APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

Retinoic acid inhibits sodium butyrate-induced monocytic differentiation of HL60 cells while synergistically inducing granulocytoid differentiation

The human myeloid leukemia cell line HL60 is an in vitro model to study myeloid differentiation. HL60 cells differentiate along different cell type lineages in response to a variety of compounds. The direction of differentiation is usually inducer-specific. However, the response of HL60 cells to sodium n-butyrate (NaB) is pleiotropic. NaB induces HL60 along the monocytic, neutrophilic, eosinophilic, and basophilic pathways. In this study we saw that physiologic concentrations of all-trans-retinoic acid (RA) switched the direction of NaB-induced differentiation from monocytic to granulocytic. We showed previously (Breitman & He, Cancer Res 1990: 50: 6268-6273) that combinations of RA and NaB synergistically induce HL60 to cells that reduce nitroblue tetrazolium. The present study shows that this synergy was even greater if the parameter measured was mature granulocytes. Our results raise the possibility that the endogenous RA in the serum used to grow cells in culture may affect the direction of differentiation of HL60 cells induced by NaB. Furthermore, our results may provide additional rationale for the use of combinations of RA and NaB in the treatment of some leukemias.

Dielectrophoretic field-flow fractionation system for detection of aquatic toxicants.

Dielectrophoretic field-flow fractionation (dFFF) was applied as a contact-free way to sense changes in the plasma membrane capacitances and conductivities of cultured human HL-60 cells in response to toxicant exposure. A micropatterned electrode imposed electric forces on cells in suspension in a parabolic flow profile as they moved through a thin chamber. Relative changes in the dFFF peak elution time, reflecting changes in cell membrane area and ion permeability, were measured as indices of response during the first 150 min of exposure to eight toxicants having different single or mixed modes of action (acrylonitrile, actinomycin D, carbon tetrachloride, endosulfan, N-nitroso- N-methylurea (NMU), paraquat dichloride, puromycin, and styrene oxide). The dFFF method was compared with the cell viability assay for all toxicants and with the mitochondrial potentiometric dye assay or DNA alkaline comet assay according to the mode of action of the specific agents. Except for low doses of nucleic acid-targeting agents (actinomycin D and NMU), the dFFF method detected all toxicants more sensitively than other assays, in some cases up to 10 (5) times more sensitively than the viability approach. The results suggest the dFFF method merits additional study for possible applicability in toxicology.

Expression of perilipin in human promyelocytic cells in response to Anaplasma phagocytophilum infection results in modified lipid metabolism

The obligate intracellular pathogen Anaplasma phagocytophilum is transmitted by ticks and causes human granulocytic anaplasmosis, tick-borne fever of ruminants, and equine and canine granulocytic anaplasmosis. In a previous study, the perilipin (PLIN) gene was identified as one of the genes differentially expressed in human promyelocytic HL-60 cells in response to infection with A. phagocytophilum. PLIN is a major adipocyte lipid droplet-associated phosphoprotein that plays a central role in lipolysis and cholesterol synthesis. Host cholesterol and other lipids are required by A. phagocytophilum for infection and multiplication in human cells. In this study, it was hypothesized that PLIN may be involved in infection of human HL-60 cells by A. phagocytophilum. To test this hypothesis, a combination of real-time RT-PCR, immunofluorescence and RNA interference was used to study the expression of PLIN. The results of these studies demonstrated that A. phagocytophilum modulates lipid metabolism by increasing PLIN mRNA levels and facilitates infection of HL-60 cells. The results of these studies expand our knowledge of the role of lipid metabolism in A. phagocytophilum infection and multiplication in HL-60 cells and suggest a mechanism by which A. phagocytophilum modulates lipid metabolism.

284 POSTER Lenalidomide and CC-4047 inhibit the proliferation of Namalwa cancer cells while expanding CD34+ progenitor cells. New insights on the combination therapy with HDAC inhibitors for hematological cancers

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader–Willi syndrome.

Doxorubicin-DNA Adducts Induce a Non-Topoisomerase II–Mediated Form of Cell Death

Doxorubicin (Adriamycin) is one of the most commonly used chemotherapeutic drugs and exhibits a wide spectrum of activity against solid tumors, lymphomas, and leukemias. Doxorubicin is classified as a topoisomerase II poison, although other mechanisms of action have been characterized. Here, we show that doxorubicin-DNA adducts (formed by the coadministration of doxorubicin with non-toxic doses of formaldehyde-releasing prodrugs) induce a more cytotoxic response in HL-60 cells than doxorubicin as a single agent. Doxorubicin-DNA adducts seem to be independent of classic topoisomerase II-mediated cellular responses (as observed by employing topoisomerase II catalytic inhibitors and HL-60/MX2 cells). Apoptosis induced by doxorubicin-DNA adducts initiates a caspase cascade that can be blocked by overexpressed Bcl-2, suggesting that adducts induce a classic mode of apoptosis. A reduction in the level of topoisomerase II-mediated double-strand-breaks was also observed with increasing levels of doxorubicin-DNA adducts and increased levels of apoptosis, further confirming that adducts exhibit a separate mechanism of action compared with the classic topoisomerase II poison mode of cell death by doxorubicin alone. Collectively, these results indicate that the presence of formaldehyde transfers doxorubicin from topoisomerase II-mediated cellular damage to the formation of doxorubicin-DNA adducts, and that these adducts are more cytotoxic than topoisomerase II-mediated lesions. These results also show that doxorubicin can induce apoptosis by a non-topoisomerase II-dependent mechanism, and this provides exciting new prospects for enhancing the clinical use of this agent and for the development of new derivatives and new tumor-targeted therapies.

Mitochondrial gene knockout HL60ρ 0 cells show preferential differentiation into monocytes/macrophages

This study compares the differentiation potential of mitochondrial gene knockout (ρ 0) and parental HL60 cells in response to 1.25% dimethylsulfoxide (DMSO) and 10 nM phorbol myristate acetate (PMA). Compared to HL60 cells, undifferentiated HL60ρ 0 cells showed partial monocyte/macrophage differentiation, with increased CD11c and CD14 expression, decreased CD71 expression, and weak non-specific esterase staining. Differentiation along the monocyte/macrophage pathway (PMA) was more pronounced in HL60ρ 0 than parental HL60 cells with increased CD11c and CD14 expression and stronger non-specific esterase staining. DMSO-exposure resulted in a poorly differentiated nuclear morphology, small respiratory burst and marginal up-regulation of CD15 expression in HL60ρ 0 cells.

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca 2+] i). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (≠HL-60 cells) and further to examine the role of Ca 2+ mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca 2+] i rise (254 ± 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 μM of N-formyl- l-methionyl- l-leucyl- l-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca 2+ (17.0 ± 4.5 arbitrary units (A.U.) in the absence of Ca 2+ versus 56.6 ± 3.9 A.U. in the presence of 1.25 mM of Ca 2+). Also, IL-8 enhanced fMLF-stimulated increase in [Ca 2+] i (375 ± 35 versus 245 ± 21 nM, 0.1 μM of fMLF). IL-8 had no effect on ≠HL-60 cells in response to 1 μM of thapsigargin (472 ± 66 versus 470 ± 60 nM). In conclusion, Ca 2+ influx is necessary for a full induction of neutrophil priming by IL-8.

Vascular endothelial growth factor receptor-1 and receptor-2 initiate a phosphatidylinositide 3-kinase–dependent clonogenic response in acute myeloid leukemia cells

ObjectiveVascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. Materials and methodsUsing KG1 (VEGFR-1+/VEGFR-2+) and HL60 (VEGFR-1+) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. ResultsRecombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF–induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 μM, whereas MEK inhibition by PD98059 (1 and 10 μM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. ConclusionThese data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.

Molecular analysis of signaling events mediated by the cytoplasmic domain of leukemia inhibitory factor receptor alpha subunit.

A chimeric receptor (130/190) containing the cytoplasmic region of leukemia inhibitory factor receptor alpha subunit (LIFRalpha, or gp190) and the extracellular transmembrane region of gp130 was generated. Expressed of the 130/190 chimera in HL-60 cells to induced the homodimerization of the cytoplasmic domains (190cyt-190cyt) with whole LIFRalpha subunit on HL-60 cells in response to LIF. Expression and activation of the signal transducer and activator of transcription factor-3 (Stat3) and inhibition of leukemia cell proliferation were evaluated in cells transfected with this chimeric molecule. Increased tyrosyl phosphorylation of Stat3 at Tyr705 was detected after 10 min LIF treatment in cells transfected with either the 130/190 or the wild type receptor. Cell proliferation was decreased upon LIF treatment in both cell types. However, expression of the C-terminal region of the cytoplasmic region of LIFRalpha subunit (190CT) in HL-60 cells resulted in lower levels of Stat3 phosphorylation induction by LIF and cell proliferation was unaffected. Immunohistochemical staining indicated an inverse correlation between Cdc25B expression and the levels of phospho-Stat3 in 190CT and 130/190 cells. Expression of CD15, a cell differentiation marker, was lower in 190CT than in 130/190 cells. Together, these results suggest that homodimerization of the 190 cytoplasmic region promotes the Tyr 705 phosphorylation, which correlates with the inhibition of proliferation and stimulation of differentiation in HL-60 cells. Our results also suggest a signal link between Stat3 and Cdc25B.

Histone acetylation-mediated regulation of genes in leukaemic cells

Histone deacetylase (HDAC) and histone acetyltransferase (HAT) functions are associated with various cancers, and the inhibition of HDAC has been found to arrest disease progression. Here, we have investigated the gene expression profiles of leukaemic cells in response to the HDAC inhibitor trichostatin A (TSA) using oligonucleotide microarrays. Nucleosomal histone acetylation was monitored in parallel and the expression profiles of selected genes were confirmed by quantitative polymerase chain reaction (PCR). A large number of genes (9% of the genome) were found to be similarly regulated in CCRF-CEM and HL-60 cells in response to TSA, and genes showing primary and secondary responses could be distinguished by temporal analysis of gene expression. A small fraction of genes were highly sensitive to histone hyper-acetylation, including XRCC1, HOXB6, CDK10, MYC, MYB, NMI and CBFA2T3 and many were trans-acting factors relevant to cancer. The most rapidly repressed gene was MKRN3, an imprinted gene involved in the Prader–Willi syndrome.

Topoisomerase II Is Required for Mitoxantrone to Signal Nuclear Factor κB Activation in HL60 Cells

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.