Human Myeloid Cell Line HL-60 Research Articles

Antitumoral Activity of 13-Demethyl or 13-Substituted Analogues of All-trans Retinoic Acid and 9-cis Retinoic Acid in the Human Myeloid Leukemia Cell Line HL-60

The antitumoral activity of 13-demethyl or 13-substituted all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9CRA) was tested using the myeloid leukemia cell line HL-60. Cell proliferation, differentiation and apoptosis were evaluated by flow cytometry and DNA fragmentation assay. The ability to bind to human RXRalpha and to activate either human retinoic acid response element (RARE)-mediated gene expression or rat CRABPII retinoid X response element (RXRalpha)-mediated gene expression were determined using luciferase reporter plasmids. In terms of the magnitude of the regulatory activity for the proliferation and differentiation of HL-60 cells, the compounds ranked as follows: ATRA>13-ethyl ATRA>13-demethyl ATRA>13-phenylethyl ATRA>13-propyl ATRA>13-butyl ATRA (ATRA analogues) and 9CRA>13-ethyl 9CRA>13-demethyl 9CRA>13-propyl 9CRA>13-phenetyl 9CRA>13-butyl 9CRA (9CRA analogues). Regarding the magnitude of the apoptosis-inducing activity, the order was: 9CRA>13-ethyl 9CRA>13-demethyl 9CRA, with ATRA and its analogues and the other 9CRA analogues virtually inactive. Similar trends were observed in binding affinity for RXRalpha and transactivation activity toward RARE- or RXRE-mediated gene expression. The results clearly indicate that the presence of a methyl group at C-13 is essential for the antitumoral activity of ATRA and 9CRA, and that bulky substituents exceeding two carbon atoms or the absence of substitution at position 13 significantly reduce the binding affinity for RAR and RXR, leading to a decreased RAR/RARE and/or RXR/RXRE-mediated gene expression.

Histone Deacetylase Inhibitor Depsipeptide (FK228) Induces Apoptosis in Leukemic Cells by Facilitating Mitochondrial Translocation of Bax, Which Is Enhanced by the Proteasome Inhibitor Bortezomib

Histone deacetylase (HDAC) inhibitors are promising candidates for molecular-targeted therapy for leukemia. In this study, we investigated the mechanisms of cytotoxic effects of depsipeptide (FK228), one of the most effective HDAC inhibitors against leukemia, using human myeloid leukemic cell lines HL-60 and K562. We found that FK228 activated caspase-9 and a subsequent caspase cascade by perturbing the mitochondrial membrane to release cytochrome c, which was almost completely blocked by overexpression of Bcl-2. The mitochondrial damage was caused by the translocation of Bax but not other pro-apoptotic Bcl-2 family proteins to the mitochondria. FK228 did not affect the interaction between Bax and Bax adaptor proteins such as 14-3-3Θ and Ku70. FK228-induced apoptosis and mitochondrial translocation of Bax were markedly enhanced by the proteasome inhibitor bortezomib. The synergistic action of FK228 and bortezomib was at least partly mediated through conformational changes in Bax, which facilitate its translocation to the mitochondria. These results suggest that the combination of HDAC inhibitors and proteasome inhibitors is useful in the treatment of leukemia especially in the context of molecular-targeted therapy. The status of Bcl-2 and Bax may influence the sensitivity of tumors to this combination and thus can be a target of further therapeutic intervention.

Sesquiterpenoids from Pulicaria canariensis and Their Cytotoxic Activities

Thirteen new sesquiterpenes, pulicanadiene A (1), B (2), and C (3), pulicanone (4), pulicanol (5), pulicanarals A (6), B (7), and C (8), pulicanadienals A (9) and B (10), pulicanadienol (11), and pulioplopanones A (12) and B (13), and seven known compounds, stigmasterol, ergosterol peroxide, calenduladiol, 7,4'-di-O-methyldihydrokaempferol, 5,7-dihydroxy-3,3',4'-trimethoxyflavone, dihydroquercetin 7,3'-dimethyl ether, and 6,15alpha-epoxy-1beta,4beta-dihydroxyeudesmane, were isolated from Pulicaria canariensis. Compound 4a showed cytotoxicity on the human myeloid leukemia cell line HL-60. The cytotoxicity was caused by induction of apoptosis as determined by microscopy of nuclear changes, activation of caspases, and the cleavage of poly(ADP-ribose) polymerase-1.

Expression of Human Enhancer of Invasion 10 (HEI10) in Human Myeloid Leukemias and Downregulation during Differentiation of Hematopoietic Stem Cells.

Background: We previously investigated the gene expression profile of drug-resistant human leukemia K562 cells by the serial analysis of gene expression (SAGE) (Ichikawa et al. Int J Hematol, 2004; 79:276), and found that the HEI10 gene was overexpressed in K562 cells. With the same technology, we also observed that human CD34+ cells expressed high levels of HEI10 gene expression compared to erythroid progenitor cells (Fujishima et al. Int J Hematol, in press). HEI10 encodes a protein which belongs to a family of E3 ubiquitin ligase,and interacts with cyclin B1 leading to the regulation of cell cycle. In the present study, we examined the gene expression of HEI10 quantitatively in human leukemias in order to learn whether there would be any association between the expression levels and leukemia subtypes. We also investigated whether the expression levels of HEI10 would be related to differentiation of myeloid leukemia cell lines and normal hematopoietic stem cells.Methods: The quantitative real-time PCR method was employed to estimate the relative gene expression levels of HEI10. Total RNA was extracted from mononuclear cells of bone marrow that had been taken for diagnostic purposes. Patients include acute myeloid leukemia (AML) (n=21) and chronic myeloid leukemia (CML) (n=7; 2 in blastic crisis, 5 in chronic phase). Differentiation of human acute myeloid leukemia cell line HL60 cells was induced by PMA. Human normal CD34+ cells were purified from G-CSF mobilized blood stem cells and were induced to differentiate into erythroid, myeloid or dendritic cells by various cytokines.Results: Quantitative real-time PCR analysis revealed the high expression of HEI10 gene in patients with leukemia with undifferentiated phenotype (3 cases of AML M1 in FAB classification and 2 cases of CML in blast crisis) and low levels of expression in acute promyelocytic leukemia and CML in chronic phase. When HL60 cells were induced to differentiation with PMA, accumulation in the G0/G1 phase was observed and the expression of HEI10 was decreased. Differentiation of normal CD34+ cells also resulted in a decrease of HEI10 expression.Conclusions: The expression of HEI10 gene in hematopoietic cells appears to be upregulated when cells are immature. The role for HEI10 in hematopoietic stem cells remains to be elucidated. Biological effects of the HEI10 gene silencing in human hematopoietic cells are currently being investigated by RNAi technology. [Display omitted] [Display omitted]

Organic Arsenic Lipid Derivatives Are More Potent and Less Toxic Than Inorganic Arsenic Trioxide in Preclinical Testing.

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is effective in patients with relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers but the risk/benefit ratio for its use still has to be determined due to its significant side effects. In contrast, organic arsenic derivatives are known to be much less toxic. We have synthesized a series of eight organic arsenic lipid derivatives and have tested them in NIH 60 cell line screen. Based on its activity, we selected GMZ27 [3-S-di(n-propyl)arsino-3-thio-1,2-propanediol] for further study in our laboratory and have confirmed its potent activity against human acute myeloid leukemia cell lines HL60 and NB4, which was significantly higher than that of arsenic trioxide. The IC50 (concentration that kills 50% of the cells) in MTS assay is 0.5 and 0.9 μm for GMZ27. Assessment of the mechanisms of action of GMZ27 in cell lines has shown that the GMZ27 was more potent inducer of superoxide than ATO. GMZ27 caused dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation, PARP cleavage, and compromise in cell membrane integrity. However, its treatment also resulted in caspase 8 cleavage, suggesting that it affects both intrinsic and extrinsic apoptotic pathway. Its activity was related to the level of glutathione in the leukemic cells as pretreatment of the cells with BSO, which depletes intracellular glutathione, results in cells sensitivity to GMZ27. On the other hand, pretreatment with DDT, which increases intracellular glutathione, results in their resistance to GMZ27. GMZ27 had no effect on cells maturation and differentiation, and cell cycle. GMZ27 when tested against healthy donor mononuclear cells in a colony forming assay showed significantly less toxicity than arsenic trioxide. In vivo toxicity testing in Swiss Webster mice showed LD50 (dose that kills 50% of mice) to be 100 mg/kg for GMZ27, comparing to 10 mg/kg for ATO. In conclusion, organic arsenic lipid derivatives, and in particular GMZ27, may have more potent antileukemic activity and significantly less toxicity in vivo and in vitro than ATO and therefore further development of these medications is warranted.

Effect of Dioxin on Normal Human Hematopoietic and Leukemic Cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of various genes, including cytokines and growth factors. TCDD has a large number of biological effects, such as long-lasting skin disease, cardiovascular disease, diabete and cancer. An increase risk of Hodgkin's disease, non-Hodgkin's lymphoma and myeloid leukemia was observed after 15 years in the population exposed to dioxin following the 1976 accident in Seveso, Italy. In this study, we analysed the effect of escalating doses of TCDD on the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia), as well as on human CD34+ progenitor cells from the leukapheresis of normal donors stimulated with G-CSF. The possible specific modulation of gene expression induced by TCDD was then tested by means of macroarray analyses (Clontech), the results of which were successfully validated by real-time quantitative PCR on five candidate genes. The dioxin dose capable of inhibiting the growth of 50% of the colonies in semisolid medium (IC50 calculated using the CalcuSyn computer program) was 33 nM for HL60, 28 nM for K562, and 20 nM for the CD34+ cells. The HL60, K562 and CD34+ cells tested before and after exposure to 20 nM of TCDD allowed us to identify a series of modulated genes. The up-regulated genes included LUCA15 putative tumour suppressor, cyclin-D binding Myb-like protein (hDMP1), MAPK/ERK kinase 6, ras-related protein RAB2, RalB GTP-binding protein, transcription factor ZFM1, and BRCA1-associated ring domain protein (BARD1). The down-regulated genes included found c-myc, TNF alpha precursor, ICAM1, MIP1-beta, interferon gamma-induced protein precursor (gamma-IP10), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein 1 precursor (MCP1). All of these genes are variously involved in the processes of proliferation, differentiation and transformation in hematological and tumoral cell models. On the basis of these data, we can hypothesise that the inhibition of clonogenic potential and the gene expression induced by TCDD exposure on normal CD34+ progenitor cells plays a role in the neoplastic transformation of hemopoietic stem cells. This supports the epidemiological data indicating an increased hematological cancer risk in the population accidentally exposed to TCDD.

Intrinsic cytotoxicity and chemomodulatory actions of novel phenethylisothiocyanate sphingoid base derivatives in HL-60 human promyelocytic leukemia cells.

The protein kinase C (PKC) isoenzyme superfamily represents a popular target in pharmacological interventions designed to elicit apoptosis directly in tumor cells or to potentiate the lethal effects of antineoplastic agents. Numerous observations support the clinical utility of PKC inhibition by experimental sphingolipid derivatives such as safingol. The present studies document the cytotoxicity and chemomodulatory capacity of phenethylisothiocyanate derivatives of sphinganine and sphingosine (PEITC-Sa and PEITC-So) in the human myeloid leukemia cell line HL-60. The biological actions of these novel derivatives were compared directly with those of the parent compounds sphinganine and sphingosine. Exposure to natural and modified sphingoid bases promoted extensive apoptotic cell death. The PEITC-sphingoid base derivatives exhibited higher cytotoxicity than their natural counterparts and were also distinctly superior to the clinically relevant sphingoid base analog safingol. In each instance, lethality was shown to correlate with inhibition of conventional and novel PKC isoforms and downstream loss of extracellular signal-regulated kinase (ERK)1/ERK2. The involvement of these signaling systems in potentiating the lethal actions of 1-(beta-D-arabinofuranosyl)cytosine (araC) was also examined with regard to the differential actions of PEITC-Sa and PEITC-So to that of the parent compounds as well as safingol. Exposure to araC alone rapidly increased PKC activity. In the presence of PEITC-Sa or PEITC-So, the therapeutic efficacy of araC increased markedly; moreover, potentiation was directly related to the loss of araC-stimulated PKC activity. These findings demonstrate that PEITC-substituted sphingoid base analogs exert potent antineoplastic effects in human leukemia cells. We suggest that these synthetic lipids represent potentially useful agents in the development of conventional PKC/novel PKC-directed chemotherapeutic strategies.

Comparative studies of resistin expression and phylogenomics in human and mouse

Resistin is a newly identified adipocytokine that has been proposed to be a link between obesity and type 2 diabetes based on animal studies. However, the role of resistin in the pathogenesis of insulin resistance associated with obesity in humans remains unclear. We comparatively and quantitatively studied the tissue distributions of resistin mRNA between human and mouse. The expression level of resistin mRNA in human adipose tissue is extremely low but detectable by real-time PCR and is about 1/250 of that in the mouse. Remarkably, resistin mRNA is abundant in human primary acute leukemia cells and myeloid cell lines U937 and HL60, but not in the Raw264 mouse myeloid cell line. Resistin expression in U937 cells was not affected by lipopolysaccharide (LPS) or by ciglitazone, a PPARγ ligand. Phylogenomics revealed that the human resistin gene is the ortholog of its murine counterpart and is located in a region of chromosome 19p13.3, which is syntenic to mouse chromosome 8A1. In addition to the resistin-like molecule (RELM) sequences already reported, bioinformatics analysis disclosed another RELM sequence in the vicinity of RELMβ on human chromosome 3q13.1, but this sequence is unlikely to encode an expressed gene. Therefore, only two RELMs, resistin and RELMβ, exist in humans, instead of the three RELMs, resistin, RELMα, and RELMβ, that exist in mice. This finding provides a possible answer to the question of why only two RELMs have been cloned in humans and suggests that the RELM family is not well conserved in evolution and may function differently between species. Therefore, caution should be exercised in interpreting resistin as a link between obesity and insulin resistance in humans. The high expression of resistin in human leukemia cells suggests a hitherto unidentified biological function of resistin in leukocytes.

IL-18 increases invasiveness of HL-60 myeloid leukemia cells: up-regulation of matrix metalloproteinases-9 (MMP-9) expression

Similar to matrix metalloproteinases (MMP-9/-2), IL-18 was overexpressed in some hematologic malignancies such as acute myeloid leukemia (AML), which is associated with a poor clinical outcome. To establish a possible functional relationship between IL-18 and MMPs in myeloid leukemia, we used semi-quantitative PCR and zymographic analysis to examine whether IL-18 stimulates human myeloid leukemia cell line HL-60 to produce MMPs and/or specific tissue inhibitors (TIMPs), and to degrade extracellular matrix (ECM) gel in vitro. In the ECM invasion assay IL-18 significantly up-regulated transmigration of HL-60 cells, which in turn was inhibited by a synthetic MMP inhibitor: O-phenanthroline (o-PE), anti-MMP-9, anti-MMP-2 as well as anti-IL-18 monoclonal antibody (McAb), respectively, suggesting that induction of gelatinases by IL-18 leads to ECM degradation by these cells. Moreover, IL-18 could significantly increase MMP-9 but not MMP-2 production at both mRNA and/or protein level, slightly up-regulate TIMP-1 mRNA, and clearly induce TIMP-2 mRNA secretion. We postulate that IL-18 may in part play a role in the clinical aggressiveness of human myeloid leukemia by stimulating MMP-9 production.

Effect of novel modulators of protein kinase C activity upon chemotherapy-induced differentiation and apoptosis in myeloid leukemic cells.

Modulation of protein kinase C (PKC) activity has been demonstrated to either prevent or enhance drug-induced apoptosis in various tissue types. We tested four novel modulators of PKC activity in comparison to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) for the capability to affect differentiation, cell cycle progression and apoptosis in the human myeloid leukemia cell lines U937 and HL-60. Farnesyl thiotriazole (FTT) and N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) are both direct activators of PKC, whereas 6-(2-(4-[(4-fluorophe-nyl)phenylmethylene]-1-piperidinyl)ethyl)-7-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quin-azolinone (R59949) are diacyl glycerol kinase inhibitors that activate PKC by enhancing the levels of the endogenous ligand diacyl glycerol. U937 cells displayed a slight reduction in the number of cells in G(2)/M cell cycle phase after exposure to FTT, SC-10, R59022 and R59949, respectively. In contrast, HL-60 cells demonstrated a largely unaltered cell cycle distribution. Whereas TPA treatment resulted in a strong induction of p21(WAF/CIP1), c-Fos and c-Jun levels, neither one of the novel PKC activators altered expression of these proteins. Consequently, we tested the ability of the activators to cause membrane translocation of PKC. While TPA treatment resulted in translocation of the PKC isoforms alpha, delta and epsilon, SC-10 and FTT failed to induce alterations in the PKC content of the membrane and cytosolic fractions, respectively. Expression of the beta(2)-integrin CD11c that is induced during TPA-mediated differentiation remained unaltered after exposure to SC-10 and was partly reduced after treatment with FTT. To further investigate the effect of these activators upon apoptosis in leukemic cells, HL-60 and U937 cells were treated with 1-beta-D-arabinofuranosylcytosine (Ara-C) or etoposide (VP-16). Whereas TPA strongly reduced apoptosis in Ara-C- or VP-16-treated U937 cells, little if any reduction was observed after pretreatment with either FTT, SC-10, R59022 or R59949, respectively, in these cells. In contrast, TPA enhanced apoptosis in Ara-C- or VP-16-treated HL-60 cells. Interestingly, FTT and SC-10 demonstrated a protective effect in Ara-C-treated HL-60 cells. Taken together, these data suggest that the novel PKC activators FTT, SC-10, R59022 and R59949 exhibit modest biological effects upon leukemic blast cells, and are not capable of enhancing the apoptotic response of these cells to cytotoxic drugs.

Evaluation of ETF1/eRF1, mapping to 5q31, as a candidate myeloid tumor suppressor gene

Interstitial deletion of the long arm of chromosome 5 is a recurrent abnormality, mainly associated with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and it has been proposed therefore that the deleted region may contain a myeloid tumor suppressor gene. We have recently mapped a human translation termination factor gene, ETF1, to band 5q31 at D5S500, and thus to the smallest commonly deleted segment. We have evaluated ETF1 as a candidate myeloid tumor suppressor gene by analysis of the human acute myeloid leukemia cell line HL60, and of patients suffering from malignant myeloid diseases with cytogenetically-defined abnormalities of chromosome 5. Fluorescence in situ hybridization analysis revealed hemizygous loss of the ETF1 locus in HL60 cells and in four of five leukemic samples, but no inactivating mutations were identified by sequencing of the remaining ETF1 allele.

A metal chelator, diphenylthiocarbazone, induces apoptosis in acute promyelocytic leukemia (APL) cells mediated by a caspase-dependent pathway without a modulation of retinoic acid signaling pathways

A metal chelator, diphenylthiocarbazone (dithizone), has been reported to induce differentiation and apoptosis of the human myeloid leukemia cell line HL-60, however, very little is known about the mechanism of dithizone-induced apoptosis. Here, we report for the first time that dithizone can induce inhibition of cellular growth of retinoic acid (RA)-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis but not differentiation. Treatment of NB4 cells with dithizone markedly-induced apoptosis, which was associated with the loss of mitochondrial transmembrane potentials (Δ Ψ m) and activation of caspase-3 and -9. Further investigation of the RA-resistant UF-1 APL cells showed that dithizone-induced apoptosis to a lesser extent. However, neither dithizone alone nor in combination with all- trans RA induced the expression of myeloid differentiation antigen CD11b. Concomitantly, the degradation of PML/RARα fusion protein was not observed after treatment with dithizone alone, and the degradation was not enhanced by the combination of dithizone and all- trans RA. We conclude that dithizone, a metal chelator, induced apoptosis without differentiation in APL cells in association with Δ Ψ m collapse and caspase-3 and -9 activation.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC 50 values of 100 μM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters ( 2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC 50/ED 50) of ca. 5 was observed. The acute i.p. toxicity LD 50 in mice for 2a, 2b, 10a and 10b was similar and in the range of 400–600 mg kg –1. The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and β-globin disorders.

Induction of apoptosis by a novel intestinal metabolite of ginseng saponin via cytochrome c-mediated activation of caspase-3 protease

Ginseng saponins exert various important pharmacological effects with regard to the control of many diseases including cancer. The novel intestinal bacterial metabolites of ginseng protopanaxadiol saponins have recently been found and isolated after the oral administration of ginseng extract in human and rats. 20- O-(β- d-Glucopyranosyl)-20( S)-protopanaxadiol (IH-901) formed from ginsenosides Rb1, Rb2, and Rc is of particular interest in cancer chemoprevention and treatment. We investigated the effects of IH-901 on the human myeloid leukemia cell line HL-60 in terms of inhibition of proliferation and induction of apoptosis. IH-901 showed a significant cytotoxic activity in HL-60 cells ( ic 50 = 24.3 μM) following a 96-hr incubation. Treatment of HL-60 cells with IH-901 resulted in the formation of internucleosomal DNA fragments. The dose- and time-dependent induction of apoptosis by IH-901 was demonstrated in sandwich enzyme immunoassay and the results were confirmed by flow cytometric analysis. Morphological examination of IH-901-treated samples showed cells with chromatin condensation, cell shrinkage, and nuclear fragmentation, all typical characteristics of apoptotic cells. The treatment of HL-60 cells with IH-901 caused activation of caspase-3 protease and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. IH-901 did not affect the expression of antiapoptotic protein Bcl-2 but did cause a release of mitochondrial cytochrome c into cytosol. In conclusion, our results demonstrate that IH-901 dramatically suppresses HL-60 cell growth by inducing programed cell death through activation of caspase-3 protease, which occurs via mitochondrial cytochrome c release independently of Bcl-2 modulation. These results may provide a pivotal mechanism for the use of IH-901 in the prevention and treatment of leukemia.

Inhibition by ascorbic acid of apoptosis induced by oxidative stress in HL-60 myeloid leukemia cells

The human myeloid leukemia cell line HL-60 transports the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulates reduced ascorbic acid. We studied the effect of ascorbic acid loading on apoptosis induced by serum- and glucose-free culture and by oxidative stress induced by H 2O 2. Uptake accumulation studies indicated that incubation of HL-60 cells with DHA resulted in the accumulation of intracellular ascorbic acid which decreased with time when cells were incubated in DHA-free medium. Exposure of HL-60 cells to increasing concentrations of H 2O 2 resulted in dose-dependent intracellular accumulation of peroxides, as determined by the use of the oxidation-sensitive fluorescent probe 2′,7′-dichlorofluorescin-diacetate (DCFH-DA), which was accompanied by a decrease in intracellular ascorbic acid and an increase in apoptosis. A dramatic decrease in intracellular ascorbic acid was noted when preloaded HL-60 cells were exposed to 150 μM H 2O 2 (the concentration dropped from 5.2 ± 0.6 mM to 3.6 ± 0.1 mM in cells preincubated with 150 μM DHA). A dose-dependent protective effect of DHA was observed. Ascorbic acid loading also provided strong protection from apoptosis associated with serum- and glucose-free culture. Flow cytometry studies showed that exposure of HL-60 cells to 150 μM H 2O 2 resulted in decreased Bcl-2 expression that was associated with enhanced apoptosis (up to 33.6 ± 2.6%). No significant variation of Bcl-2 expression was measured following exposure of HL-60 cells, loaded with ascorbic acid, to 150 μM H 2O 2 and only a slight increase (up to 10.1 ± 3.1%) in apoptosis. These findings indicate that ascorbic acid can inhibit apoptosis induced by oxidative stress in HL-60 cells.

Enhancement of myeloid cell growth by benzene metabolites via the production of active oxygen species

In low concentrations, benzene and its metabolite hydroquinone are known to have diverse biological effects on cells, including the synergistic stimulation with GM-CSF of hematopoietic colony formation in vitro, stimulation of granulocytic differentiation in vitro and in vivo, and general suppression of hematopoiesis in vivo. These chemicals are also known to be active in the induction of active oxygen species. We used several assays to determine the effects of benzene metabolites (hydroquinone, benzenetriol, benzoquinone) and active oxygen species (xanthine/xanthine oxidase) on cell growth and cell cycle kinetics of the human myeloid cell line HL-60. HL-60 cells treated with these chemicals for 2 h in PBS showed increased growth over untreated controls in a subsequent 18h growth period in complete media. Incorporation of 3H-thymidine was also increased proportionately by these treatments. Catalase treatment abrogated the increased cell growth of all chemicals, suggesting an oxidative mechanism for the effect of all treatments alike. Cell cycle kinetics assays showed that the growth increase was caused by an increased recruitment of cells from G0/G1 to S-phase for both hydroquinone and active oxygen, rather than a decrease in the length of the cell cycle. Benzene metabolite's enhancement of growth of myeloid cells through an active oxygen mechanism may be involved in a number of aspects of benzene toxicity, including enhanced granulocytic growth and differentiation, stimulation of GM-CSF-induced colony formation, apoptosis inhibition, and stimulation of progenitor cell mitogenesis in the bone marrow. These effects in sum may be involved in the benzene-induced “promotion” of a clonal cell population to the fully leukemic state.

Integrin receptor-targeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides.

Integrin receptor-targeted transfer of oligodeoxynucleotides (ODNs) by small synthetic peptides was used for improving delivery of antisense ODNs. An 18-mer phosphodiester bond containing ODN complementary to c-myb-encoded mRNA was complexed with several transfer peptides, containing as their parts two modules: (a) an RGD-motif as targeting sequence for integrin receptor and (b) nucleocapsid protein (NCp) 7 of HIV-1 or NCp7-derived peptides for complex formation with the ODNs. The amount of antisense ODN required for the inhibition of proliferation of human myeloid cell line HL-60 in vitro can be more than 50-fold reduced by complexing with transfer peptides. The efficiency of antisense delivery was increased by multimerization of the targeting sequence for the integrin receptor. Competition with integrin peptide abolished the effect, indicating that the integrin receptor is indeed responsible for the reaction.

Ara-C induced apoptosis in human myeloid leukemia cell line HL-60: Inducing apoptosis is the primary mechanism of chemotherapy

Objective: To elucidate the pattern of chemotherapy drugs induced apoptosis and its role in chemotherapy of acute leukemia. Methods: Apoptosis induced by Ara-C in human myeloid leukemia cell line HL-60 was investigated by applying light microscope, electron microscopy combined with DNA electrophoresis and flow cytometry analysis techniques. Results: Apoptosis persisted throughout 36 h following addition of Ara-C with a gradual augmentation. Efficiency of apoptosis was enhanced in a dosedependent pattern, HL-60 treated with six other chemotherapy drugs and peripheral white blood cells from a AML case undergoing DA regimen chemotherapy exhibited typical DNA ladder pattern. Further investigation indicated that chemotherapy drugs apoptosis came into being possibly by downregulating the expression of c-myc and bcl-2 oncogenes. Conclusion: Chemotherapy induced apoptosis is the primary mechanism of chemotherapy.

Myeloperoxidase mediates cell adhesion via the alpha M beta 2 integrin (Mac-1, CD11b/CD18).

Myeloperoxidase is a leukocyte component able to generate potent microbicidal substances. A homologous invertebrate blood cell protein, peroxinectin, is not only a peroxidase but also a cell adhesion ligand. We demonstrate in this study that human myeloperoxidase also mediates cell adhesion. Both the human myeloid cell line HL-60, when differentiated by treatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid, and human blood leukocytes, adhered to myeloperoxidase; however, undifferentiated HL-60 cells showed only minimal adhesion. No cells adhered to horseradish peroxidase, and cell adhesion to myeloperoxidase was not decreased by catalase, thus showing that peroxidase activity, per se, was neither sufficient nor necessary for the adhesion activity. Mannan, which has been reported to inhibit the binding of peroxidases to cells, did not affect adhesion to myeloperoxidase. However, adhesion to myeloperoxidase was inhibited by monoclonal antibodies to alpha M (CD11b) or to beta2 (CD18) integrin subunits, but not by antibodies to alpha L (CD11a), alpha M (CD11c), or to other integrins. Native myeloperoxidase mediated dose-dependent cell adhesion down to relatively low concentrations, and denaturation abolished the adhesion activity. It is evident that myeloperoxidase supports cell adhesion, a function which may be of considerable importance for leukocyte migration and infiltration in inflammatory reactions, that alpha M beta2 integrin (Mac-1 or CD11b/CD18) mediates this adhesion, and that the alphaM beta2 integrin-mediated adhesion to myeloperoxidase is distinct from the previously reported ability of this integrin to bind to certain denatured proteins at high concentrations.

N-4-Hydroxyphenylretinamide Enhances Retinoic Acid-Induced Differentiation and Retinoylation of Proteins in the Human Acute Promyelocytic Leukemia Cell Line, NB4, by a Mechanism That May Involve Inhibition of Retinoic Acid Catabolism

All-trans-retinoic acid (RA) induces differentiation of acute promyelocytic leukemia cells bothin vitroandin vivoand is an alternative to cytotoxic chemotherapy in the treatment of acute promyelocytic leukemia. However, despite a complete remission rate of about 90%, most patients relapse and are resistant to further treatment with RA. This resistance primarily is due to an increased systemic catabolism of RA. In this study we examined the catabolism of RA by the human acute promyelocytic leukemia cell line NB4 and the human myeloid leukemia cell line HL60. NB4 cells converted RA to 4-hydroxy-RA, 4-oxo-RA and more polar unidentified retinoids at a much greater rate than HL60 cells. Exposure of NB4 cells to RA induced RA catabolism. We found that 4-hydroxyphenylretinamide (4-HPR) inhibited the catabolism of RA. This inhibition was dose-dependent and greater, on a molar basis, than the inhibition seen with the cytochrome P450 inhibitor, ketoconazole, or two synthetic retinoids, Ch55 and Am80. 4-HPR alone was a poor inducer of differentiation of NB4 cells. However, it markedly enhanced RA-induced differentiation and increased the level of retinoylation, the covalent binding of RA to proteins. These results suggest some retinoid analogs, including 4-HPR, may have clinical utility because of their ability to increase the biological half-life of RA.