Processing of the formyl peptide receptor by HL-60 cells

Processing of the formyl peptide receptor by differentiated HL-60 cells has been studied using the photoaffinity label N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys-N epsilon-6-(4'-azido-2' -nitrophenylamino)-hexanoate. The receptor on live cells has an apparent molecular weight of 60,000 to 80,000 and possesses one predominant papain cleavage site on the cell exterior yielding a 35,000-Da fragment that contains the binding site. The affinity-labeled receptor was internalized with a t1/2 = 3.2 min at 37 degrees C, a t1/2 = 12 min at 24 degrees C, and was not internalized at 15 degrees C. The internalized receptor was localized in two intracellular compartments with buoyant densities less than that of the plasma membrane. The compartment with the lowest buoyant density was coincident with the Golgi marker galactosyltransferase. Intracellular dissociation of noncovalently bound peptide from the receptor occurred with a t1/2 = 25-28 min. Following a 3-h lag period, internalized affinity-labeled receptor was degraded by a first-order process with a t1/2 = 7 h.     

New ferrocene containing peptide conjugates: synthesis and effect on human leukemia (HL-60) cells

Data reported in this article describe the synthesis of Arg-rich oligopeptide conjugates of ferrocenecarboxylic acid on solid support with two different strategies and for the first time, the successful preparation of peptide conjugates of ferrocenylacrylic acid in solution. The antitumor effect of conjugates was analyzed by MTT assay in vitro. We demonstrated that ferrocenylacrylic acid possessing an enone (--CH==CH--CO--) moiety exhibited remarkable antiproliferative effect against human leukemia cells (HL-60) in vitro, but its effect was not improved by conjugation with hexa- or octaarginines. However, we observed highly increased water-solubility. In contrast, the results provide evidence that conjugation of ferrocenecarboxylic acid to Arg(n) (n = 6, 8) improved not only its water-solubility, but also antitumor effect on human leukemia cells in vitro.     

Variables that regulate production of insulin-like peptide(s) in human leukemia cell line (HL-60)

A human myeloid leukemia cell line (HL-60) produces a peptide or peptides with insulin-like activity which is distinct from insulin or insulin-like growth factors (somatomedins). Factors regulating the production of this peptide (HL-ILP) were explored in the present study. The production of HL-ILP was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages, induced by dihydroxyvitamin D3 or phorbol esters, was also associated with a decrease in HL-ILP production. Glucose consumption by the cells in the early log phase was closely related with HL-ILP production, and HL-ILP was found to stimulate glucose consumption by HL-60 cells. Production of HL-ILP was dependent on glucose concentrations in the culture medium and glucose concentrations higher than 1mg/d1 suppressed the release of HL-ILP. These observations are not inconsistent with a hypothesis that HL-ILP is involved in the glucose metabolism of the HL-60 cells that produce this peptide.     

Cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) in human promyelocytic leukemia cells HL-60

12-O-Tetradecanoylphorbol 13-acetate (TPA) is a potent tumor promoter and is known to induce terminal differentiation of human promyelocytic leukemia cells HL-60 to mature monocytes. To investigate the molecular mechanism of TPA actions, TPA-specific binding proteins in HL-60 were analyzed. Anion exchange high-performance liquid chromatography revealed that HL-60 cells possess TPA-specific binding proteins other than protein kinase C (PKC). One of these TPA-specific binding proteins exists in the cytosolic fraction of HL-60 cells, but translocates into the nuclear fraction of HL-60 cells after the treatment of the cells with TPA. The results suggest that HL-60 cells take up TPA into the nuclei via the TPA-specific binding protein. The TPA-specific binding protein binds TPA, phorbol 12,13-di-butylate, teleocidin B-2, teleocidin B-3, and debromoaplysiatoxin in a mutually competitive manner. However, the protein does not bind to okadaic acid, olivoretin C, retinoic acid, or dioxin. This cytosolic-nuclear tumor promoter-specific binding protein (CN-TPBP) might play an essential role in the action of tumor promoters.     

Retinoic acid-induced expression of tissue transglutaminase in human promyelocytic leukemia (HL-60) cells

Addition of retinoic acid to human promyelocytic leukemia cells results in a dramatic increase in cellular transglutaminase activity. This increase is due to the induction of a specific intracellular transglutaminase, tissue transglutaminase. Retinoic acid-induced expression of tissue transglutaminase is potentiated by analogues of cyclic AMP. The induction of the enzyme can be detected within 6 h of the addition of the retinoid to the cell and results in increases of the enzyme of at least 50-fold. The induction of HL-60 transglutaminase is a specific response of the cells to retinoic acid and is not seen with other agents that induce HL-60 differentiation. We believe that the induction of tissue transglutaminase is a useful index of the early events in retinoid-regulated gene expression in both normal and transformed cells.     

Chitooligosaccharides induce apoptosis in human myeloid leukemia HL-60 cells

In this study we propose a novel anticancer agent using hetero-chitooligosaccharide (hetero-COS). To examine the possibility of the hetero-COS as a anticancer agent, we prepared nine kinds of hetero-COS with relatively higher molecular weights (90, 75 and 50-COS I, 5-10kDa), medium molecular weights (90, 75 and 50-COS II, 1-5kDa), and lower molecular weights (90, 75 and 50-III, below 1kDa), and their anticancer properties were investigated on HL-60 cells using flow cytometry and morphological analysis. The results obtained indicate that 90-COS III, which is relatively higher degree of deacetylation and lower molecular weights, showed the highest anticancer activity, and the data showed the anticancer property of the hetero-COSs depended on their degree of deacetylation values and molecular weight.     

Humic acid induces apoptosis in human premyelocytic leukemia HL-60 cells

It has been shown that humic acid (HA), a phenolic polymer, exhibits pro-oxidant and cytotoxic effects. In this study, HA induction of apoptosis was studied using cultured human premyelocytic leukemia HL-60 cells. Treatment at a range of HA concentrations (50-400 microg/ml) resulted in dose-and time-dependent sequences of events marked by apoptosis, as demonstrated through by apoptotic features such as loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. This HA-induced apoptosis in the HL-60 cells was mainly associated with the release of cytochrome c from the mitochondria. Furthermore, apoptosis in the HL-60 cells was accompanied by the activation of caspase-3 and the specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), a major component in the apoptotic cell death mechanism. Although the HA-induced apoptosis was associated with Bax protein levels, negligible Bcl-2 reduction was observed. Analysis of the data reported herein reveals that HA exerts antiproliferative action and growth inhibition on HL-60 cells through induction of apoptosis, which may have anticancer properties potentially useful for the development of new drug products.     

Detection of a novel cyclooxygenase metabolite produced by human promyelocytic leukemia (HL-60) cells

Arachidonic acid metabolism via the lipoxygenase pathway was examined in HL-60 cells before and after N,N-dimethylformamide induced differentiation along granulocytic lines. Untreated HL-60 cells produced small amounts of the 5-lipoxygenase products, 5-hydroxy-eicosatetraenoic acid and leukotriene B4 upon stimulation with calcium ionophore A23187. N,N-dimethylformamide treatment, caused a 10 to 20 fold increase in the amount of ionophore A23187-induced 5-lipoxygenase metabolites. An additional, and as yet unidentified arachidonic acid metabolite was routinely observed during reverse-phase high pressure liquid chromatography analyses of lipoxygenase products. Sensitivity to inhibition by less than 10(-7)M indomethacin coupled with other characteristics of its production, strongly suggest the compound is a cyclooxygenase product. The unusual UV absorbance and chromatographic elution pattern, however, suggest that it is not a typical prostaglandin, thromboxane or prostacyclin product.     

Identification and signal transduction mechanism of elastin peptide receptor in human leukocytes

The existence of a novel receptor on human polymorphonuclear leukocytes (PMNLs) and monocytes was demonstrated, named soluble elastin peptide receptor. Soluble elastin peptides, like K-elastin, which are liberated from elastin fibres, can be found in the sera, and they possess several biological activities such as chemotaxis. Studying the effects of elastin peptides on leukocytes, it was found that: (i) the elastin peptide stimulates the oxidative burst, the intracellular free Ca2+ elevation through a specific receptor; and (ii) in the signal transduction mechanism of this elastin peptide receptor, the phosphatidylinositol breakdown is involved.     

Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells

Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2alpha, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress.     

Gene expression spectra in human leukemia HL-60 cells treated with EGCG

To decipher the molecular mechanism of EGCG induced HL-60 cell apoptosis, alterations of gene expression spectra in HL-60 cell line cells after treatment with EGCG were screened by cDNA chip, and analyzed with the GenePix 3.0 twice; and the cDNA chip results further identified by RT-PCR. Ninety-seven genes among the total 8398 (1.15%) showed consistent significant differential expression in the duplicated cDNA chip assessments. Thirty-nine genes (40.2%) were up-regulated and 58 genes (59.8%) were down-regulated; and the randomly selected four performed RT-PCR results agreed with the cDNA chip data. The results suggest that the apoptosis of HL-60 cells induced by EGCG is a progressive transformation process regulated by a variety of genes.     

Arsenic suppresses necrosis induced by selenite in human leukemia HL-60 cells

Selenium, an essential trace element for humans, has been shown to have anticancer effects. Arsenic, a possibly essential ultratrace element for humans, has been used in the treatment of leukemia. Anticancer effects of selenium and arsenic have been related to their ability to induce apoptosis. Because humans are exposed to diverse trace elements simultaneously, it is important to learn their interrelationship. In this study, we demonstrate that sodium selenite (Na₂SeO₃) causes apoptosis at 3 μM and necrosis at high concentrations (>3 μM) in HL-60 cells. Similarly, both sodium arsenite (NaAsO₂) at 50 μM and sodium arsenate (Na₂HAsO₄) induce apoptosis at 500 μM and necrosis at higher concentrations (>50 μM and >500 μM, respectively) in HL-60 cells. Arsenite/arsenate, but not selenite, enhances AP-1 DNA-binding activity. This finding indicates different mechanisms through which apoptosis is induced by these two elements. Interestingly, we observed that HL-60 cell necrosis induced by a high concentration (>3 μM) of selenite was essentially inhibited by arsenic (50 μM of NaAsO₂ or 500 μM of Na₂HAsO₄), which resulted in a net effect of apoptosis. Because AP-1 DNA-binding activity was not induced in the presence of a combination of necrotic amount of selenite and apoptotic amount of arsenite/arsenate, the observed apoptosis apparently was through the mechanism used by selenite. Our results suggest, for the first time, that the toxic necrotic effect of selenite can be neutralized by arsenite/arsenate at the cellular level. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Effects of tunicamycin on the expression and function of formyl peptide chemotactic receptors of differentiated HL-60 cells

We previously showed that formyl peptide chemotactic receptors (FPCR) of human phagocytic cells contain at least two asparagine-linked oligosaccharide chains located at the distal end of the receptor. The requirement of these N-linked oligosaccharide chains for expression and function of FPCR was investigated in HL-60 cells induced to differentiate by N6,O2-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) in the presence or absence of 5 micrograms/ml tunicamycin. Tunicamycin did not prevent the changes in morphology associated with Bt2cAMP-induced differentiation of HL-60 cells. Autoradiographic analysis after SDS-PAGE of FPCR affinity labeled with N-formyl-Nle-Leu-Phe-Nle-[125I]iodo-Tyr-Lys (formyl 125I-hexapeptide) and ethylene glycol bis(succinimidyl succinate) demonstrated that greater than 95% of FPCR expressed by tunicamycin-treated cells completely lacked N-linked oligosaccharide (Mr 32,000), and no fully glycosylated FPCR (Mr 62,000 to 85,000) was detectable. Scatchard analysis of formyl 125I-hexapeptide binding indicated the presence of two classes of binding sites for both control and tunicamycin-treated cells (control cells, 82,000 +/- 32,000 sites/cell with Kd 10.0 +/- 4.3 nM and 520,000 +/- 40,000 sites/cell with Kd 250 +/- 80 nM; tunicamycin-treated cells, 11,000 +/- 5000 sites/cell with Kd 3.0 +/- 1.9 nM and 470,000 +/- 70,000 sites/cell with Kd of 500 +/- 140 nM). Both control and tunicamycin-treated cells augmented superoxide anion release, exhibited a migratory response, and showed a transient rise in intracellular free Ca2+ upon stimulation with N-formyl-Nle-Leu-Phe. However, the responses of the tunicamycin-treated cells were less than that of the control cells. The present studies demonstrate that N-glycosylation of FPCR is not essential for cell surface expression or for several FPCR-mediated cell responses.     

RGD peptide-induced apoptosis in human leukemia HL-60 cells requires caspase-3 activation

RGD motif-containing peptides have been used in various studies of cell adhesion and growth. We report that RGD triggered apoptosis at a concentration of 1 mmol/L, whereas RAD-containing peptides failed to induce apoptosis in HL-60 cells. RGD-treated cells revealed internucleosomal DNA fragmentation. Western blot reveals caspase-3 activation in RGD peptide-treated cells. A caspase-3 inhibitor z-VAD-FMK completely blocked the apoptosis, but a caspase-1 inhibitor (Ac-YVAD-CMK) and caspase-2 inhibitor (z-VDVAD-FMK) did not block the apoptosis, suggesting that caspase-3 might have a critical role in the execution process of apoptosis induced by RGD. RGD peptides have been used extensively to inhibit tumor metastasis. Our results should help in further understanding the RGD peptide-induced apoptosis, which is important since RGD peptides have a potential role in therapies of the future. This revised version was published online in July 2006 with corrections to the Cover Date.