Myeloid Leukemia HL-60 Research Articles

Raf‐1 signaling is required for the later stages of 1,25‐dihydroxyvitamin D3‐induced differentiation of HL60 cells but is not mediated by the MEK/ERK module

We are interested in determining the signaling pathways for 1,25-dihydroxyvitamin D3 (1,25D)-induced differentiation of HL60 leukemic cells. One possible candidate is Raf-1, which is known to signal cell proliferation and neoplastic transformation through MEK, ERK, and downstream targets. It can also participate in the regulation of cell survival and various forms of cell differentiation, though the precise pathways are less well delineated. Here we report that Raf-1 has a role in monocytic differentiation of human myeloid leukemia HL60, which is not mediated by MEK and ERK, but likely by direct interaction with p90RSK. Specifically, we show that Raf-1 and p90RSK are increasingly activated in the later stages of differentiation of HL60 cells, at the same time as activation of MEK and ERK is decreasing. Transfection of a wild-type Raf-1 construct enhances 1,25D-induced differentiation, while antisense Raf-1 or short interfering (si) Raf-1 reduces 1,25D-induced differentiation. In contrast, antisense oligodeoxynucleotides (ODN) and siRNAs to MEK or ERK have no detectable effect on differentiation. In late stage differentiating cells Raf-1 and p90RSK are found as a complex, and inhibition of Raf-1, but not MEK or ERK expression reduces the levels of phosphorylated p90 RSK. These findings support the thesis that Raf-1 signals cell proliferation and cell differentiation through different intermediary proteins.

Myeloperoxidase-Catalyzed Metabolism of Etoposide to Its Quinone and Glutathione Adduct Forms in HL60 Cells

Etoposide is a widely used antineoplastic agent that has provided great success in the treatment of childhood leukemias and other malignancies. Unfortunately, its use is associated with the increased risk of development of secondary acute myelogenous leukemias involving translocations at the MLL gene in chromosome band 11q23. Previous studies showed that the phenoxyl radical of etoposide can be generated by myeloperoxidase (MPO), an enzyme prevalent in myeloid progenitor cells that can derive myelogenous leukemias. Disproportionation of this radical leads to formation of the redox active etoposide ortho-quinone metabolite. We hypothesized that etoposide ortho-quinone could therefore form in myeloid progenitor cells and might be a contributor to the development of treatment-related secondary leukemias. Etoposide ortho-quinone is an inherently unstable compound and readily reacts with glutathione in aqueous media without any requirement for catalytic assistance from glutathione S-transferase. We looked for the presence of its glutathione adduct as an indicator of etoposide ortho-quinone in cells. MPO-expressing human myeloid leukemia HL60 cells were treated with etoposide for 0.5 h in the presence and absence of the cosubstrate of MPO, hydrogen peroxide. Cell lysates and medium were analyzed by LC-ESI-ion trap-MS and MS/MS, which yielded clear evidence of the intracellular formation of the etoposide ortho-quinone-glutathione adduct. A stable isotope-labeled form of the GSH adduct was synthesized and employed as an isotope dilution internal standard in LC-ESI-quadrupole-MS analyses. The glutathione adduct level was dependent on the concentration of etoposide added to the cells. More importantly, the formation of the glutathione adduct was significantly suppressed by the pretreatment of HL60 cells with the heme synthesis inhibitor succinylacetone (p < 0.001), which resulted in a decreased level and activity of MPO. These results are consistent with the idea that MPO is responsible for the conversion of etoposide to its ortho-quinone in these cells.

Differential Targets and Subcellular Localization of Antitumor Alkyl-lysophospholipid in Leukemic Versus Solid Tumor Cells

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

The Mechanism Study of Etoposide Related Secondary Leukemia: Myeloperoxidase‐Catalyzed Formation of Etoposide Ortho‐Quinone Glutathione Adduct in HL60 Cells

Etoposide is a widely used antineoplastic agent in the treatment of leukemia, but its use is associated with the increased risk of development of secondary acute leukemia. We hypothesize that one of the etoposide intermediate metabolites, etoposide ortho-quinone, can react with glutathione to form the GSH adduct in HL60 cells and therefore increase oxidative stress which may lead to DNA breaks. Myeloperoxidase (MPO)-expressing human myeloid leukemia HL60 cells were treated with etoposide in the presence and absence of a cosubstrate of MPO, hydrogen peroxide. Cell lysates and medium were analyzed by LC-ESI-ion trap-MS and MS/MS, which yielded clear evidence of the intracellular formation of the etoposide ortho-quinone-glutathione adduct. A stable isotope-labeled form of the GSH adduct was synthesized and employed as an isotope dilution internal standard in LC-ESI-quadrupole-MS analyses. The glutathione adduct formation was dependent on the concentration of etoposide. The formation of the glutathione adduct was significantly suppressed by pretreatment of HL60 with the heme synthesis inhibitor succinylacetone (p < 0.001), consistent with the idea that MPO is responsible for the conversion of etoposide to its ortho-quinone in cells. This research was supported by NIH Grant R01 CA090787

Novel valproic acid derivatives with potent differentiation-inducing activity in myeloid leukemia cells

The anti-epileptic drug valproic acid harbors anti-tumoral activity in solid and leukemic tumor cell models and is currently evaluated in clinical trials. However, the plasma trough concentrations obtained in patients by common anti-epileptic dose regimens are below concentrations required for exerting anti-tumor effects in vitro. Here, we describe the identification of three novel valproic acid derivatives with superior differentiation-inducing and anti-proliferative activities in K562 bcr/abl-positive chronic myeloid leukemia cells and HL60 promyelocytic leukemia cells at achievable therapeutic VPA concentrations. These compounds reveal potent inhibition of histone deacetylase activity, induction of p21 Cip/Waf expression as well as low toxicity on CD34 + bone marrow cells.

JWA, a novel signaling molecule, involved in all-trans retinoic acid induced differentiation of HL-60 cells

JWA (AF070523) was originally identified as a novel all-trans retinoic acid (ATRA) responsible gene in primary human tracheal bronchial epithelial cells. For the notable performance achieved by ATRA in the differentiation induction therapy, we investigated the role of JWA in ATRA-mediated differentiation of the human myeloid leukemia HL-60 cells. We found that concomitant with the progressive cell differentiation, JWA expression was up-regulated by ATRA in a dose- and time-dependent manner. Inhibition of JWA expression by RNA interference partially blocked ATRA-induced differentiation and growth inhibition of HL-60 cells. Pre-treatment of phorbol-12-myristate-13-acetate (TPA), a PKC activator, decreased ATRA-mediated differentiation, companied with the down-regulation of JWA expression. Arsenic trioxide (As(2)O(3), 0.5 microM) enhanced the cellular differentiation induced by 0.01 microM ATRA, but had no noticeable effect on the differentiation induced by 0.1 microM ATRA. Concurrent with the enhancement, JWA expression was up-regulated. All the data suggest that up-regulation of JWA expression is essential for ATRA-induced differentiation of HL-60 cells. And JWA, associated with PKC, is involved in its signal pathways. Ideal therapeutic efficacy with low toxicity may be obtained if low doses of ATRA (0.01 microM) and As(2)O(3) (0.5 microM) are combined. These findings may present a novel mechanism that cellular differentiation and growth inhibition induced by ATRA are mediated at least in part through regulation of JWA expression. JWA may be a novel molecular marker for ATRA-induced HL-60 cell differentiation. ATRA up-regulates JWA expression by stimulating the transcriptional activity of JWA gene promoter.

Cotylenin A-induced differentiation is independent of the transforming growth factor-β signaling system in human myeloid leukemia HL-60 cells

Cotylenin A, which has been isolated as a plant growth regulator, potently induces the differentiation of human myeloid leukemia cells. Treatment of HL-60 cells with a combination of transforming growth factor (TGF)-β and 1α, 25-dihydroxyvitamin D3 (VD3) resulted in increased differentiation compared to separate treatments, but TGF-β did not affect the cotylenin A-induced differentiation of HL-60 cells. It is possible that the signal transduction pathway used by cotylenin A for inducing the differentiation of leukemia cells is the same as that used by TGF-β. However, cotylenin A did not affect the expression of TGF superfamily or Smad genes in HL-60 cells. Treatment with neutralizing anti-TGF-β antibody or an inhibitor of TGF-β signaling did not inhibit cotylenin A-induced differentiation, although VD3-induced differentiation was significantly suppressed by these treatments. The subcellular distribution of Smad3 was also unaffected by cotylenin A. These results suggest that the cotylenin A-induced differentiation of leukemia cells is independent of the TGF-β signaling system, although TGF-β acts as an autocrine mediator of the growth arrest and differentiation of leukemia cells induced by VD3 and other inducers.

Enhancement of TPA-induced growth inhibition and apoptosis in myeloid leukemia cells by BAY 11-7082, an NF-κB inhibitor

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation and apoptosis in myeloid leukemia cells. In the present study, we investigated the role of the transcription factor NF-kappaB in TPA-induced growth inhibition and apoptosis in the myeloid leukemia HL-60 cell line and its TPA-resistant cell variant HL-525. Unlike the parental cell line, HL-525 cells are protein kinase C (PKC)-beta deficient and resistant to TPA-induced differentiation and apoptosis. We found that treatment of HL-60 cells with TPA resulted in a concentration-dependent growth inhibition and an increase in apoptotic cells. TPA only had a small effect on growth and apoptosis in HL-525 cells. Treatment of HL-60 cells with TPA (0.64-3.2 nM) caused a rapid activation of NF-kappaB as determined by electrophoresis mobility shift assay (EMSA) and immunocytochemistry. Although the basal level of NF-kappaB activity was low in HL-60 cells, TPA-resistant HL-525 cells had a high basal level of NF-kappaB activity. Treatment of HL-525 cells with higher concentrations of TPA (16-80 nM) resulted in a further increase in NF-kappaB activity. (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), which inhibits IkappaB alpha phosphorylation and thus decreases NF-kappaB activation, was found to decrease TPA-induced nuclear translocation of NF-kappaB. Furthermore, BAY enhanced TPA-induced growth inhibition and apoptosis in both HL-60 and HL-525 cells. Results from the present study indicate that inhibition of NF-kappaB by BAY was associated with enhanced TPA-induced growth inhibition and apoptosis in human myeloid leukemia cells. TPA in combination with pharmacological inhibitors of NF-kappaB may improve the therapeutic efficacy of TPA and overcome the resistance to TPA in some myeloid leukemia patients.

Inhibition of Growth ofStreptococcus mutans, Methicillin-ResistantStaphylococcus aureus, and Vancomycin-Resistant Enterococci by Kurarinone, a Bioactive Flavonoid Isolated fromSophora flavescens

Inhibition of Growth of<i>Streptococcus mutans</i>, Methicillin-Resistant<i>Staphylococcus aureus</i>, and Vancomycin-Resistant Enterococci by Kurarinone, a Bioactive Flavonoid Isolated from<i>Sophora flavescens</i>

Cell apoptosis induced by a synthetic carbazole compound LCY-2-CHO is mediated through activation of caspase and mitochondrial pathways

The mechanisms involved in the apoptotic effect of LCY-2-CHO [9-(2-chlorobenzyl)-9 H-carbazole-3-carbaldehyde], a synthetic carbazole derivative identified as an anti-inflammatory compound, were studied. Cell cycle analysis by propidium iodide staining in human THP-1 monocytic leukemia cells showed the ability of LCY-2-CHO to increase cell population in sub-G1 stage with time- and concentration-dependent manners. LCY-2-CHO-mediated cell death was also demonstrated by DNA laddering and was not related to the release of lactate dehydrogenase. Apoptosis in THP-1 cells induced by LCY-2-CHO was accompanied by the Bid cleavage, collapse of mitochondrial transmembrane potential, the release of cytochrome c and the activation of caspase-3. The apoptotic effect of LCY-2-CHO was diminished by the presence of zVEID-fmk (caspase-6 inhibitor), zIETD-fmk (caspase-8 inhibitor), and zVAD-fmk (non-selective caspase inhibitor), but was not altered by several antioxidants, and cathepsin inhibitor. The Bid cleavage and loss of mitochondrial transmembrane potential, but not the cytochrome c release, were reversed by zIETD-fmk. Comparing the cell selectivity of LCY-2-CHO, we found T-cell acute lymphoblastic CEM leukemia cells were sensitive to 1 μM LCY-2-CHO, acute myeloid leukemia HL-60 cells underwent apoptosis at 10 μM, while adherent cancer cells, such as PC3, HT29 and MCF-7, were resistant to 30 μM LCY-2-CHO within 24-h incubation. Taken together in the present study, we demonstrated LCY-2-CHO might be apoptotic for malignant hematopoietic cells but not anchorage-dependent cells. This action is mediated by an intrinsic caspase-dependent apoptotic event involving mitochondria.

Dicoumarol relieves serum withdrawal-induced G 0/1 blockade in HL-60 cells through a superoxide-dependent mechanism

This work was set to study how dicoumarol affects the cell cycle in human myeloid leukemia HL-60 cells. Cells were accumulated in G 0/1 after serum deprivation. However, when cells were treated with 5 μM dicoumarol in serum-free medium, a significant increment in the number of cells in S-phase was observed. Inhibition of G 0/1 blockade was confirmed by the increase of thymidine incorporation, the phosphorylation of retinoblastoma protein, and the promotion of cell growth in long-term treatments in the absence of serum. Dicoumarol treatment increased superoxide levels, but did not affect peroxide. Increase of cellular superoxide was essential for inhibition of G 0/1 blockade, since scavenging this reactive species with a cell-permeable form of SOD and the SOD mimetics 2-amino-3,5-dibromo- N-[ trans-4-hydroxycyclohexyl]benzylamine (ambroxol, 100 μM) and copper[II]diisopropyl salicylate (CuDIPS, 10 μM) completely abolished the effect of dicoumarol. However, N-acetyl-cysteine, overexpression of Bcl-2 or a cell-permeable form of catalase were not effective. 5-Methoxy-1,2-dimethyl-3-[(4-nitrophenol)methyl]-indole-4,7-dione (ES936), a mechanism-based irreversible inhibitor of NAD(P)H:quinone oxidoreductase 1 (NQO1), did not promote S phase entry, and dicoumarol still inhibited G 0/1 blockade in the presence of ES936. We demonstrate that dicoumarol inhibits the normal blockade in G 0/1 in HL-60 cells through a mechanism involving superoxide, but this effect is not dependent solely on the inhibition of the NQO1 catalytic activity. Our results send a precautionary message about use of dicoumarol to elucidate cellular processes involving oxidoreductases.

Immediate up-regulation of the calcium-binding protein S100P and its involvement in the cytokinin-induced differentiation of human myeloid leukemia cells

Cytokinins are important purine derivatives that act as redifferentiation-inducing hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA) and kinetin are very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. We examined the gene expression profiles associated with exposure to IPA using cDNA microarrays and compared the results with those obtained with other inducers of differentiation, such as all- trans retinoic acid (ATRA), 1α,25-dihydroxyvitamin D 3 (VD3) and cotylenin A (CN-A). Many genes were up-regulated, and only a small fraction were down-regulated, upon exposure to the inducers. IPA and CN-A, but not ATRA or VD3, immediately induced the expression of mRNA for the calcium-binding protein S100P. The up-regulation of S100P was confirmed at the protein expression level. We also examined the expression of other S100 proteins, including S100A8, S100A9 and S100A12, and found that IPA preferentially up-regulated S100P at the early stages of differentiation. IPA-induced differentiation of HL-60 cells was suppressed by treatment with antisense oligonucleotides against S100P, suggesting that S100P plays an important role in cell differentiation.

Synergistic stimulatory effect of 12-O-tetradecanoylphorbol-13-acetate and capsaicin on macrophage differentiation in HL-60 and HL-525 human myeloid leukemia cells

Our previous studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) had pharmacological activity for the treatment of myeloid leukemia patients. In the present study, we investigated the effects of TPA alone or in combination with capsaicin (8-methyl-N-vanillyl-6-nonenamide) on growth and differentiation in myeloid leukemia HL-60 cells and in a TPA-resistant HL-60 variant cell line termed HL-525. Treatment of HL-60 cells with TPA (0.16-1.6 nM) for 48 h resulted in concentration-dependent growth inhibition and cell differentiation (via the macrophage pathway). Capsaicin (5-50 microM) inhibited the growth of HL-60 cells in a concentration-dependent manner. Treatment of HL-60 cells with capsaicin alone only resulted in a small increase in the number of differentiated cells but treatment of the cells with TPA in combination with capsaicin synergistically increased differentiation. Moreover, inhibitors of protein kinase C (PKC), 7-hydroxystaurosporin (UCN-01; 100 nM) and chelerythrine (0.5 microM), significantly decreased HL-60 cell differentiation induced by the combination of TPA and capsaicin. These results suggest that PKC may be involved in HL-60 cell differentiation induced by TPA in combination with capsaicin. Capsaicin alone caused a very small increase in differentiation in the TPA-resistant HL-525 cells. However, treatment of HL-525 cells with combinations of TPA (0.16 nM) and capsaicin (10-50 microM) caused a strong synergistic increase in differentiation. Results from the present study suggest that a combination of TPA and capsaicin may improve the therapeutic efficacy of TPA and overcome resistance to TPA in some myeloid leukemia patients.

Expression of survivin and bax/bcl-2 in peroxisome proliferator activated receptor-γ ligands induces apoptosis on human myeloid leukemia cells in vitro

The present study was undertaken to investigate the mechanisms of peroxisome proliferator activated receptor-gamma (PPAR-gamma) ligand-induced apoptosis on human myeloid leukemia K562 and HL-60 cell lines. The results revealed that both 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and troglitazone (TGZ) have significant anti-proliferation- and apoptosis-inducing effects on these two kinds of leukemia cells. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly using Wright's and Hoechst 33258 staining. Reverse transcription-PCR and western blot analyses demonstrated that both survivin and bcl-2 expression were downregulated markedly, while bax expression was upregulated concurrently when apoptosis occurred. We therefore conclude that 15d-PGJ2 and TGZ have significant apoptosis effects on K562 and HL-60 cells in vitro, and that upregulation of bax as well as downregulation of survivin and bcl-2 expression may be the important apoptosis-inducing mechanisms. The results suggest that PPAR-gamma ligands may serve as potential therapeutic agents for both acute and chronic myeloid leukemia.

Coenzyme Q and the regulation of intracellular steady‐state levels of superoxide in HL‐60 cells

The present work was set to study how CoQ concentrations affected steady-state levels of superoxide in a cellular model of partial CoQ(10) deficiency in cultured human myeloid leukemia HL-60 cells. Culturing HL-60 cells in the presence of p-aminobenzoate, a competitive inhibitor of polyprenyl-4-hydroxybenzoate transferase (Coq2p), produced a significant decrease of CoQ(10) levels without affecting cell viability. Concomitant decreases in CoQ-dependent electron transport activity and mitochondrial membrane potential were observed under these conditions. Intracellular superoxide was significantly elevated in cells treated with p-aminobenzoate, both under serum-containing and serum-free conditions, and this effect was reversed by exogenous CoQ(10). A slight increase of superoxide was also observed in CoQ(10)-supplemented cells in the absence of serum. Our results support a requirement for CoQ(10) to control superoxide levels in HL-60 cells. The importance of extramitochondrial sources of superoxide in cells with impaired CoQ(10) biosynthesis is discussed.

Aerolysin Cytotoxicity Can Serve as a Surrogate Marker for Immune Sensitivity of Tumor Cells to Granule-Exocytosis Pathway in Vitro.

Background: Aerolysin is a pore-forming toxin produced by Aeromonas hydrophila and interacts with target mammalian cells through binding to glycosylphosphatidyl inositol (GPI)-anchored proteins, subsequently forming a pore in the plasma membrane. Both osmotic lysis of the cell secondary to fluid influx thru pores and a rapid increase in intracellular calcium, which may have been the signal for apoptosis, when a small number of channels were opened appear to be operational in aerolysin cytotoxicity. Perforin (PFP), a prototype of pore-forming proteins is closely linked to granzyme B effect and the central element of natural killer (NK) and T cell mediated cytotoxicity, and induces apoptosis of target cells through granule-exocytosis pathway. Since perforin/granzyme B pathway is pivotal to immune mediated elimination and due to similarities between PFP and aerolysin action, we investigated the relationship between aerolysin cytotoxicity and lymphokine activated killer (LAK) cell-mediated elimination of human tumor cells.Material and Methods: We studied the effect of active aerolysin (2x10−10M) against eight tumor cell lines and five acute myeloid leukemia patient samples. Aerolysin cytotoxicity was assessed at eight different time points: first at ½ to 4 hours of incubation consistent with the known optimal cell kill mediated by PFP in NK cytotoxicity, then lastly at 18 hours to be able see overall effect. Cytotoxicity was measured by flow cytometric detection of annexin V/PI-positive cells, DiOC6 (3) /PI staining and further quantification of fluorosphere-adjusted events. Tumor cell type selection was based on their sensitivity against LAK cell-mediated cytotoxicity. Acute myeloid leukemia cells K562, U937, CMK, Meg-01, HL-60 and resistant central nervous system tumor cells (HTB-11, HTB-12 and HTB-14) were included in this study. LAK cell-mediated cytotoxicity against tumor cell lines and patient samples was measured by flow cytometric cell mediated cytotoxicity assay. Drug cytotoxicity was also evaluated in all cell lines and patient samples.Results: In timing studies, aerolysin treatment reached peak cytotoxicity around 2 hours of incubation. U937 was the most sensitive cell type to both LAK cells and aerolysin. Some cells were very resistant to LAK killing and aerolysin at the concentration used. There was significant correlation between aerolysin cytotoxicity and LAK cell-mediated kill at 2 hours in cell lines and patient samples (r = 0.99; p < 0.001, r = 0.85; p = 0.035, respectively). There was also a statistically significant correlation between optimal dose aerolysin killing at 18 hours of incubation and LAK cell-mediated cytotoxicity (r = 0.94; p = 0.04). However, there was not any correlation between drug cytotoxicity and LAK cell-mediated cytotoxicity or aerolysin-induced cell kill.Conclusion: Aerolysin sensitivity may be used as a surrogate marker for the assessment of in vitro LAK cell-mediated tumor cell kill through granule/exocytosis pathway.

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.

Docosahexaenoic Acid Induces Apoptosisviathe Bax-Independent Pathway in HL-60 Cells

We attempted to determine whether docosahexaenoic acid (DHA)-induced apoptosis is mediated via the Bax-mediated pathway in human myeloid leukemia HL-60 cells. DHA-induced apoptosis was confirmed by morphological analysis and caspase-3 activation. But, cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition (MPT), did not inhibit DHA-induced Bax translocation to mitochondria or caspase-3 activation. These data suggest that DHA can induce apoptosis via the Bax-independent pathway.

PC-SPES decreases proliferation and induces differentiation and apoptosis of human acute myeloid leukemia cells

PC-SPES is an eight herbal mixture which has been shown to be active against prostate cancer cells in vitro as well as in patients. In this study, we discovered that it has anti-leukemia activity. HL-60, NB4, U937 and THP-1 human acute myeloid leukemia cells were cultured in the presence of various concentrations of PC-SPES (0.06-0.5 micro l/ml) for 4 days, and cell numbers were counted by Trypan blue exclusion. PC-SPES inhibited proliferation of these cells with an ED50 of 0.17, 0.09, 0.18, 0.32 micro l/ml, respectively. In clonogenic assay, PC-SPES inhibited growth of HL-60 cells (ED50, 0.043 micro l/ml). On the other hand, PC-SPES (0.1 micro l/ml) stimulated growth of normal myeloid committed stem cells (CFU-GM) by 1.4-fold of control (p=0.03). Anti-leukemia effects also occurred against freshly isolated leukemia cells from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) patients. Interestingly, when PC-SPES was combined with ATRA, the antiproliferative effect was markedly enhanced. For example, PC-SPES (0.125 micro l/ml) or ATRA (10(-8) mol/l) inhibited growth of HL-60 cells after 4 days of culture, by approximately 40 and 30%, respectively; simultaneous treatment with both, suppressed growth by 80%. In addition, PC-SPES induced differentiation of HL-60 and NB4 cells, as measured by expression of CD11b and reduction of NBT. ATRA synergistically enhanced this activity. For example, either PC-SPES (0.5 micro l/ml) or ATRA (10(-8) mol/l) induced 23 and 18% of HL-60 cells, respectively to express CD11b on day 2 of culture; and when both were combined, 60% of HL-60 cells were stimulated to express CD11b antigen. Furthermore, PC-SPES (0.5 micro l/ml) produced apoptosis of HL-60 and NB4 cells, as measured by TUNEL assay, with 17% of HL-60 cells and 52% of NB4 cells becoming apoptotic on their third day of culture. Importantly, PC-SPES stimulated expression of the novel myeloid specific transcription factor C/EBPepsilon in HL-60 and NB4 cells. Taken together, PC-SPES inhibits growth and induces differentiation and apoptosis of myeloid leukemia cells, and enhances the antiproliferative and prodifferentiative effects of ATRA on these cells. PC-SPES might be useful with ATRA for treatment of patients with acute promyelocytic leukemia (APL), and it could have a role in other types of cancers including MDS.

Cytokinin-induced differentiation of human myeloid leukemia HL-60 cells is associated with the formation of nucleotides, but not with incorporation into DNA or RNA

Cytokinins are important purine derivatives that act as hormones to control many processes in plants. Cytokinins such as isopentenyladenine (IPA), kinetin and benzyladenine were very effective at inducing the granulocytic differentiation of human myeloid leukemia HL-60 cells. The metabolism of cytokinins to their nucleotides was closely associated with cytokinin-induced differentiation and growth inhibition. When the cells were incubated with [ 14C]-benzyladenine, radioactivity was significantly incorporated into RNA and DNA. However, the radioactive nucleotides in RNA or DNA were adenine nucleotides, not benzyladenine nucleotides, suggesting that cytokinins were not incorporated into RNA and DNA. The benzyladenine nucleotides were not stably released into the medium in intact form. Cytokinins effectively induced a phosphorylated (active) form of mitogen-activated protein kinase (MAPK). MAPK activation was necessary for cytokinin-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by cytokinins. These results suggest that cytokinin nucleotides themselves play an important role in inducing the differentiation of HL-60 cells.