Increased dihydrofolate reductase activity in methotrexate-resistant human promyelocytic-leukaemia (HL-60) cells. Lack of correlation between increased activity and overproduction.
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ABSTRACT:
SUBMITTER: Dedhar S
PROVIDER: S-EPMC1144635 | biostudies-other | 1985 Feb
REPOSITORIES: biostudies-other
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Project description:Cyclo-oxygenase (COX) production in human promyelocytic leukaemia (HL-60) cells was studied during monocytic differentiation induced by 1 alpha, 25-dihydroxyvitamin D3 (24 nM; 3 days) or phorbol 12-myristate 13-acetate (100 nM; 1 day), or during granulocytic differentiation induced by retinoic acid (1 microns; 4 days). Undifferentiated or differentiated HL-60 cells were labelled with [35S]methionine, and membrane-bound COX was solubilized and quantified by SDS/PAGE. Immunoprecipitated 35S-labelled COX from cells induced to differentiate into monocytic or granulocytic lineage were clearly detected on the autoradiograms as a protein of approx. 70 kDa molecular size, whereas only a very faint COX band was detected in untreated HL-60 cells. During both monocytic and granulocytic differentiation, COX activity (measured by the conversion of exogenous arachidonic acid into prostaglandin E2) was dramatically increased. In addition, thromboxane synthesis was preferentially enhanced during monocytic differentiation. HL-60 cells, induced to differentiate into the monocytic or granulocytic lineage, provide a useful tool to investigate the cellular mechanisms involved in regulation of the synthesis of individual prostanoid-metabolizing enzymes.
| S-EPMC1149687 | biostudies-other
Project description:The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.
| S-EPMC5625320 | biostudies-literature
Project description:Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which has been shown to play a role in the differentiation of haematopoietic cells. We report here that neutrophils are the first nucleated mammalian cell type demonstrated to be devoid of immunoreactive PARP. Both NB4 acute promyelocytic leukaemia and HL-60 (acute myelocytic leukaemia) cells were differentiated into non-malignant neutrophils with all-trans-retinoic acid (ATRA). Western blot analysis demonstrated that ATRA had no effect on PARP expression in HL-60 cells. However, PARP was completely down-regulated in NB4 cells within 36 h of treatment initiation. This decrease in PARP polypeptide coincided with growth arrest and preceded the appearance of neutrophilic differentiation features. NB4 cells require a combination of 1,25-dihydroxyvitamin D3 (1,25-D3) and phorbol 12-myristate 13-acetate (PMA) to differentiate completely into monocyte/macrophages, whereas HL-60 cells can be made to differentiate by combined or single agents. PARP expression was up-regulated 90-fold when NB4 cells were treated with PMA and 1,25-D3 together, and this increase accompanied expression of the monocyte/macrophage phenotype. Only modest changes in PARP expression were observed when each agent was used alone in NB4 cells or when HL-60 cells were differentiated along the monocyte/macrophage pathway. In addition, PARP activity was modulated in a pattern similar to protein levels when NB4 cells were induced to differentiate along the neutrophilic and monocyte/macrophage pathways. This suggests that the activity of PARP may be controlled through regulation of protein levels during NB4 cell differentiation. We conclude that PARP levels are dramatically modulated during monocyte/macrophage and neutrophilic differentiation. On the basis of the tremendous changes in PARP polypeptide and total activity during myeloid differentiation, we propose that modulation of PARP gene expression is required for cellular maturation in both lineages.
| S-EPMC1136853 | biostudies-other
Project description:Tumor necrosis factor alpha (TNF-α) is a potent cytokine involved in systemic inflammation and immune modulation. Signaling responses that involve TNF-α are context dependent and capable of stimulating pathways promoting both cell death and survival. TNF-α treatment has been investigated as part of a combined therapy for acute myeloid leukemia due to its modifying effects on all-trans retinoic acid (ATRA) mediated differentiation into granulocytes. To investigate the interaction between cellular differentiation and TNF-α, we performed RNA-sequencing on two forms of the human HL-60/S4 promyelocytic leukemia cell line treated with TNF-α. The ATRA-differentiated granulocytic form of HL-60/S4 cells had an enhanced transcriptional response to TNF-α treatment compared to the undifferentiated promyelocytes. The observed TNF-α responses included differential expression of cell cycle gene sets, which were generally upregulated in TNF-α treated promyelocytes, and downregulated in TNF-α treated granulocytes. This is consistent with TNF-α induced cell cycle repression in granulocytes and cell cycle progression in promyelocytes. Moreover, we found evidence that TNF-α treatment of granulocytes shifts the transcriptome toward that of a macrophage. We conclude that TNF-α treatment promotes a divergent transcriptional program in promyelocytes and granulocytes. TNF-α promotes cell cycle associated gene expression in promyelocytes. In contrast, TNF-α stimulated granulocytes have reduced cell cycle gene expression, and a macrophage-like transcriptional program.
| S-EPMC6686940 | biostudies-literature
Project description:We previously reported that human alveolar macrophages rapidly metabolize the chemotactic active lipid mediator leukotriene B4 (LTB4) into the dihydro-LTB4 by reduction of one of the conjugated double bonds. We herein report that human HL-60 cells (a myeloid precursor which can be differentiated into granulocyte- as well as monocyte-like cells by dimethyl sulphoxide or phorbol myristate acetate) express a highly active LTB4 reductase in the undifferentiated state. Differentiation by dimethyl sulphoxide (1.3%) along the granulocyte lineage, as confirmed by light microscopy, conversion of NitroBlue Tetrazolium into formazan, failed to induce a substantial capacity for omega-oxidation of LTB4; this reaction is exclusively found in mature granulocytes. Studies with the cell homogenate of undifferentiated HL-60 cells indicated that the activity of the enzyme depends on the presence of NADPH, Ca2+ and Mg2+, with a pH optimum of 7.5 at 37 degrees C. The enzyme was not released into the supernatant after stimulation of HL-60 cells with phorbol myristate acetate (100 ng) or Ca2+ ionophore (7.5 microM). Subcellular fractionation revealed evidence that the LTB4 reductase is located within the membrane fraction. Purification of the enzyme by gel filtration and gel electrophoresis suggests an apparent molecular mass of 40 kDa.
| S-EPMC1151577 | biostudies-other
Project description:We have examined the sterol composition and metabolism of promyelocytic leukaemia cell lines (HL-60) after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). A variant cell line (Blast II cells) which is resistant to TPA was used as control. Analysis of the sterols of TPA-sensitive cells radiolabelled with [3H]leucine, [14C]acetate or [14C]pyruvate showed a high incorporation into cholesterol and a low incorporation in lanosterol + dihydrolanosterol. The inverse relationship was observed in TPA-resistant cells. Experiments with other cellular variants representing TPA-sensitive and TPA-resistant classes gave similar results. Analysis of the cellular sterol composition by gas chromatography confirmed that TPA-resistant cells are particularly rich in lanosterol/dihydrolanosterol. TPA treatment enhanced the incorporation of [14C]pyruvate into the sterol fraction of both cell types. This was accompanied by an alteration of incorporation into several lipids, particularly phospholipids. Pulse-chase studies with [14C]acetate revealed that TPA induced the release of radioactive lipids into the medium from HL-60 and Blast II cells. However this treatment released phospholipids from the TPA-sensitive cells and sterols and fatty acids from the TPA-resistant cells. We conclude that the sterol composition can regulate specific biochemical processes in the membrane and can be considered as a factor that plays a role in the responsiveness of HL-60 cells to TPA.
| S-EPMC1148862 | biostudies-other
Project description:Human promyelocytic leukaemia (HL-60) cells were employed to study the induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme in controlling prostaglandin inactivation. Phorbol 12-myristate 13-acetate (PMA) stimulated 15-PGDH activity in a time- and concentration-dependent manner. Dimethyl sulphoxide (DMSO) also stimulated the enzyme activity, although a much delayed stimulation was observed. Western blot studies indicated that PMA increased significantly a 28 kDa immunoreactive protein characteristic of 15-PGDH. L-[35S]Methionine labelling of the PMA-treated cells showed a similar enhancement over the control cells. These studies indicate that PMA induced synthesis of 15-PGDH. Stimulation of 15-PGDH activity by PMA or DMSO appears to be mediated by protein kinase C activation, since an inactive analogue of PMA failed to induce the effect, and both staurosporine and H-7 blocked the stimulation. Stimulation by PMA was optimal at 10 nM and less effective at higher concentrations. Western blot studies indicated that a similar, if not greater, amount of enzyme protein was induced at high concentrations of PMA, suggesting that enzyme inactivation might be occurring. Possible enzyme inactivation by protein kinase C activation was further examined by incubating DMSO-treated cells with a high concentration of PMA (50 nM). Time-dependent inactivation of 15-PGDH within the first 1 h was observed and this inactivation was partially blocked by staurosporine and H-7. Pulse-chase experiments indicated that 15-PGDH had a rapid turnover rate (t 1/2 = 47 min), and PMA shortened the half-life of the enzyme (t 1/2 = 33 min), suggesting that PMA might have an additional effect on 15-PGDH degradation. The rapid turnover of 15-PGDH indicates that the enzyme activity depends on continued enzyme synthesis, and this could be susceptible to hormone and drug control mechanisms.
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Project description:Arsenic trioxide has shown the excellent therapeutic efficiency for acute promyelocytic leukemia. Nowadays, more and more research focuses on the design of the arsenic drugs, especially organic arsenicals, and on the mechanism of the inducing cell death. Here we have synthesized some organic arsenicals with Schiff base structure, which showed a better antitumor activity for three different kinds of cancer cell lines, namely HL-60, SGC 7901 and MCF-7. Compound 2a (2-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) and 2b (2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) were chosen for further mechanism study due to their best inhibitory activities for HL-60 cells, of which the half inhibitory concentration (IC50) were 0.77 μM and 0.51 μM, respectively. It was illustrated that 2a or 2b primarily induced the elevation of reactive oxygen species, decrease of glutathione level, collapse of mitochondrial membrane potential, release of cytochrome c, activation of Caspase-3 and apoptosis, whereas all of the phenomena can be eliminated by the addition of antioxidants. Therefore, we concluded that compound 2a and 2b can induce the oxidative stress-mediated intrinsic apoptosis in HL-60 cells. Both the simplicity of structure with Schiff base group and the better anticancer efficiency demonstrate that organic arsenicals are worthy of further exploration as a class of potent antitumor drugs.
| S-EPMC4949440 | biostudies-literature
Project description:Furoquinolone and its derivatives exhibit antimicrobial, anti-allergic, anti-inflammatory and anticancer properties. The present study investigated the anti-tumor activity of synthesized intermediates of furoquinolone in human promyelocytic leukemia HL-60 cells. The biological effects of the active compound ethyl 2-anilino-4-oxo-4,5-dihydrofuran-3-carboxylate (compound 131) were examined in HL-60 cells. The following properties were analyzed: Cell survival, cell cycle profile, caspase-3 activity, Bax and Bcl-2 expression, the amount of intracellular Ca2+, the number of reactive oxygen species (ROS) and the mitochondrial membrane potential. Compound 131 (50% cytotoxic concentration, 23.5 µM) significantly reduced the proliferation of HL-60 cells and was revealed to induce apoptosis in HL-60 cells in a concentration-dependent manner. Moreover, this was associated with the activation of caspase-3, upregulation of Bax, an increase in intracellular Ca2+ and ROS production, and a decrease in mitochondrial membrane potential and Bcl-2 expression levels. Compound 131, a novel 4,5-dihydrofuran-3-carboxylate, induced apoptosis in HL-60 cells via the increase of intracellular Ca2+ and ROS to alter the mitochondrial membrane potential and the protein level of Bax and Bcl-2, as well as activating caspase-3. The results of the current study indicate that compound 131 may represent a promising compound for the development of anti-leukemia therapeutics.
| S-EPMC7039143 | biostudies-literature
Project description:The present study demonstrated that 2'-hydroxycinnamaldehyde (2'-HCA) induced apoptosis in human promyelocytic leukemia HL-60 cells through the activation of mitochondrial pathways including (1) translocation of Bim and Bax from the cytosol to mitochondria, (2) downregulation of Bcl-2 protein expression, (3) cytochrome c release into the cytosol, (4) loss of mitochondrial membrane potential (ΔΨm), and (5) caspase activation. 2'-HCA also induced the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase1/2 (ERK1/2) in HL-60 cells. The pharmacological and genetic inhibition of JNK effectively prevented 2'-HCA-induced apoptosis and activator protein-1 (AP-1)-DNA binding. In addition, 2'-HCA resulted in the accumulation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH) and protein thiols (PSH) in HL-60 cells. NAC treatment abrogated 2'-HCA-induced JNK phosphorylation, AP-1-DNA binding, and Bim mitochondrial translocation, suggesting that oxidative stress may be required for 2'-HCA-induced intrinsic apoptosis. Xenograft mice inoculated with HL-60 leukemia cells demonstrated that the intraperitoneal administration of 2'-HCA inhibited tumor growth by increasing of TUNEL staining, the expression levels of nitrotyrosine and pro-apoptotic proteins, but reducing of PCNA protein expression. Taken together, our findings suggest that 2'-HCA induces apoptosis via the ROS-dependent JNK pathway and could be considered as a potential therapeutic agent for leukemia.
| S-EPMC8618870 | biostudies-literature