Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADPH oxidase was induced in HL-60 human promyelocytic leukemia cells when these cells were treated with 10(-7) M 1,25-dihydroxyvitamin D3 (VD3) for 4 days. The treated cells were disrupted by sonication, and the postnuclear fraction was separated into 48,000 X g supernatant (cytosol) and precipitate (membrane) fractions. Membrane-bound NADPH oxidase was activated in vitro with SDS and cytosol. However, the cytosol from untreated HL-60 cells could not activate NADPH oxidase. The cytosolic activity was induced 2 days after VD3 treatment and fully expressed on day 4. The activity was heat-sensitive and destroyed by trypsin. The possibility that the cytosolic activation factor is a protein kinase C (PKC) was then tested. Ca2+- and phospholipid-dependent PKC activity was low in the cytosol of untreated HL-60 cells but increased in the cytosols of VD3-treated cells 4 and 11 times, respectively, 2 days and 4 days after treatment. H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride], inhibited PKC activity in a dose-dependent manner at 1-100 microM. Cytosolic activity of NADPH oxidase was not inhibited at all at those concentrations. Furthermore, PKC activity was lost when Ca2+ was omitted from the assay mixture, but NADPH oxidase was activated in the presence of EGTA. These results indicated that the cytosolic factor is not a PKC, and that NADPH oxidase in this cell-free system is activated by a mechanism that does not involve PKC.
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PMID:Induction of cytosolic activation factor for NADPH oxidase in differentiated HL-60 leukemia cells. 316 10

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation. NADPH oxidase activities in phagocytic vesicles from induced HL-60 cells and polymorphonuclear leukocytes were equal, indicating the equivalence of the enzyme system in active portions of their cell membranes. Separation by centrifugal elutriation of the HL-60 cell population into fractions of varying maturity confirmed the relationship of NADPH oxidase activity to advancing differentiation in both dimethylformamide-induced and spontaneously maturing cells. Membrane potential change, an early event related to activation of the oxidase, was followed by 3,3'-dipropylthiodicarbocyanine dye fluorescence. The depolarization response increased dramatically in both magnitude and initial rate of change during differentiation. The cells' cytochrome b content increased 3-fold with induction of differentiation, in proportion to the change in NADPH oxidase activity.
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PMID:Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line. 632 39

A flavoprotein dehydrogenase assayed for the activity of electron transfer from NADPH to cytochrome c was highly purified from the cytosolic fraction of differentiated human promyelocytic leukemia HL-60 cells. The purified enzyme had an apparent molecular mass of 68 kDa by sodium dodecyl sulfate gel electrophoresis and an equimolar amounts of flavin mononucleotide and flavin-adenine dinucleotide. The purification factor of the enzyme with respect to the cytosolic fraction was close to 1100 and the recovery of activity was approximately 18%. Reduction of cytochrome c by NADPH indicated Michaelis-Menten kinetics with a Km value of 1.50 microM for NADPH. When cytochrome c was the varied substrate, a Km value of 4.10 microM was obtained. NADH was not an effective electron donor for cytochrome c reduction and NADPH-dependent reduction of nitroblue tetrazolium was negligibly small. The purified enzyme alone did not exhibit superoxide production, and NADPH oxidase activity was not markedly stimulated upon incubation of the reductase with cytochrome b558 purified from porcine neutrophils. The purified flavoprotein gave a positive cross-reactivity to polyclonal antibodies raised to microsomal NADPH-cytochrome P450 reductase, indicating structural homology between these enzymes. The catalytic properties of the purified NADPH-cytochrome c reductase have similarities to those of liver NADPH-cytochrome P450 reductase.
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PMID:Characterization of superoxide dismutase-insensitive cytochrome c reductase activity in HL-60 cytosol as NADPH-cytochrome P450 reductase. 848 36

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 microM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 microM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 microM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.
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PMID:Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA in HL-60 cells. 1120 61

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O(2)(-) concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA- induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K(d) value of 6.7 nm.
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PMID:Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid. 1202 2

Activated inflammatory leukocytes generate a variety of reactive oxygen and nitrogen species (RONS) that may have roles in mutagenesis and carcinogenesis. The purpose of the present study was to explore the relationship between inflammatory leukocyte activation and mutagenesis using co-culture systems. We investigated the mutagenic potentials of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated differentiated HL-60 (human promyelocytic leukemia cells), and RAW 264.7 cells (murine macrophages) stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma by co-culturing each cell line with AS52 cells, a transgenic Chinese hamster ovary cell line. HL-60 cells rapidly generated superoxide (O(2)(-)) 15 min to 1 h (peak at 30 min) following TPA stimulation. RAW 264.7 cells stimulated with LPS and IFN-gamma produced O(2)(-), nitric oxide (NO) and peroxynitrite (ONOO(-)) continuously for 5-25 h. There was a 2.0-fold increase in the mutation frequency of the gpt gene in AS52 cells co-cultured with TPA stimulated HL-60 cells, when compared with non-treated cells. Importantly, this increase in mutation frequency was significantly suppressed by antioxidants, such as superoxide dismutase (SOD) and diphenylene iodonium (DPI), an NADPH oxidase inhibitor (inhibition rates: IRs = 18.2 and 35.1%, respectively). Similarly, co-culture of AS52 cells with LPS/IFN-gamma-stimulated RAW 264.7 cells also increased the mutation frequency of the gpt gene by 2.6-fold, and this increase in mutation frequency was suppressed by SOD, DPI and N(5)-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO), an specific iNOS inhibitor (IRs = 58.3, 70.8 and 70.8%, respectively). In co-culture experiments, activated HL-60 and RAW 264.7 cells increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in AS52 cells when compared with non-treated controls (1.7- and 1.6-fold, respectively). Treatment of AS52 cells with hydrogen peroxide (H(2)O(2), 100 micro M), ONOO(-) (100 micro M) and SIN-1 (100 micro M), a ONOO(-) generator, also increased the mutation frequency of the gpt gene (4.6-, 5.4- and 2.8-fold, respectively). Taken together, these results support the hypothesis that RONS, derived from activated inflammatory leukocytes, are mutagenic in the biological systems, and that RONS generation inhibitors are potentially anti-mutagenic, and thus may be useful in cancer preventive strategies.
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PMID:Mutagenicity of reactive oxygen and nitrogen species as detected by co-culture of activated inflammatory leukocytes and AS52 cells. 1258 72

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.
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PMID:Promyelocytic HL60 cells express NADPH oxidase and are excellent targets in a rapid spectrophotometric microplate assay for extracellular superoxide. 1451 66

Rotenone, an inhibitor of NADH dehydrogenase complex, is a naturally occurring insecticide, which is capable of inducing apoptosis. Rotenone-induced apoptosis is considered to contribute to its anticancer effect and the etiology of Parkinson's disease (PD). We demonstrated that rotenone induced internucleosomal DNA fragmentation, DNA ladder formation, in human cultured cells, HL-60 (promyelocytic leukemia) and BJAB cells (B-cell lymphoma). Flow cytometry showed that rotenone induced H2O2 generation, followed by significant changes in the mitochondrial membrane potential (DeltaPsim). Caspase-3 activity increased in HL-60 cells in a time-dependent manner. These apoptotic events were delayed in HP100 cells, an H2O2-resistant clone of HL-60, confirming the involvement of H2O2 in apoptosis. Expression of anti-apoptotic protein, Bcl-2, in BJAB cells drastically inhibited DeltaPsim change and DNA ladder formation but not H2O2 generation, confirming the participation of mitochondrial dysfunction in apoptosis. NAD(P)H oxidase inhibitors prevented H2O2 generation and DNA ladder formation. These results suggest that rotenone induces O2(-)-derived H2O2 generation through inhibition of NADH dehydrogenase complex and/or activation of NAD(P)H oxidase, and H2O2 generation causes the disruption of mitochondrial membrane in rotenone-induced apoptosis.
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PMID:Mechanism for generation of hydrogen peroxide and change of mitochondrial membrane potential during rotenone-induced apoptosis. 1456 32

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) mediated generation of reactive oxygen species (ROS) was originally identified as the powerful host defense machinery against microorganism in phagocytes. But recent reports indicated that some non-phagocytic cells also have the NADPH oxidase activity, and the ROS produced by it may act as cell signal molecule. But as far as today, whether the NADPH oxidase also plays similar role in phagocyte has not been paid much attention. Utilizing the undifferentiated HL-60 promyelocytic leukemia cells as a model, the aim of the present study was to determine whether NADPH oxidase plays a role on ROS generation in undifferentiated HL-60, and the ROS mediated by it was essential for cell's survival. For the first time, we verified that the release of ROS in undifferentiated HL-60 was significantly increased by the stimulation with Calcium ionophore or opsonized zymosan, which are known to trigger respiration burst in phagocytes by NADPH oxidase pathway. Diphenylene iodonium (DPI) or apocynin (APO), two inhibitors of NADPH oxidase, significantly suppressed the increasing of ROS caused by opsonized zymosan. Cell survival assay and fluorescence double dyeing with acridine orange and ethidium bromide showed that DPI and APO, as well as superoxide dismutase (SOD) and catalase (CAT) concentration-dependently decreased the viability of undifferentiated HL-60 cells, whereas exogenous H2O2 can rescue the cells from death obviously. Our results suggested that the ROS, generated by NADPH oxidase play an essential role in the survival of undifferentiated HL-60 cells.
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PMID:NADPH oxidase-mediated generation of reactive oxygen species is critically required for survival of undifferentiated human promyelocytic leukemia cell line HL-60. 1534 54

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.
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PMID:Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells. 1611 42


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