Absence Of Hemin Research Articles

Brazilin From Caesalpinia sappan L. Induced Apoptosis via mTOR and HO-1 Pathway in SW480 Human Colon Cancer Cells

The mTOR pathway is a crucial biological regulatory mechanism of cell growth, proliferation and cell death, and its inhibitors were new candidates of anticancer drugs through regulation of energy balance and metabolism. In the present study, whether brazilin and mTOR inhibitor (Torin1) exerts anti-cancer effects was evaluated and the mechanism of its regulation in colorectal cancer cells investigated. Brazilin showed dose- and time-dependent cytotoxicity of colorectal cancer cells (SW480 cells) through apoptosis pathways such as Bcl-2, Bax, as well as cleavage of caspase 3, caspase 9, and PARP1. In addition, brazilin reduced mammalian target of rapamycin (mTOR) phosphorylation in a dose- and time-dependent manner, and the mTOR inhibitor torin 1 blocked this phosphorylation. Brazilin also decreased heme oxygenase-1 (HO-1) expression in a dose- and time-dependent manner; however, hemin, a specific HO-1 substrate, markedly increased HO-1 expression. Torin 1 reduced hemin-induced HO-1 expression and increased colorectal cell death in a dose-dependent manner in the presence and absence of hemin. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2) translocation into nucleus fraction was crucial role in brazilin-mediated apoptosis of colorectal cancer cells. These results showed that brazilin and torin1 might regulate the mTOR signaling pathway by decreasing mTOR phosphorylation. Furthermore, mTOR signaling was associated with brazilin-regulated HO-1 expression, which induced apoptosis in colorectal cancer cells. These results suggest that synthetic and/or natural mTOR inhibitors were useful candidate for treatment of colorectal cancer cells.

The Effect of Respiration, pH, and Citrate Co-Metabolism on the Growth, Metabolite Production and Enzymatic Activities of Leuconostoc mesenteroides subsp. cremoris E30.

Leuconostoc mesenteroides includes strains used as starter and/or adjunct cultures for the production of several fermented foods. In this study, the effect of anaerobic and respiratory cultivations, as well as of citrate supplementation and different pH values, was evaluated on growth, biomass, metabolite, and enzymatic activities (pyruvate oxidase, POX; NADH-dependent oxidase, NOX; NADH-dependent peroxidase, NPR) of Leuconostoc mesenteroides subsp. cremoris E30. We compared the respiration-increased growth rate and biomass production of Leuc. mesenteroides E30 to anaerobic cultivation. A supplementation of citrate impaired the growth rate of the respiratory cells. As expected, anaerobic cultures did not consume oxygen, and a similar trend in oxygen uptake was observed in respiratory cultures. The aerobic incubation caused changes in the metabolic pattern, reducing the production of ethanol in favour of acetic acid. Citrate was already exhausted in the exponential phase and did not affect the yields in acetic acid and ethanol. NOX activity increased in the presence of oxygen, while catalase was also detected in the absence of hemin. The absence of H2O2 suggested its degradation by NPR and catalase. Respiratory cultivation provided benefits (increase in growth rate, biomass, and activity in antioxidant enzymes) for Leuc. mesenteroides E30. Therefore, the exploitation of respiratory phenotypes may be useful for the formulation of competitive starter or adjunct cultures.

Self-Assembling Catalytic Peptide Nanomaterials Capable of Highly Efficient Peroxidase Activity.

The self-assembly of short peptides gives rise to versatile nanomaterials capable of promoting efficient catalysis. We have shown that short, seven-residue peptides bind hemin to produce functional catalytic materials which display highly efficient peroxidation activity, reaching a catalytic efficiency of 3×105 m-1 s-1 . Self-assembly is essential for catalysis as non-assembling controls show no activity. We have also observed peroxidase activity even in the absence of hemin, suggesting the potential to alter redox properties of substrates upon association with the assemblies. These results demonstrate the practical utility of self-assembled peptides in various catalytic applications and further support the evolutionary link between amyloids and modern-day enzymes.

Hydroxyurea Scavenges Free Radicals and Induces the Expression of Antioxidant Genes in Human Cell Cultures Treated With Hemin.

The excessive release of heme during hemolysis contributes to the severity of sickle cell anemia (SCA) by exacerbating hemoglobin S (HbS) autoxidation, inflammation and systemic tissue damage. The present study investigated the effect of hydroxyurea (HU) on free radical neutralization and its stimulation of antioxidant genes in human peripheral blood mononuclear cells (PBMC) and human umbilical vein endothelial cells (HUVEC) in the presence or absence of hemin. HU (100 and 200 μM) significantly reduced the production of intracellular reactive oxygen species (ROS) induced by hemin at 70 μM in HUVEC. HUVECs treated with HU+hemin presented significant increases in nitric oxide (NO) production in culture supernatants. HU alone or in combination with hemin promoted the induction of superoxide dismutase-1 (SOD1) and glutathione disulfide-reductase (GSR) in HUVECs and PBMCs, and glutathione peroxidase (GPX1) in PBMCs. Microarray analysis performed in HUVECs indicated that HU induces increased expression of genes involved in the antioxidant response system: SOD2, GSR, microsomal glutathione S-transferase (MGST1), glutathione S-transferase mu 2 (GSTM2), carbonyl reductase 1 (CBR1) and klotho B (KLB). Significant increases in expression were observed in genes with kinase activity: protein kinase C beta (PRKCB), zeta (PRKCZ) and phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (PIK3C2B). HU also induced a significant increase in expression of the gene p62/sequestosome (p62/SQSTM1) and a significant decrease in the expression of the transcriptional factor BACH1 in HUVECs. Upstream analysis predicted the activation of Jun, miR-155-5p and mir-141-3p. These results suggest that HU directly scavenges free radicals and induces the expression of antioxidant genes via induction of the Nrf2 signaling pathway.

Application of hemin-induced growth and biochemical modifications in Hassawi okra (Abelmoschus esculentus L.) grown in seawater salinity

The present study investigated the possible protective role of hemin (75 µM) on okra (Abelmoschus esculentus L.) plants grown under diluted seawater (seawater/normal water) in plastic bags on loam soil and compost (1:1). Four levels of seawater were prepared by diluting seawater to give 1.8, 3.6, 5.4 and 7.2 dsm-1. Okra plants were irrigated with these concentrations of seawater. The effects of seawater salinity on okra plants were evaluated by determining growth parameters, superoxide dismutase (SOD) activity, catalase (CAT) activity ascrobate content, α-amylase, protease and lipid peroxidation in the presence or absence of hemin. The study found that seawater salinity resulted in a high level of lipid peroxidation, which was associated with significant decrease in growth parameters and protease activity. The activity of SOD, CAT and ascorbate content were increased significantly, compared to control plants. Alleviation effect of hemin was obvious on growth parameters at most salinity levels. This was associated by enhancement of CAT and α-amylase activities and reduction of lipid peroxidation compared to the corresponding untreated salinized plants. So hemin could play a central function as a signal molecule in salt tolerance of okra plants. The results of this study demonstrated that okra can be grown successfully using diluted seawater and the different antioxidants could partially alleviate the harmful effects of seawater stress that reflected on growth and some physiological changes of okra plant. According to these findings, it can be pronounced that the treatment of salinized okra with hemin (75 µM) may reduce the negative impact of light salinity stress.

Heme oxygenase-1 induction attenuates senescence in chronic obstructive pulmonary disease lung fibroblasts by protecting against mitochondria dysfunction.

Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by an irreversible proliferation arrest associated with secretion of inflammatory mediators. ROS production, known to induce senescence, is increased in COPD fibroblasts and mitochondria dysfunction participates in this process. Among the battery of cellular responses against oxidative stress damage, heme oxygenase (HO)‐1 plays a critical role in defending the lung against oxidative stress and inflammation. Therefore, we investigated whether pharmacological induction of HO‐1 by chronic hemin treatment attenuates senescence and improves dysfunctional mitochondria in COPD fibroblasts. Fibroblasts from smoker controls (S‐C) and COPD patients were isolated from lung biopsies. Fibroblasts were long‐term cultured in the presence or absence of hemin, and/or ZnPP or QC‐15 (HO‐1 inhibitors). Lung fibroblasts from smokers and COPD patients displayed in long‐term culture a senescent phenotype, characterized by a reduced replicative capacity, an increased senescence and inflammatory profile. These parameters were significantly higher in senescent COPD fibroblasts which also exhibited decreased mitochondrial activity (respiration, glycolysis, and ATP levels) which led to an increased production of ROS, and mitochondria biogenesis and impaired mitophagy process. Exposure to hemin increased the gene and protein expression level of HO‐1 in fibroblasts and diminished ROS levels, senescence, the inflammatory profile and simultaneously rescued mitochondria dysfunction by restoring mitophagy in COPD cells. The effects of hemin were abolished by a cotreatment with ZnPP or QC‐15. We conclude that HO‐1 attenuates senescence in COPD fibroblasts by protecting, at least in part, against mitochondria dysfunction and restoring mitophagy.

G-Quadruplex Forming Sequences Found In The Beta-Globin Gene Cluster Regulate Erythroid Differentiation Of K562 Cells

IntroductionGuanine-rich DNA sequences can form stable four stranded structures by folding of the DNA strands, forming stacks of G-tetrads called G-quadruplex. G-quadruplex forming sequences are found in eukaryotic telomeres, in promoter regions and in noncoding regions of the genome. Although the function of these structures has not yet been identified it has been suggested that they play a negative regulatory role at the transcription level. The most studied G-quadruplex sequences (AS1411 and PU27) show activity in reducing growth and invasiveness of malignant cells, as well as increasing cell death of a large array of tumors with promising therapeutic applications. We identified two G-quadruplex forming sequences in the human beta globin cluster (Hbd and Hbg2). We confirmed quadruplex formation by these sequences using circular dichroism spectroscopy. We also observed in Southern blot assay that these oligo-sequences bind to specific β-globin gene fragments after EcoRI digestion. Finally we demonstrate that neither Hbd nor Hbg2 (5 or 10µM) were growth inhibitory to four different leukemia cell lines after 6 days culture measured by MTT. ResultsThe β-globin cluster contains 5 functional globin-like genes and long range regulator elements, the expression of each of these genes is tightly regulated during development (from embryo to fetus to adult), in cell type and in the course of differentiation/maturation of the erythroid lineage. Recent studies have demonstrated the importance of folding and looping of the DNA in the control of globin genes expression. We hypothesized that the G-quadruplex forming sequences within the β-globin gene cluster participate in regulation and splicing of the different globin genes during erythroid maturation. The erythroleukemia cell line K562 was used in this study because of its ability to differentiate into erythrocytes under Hemin stimulation. K562 cells were divided into 6 groups of treatment and cultured for 3 days in the presence or absence of Hemin (30µM) with or without either Hbd or Hbg2 oligonucleotides (5µM). Erythroid differentiation was evaluated by quantitative QRT-PCT for the expression of β-, γ- and ε-globin genes as well as α-globin and by flow-cytometry for the presence of β-, γ- and ε-globin; α-globin and CD235a (Glycophorin A) were used as controls. The data reveal a difference in gene expression for all globin genes when K562 were exposed to Hbd or Hbg2 only, interestingly, a significant up-regulation of β-globin was observed only in Hbg2 treated cells. Similarly in K562 induced to differentiate with Hemin show up-regulation of γ-, ε- and α- globin, the addition of Hbd or Hbg2 oligonucleotides to Hemin at the beginning of the culture slightly improve the expression of γ- and ε- however there was no difference for the expression of β- globin in the hemin +/- Hb oligonucleotides treated cells compared to control untreated cells. Flow-cytometry experiments confirmed the increase in the expression of each globin gene in all treatments compared to control. Altogether the data suggest a role for Hbd and Hbg2 in the regulation of β-globin gene transcription and therefore in the differentiation of K562 in erythroid lineage. ConclusionsThe implications of our findings are multiple. 1) the presence of G-quadruplex sequences in a gene complex not involved in growth regulation or oncogenesis; 2) to our knowledge this is the first time that G-quadruplex sequences are shown to specifically alter the expression of the gene in which they are located; and 3) the potential use of globin G-quadruplex to modify the globin gene expression therapeutically. Disclosures:No relevant conflicts of interest to declare.

Hemin Binds to Human Cytoplasmic Arginyl-tRNA Synthetase and Inhibits Its Catalytic Activity

The free form of human cytoplasmic arginyl-tRNA synthetase (hcArgRS) is hypothesized to participate in ubiquitin-dependent protein degradation by offering arginyl-tRNA(Arg) to arginyl-tRNA transferase (ATE1). We investigated the effect of hemin on hcArgRS based on the fact that hemin regulates several critical proteins in the "N-end rule" protein degradation pathway. Extensive biochemical evidence has established that hemin could bind to both forms of hcArgRS in vitro. Based on the spectral changes of the Soret band on site-directed protein mutants, we identified Cys-115 as a specific axial ligand of hemin binding that is located in the Add1 domain. Hemin inhibited the catalytic activity of full-length and N-terminal 72-amino acid-truncated hcArgRSs by blocking amino acid activation. Kinetic analysis demonstrated that the K(m) values for tRNA(Arg), arginine, and ATP in the presence of hemin were not altered, but k(cat) values dramatically decreased compared with those in the absence of hemin. By comparison, the activity of prokaryotic ArgRS was not affected obviously by hemin. Gel filtration chromatography suggested that hemin induced oligomerization of both the isolated Add1 domain and the wild type enzyme, which could account for the inhibition of catalytic activity. However, the catalytic activity of an hcArgRS mutant with Cys-115 replaced by alanine (hcArgRS-C115A) was also inhibited by hemin, suggesting that hemin binding to Cys-115 is not responsible for the inhibition of enzymatic activity and that the specific binding may participate in other biological functions.

Flow-based method for epinephrine determination using a solid reactor based on molecularly imprinted poly(FePP–MAA–EGDMA)

A solid phase reactor based on molecularly imprinted poly(iron (III) protoporphyrin-methacrylic acid-ethylene glycol dimethacrylate) (MIP–MAA) has been synthesized by bulk method and applied as an selective material for the epinephrine determination in the presence of hydrogen peroxide. In order to prove the selective behaviour of MIP, two blank polymers named non-imprinted polymer (NIP1), non-imprinted polymer in the absence of hemin (NIP2) as well as a poly(iron (III) protoporphyrin-4-vynilpyridine-ethylene glycol dimethacrylate) (MIP–4VPy) were synthesized. The epinephrine-selective MIP–MAA reactor was used in a flow injection system, in which an epinephrine solution (120μL) at pH 8.0 percolates in the presence of hydrogen peroxide (300μmolL−1) through MIP–MAA. The oxidation of epinephrine by hydrogen peroxide is increased by using MIP–MAA, being the product formed monitored by amperometry at 0.0V vs. Ag/AgCl. The MIP–MAA showed better selective behaviour than NIP1, NIP2 and MIP–4VPy, demonstrating the effectiveness of molecular imprinting effect. Highly improved response was observed for epinephrine in detriment of similar substances (phenol, ascorbic acid, methyl-l-DOPA, p-aminophenol, catechol, l-DOPA and guaiacol). The method provided a calibration curve ranging from 10 to 500μmolL−1 and a limit of detection of 5.2μmolL−1. Kinetic data indicated a value of maximum rate Vmax (0.993μA) and apparent Michaelis–Menten constant of Kmapp(725.6μmolL−1). The feasibility of biomimetic solid reactor was attested by its successful application for epinephrine determination in pharmaceutical formulation.

The protective effects of ginsenosides on human erythrocytes against hemin-induced hemolysis

Panax ginseng has been used in traditional Chinese medicine to enhance stamina and capacity to deal with fatigue and physical stress. Many reports have been devoted to the effects of ginsenosides on many in vitro or in vivo experimental systems. The major aim of this work is to investigate the protective effects of 12 individual ginsenosides including Rb1, Rb3, Rc, Rd, Re, Rg1, Rg2, Rg3, Rh1, Rh2, R1 and pseudoginsenoside F11, together with the central structures of aforementioned ginsenosides, 20(S)-protopanaxadiol (PD) and 20(S)-protopanaxatriol (PT), on hemin-induced hemolysis of human erythrocytes. This is because hemin can induce hemolysis by accelerating the potassium leakage, dissociating skeletal proteins and prohibiting some enzymes in the membrane of erythrocyte. Thus, the structure-activity-relationship (SAR) between ginsenosides and protective effects has been screened in this in vitro experimental system. It is found that Rh2 and Rg3 intensify hemolysis in the presence of hemin, and initiate hemolysis even in the absence of hemin. All the other ginsenosides protect human erythrocytes against hemin-induced hemolysis more or less. The overall sequence is Rc > Rd > Re ∼ Rb1 > Rg1 ∼ Rh1 > Rb3 ∼ Rg2 ∼ R1 ∼ F11 ∼ PT. In addition, the protective effects of PD and PT have been detected, and found that PD promotes hemolysis appreciably, whereas PT protects erythrocytes efficiently. Moreover, the protective effects of PT ginsenosides are similar to PT itself, and the protective effects of PD ginsenosides vary remarkably, demonstrating that the positions of the sugar moieties make the protective activities of ginsenosides complicated. Especially, sugar moiety at 20-position is critical for PD ginsenosides to inhibit hemolysis, whereas hydroxyl group at 3-position is important for PT ginsenosides. The present result may be useful for understanding the SAR of ginsenosides.

Non-heme Induction of Heme Oxygenase-1 Does Not Alter Cellular Iron Metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.

Hemin Confers Resistance to Imatinib (Glivec)-Induced Cell Death but Enhances Nilotinib (AMN107) Cytotoxicity in Human K-562 CML Leukemia Cells.

Imatinib (Glivec) and Nilotinib (AMN107) are selective inhibitors of the Bcr-Abl, Kit and PDGFR tyrosine kinases, developed by Novartis Pharma AG to treat CML and other malignancies by promoting apoptosis (Manley P.W., et al. Biochim Biophys Acta 1754(1–2):3–13, 2005). Heme (iron protoporphyrin-IX) and its oxidized form Hemin, are key regulators of key cellular functions in mammalian cells and Hemin protects normal and leukemia cells from cytotoxic agents (Tsiftsoglou A.S. et al., Pharmacology & Therapeutics 111(2):327–345, 2006). We have recently observed that Hemin, like Erythropoietin (EPO), protects human Ph+ K-562 CML leukemia cells from Imatinib-induced cell death. We extended this observation and investigated whether Hemin also counteracts Nilotinib-induced cytotoxicity in cells. K-562 CML leukemia cells were treated (from 0h to 96h) separately with each of the inhibitors added at different concentrations (0.01 μM to 1 μM) in the presence and absence of Hemin (90 μM) and assessed for their ability to proliferate, differentiate into erythroid lineage or undergo cell death. K-562 cells pre-treated with Hemin prior to be being exposed to Imatinib and/or Nilotinib were used to demonstrate whether induction of differentiation by Hemin alters their developmental stage and susceptibility to either inhibitor. Our results thus indicate that:Hemin confers resistance to Imatinib in a substantial proportion of cells (20–30%). Priming cells with Hemin gradually decreased susceptibility to Imatinib-induced cell death. Considering that the intracellular Heme in CML cells may overcome the Imatinib effect, this resistance could affect the clinical outcome of CML patients treated with Imatinib.Conversely, Hemin enhanced cell death induced by Nilotinib in the concentration range 0.01 μM to 1 μM.These findings indicate that Imatinib and Nilotinib induce cell death in K-562 CML leukemia cells, via pathways affected differently by Hemin. Experiments are now in progress to delineate the mechanisms via which Hemin exerts such disparate action (antagonistic and synergistic) on these inhibitors in human leukemias.

Kinetics ofLactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.

Hemin Potentiates Nitric Oxide-Mediated Nitrosation of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) to 2-Nitrosoamino-3-methylimidazo[4,5-f]quinoline

Heme has been reported to be an important contributor to endogenous N-nitrosation within the colon and to the enhanced incidence of colon cancer observed with increased intake of red meat. This study uses the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) as a target to evaluate hemin potentiation of nitric oxide (NO)-mediated nitrosation. Formation of 14C-2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ) was monitored by HPLC following incubation of 10 microM IQ with the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 in the presence or absence of hemin. N-NO-IQ formation due to autoxidation of NO was at the limit of detection (0.1 microM) and increased 22-fold in the presence of 10 microM hemin and an in situ system for generating H2O2 (glucose oxidase/glucose). A linear increase in N-NO-IQ formation was observed from 1 to 10 microM hemin. Significant nitrosamine formation occurred at fluxes of NO and H2O2 as low as 0.024 and 0.25 microM/min, respectively. Potentiation by hemin was not affected by a 400-fold excess flux of H2O2 over NO or a 4.8-fold excess flux of NO over H2O2. Reactive nitrogen species produced by hemin potentiation had a 46-fold greater affinity for IQ than those produced by autoxidation. Azide inhibited autoxidation, suggesting involvement of the nitrosonium ion, NO+. Hemin potentiation was inhibited by NADH, but not azide, suggesting oxidative nitrosylation with NO2* or a NO2*-like species. IQ and 2,3-diaminonaphthylene were much better targets for nitrosation than the secondary amine morpholine. Apc(min) mice with dextran sulfate sodium-induced colitis demonstrated increased levels of urinary nitrite and nitrate consistent with increased expression of iNOS and NO synthesis. As reported previously, identical conditions increased fecal N-nitroso compounds. Thus, hemin potentiation of NO-mediated nitrosation of heterocyclic amines provides a testable mechanism by which red meat consumption can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.

Role of Transcription Factor HBP1 in Human Leukaemic Cell Proliferation, Apoptosis and Differentiation.

HMG-box containing protein 1 (HBP1) is a recently described member of the high mobility group of transcription factors. We showed that HBP1 is expressed at low levels in immature myeloid cell lines, and at greatly increased levels following induction of differentiation. We also showed that HBP1 protein interacts physically and functionally with the retinoblastoma (RB) protein. We demonstrated that in human myeloid cell lines which express the myeloperoxidase (MPO) gene, HBP1 enhances the activity of the MPO promoter by binding to promoter DNA. However, its role in regulating myeloid development and differentiation remains to be elucidated. To begin to explore this question, we stably transfected the myeloid cell line, K562, with plasmid constructs derived from pBudCE4.1 (Invitrogen), which overexpress HBP1. K562 cells are a myeloid cell line which can be induced to differentiate (by appropriate chemicals) along erythroid or megakaryocytic pathways. Five sets of bulk cultures and multiple clones derived from each of them were obtained: 1) cell only; 2) CAT control; 3) HBP1 driven by CMV promoter (low level overexpression); 4) HBP1 driven by EF1 promoter (high level overexpression); and 5) HBP1 driven by CMV promoter plus RB driven by EF1 promoter. Overexpression of HBP1 with or without co-overexpression of RB resulted in slower cell growth rates compared with control cells. Cell cycle analysis revealed that HBP1 transfectants displayed marked retardation of S-phase progression by a 5-bromo-2-deoxyuridine (BrdU) incorporation method. Real-time RT-PCR showed that expression of cyclin D1 and cyclin D3 mRNAs significantly decreased in HBP1 transfectants. The D-type cyclins (cyclin D1, D2, and D3) promote cell cycle progression from G1 to S phase by binding to and activating the cyclin dependent kinases Cdk4 and Cdk6. HBP1 transfectants underwent increased apoptosis as demonstrated by intracellular DNA fragmentation, and the binding of annexin V to the cell surface. Consistent with this, substantial increases in Fas ligand mRNA levels were noted in HBP1 transfectants suggesting that apoptosis may be occurring by the Fas/Fas ligand pathway. HBP1 overexpression induced terminal, irreversible erythroid differentiation of K562 cells in the absence of hemin and enhanced hemin-induced erythroid differentiation, as evidenced by hemoglobin production and increased expression of glycophorin A on the K562 cell membrane. During this erythroid differentiation, c-Myc and c-Myb transcripts were downregulated whereas JunB transcription was upregulated. HBP1 overexpression increased TPA-induced induction of megakaryocytic markers such as CD41a and increased cell size as determined by flow cytometry. Similar effects were seen in clones derived from bulk cultures of transfected cells. In summary, overexpression of HBP1 in the myeloid cell line K562 resulted in reduced cell growth rates, increased rates of apoptosis, and increased differentiation of cells toward erythroid or megakaryocytic lineages. Real-time RT-PCR studies suggested that these changes resulted, at least in part, from changes in transcription of critical genes which control cell growth, apoptosis, and differentiation in myeloid cells. Our results support the hypothesis that HBP1 is intimately involved in the control of cell growth, apoptosis, and terminal differentiation in developing myeloid cells.

Oxidation of melatonin by carbonate radicals and chemiluminescence emitted during pyrrole ring cleavage.

Oxidation of melatonin was followed by measuring chemiluminescence emitted during pyrrole ring cleavage, a process leading to the main oxidation product of this indoleamine, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). Radical reactions of melatonin were studied in two variants of a moderately alkaline (pH 8) H2O2 system, one of which contained hemin as a catalyst. In both systems, light emission from melatonin oxidation lasted several hours. Time courses and turnover rates depended on the presence or absence of hemin; the catalyst enhanced light emission many-fold. In the two reaction systems, the presence of hydrogen carbonate (HCO)(3)(-) enhanced chemiluminescence by more than 10-fold, indicating scavenging of carbonate radicals. In the presence of 10% dimethylsulfoxide (DMSO) or 1 m mannitol, HCO(3)(-)-dependent as well as independent light emissions were only partially inhibited. With regard to the stimulatory effect of HCO(3)(-), this implies a formation of carbonate radicals (CO)(3)(-) independent of hydroxyl (OH) radicals, presumably involving superoxide anions abundantly present in the system. Tiron, a scavenger of superoxide anions, strongly and almost instantaneously inhibited chemiluminescence, in accordance to the requirement of this reactive oxygen species for AFMK formation and its involvement in -radical formation. Melatonin's capability of scavenging CO(3)(-) may contribute to its protective potency.

Modulation of transduced erythropoietin expression by iron

Objective Future prospects for gene therapy of chronic anemias involve expression of the erythropoietin transgene, which is regulated by oxygen tension. However, other factors such as cytokines or the iron load of erythropoietin-expressing cells can concomitantly modulate transgene expression, as shown for the expression of the endogenous erythropoietin gene in human cell lines and in animals. We tested the effects of iron overload or depletion on the expression of the mouse erythropoietin transgene (cDNA), driven by the hypoxia-regulated phosphoglycerate kinase 1 promoter. Materials and Methods Retrovirally transduced mouse cells (C3H fibroblasts or C2C12 myoblasts) were cultured in normoxia (room air, O 2: 21%) or hypoxia (O 2: 1.5%) in the presence or absence of hemin (an iron donor) or deferiprone (an iron chelator), both of which easily enter the cell. Results Hemin inhibited the hypoxia-induced expression of the transgene. In contrast, deferiprone enhanced the hypoxia-induced expression of the erythropoietin transgene and induced its expression in normoxia. Conclusion These results show that, in addition to oxygen partial pressure, the intracellular iron content is critical in the modulation of hypoxia-regulated erythropoietin transgene expression.

Translocation of chicken heart apocytochrome c and its mutants (C17S, H18D) across mitochondrial membrane

The dependence of import of chicken heart apocytochrome c on its transformation to holoform by heme attachment was studied. Results showed that there was no difference in the translocation of apocytochrome c across the mitochondrial membrane in the presence or absence of hemin + dithionite. Furthermore, two heme unattached mutants (H18D. C17S) were prepared, which could still be accumulated in mitochondria, but their import velocity was obviously reduced.

Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.

The Effects of Pyrroloquinoline Quinone on Heme-Regulated eIF-2α Kinase and eIF-2B Activities in Eukaryotic Protein Synthesis

ABSTRACT: Pyrroloquinoline quinone (PQQ), a novel cofactor of biological redox processes, is ubiquitous in animal cells. We have examined the effects of PQQ on protein synthesis. PQQ inhibits protein synthesis in hemin-supplemented rabbit reticulocyte lysates. This inhibition is characterized by increased phosphorylation of eIF-2α and by diminished guanine nucleotide exchange activity of eIF-2B. The increased eIF-2α phosphorylation is the result of activation by PQQ of the heme-regulated eIF-2α kinase (HRI). The addition of 10 μM PQQ completely inhibits the increase in protein synthesis that occurs on the addition of hemin (20 μM) to heme-deficient lysates, whereas a lower concentration of PQQ (100 nM) causes a very slight stimulation of protein synthesis. The increased eIF-2α phosphorylation that occurs at high concentrations of PQQ inhibits eIF-2B activity, presumably due to formation of a 15S complex [eIF-2(αP).eIF-2B] in which eIF-2B becomes non-functional. Low concentrations of PQQ (0.1-1 μM) do not affect eIF-2α phosphorylation, but rather enhance the guanine nucleotide exchange activity of eIF-2B in reticulocyte lysates. In Chinese hamster ovary cell extract which is devoid of significant eIF-2α kinase activity, addition of both low and high concentrations of PQQ results in an increase in eIF-2B activity. The addition of PQQ to reticulocyte lysates activates HRI whereas addition of PQQ to purified HRIin vitroinhibits the autokinase and eIF-2α kinase activity of the HRI; the inhibition of purified HRI by PQQ is observed both in the presence and absence of hemin. These findings suggest that PQQ inhibits purified HRI by acting as an oxidant whereas in lysates in which PQQ is readily reduced, the PQQ acts as a reductant and increases the activities of both HRI and eIF-2B.