A novel nuclear protein which binds to G gamma and A gamma globin promoters and modulates hemoglobin synthesis in K562 cells.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.     

A Role for the MEK/MAPK Pathway in PMA-Induced Cell Cycle Arrest: Modulation of Megakaryocytic Differentiation of K562 Cells

In vitromegakaryocytic differentiation of the pluripotent K562 human leukemia cell line is induced by PMA. Treatment of K562 cells with PMA results in growth arrest, polyploidy, morphological changes, and increased cell–cell and cell–substrate adhesion. These PMA-induced changes in K562 cells are preceded by a rapid rise in the activity of MEK (MAP kinase/extracellular regulated kinases) that leads to a sustained activation of ERK2 (extracellular regulated kinase; MAPK). Blockade of MEK1 activation by PD098059, a recently described specific MEK inhibitor [D. T. Dudleyet al.(1995).Proc. Natl. Acad. Sci. USA92, 7686–7689], reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment. These changes are not associated with a disruption of PMA-induced down-regulation of BCR-ABL kinase or early integrin signaling events but are associated with a block of the cell-surface expression of the gpIIb/IIIa (CD41) integrin, a cell marker of megakaryocytic differentiation. These results demonstrate that the PMA-induced signaling cascade initiated by protein kinase C activation requires the activity of the MEK/ERK signaling complex to regulate cell cycle arrest, thus regulating the program that leads to the cell-surface expression of markers associated with megakaryocytic differentiation.     

Rapid Stimulation of EAAC1-Mediated Na+-Dependent l-Glutamate Transport Activity in C6 Glioma Cells by Phorbol Ester

Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na⁺-dependent l -[³H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l -[³H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC₅₀ value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in V_max with no change in K_m. PMA also increased the transport of the nonmetabolizable analogue, d -[³H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na⁺-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l -[³H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca²⁺ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na⁺/H⁺ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na⁺-dependent l -[³H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.     

Acceptors of poly(ADP-ribosylation) in differentiation inducer-treated and untreated Friend erythroleukemia cells

Acceptors of poly(ADP-ribosylation) were identified and compared between inducer-treated and untreated Friend erythroleukemia cells. When permeabilized Friend cells were pulse labeled with 0.6 μM [³²P]NAD for 1 min and labeled proteins analyzed by SDS-polyacrylamide gel electrophoresis, nucleosome core histones were found to be the primary acceptors, with an additional minor radioactive peak at a position corresponding to M_r = 170 000. Friend cells induced to differentiate by DMSO treatment showed a similar distribution of radioactivity, but with a 60% reduction in the overall level of poly(ADP-ribosylation) under identical labeling conditions. When isolated nuclei were pulse labeled with 0.6 μM [³²P]NAD, radioactive peaks were not restricted mainly at the positions of core histones but widely dispersed in the area from 10 to 50 kDa with another peak at 170 kDa. Increase of NAD concentration resulted in the overall shift of peaks to higher molecular weight positions. When pulse-labeled nuclei or permeable cells were chased with 1 mM NAD, radioactive peaks migrated to positions of very high molecular weight (>M_r = 180 000). Remarkable suppression of poly(ADP-ribose) synthesis was observed when DMSO, hexamethylene bisacetamide, butyric acid, or hemin were used as the inducers.     

Apoptosis induced by 3,7-dinitrodibenzobromonium salts in K562 cells

Abstract We evaluated the inhibitory effect of 3,7-dinitrodibenzobromonium salts (cBr) on the proliferation of human chronic myelogenous leukemia K₅₆₂ cell by trypan blue exclusion test and MTT colorimetric assay. The degree of DNA damage in K₅₆₂ cells treated with cBr, was detected by isotopic tracer method (³H-TdR). The morphological changes of these K₅₆₂ cells were examined by fluorescence and electron microscopy. Biochemical characteristics of K₅₆₂ cells were detected by flow cytometry and ³H-thymidine incorporation assay. Findings indicated that cBr could significantly inhibit cell proliferation and result in DNA damage of K₅₆₂ cells. cBr is a new type of immunostimulant and can induce cell apoptosis. __________ Translated from Journal of Lanzhou University (Natural Sciences), 2006, 42(6): 49–61 [译自: 兰州大学学报(自然科学版)]     

Expression,Purification, and Characterization of the Recombinant Putative Periplasmic Hemin-Binding Protein (HutB) of Photobacterium damselae subsp. piscicida

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K _d) of 10 μM.     

The outcome of poliovirus infections in K562 cells is cytolytic rather than persistent after hemin-induced differentiation.

K562-Mu erythroleukemia cells readily establish a long-term persistent poliovirus infection characterized by continuous virus production in the absence of complete p220 cleavage and host translation shutoff (R. E. Lloyd and M. Bovee, Virology 194:200-209, 1993). The mechanism of resistance appears to be modulated at the intracellular level and to be related to decreased virus-mediated cytopathic effects (P. A. Benton, J. W. Murphy, and R. E. Lloyd Virology 213:7-18, 1995). It is well documented that hemin induces the differentiation of K562 cells and alters the expression of several host proteins. We report here that growth of K562 cells in hemin prior to poliovirus infection results in a dose-dependent increase in virus-induced cell lysis and thereby alters the normally persistent outcome of infection to a more lytic phenotype. K562 cells infected after hemin treatment displayed increased host translation shutoff, p220 cleavage, viral protein synthesis, and viral RNA accumulation compared with nontreated cells. Since hemin treatment of K562 cells also induced the increased expression of several heat shock proteins (Hsp70, Hsc70, Hsp90, and cohort p60), we tested the hypothesis that their increased expression may play a role in altering poliovirus infection in hemin-treated K562 cells. However, neither heat stress nor oxidative stress, inducers of heat shock protein synthesis, altered the outcome (of virus infections. In addition, we report the novel finding that subunits of two translation initiation factors, p220 (eIF-4G) and eIF-2alpha, are cleaved as a result of hemin treatment of K562 cells. It is proposed that hemin alters the expression of specific host proteins in K562 cells, probably other than heat shock proteins, which changes the initial response to poliovirus infections from persistent to lytic.     

Induction to erythroid differentiation of K562 cells by 1-beta-D-arabinofuranosylcytosine is inhibited by iron chelators: reversion by treatment with hemin

Erythroid differentiation of human leukemic K 562 cells is inhibited by the iron chelator desferrioxamine (DF). In addition, desferrioxamine induces an increase of uptake of hemin. When hemin is added to the culture medium, the DF-mediated inhibitory effects on erythroid induction are reversed. Briefly, hemin allows hemoglobin synthesis by K 562 cells induced to erythroid differentiation by 1-beta-D-arabinofuranosylcytosine (ara-C) and treated with 12.5 micrograms/ml DF. In addition, it was found that hemin treatment leads to a reversion of inhibition of K 562 cell proliferation mediated by 50-75 micrograms/ml DF. This effect of hemin was also detected in other cultured human tumor cell lines (B-lymphoid, erythroleukemic and from breast carcinomas, melanomas and kidney carcinomas).     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Brefeldin a down-regulates the transferrin receptor in K562 cells

The fungal metabolite brefeldin A (BFA) induces profound alterations in the morphology of intracellular organelles. Although BFA promotes the formation of extensive tubular endosomal domains, our understanding of the effects of the antibiotic on vesicle traffic events associated with endocytosis is limited. Thus, alterations in the transferrin (Tf) receptor's endocytic/recycling pathway upon treatment of human erythroleukemia K562 cells with BFA were studied as a pharmacological response. Treatment of K562 cells with BFA caused a down-regulation in the number of cell surface Tf receptors. This effect is highly reminiscent of the well-known action of phorbol 12-myristate 13-acetate (PMA) on Tf receptor traffic in K562 cells. However, our results demonstrate that these two agents down-regulate the Tf receptor via different mechanisms. The effects of BFA and PMA were additive when K562 cells were incubated with both together. Using the In/Sur method, the endocytic rate constant for Tf internalization was determined and PMA was found to greatly enhance k_e, from 0.28 min^–1 to 0.43 min^–1, while BFA had little effect (K_e=0.20 min^–1). In contrast, BFA-treatment alters the exocytic rate constant for return of internalized receptors to the cell surface, with the largest effect exerted on a slow-release, monensin-sensitive, compartment. The sum of the endocytic and exocytic kinetic data support a model in which BFA and PMA down-regulate the Tf receptor in K562 cells by mechanistically distinct actions, with BFA targeting exocytic monensin-sensitive intracellular compartments and PMA acting to exert a profound influence on elements of receptor internalization.Abbreviations BFA brefeldin A - ARF ADP-ribosylation factor - HRP horseradish peroxidase - Tf transferrin - PMA phorbol 12-myristate 13-acetate - DMSO dimethyl sulfoxide - PBS phosphate-buffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin - FITC-Tf fluorescein isothiocyanate-labelled transferrin     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

Phosphatase antagonist okadaic acid inhibits steady-state K+ currents in guard cells of Vicia faba

The effects of protein phosphatase inhibitors on steady-state K⁺ currents in the plasma membrane of Vicia faba guard cells were studied. Cells were impaled with double barrelled electrodes to monitor membrane voltage and K⁺ currents under voltage clamp. Okadaic acid (OA) (1 μM), a specific inhibitor of phosphatase 1 and 2A activity, blocks inward (l_K⁺_(in)) and outward (l_K⁺_(out)) rectifying K⁺ channels. Both currents decreased in parallel with a sigmoidal time course with 50% inhibition at about 8 min. With 0.2 μM OA inhibition became slower and more variable (4–34 min). Inhibition did not recover by washing cells ≤ 20 min in OA-free solution. In five out of seven cells OA also induced a rise in the background conductance, which lagged behind the inhibition of K⁺ current. Both decaying l_K*_(out) and rising leak conductance caused a depolarization. OA-induced inhibition of l_K⁺_(in) and l_K⁺_(out) was without a significant effect on the kinetics of voltage-dependent current activation and deactivation. In an alternative approach, guard cells were loaded from the voltage recording pipette with the non-specific phosphatase inhibitor naphthylphosphate. After an impalement of some minutes l_K⁺_(in) and l_K⁺_(out) were small or undetectable. In conclusion inward and outward K⁺ channels in guard cells have a common voltage-independent mode of control which is sensitive to phosphatase inhibitors. The known specificity of OA points to a mode of action in which a net increase of protein phosphorylation through inhibition of phosphatase 1 and/or 2A activity blocks conductance of both, l_K⁺_(in) and l_(out)     

Functional Expression and Purification of Histidine-Tagged Rat Renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) Cotransporters

Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P _i -cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/P _i -cotransport exhibited a K _m of P _i of 0.21 mm and an apparent K _m of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K _m of SO²⁻ ₄ of about 0.3–0.4 mm and a K _m of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS _i -1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography. Received: 24 March 1997/Revised: 8 July 1997     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

Voltage-Gated K+ Channel,Kv3.3 Is Involved in Hemin-Induced K562 Differentiation

Voltage-gated K⁺ (K_v) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between K_v channels and cell differentiation remains poorly investigated. This study demonstrates that K_v3.3 is involved in K562 cell erythroid differentiation. Down-regulation of K_v3.3 using siRNA-K_v3.3 increased hemin-induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element-binding protein, and c-fos. Down-regulation of K_v3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The K_v channels, or at least K_v3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of K_v channel regulation of cell differentiation would provide important information regarding vital cellular processes.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Taurine Stimulation of Isolated Hamster Brain Na+, K+-ATPase: Activation Kinetics and Chemical Specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na⁺, K⁺-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic “free” calcium activity, and neuronal excitability. Taurine may act in part by modulating Na⁺, K⁺-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na⁺, K⁺-ATPase by taurine. Normal whole brain homogenate Na⁺, K⁺-ATPase activity is 5.1 ± 0.4 (4) μmol P_i± h^?1± mg^?1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na⁺, K⁺-ATPase activity of 204.6 ± 5.8 (4) mol P_i± h^?1± mg^?1 Lowry protein. Taurine activates the Na⁺, K⁺-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K_1/2= 39 mM taurine, activation maximum =+87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid > hypotaurine > no activation =β-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na⁺, K⁺-ATPase. Its action is mediated by a membrane-bound protein.     

dr and spr/sr mutations of Chlamydomonas reinhardtii affecting D1 protein function and synthesis define two independent steps leading to chronic photoinhibition and confer differential fitness

The effects of introduced chloroplast gene mutations affecting D1 synthesis, turnover and function on photosynthesis, growth and competitive ability were examined in autotrophic cultures of Chlamydomonas reinhardtii (Chlorophyta) adapted to low or high irradiance. Few discernible effects were evident when the mutants were grown in low light (LL, 70 μmol m^?2 s^?1). The herbicide-resistant psbA mutation Ser²⁶⁴→ Ala (dr) slowed electron transfer and accelerated D1 degradation in cells grown under high light (HL, 600 μmol m^?2 s^?1). The maximum rate of light-and CO₂-saturated photosynthesis, cell growth rate and competitive ability in the dr mutant were reduced compared to wild type under HL. However, the wild-type rate of D1 synthesis in dr was adequate to compensate for accelerated D1 degradation. 16S rRNA mutations conferring resistance to streptomycin and spectinomycin (spr/sr) that altered chloroplast ribosome structure and assembly were used to inhibit chloroplast protein synthesis. In spr/sr cells grown under HL, D1 synthesis was reduced by 40–60% compared to wild type and D1 degradation was accelerated, leading to a 4-fold reduction in D1 pool size. The reduced D1 levels were accompanied by an elevation of F_o and a decline in F_v/F_m, quantum yield and maximum rate of CO₂-saturated photosynthesis. Chemostat experiments showed that the growth rate and competitive ability of spr/sr were reduced against both wild type and dr.