Synergistic effect of hydrogen peroxide on polyploidization during the megakaryocytic differentiation of K562 leukemia cells by PMA

The human myelogenous cell line, K562 has been extensively used as a model for the study of megakaryocytic (MK) differentiation, which could be achieved by exposure to phorbol 12-myristate 13-acetate (PMA). In this study, real-time PCR analysis revealed that the expression of catalase (cat) was significantly repressed during MK differentiation of K562 cells induced by PMA. In addition, PMA increased the intracellular reactive oxygen species (ROS) concentration, suggesting that ROS was a key factor for PMA-induced differentiation. PMA-differentiated K562 cells were exposed to hydrogen peroxide (H₂O₂) to clarify the function of ROS during MK differentiation. Interestingly, the percentage of high-ploidy (DNA content >4N) cells with H₂O₂ was 34.8±2.3% at day 9, and was 70% larger than that without H₂O₂ (21.5±0.8%). Further, H₂O₂ addition during the first 3 days of PMA-induced MK differentiation had the greatest effect on polyploidization. In an effort to elucidate the mechanisms of enhanced polyploidization by H₂O₂, the BrdU assay clearly indicated that H₂O₂ suppressed the division of 4N cells into 2N cells, followed by the increased polyploidization of K562 cells. These findings suggest that the enhancement in polyploidization mediated by H₂O₂ is due to synergistic inhibition of cytokinesis with PMA. Although H₂O₂ did not increase ploidy during the MK differentiation of primary cells, we clearly observed that cat expression was repressed in both immature and mature primary MK cells, and that treatment with the antioxidant N-acetylcysteine effectively blocked and/or delayed the polyploidization of immature MK cells. Together, these findings suggest that MK cells are more sensitive to ROS levels during earlier stages of maturation.     

Autocrine signaling of platelet-derived growth factor regulates disabled-2 expression during megakaryocytic differentiation of K562 cells

Platelet-derived growth factor (PDGF) is involved in megakaryocytopoiesis and is secreted into the culture medium during megakaryocytic differentiation of human leukemic cells. We investigate whether PDGF plays a role in the regulation of the adapter protein Disabled-2 (DAB2) that expresses abundantly in platelets and megakaryocytes. Western blot analysis revealed that conditioned medium from 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated, megakaryocytic differentiating K562 cells upregulated DAB2 expression. DAB2 induction and megakaryocytic differentiation was abrogated when cells were co-treated with the PDGF receptor inhibitor STI571 or when the conditioned medium was derived from TPA-plus STI571-treated cells. Although the level of PDGF mRNA was not altered by STI571, an approximate 44% decrease in PDGF in the conditioned medium was observed. Consistent with these findings, interfering PDGF signaling by PDGF neutralization antibody or dominant negative PDGF receptors attenuated DAB2 expression. Accordingly, transfection of an expression plasmid encoding secreted PDGF upregulated DAB2. This study shows for the first time that PDGF autocrine signaling regulates DAB2 expression during megakaryocytic differentiation.     

Erythroid differentiation sensitizes K562 leukemia cells to TRAIL-induced apoptosis by downregulation of c-FLIP

Regulation of the apoptotic threshold is of great importance in the homeostasis of both differentiating and fully developed organ systems. Triggering differentiation has been employed as a strategy to inhibit cell proliferation and accelerate apoptosis in malignant cells, in which the apoptotic threshold is often characteristically elevated. To better understand the mechanisms underlying differentiation-mediated regulation of apoptosis, we have studied death receptor responses during erythroid differentiation of K562 erythroleukemia cells, which normally are highly resistant to tumor necrosis factor (TNF) alpha-, FasL-, and TRAIL-induced apoptosis. However, upon hemin-mediated erythroid differentiation, K562 cells specifically lost their resistance to TNF-related apoptosis-inducing ligand (TRAIL), which efficiently killed the differentiating cells independently of mitochondrial apoptotic signaling. Concomitantly with the increased sensitivity, the expression of both c-FLIP splicing variants, c-FLIP(L) and c-FLIP(S), was downregulated, resulting in an altered caspase 8 recruitment and cleavage in the death-inducing signaling complex (DISC). Stable overexpression of both c-FLIP(L) and c-FLIP(S) rescued the cells from TRAIL-mediated apoptosis with isoform-specific effects on DISC-recruited caspase 8. Our results show that c-FLIP(L) and c-FLIP(S) potently control TRAIL responses, both by distinct regulatory features, and further imply that the differentiation state of malignant cells determines their sensitivity to death receptor signals.     

A comparison of the neurotrophic activities of the flavonoid fisetin and some of its derivatives

Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classical neurotrophic factors are proteins and thus not well-suited for therapeutic purposes. Recently, we showed that specific flavonoids such as fisetin (3, 7, 3', 4' tetrahydroxyflavone) promote the differentiation of nerve cells in culture through the activation of extracellular signal-regulated kinase (ERK) suggesting that flavonoids could substitute for neurotrophic factors. It has also been shown that fisetin promotes nerve cell survival following exposure to toxic oxidative insults. To determine whether or not this is unique to fisetin, a series of related compounds were assayed for neurotrophic activities. Many of these related compounds also promote nerve cell differentiation and are neuroprotective against toxic oxidative insults. However, the mechanisms underlying these neurotrophic effects differ among the compounds.     

A comparison of the neurotrophic activities of the flavonoid fisetin and some of its derivatives

Neurotrophic factors promote the development, maintenance and regeneration of nerve cells. Classical neurotrophic factors are proteins and thus not well-suited for therapeutic purposes. Recently, we showed that specific flavonoids such as fisetin (3, 7, 3′, 4′ tetrahydroxyflavone) promote the differentiation of nerve cells in culture through the activation of extracellular signal-regulated kinase (ERK) suggesting that flavonoids could substitute for neurotrophic factors. It has also been shown that fisetin promotes nerve cell survival following exposure to toxic oxidative insults. To determine whether or not this is unique to fisetin, a series of related compounds were assayed for neurotrophic activities. Many of these related compounds also promote nerve cell differentiation and are neuroprotective against toxic oxidative insults. However, the mechanisms underlying these neurotrophic effects differ among the compounds.     

Mechanisms of vitamin C stabilization by K562 erythroleukemic cells

Summary K562 cells display several possibilities to keep ascorbic acid in the surrounding medium in the reduced state and prevent its loss by degradation of the oxidized form, dehydroascorbic acid: (1) A semidehydroascorbic acid reductase with high affinity for the ascorbate radical scavenges this before it disproportionates into the two parent forms of vitamin C (ascorbate and dehydroascorbic acid). (2) Dehydroascorbic acid in the extracellular medium is slowly converted to ascorbate by a different mechanism with low affinity which may or may not involve uptake of the oxidized and release of the reduced form. (3) Ascorbate remains relatively stable in the cell culture medium in presence, but also in absence of the cells after their removal, This is most probably due to the presence of released peptides in the cell-conditioned medium which can chelate transition metal ions and thus prevent catalytic autoxidation of ascorbate.     

Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells

Embryonic stem (ES) cells represent an ideal source for cell engraftment in the damaged central nervous system (CNS). Understanding key signals that control ES cell differentiation may improve cell type-specific differentiation that is suitable for transplantation therapy. We tested the hypothesis that extracellular signal-regulated kinase (ERK) 1/2 phosphorylation is an early signaling event required for the neuronal differentiation of ES cells. Cultured mouse ES cells were treated with an all-trans-retinoic-acid (RA) protocol to generate neurally induced progenitor cells. Western blot analysis showed a dramatic increase in ERK 1/2 phosphorylation (p-ERK 1/2) 1-5 days after RA induction, which was attenuated in the presence of the p-ERK 1/2-specific inhibitor UO126. Phospho-ERK 1/2 inhibition significantly reduced the number of NeuN-positive cells and the expression of associated cytoskeletal proteins. In differentiating ES cells, there was increased nuclear translocation of STAT3 and decreased protein expression levels of GDNF, BDNF and NGF. STAT3 translocation was attenuated by UO126. Finally, caspase-3 activation was observed in the presence of UO126, suggesting that the ERK pathway also contributes to the survival of differentiating ES cells. These data indicate that ERK 1/2 phosphorylation is a key event required for early neuronal differentiation and survival of ES cells.     

一种用作阴性对照的siRNA可诱导人K562细胞向红系分化

我们发现,一种在RNA干扰实验中用作阴性对照的商业化siRNA具有明显的诱 导人慢性髓性白血病K562细胞系向红系方向分化的作用.它表现为K562细胞瞬时转 染该siRNA后,红系分化的特异表面标志CD235及ε 、γ 和β 珠蛋白的表达升高,GATA-2的表达降低,细胞增殖速度减慢,软琼脂克隆形成率降低,并且此 过程不伴随细胞凋亡. 而生物信息学分析显示,该siRNA序列与目前所有已知人类 基因均无明显同源性.研究结果提示,该siRNA不适于用作红系分化实验中的阴性 对照. siRNA的作用远比人们目前所知的要复杂得多,siRNA的脱靶效应应当引起 研究者的足够重视,在RNA干扰实验中阴性对照siRNA的选择会极大地影响对实验 结果的判读.     

Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB

Signaling pathways involved in oxidative stress-induced inhibition of osteoblast differentiation are not known. We showed in this report that H(2)O(2) (0.1-0.2mM)-induced oxidative stress suppressed the osteoblastic differentiation process of primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblasts, manifested by a reduction of differentiation markers including alkaline phosphatase (ALP), type I collagen, colony-forming unit-osteoprogenitor (CFU-O) formation, and nuclear phosphorylation of Runx2. H(2)O(2) treatment stimulated phospholipase C-gamma1 (PLC-gamma1), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-kappaB signaling but inhibited p38 mitogen-activated protein kinase (MAPK) activation. In the presence of 20microM PD98059 or 50microM caffeic acid phenethyl ester (CAPE), specific inhibitor for ERKs or NF-kappaB, respectively, could significantly reverse the decrease of above-mentioned osteoblastic differentiation markers elicited by H(2)O(2) (0.1mM). Furthermore, PD98059 also suppressed H(2)O(2)-stimulated NF-kappaB signaling in this process. These data suggest that ERK and ERK-dependent NF-kappaB activation is required for oxidative stress-induced inhibition of osteoblastic differentiation in rabbit BMSC and calvarial osteoblasts.     

Induction of PC12 cell differentiation by flavonoids is dependent upon extracellular signal-regulated kinase activation

Many of the physiological benefits attributed to flavonoids are thought to stem from their potent antioxidant and free radical scavenging properties. Recently, it was shown that flavonoids protect nerve cells from oxidative stress by multiple mechanisms, only one of which is directly related to their antioxidant activity, suggesting that specific flavonoids may have other properties that could make them useful in the treatment of conditions that lead to nerve cell death. In particular, it was asked if any flavonoid could mimic neurotrophic proteins. To examine this possibility, we looked at the ability of flavonoids to induce nerve cell differentiation using PC12 cells. PC12 cells were treated with a variety of flavonoids to determine if there was a correlation between their neuroprotective activity and their neurite outgrowth-promoting activity. In addition, the signaling pathways required for flavonoid-induced differentiation were examined. We found that only a small subset of the flavonoids that were neuroprotective could induce neurite outgrowth by an extracellular signal-regulated kinase-dependent process. There was a strong correlation between the concentrations of the flavonoids that were neuroprotective and the concentrations that induced differentiation. These results suggest that the consumption of specific flavonoids could have further beneficial effects on nerve cells following injury, in pathological conditions or in normal aging.     

Substantial changes of cellular iron homeostasis during megakaryocytic differentiation of K562 cells

We investigated the remodeling of iron metabolism during megakaryocytic development of K562 cells. Differentiation was successfully verified by increase of the megakaryocytic marker CD61 and concomitant decrease of the erythroid marker γ-globin. The reduction of erythroid properties was accompanied by changes in the cellular iron content and in the expression of proteins regulating cellular iron homeostasis. Independent of available inorganic or transferrin-bound extracellular iron, total intracellular iron increases while the iron-to-protein ratio decreases. The iron exporter ferroportin is downregulated within 1-6 h, followed by downregulation of transferrin receptor-1 (TfR1) and ferritin heavy chain (H-ferritin) mainly after 24-48 h. The hemochromatosis protein-1, a ligand of TfR1, peaked after 24 h. All effects were independent of iron supply with the exception of H-ferritin, which was restored by excess iron. While alterations of CD61, TfR1 and ferritin expression were revoked by a protein kinase C inhibitor, downregulation of ferroportin remained unaffected.     

Essential role for ALCAM gene silencing in megakaryocytic differentiation of K562 cells

Background  

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is expressed by hematopoietic stem cells. However, its role in hematopoietic differentiation has not previously been defined.     

Erythroid differentiation is augmented in Reelin-deficient K562 cells and homozygous reeler mice

Reelin is an extracellular glycoprotein that is highly conserved in mammals. In addition to its expression in the nervous system, Reelin is present in erythroid cells but its function there is unknown. We report in this study that Reelin is up-regulated during erythroid differentiation of human erythroleukemic K562 cells and is expressed in the erythroid progenitors of murine bone marrow. Reelin deficiency promotes erythroid differentiation of K562 cells and augments erythroid production in murine bone marrow. In accordance with these findings, Reelin deficiency attenuates AKT phosphorylation of the Ter119⁺CD71⁺ erythroid progenitors and alters the cell number and frequency of the progenitors at different erythroid differentiation stages. A regulatory role of Reelin in erythroid differentiation is thus defined.