HERG1 K+通道在肝癌细胞系BEL-7402中的表达及其意义

[目的]观察HERG1 K 通道在肝癌细胞系BEL-7402的表达,探索其在肝癌发生与发展中的作用。[方法]RT-PCR方法检测hepg1在肝癌细胞系BEL-7402细胞和正常肝细胞系L-02细胞中表达;四甲基偶氮唑盐比色法(MTT法)研究HERG1 K 通道对BEL-7402细胞和L-02细胞增殖作用;以流式细胞仪检测该通道对肝癌细胞系BEL-7402细胞周期的影响。[结果]HERG1 K 通道在肝癌细胞中表达,而在正常肝细胞中不表达,该通道特异性抑制剂E-4031可以显著抑制BEL-7402细胞的增殖,而对不表达herg1基因的L-02细胞增殖不起作用;E-4031抑制BEL-7402细胞HERG1 K 通道后可导致G1期细胞显著上升。[结论]HERG1 K 通道可能是一个潜在的癌基因,可促进肝癌细胞的增殖;参与调控BEL-7402细胞周期的G1期进程。通过抑制该通道的功能,或下调其表达抑制该肿瘤的发生与发展;HERG1 K 通道可能是肝癌治疗的一个潜在的药物靶点和诊断性生物标志。     

Role of K(+) channels in frequency regulation of spontaneous action potentials in rat pituitary GH(3) cells

The frequency of spontaneous action potentials (SAP) is important in the regulation of hormone secretion. The decrease in K(+) conductance is known as a primary mechanism for increasing SAP frequency. To investigate the nature of K(+) channels that contribute to the frequency regulation of the SAP in rat clonal pituitary GH(3) cells, the effect of various K(+) channel blockers on the SAP and membrane currents were recorded using the patch-clamp technique. A classical inward rectifying K(+) channel blocker, Cs(+) (5 mM), caused an increase in firing frequency and depolarization in after-hyperpolarization (AHP) voltage. An ETHER-A-GO-GO(ERG) type K(+) channel blocker, E-4031 (5 microM), caused no significant effect on the SAP. Tetraethylammonium (TEA, 10 mM) decreased firing frequency and increased the duration of SAP. These effects were not changed by the presence of high concentration of Ca(2+) buffer (10 mM EGTA or BAPTA) in pipette solutions. In voltage-clamp experiments, Cs(+) and E-4031 did not affect outwardly rectifying K(+) currents, but significantly inhibited inwardly rectifying K(+) currents recorded in isotonic K(+) solution. However, the kinetics of Cs(+)-sensitive current and E-4031-sensitive current were distinctive: the time to peak was more immediate and the decay rate was slower in Cs(+)-sensitive current than in E-4031-sensitive current. These results imply that Cs(+) and E-4031 inhibit the distinct components of inwardly rectifying K(+) currents, and that the contribution of the Cs(+)-sensitive current can be immediate on repolarization and can last more effectively over pacemaking potential range than E-4031-sensitive current.     

VEGFR-1 (FLT-1), beta1 integrin, and hERG K+ channel for a macromolecular signaling complex in acute myeloid leukemia: role in cell migration and clinical outcome

Leukemia cell motility and transendothelial migration into extramedullary sites are regulated by angiogenic factors and are considered unfavorable prognostic factors in acute leukemias. We have studied cross talk among (1) the vascular endothelial growth factor receptor-1, FLT-1; (2) the human eag-related gene 1 (hERG1) K(+) channels; and (3) integrin receptors in acute myeloid leukemia (AML) cells. FLT-1, hERG1, and the beta(1) integrin were found to form a macromolecular signaling complex. The latter mostly recruited the hERG1B isoform of hERG1 channels, and its assembly was necessary for FLT-1 signaling activation and AML cell migration. Both effects were inhibited when hERG1 channels were specifically blocked. A FLT-1/hERG1/beta(1) complex was also observed in primary AML blasts, obtained from a population of human patients. The co-expression of FLT-1 and hERG1 conferred a pro-migratory phenotype to AML blasts. Such a phenotype was also observed in vivo. The hERG1-positive blasts were more efficient in invading the peripheral circulation and the extramedullary sites after engraftment into immunodeficient mice. Moreover, hERG1 expression in leukemia patients correlated with a higher probability of relapse and shorter survival periods. We conclude that in AML, hERG1 channels mediate the FLT-1-dependent cell migration and invasion, and hence confer a greater malignancy.     

Comparative pharmacology of guinea pig cardiac myocyte and cloned hERG (I(Kr)) channel

INTRODUCTION: This study used whole-cell, patch clamp techniques on isolated guinea pig ventricular myocytes and HEK293 cells expressing cloned human ether-a-go-go-related gene (hERG) to examine the action of drugs causing QT interval prolongation and torsades de pointes (TdP) in man. Similarities and important differences in drug actions on cardiac myocytes and cloned hERG I(Kr) channels were established. Qualitative actions of the drugs on cardiac myocytes corresponded with results obtained from Purkinje fibers and measurement of QT interval prolongation in animal and human telemetry studies. METHODS AND RESULTS: Adult guinea pig ventricular myocytes were isolated by enzymatic digestion. Cells were continuously perfused with Tyrode's solution at 33-35 degrees C. Recordings were made using the whole-cell, patch clamp technique. Action potentials (APs) were elicited under current clamp. Voltage clamp was used to study the effect of drugs on I(Kr) (rapidly activating delayed rectifier potassium current), I(Na) (sodium current), and I(Ca) (L-type calcium current). Dofetilide increased the myocyte action potential duration (APD) in a concentration-dependent manner, with a pIC50 of 7.3. Dofetilide 1 microM elicited early afterdepolarizations (EADs) but had little affect on I(Ca) or I(Na). E-4031 increased APD in a concentration-dependent manner, with a pIC50 of 7.2. In contrast, 10 microM loratadine, desloratadine, and cetirizine had little effect on APD or I(Kr). Interestingly, cisapride displayed a biphasic effect on myocyte APD and inhibited I(Ca) at 1 microM. Even at this high concentration, cisapride did not elicit EADs. A number of AstraZeneca compounds were tested on cardiac myocytes, revealing a mixture of drug actions that were not observed in hERG currents in HEK293 cells. One compound, particularly AR-C0X, was a potent blocker of myocyte AP (pIC50 of 8.4). AR-C0X also elicited EADs in cardiac myocytes. The potencies of the same set of drugs on the cloned hERG channel also were assessed. The pIC50 values for dofetilide, E-4031, terfenadine, loratadine, desloratadine, and cetirizine were 6.8, 7.1, 7.3, 5.1, 5.2, and <4, respectively. Elevation of temperature from 22 to 35 degrees C significantly enhanced the current kinetics and amplitudes of hERG currents and resulted in approximately fivefold increase in E-4031 potency. CONCLUSION: Our study demonstrates the advantages of cardiac myocytes over heterologously expressed hERG channels in predicting QT interval prolongation and TdP in man. The potencies of some drugs in cardiac myocytes were similar to hERG, but only myocytes were able to detect important changes in APD characteristics and display EADs predictive of arrhythmia development. We observed similar qualitative drug profiles in cardiac myocytes, dog Purkinje fibers, and animal and human telemetry studies. Therefore, isolated native cardiac myocytes are a better predictor of drug-induced QT prolongation and TdP than heterologously expressed hERG channels. Isolated cardiac myocytes, when used with high-throughput patch clamp instruments, may have an important role in screening potential cardiotoxic compounds in the early phase of drug discovery. This would significantly reduce the attrition rate of drugs entering preclinical and/or clinical development. The current kinetics and amplitudes of the cloned hERG channel were profoundly affected by temperature, significantly altering the potency of one drug (E-4031). This finding cautions against routine drug testing at room temperature compared to physiologic temperature when using the cloned hERG channel.     

HERG K^＋通道对VEGF诱导的肝癌细胞迁移和侵袭的影响

目的研究HERG K＋通道对VEGF诱导的肝癌细胞在侵袭和迁移方面的调节作用。方法运用膜片钳技术分别检测正常肝细胞系L-02和肝癌细胞系SMMC-7721中HERG K＋通道的表达情况;采用Bodyen-Chamber系统检测在HERG K＋通道特异性抑制剂E-4031作用后,对VEGF诱导的SMMC-7721细胞侵袭力和迁移潜能方面的影响;ELISA法检测E-4031处理SMMC-7721细胞后,培养基上清中的VEGF水平的变化。结果 HERG K＋通道在SMMC-7721细胞中表达,而在L-02细胞中不表达;且VEGP诱导的SMMC-7721细胞侵袭和迁移现象可被E-4031呈剂量依赖性地抑制;在阻断HERG K＋通道后上清中VEGF水平明显降低。结论肝癌细胞中存在HERG K＋通道,它可通过调控VEGF分泌水平来影响肝癌细胞的侵袭和迁移能力。由此,HERG K＋通道将有可能成为诊断肝癌和判断预后的新标志物及治疗的新靶位。     

Chemotherapy resistance in acute lymphoblastic leukemia requires hERG1 channels and is overcome by hERG1 blockers

Bone marrow mesenchymal cells (MSCs) can protect leukemic cells from chemotherapy, thus increasing their survival rate. We studied the potential molecular mechanisms underlying this effect in acute lymphoblastic leukemia (ALL) cells. Coculture of ALL cells with MSCs induced on the lymphoblast plasma membrane the expression of a signaling complex formed by hERG1 (human ether-à-go-go-related gene 1) channels, the β(1)-integrin subunit, and the chemokine receptor CXC chemokine receptor-4. The assembly of such a protein complex activated both the extracellular signal-related kinase 1/2 (ERK1/2) and the phosphoinositide 3-kinase (PI3K)/Akt prosurvival signaling pathways. At the same time, ALL cells became markedly resistant to chemotherapy-induced apoptosis. hERG1 channel function appeared to be important for both the initiation of prosurvival signals and the development of drug resistance, because specific channel blockers decreased the protective effect of MSCs. NOD/SCID mice engrafted with ALL cells and treated with channel blockers showed reduced leukemic infiltration and had higher survival rates. Moreover, hERG1 blockade enhanced the therapeutic effect produced by corticosteroids. Our findings provide a rationale for clinical testing of hERG1 blockers in the context of antileukemic therapy for patients with ALL.     

SDF-1alpha/CXCR4 decreases endothelial progenitor cells apoptosis under serum deprivation by PI3K/Akt/eNOS pathway

Recent studies have demonstrated that stromal cell-derived factor-1alpha (SDF-1alpha)/CXCR4 interaction regulates multiple cell signal pathways and a variety of cellular functions such as cell migration, proliferation, survival and angiogenesis. In present study, we aimed to determine the effect of SDF-1alpha on endothelial progenitor cells (EPCs) apoptosis induced by serum deprivation and the implication of phosphoinositide 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signaling in this effect. EPCs were isolated and characterized. SDF-1alpha decreased EPCs apoptosis induced by serum deprivation in a dose-dependent manner and the inhibitory effect was CXCR4 dependent as confirmed by the total abolishment by AMD3100, a CXCR4-specific peptide antagonist. SDF-1alpha treatment also significant decreased caspase-3 expression and activity. The inhibitory effect of SDF-1alpha on EPCs apoptosis was nearly completely abolished by PI3K inhibitors (either Wortmannin or LY294002) and partially abolished by NOS inhibitor, N(G)-nitro-arginine methyl ester, whereas inhibitors of MAPKs had no significant effect on this inhibitory effect. The treatment of EPCs with SDF-1alpha resulted in time-dependent Akt, eNOS, extracellular-regulated kinase (ERK1/2), p38 MAPK and c-Jun N-terminal kinase (JNK) phosphorylations. These findings suggest that PI3K/Akt/eNOS activation, but not MAPKs activation, is required for the inhibitory effect of SDF-1alpha on EPCs apoptosis.     

Ph+/VE-cadherin+ identifies a stem cell like population of acute lymphoblastic leukemia sustained by bone marrow niche cells

Although leukemic stem cells (LSCs) show a symbiotic relationship with bone marrow microenvironmental niches, the mechanism by which the marrow microenvironment contributes to self-renewal and proliferation of LSCs remains elusive. In the present study, we identified a unique subpopulation of Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL) cells coexpressing markers of endothelial cells (including VE-cadherin, PECAM-1, and Flk-1) and committed B-lineage progenitors. After long-term coculture with bone marrow stromal cells, tumor cells formed hematopoietic colonies and cords, expressed early stem- cell markers, and showed endothelial sprouting. Gene expression profiles of LSCs were altered in the presence of stromal cell contact. Stromal cell contact promoted leukemic cell VE-cadherin expression, stabilized beta-catenin, and up-regulated Bcr-abl fusion gene expression. Our study indicates that these specific tumor cells are uniquely positioned to respond to microenvironment-derived self-renewing and proliferative cues. Ph(+)/VE-cadherin(+) tumor subpopulation circumvents the requirement of exogenous Wnt signaling for self-renewal through stromal cell support of leukemic cell VE-cadherin expression and up-regulated Bcr-abl tyrosine kinase activity. These data suggest that strategies targeting signals in the marrow microenvironment that amplify the Bcr-abl/VE-cadherin/beta-catenin axis may have utility in sensitizing drug-resistant leukemic stem cells.     

Modeling SDF-1-induced mobilization in leukemia cell lines

The stromal cell-derived factor 1 (SDF-1) is essential for circulation, homing, and retention of hematopoietic stem cells in the bone marrow. Present evidence indicates that this factor might play an important role in leukemia cells as well. The aim of this study is to present a model of SDF-1-induced mobilization using leukemia cell lines. CXCR4 expression was compared in Kasumi-1, Jurkat, HL-60, KG-1a, and K562 cells by flow cytometry and Western blot. Migration was analyzed with Transwell assays, and adhesive cell-cell interaction was quantified with a standardized adhesion assay and flow cytometry. CXCR4 was expressed by all leukemic cell lines analyzed, although surface expression of this receptor was found in Kasumi-1 and Jurkat cells only. Correspondingly, SDF-1α effects on migration and cell-cell adhesion were observed in Kasumi-1 and Jurkat cells only, and this could be blocked by AMD3100 in a reversible manner. We have provided evidence that SDF-1α acts as a chemotactic and chemokinetic agent. In addition, surface expression of integrin-β2, activated leukocyte cell adhesion molecule and N-cadherin decreased after stimulation with SDF-1α. SDF-1α affects cell-cell adhesion and migration only in leukemia cells on which the CXCR4 receptor is present on the surface. An SDF-1 gradient is not necessarily required to induce migration, as chemokinesis can also occur. Upon stimulation with SDF-1, CXCR4 promotes modifications on the surface pattern of adhesion molecules, which have an influence on adhesion and migration.     

Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G(0)/G(1) transition in CD34(+) cells: evidence for an autocrine/paracrine mechanism

The stromal cell-derived factor 1 (SDF-1) chemokine has various effects on hematopoietic cell functions. Its role in migration and homing of hematopoietic progenitors is currently well established. Previously it was shown that SDF-1 stimulates myeloid progenitor proliferation in synergy with cytokines. Results of this study indicate that SDF-1 alone promotes survival of purified CD34(+) cells from human unmobilized peripheral blood (PB) by counteracting apoptosis as demonstrated by its capacity to reduce DNA fragmentation, annexin-V(+) cell number, and APO2.7 detection and to modulate bcl-2 homolog protein expression. The study demonstrates that SDF-1, produced by sorted CD34(+)CD38(+) cells and over-released in response to cell damage, exerts an antiapoptotic effect on CD34(+) cells through an autocrine/paracrine regulatory loop. SDF-1 participates in the autonomous survival of circulating CD34(+) cells and its effect required activation of the phosphotidyl inositol 3 kinase (PI3-K)/Akt axis. Cell sorting based on Hoechst/pyroninY fluorescences shows that SDF-1 production is restricted to cycling CD34(+) cells. SDF-1 triggers G(0) quiescent cells in G(1) phase and, in synergy with thrombopoietin or Steel factor, makes CD34(+) cells progress through S+G(2)/M phases of cell cycle. By assessing sorted CD34(+)CD38(-) and CD34(+)CD38(+) in semisolid culture, the study demonstrates that SDF-1 promotes survival of clonogenic progenitors. In conclusion, the results are the first to indicate a role for endogenous SDF-1 in primitive hematopoiesis regulation as a survival and cell cycle priming factor for circulating CD34(+) cells. The proposal is made that SDF-1 may contribute to hematopoiesis homeostasis by participating in the autonomous survival and cycling of progenitors under physiologic conditions and by protecting them from cell aggression in stress situations.     

Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival

The chemokine stromal cell-derived factor-1 (SDF-1), and its receptor, CXCR-4, have been implicated in the homing and mobilization of human CD34(+) cells. We show here that SDF-1 may also be involved in hematopoiesis, promoting the proliferation of human CD34(+) cells purified from normal adult peripheral blood (PB). CXCR-4 was expressed on PB CD34(+) cells. The amount of CXCR-4 on PB CD34(+) cells was 10 times higher when CD34(+) cells were purified following overnight incubation. CXCR-4 overexpression was correlated with a primitive PB CD34(+) cell subset defined by a CD34(high) CD38(low)CD71(low)c-Kit(low)Thy-1(+) antigenic profile. The functional significance of CXCR-4 expression was ascertained by assessing the promoting effect of SDF-1alpha on cell cycle, proliferation, and colony formation. SDF-1 alone increased the percentage of CD34(+) cells in the S+G(2)/M phases and sustained their survival. In synergy with cytokines, SDF-1 increased PB CD34(+) and CD34(high)CD38(low) cell expansion and colony formation. SDF-1 also stimulated the growth of colonies derived from primitive progenitors released from quiescence by anti-TGF-beta treatment. Thus, our results shed new light on the potential role of this chemokine in the stem cell engraftment process, which involves migration, adhesion, and proliferation. Furthermore, both adhesion-induced CXCR-4 overexpression and SDF-1 stimulating activity may be of clinical relevance for improving cell therapy settings in stem cell transplantation.     

Pantoprazole Induces Apoptosis of Leukemic Cells by Inhibiting Expression of P-Glycoprotein/Multidrug Resistance-Associated Protein-1 Through PI3K/AKT/mTOR Signaling

This study aims to investigate the effects and mechanism of pantoprazole on multidrug resistant leukemia K562/A02 and K562/ADM cell lines. K562/A02 and K562/ADM cells at logarithmic growth phase were pre-treated with different concentration of pantoprazole (0, 50, 100, 200 μg/mL) for 24 h. Flow cytometry was used to measure the cell growth cycle and apoptosis. RT-PCR and Western blot were used to measure the expression of p-PI3K, p-AKT, p-mTOR, P-glycoprotein (P-gp) and multidrug resistance-associated protein-1 (MRP1). Pantoprazole pretreatment significantly increased the ratio of G0/G1 phase but decreased the S phase of K562/A02 and K562/ADM cells in dose-dependent manner (p < 0.05). Flow cytometry analysis indicated that pretreatment of leukemic cells with pantoprazole induced apoptosis in a dose-dependent manner. RT-PCR and Western blot analysis indicated that pantoprazole pretreatment inhibited the mRNA and protein expression of p-PI3K, p-Akt, p-mTOR, P-gp and MRP1 in K562/A02 and K562/ADM cells in a dose-dependent manner (p < 0.05). Pantoprazole arrested cell cycle and induced apoptosis of multidrug resistant leukemic cells by inhibiting the expression of P-gp and MRP1 through PI3K/Akt/mTOR signaling pathway.     

Physiological properties of hERG 1a/1b heteromeric currents and a hERG 1b-specific mutation associated with Long-QT syndrome

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.     

CXCR4/SDF-1α信号对乳腺癌细胞的体外增殖和迁移的促进作用

目的探讨CXCR4/SDF-1α信号在乳腺癌细胞体外增殖和迁移中的作用。方法免疫组织化学染色法检测CXCR4在乳腺癌组织中的表达;采用免疫荧光标记法和RT-PCR方法检测CXCR4在乳腺癌细胞株上的表达;MTT法研究SDF-1α细胞因子对乳腺癌细胞株体外增殖的影响及抗体12G5的阻断作用;体外微孔隔离室迁移技术研究CXCR4/SDF-1α信号对乳腺癌细胞株体外迁移能力的影响和抗体12G5的阻断作用。结果 CXCR4表达于乳腺癌组织,也在乳腺癌细胞株表达。SDF-1α细胞因子可有效促进乳腺癌细胞株MDA-M231的体外增殖和迁移,而阻断型CXCR4单抗12G5可有效抑制其增殖和迁移,并呈浓度依赖性。结论 CXCR4/SDF-1α信号参与了乳腺癌细胞株的体外生长和转移,与乳腺癌的侵袭和转移有关。     

白血病细胞系WT1的表达及反义核苷酸对其的抑制作用

目的:探讨RT-PCR方法测定白血病细胞系WT1基因的表达以及WT1反义寡核苷酸对白血病细胞系增殖的抑制作用。方法:应用RT-PCR方法测定K562、HL-60、TF-1及U937 4株白血病细胞系WT1基因的表达,然后应用针对WT1翻译起始位点的反义寡核苷酸(WT1 ASO)处理K562细胞系、U937细胞系,采用锥虫蓝拒染法确定白血病细胞的增殖。结果:K562、HL-60及TF-1 3株白血病细胞系均高度表达WT1,而U937细胞系未测到WT1。WT1 ASO能够抑制WT1表达阳性的K562细胞的生长,对WT1表达阴性的U937则无抑制作用;而WT1有义寡核苷酸对K562细胞系、U937细胞系均无抑制作用。结论:RT-PCR测定方法可以检测WT1基因在白血病细胞系表达,WT1反义寡核苷酸对白血病细胞有特异的抑制作用。WT1基因在白血病细胞增殖过程中起一定作用。     

PI-103在体外抗急性髓细胞白血病效应的实验研究

目的研究急性髓细胞白血病（AML）细胞P13K-Akt—mTOR信号转导通路各基因的表达及PI-103在体外对AML细胞增殖、凋亡及细胞周期的影响。方法RT-PCR法检测AML细胞P13K、Akt、mTORmRNA的表达;四甲基偶氮唑蓝法检测PI-103对AML细胞的增殖作用;流式细胞仪检测PI-103对AML细胞的凋亡率和细胞周期的影响。结果①81．25％的AML患者表达P13K基因,87．5％的患者表达mTOR基因,50％的患者同时表达此两种基因。②PI-103能明显提高PI3K—Akt-mTOR信号通路持续活化的AML细胞的增殖抑制率和凋亡率、阻滞细胞于G0／G1期（P均〈0．05）,且与剂量显著相关（P均〈0．05）。结论大部分AML患者存在PI3K-Akt-mTOR信号转导通路的异常活化;PI-103可通过PI3K、mTOR信号通路抑制AML细胞增殖、促进其凋亡。     

Motility, proliferation, and egress to the circulation of human AML cells are elastase dependent in NOD/SCID chimeric mice

The role of the proteolytic enzyme elastase in motility and proliferation of leukemic human acute myeloblastic leukemia (AML) cells is currently unknown. We report a correlation between abnormally high levels of elastase in the blood of AML patients and the number of leukemic blast cells in the circulation. In AML cells, we observed expression of cell-surface elastase, which was regulated by the chemokine stromal cell-derived factor-1 (SDF-1). In vitro inhibition of elastase prevented SDF-1-induced cell polarization, podia formation, and reduced migration of human AML cells as well as their adhesion. Elastase inhibition also significantly impaired in vivo homing of most human AML cells to the bone marrow (BM) of nonobese diabetic-severe combined immunodeficient (NOD/SCID)/beta-2 microglobulin knock-out (B2m null) mice that underwent transplantation. Moreover, in vitro proliferation of AML cells was elastase dependent. In contrast, treatment with elastase inhibitor enhanced the proliferation rate of human cord blood CD34+ cells, including primitive CD34+/CD38- cells, and their in vivo homing. Finally, NOD/SCID mice previously engrafted with human AML cells and treated with elastase inhibitor had significantly reduced egress of leukemic cells into the circulation. Taken together, our data demonstrate that human AML cells constitutively secrete and express SDF-1-dependent cell-surface elastase, which regulates their migration and proliferation.     

Harmine,a Novel DNA Methyltransferase 1 Inhibitor in the Leukemia Cell Line

DNA methylation followed by tumor suppressor gene repression plays a critical role in the leukemia development. So, DNA methyl transferase inhibitors have great importance in treatment of theses malignancies. Harmine, A beta carboline alkaloid derivative of Peganum harmala, had shown anti- proliferative effects on leukemic cell line. This study aimed to evaluate the effect of Harmine on DNMT1 (DNA methyl transferase 1) expression in a leukemic cell line. Cell proliferation and cell cycle analysis were studied in NB4 cell line after treatment with Harmine for 72 h. DNMT1 expression in treated cells was analyzed by real time PCR. Tumor suppressor gene hypometylation and reactivation was evaluated via MSP analysis and also real time PCR. Harmine reduced cell proliferation in NB4 cell line in a time and dose-dependent manner. 102 µg/ml of Harmine was increased amount of cells in G1 Phase of cell cycle (p < 0.05). Anti proliferative doses of Harmine, has suppressed DNMT1 gene in NB4 cell line. Down-regulated DNMT1 induced p15 tumor suppressor promoter hypomethylation and reactivation. Our data indicate that Harmine can be considered as a potential treatment for AML (Acute Myeloid Leukemia), and future studies are required to test the clinical efficacy of Harmine—whether used as a single agent or as an adjuvant—for AML treatment.     

A requirement for K+-channel activity in growth factor-mediated extracellular signal-regulated kinase activation in human myeloblastic leukemia ML-1 cells.

Voltage-gated K+ channels have been shown to be required for proliferation of various types of cells. Much evidence indicates that K+-channel activity is required for G1 progression of the cell cycle in different cell backgrounds, suggesting that K+-channel activity is required for early-stage cell proliferation in these cells. However, little is known about the molecular mechanisms that underlie this phenomenon. We have shown in human myeloblastic leukemia ML-1 cells that K+ channels are activated by epidermal growth factor (EGF), whereas serum starvation deprivation suppressed their activity. In addition, voltage-gated K+ channels are required for G1/S-phase transition of the cell cycle. We report here that suppression of K+ channels prevented the activation of extracellular signal-regulated protein kinase 2 (ERK-2) in response to EGF and serum. However, blockade of K+ channels did not prevent ERK-2 activation induced by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Elimination of extracellular Ca2+ did not alter either ERK-2 activation or the effect of K+-channel blockade on ERK-2 activation. Our data demonstrate that the K+ channel is a part of the EGF-mediated mitogenic signal-transduction process and is required for initiation of the EGF-mediated mitogen-activated protein kinase (MAPK) pathways. Our findings may thus explain why an increase in K+-channel activity is associated with cell proliferation in many types of cells, including ML-1 cells.