Twist 1通过MPL促进髓系白血病细胞增殖和耐药

目的:检测Twist-1与骨髓增生性白血病病毒癌基因(myeloproliferative leukemia virus oncogene,MPL)在髓系白血病患者和髓系造血系统恶性肿瘤细胞系中表达的相关性,并探讨Twist-1是否通过MPL对髓系白血病细胞增殖、耐药发挥促进作用.方法:选取中国医学科学院血液病医院2005年1月至2008年12月初次诊断为急性髓系白血病(acute myeloid leu-kemia,AML)、慢性粒细胞白血病(chronic myeloid leukemia,CML)患者的骨髓标本41例(其中AML 23例、CML 18例),用Re-al-time PCR检测AML、CML患者以及髓系造血系统恶性肿瘤细胞系中Twist-1和MPL mRNA的表达情况,并分析其相关性.构建MPL过表达载体,制备慢病毒并感染髓系白血病细胞系K562、U937,通过细胞计数实验、集落形成实验以及药物敏感实验评价其对白血病细胞增殖、集落形成能力以及耐药的影响,并进一步确定Twist-1是否通过MPL发挥白血病促进作用.结果:在U937和K562细胞中过表达Twist-1明显增加MPL蛋白水平(P＜0.05),敲降Twist-1后MPL mRNA的表达水平明显下降(P＜0.01);AML、CML患者骨髓单核细胞(bone marrow mononuclear cells,BMMCs)中Twist-1 mRNA表达与MPL mRNA表达水平呈显著正相关(P＜0.05).过表达MPL使K562和U937细胞对化疗药物柔红霉素及伊马替尼的敏感性显著降低(P＜0.01),且提高K562-MPL、U937-MPL的细胞增殖、集落形成数目(均P＜0.01);干扰Twist-1并过表达MPL显著降低髓系白血病细胞增殖和集落形成(均P＜0.01).结论:Twist-1通过MPL促进AML、CML白血病细胞的增殖、存活和耐药.     

胚胎发育相关基因精子转录本在白血病细胞系中的表达及其调节

 目的 探讨胚胎发育相关基因（EDAG）在白血病细胞中的调控机制。方法 用半定量RT-PCR方法检测白血病细胞系中EDAG精子转录本EDAG-t的表达;使用不同的分化诱导剂诱导K562和U937细胞分化,检测EDAG-t的表达改变情况。结果 在白血病细胞系K562和U937中也可检测到EDAG-t的表达;使用不同的分化诱导剂诱导K562和U937细胞分化,发现EDAG精子转录本和造血转录本的表达都会随细胞分化而下调,但它们的下调步伐不一致,总的说来,EDAG-t的减弱较EDAG-h慢。结论 EDAG精子转录本在白血病细胞中的异常表达值得进一步研究。     

Renin expression in hematological malignancies and its role in the regulation of hematopoiesis

It has been demonstrated that some myeloid blasts express renin, but normal bone marrow (BM) does not display this expression. The aim of the present work was to analyze the renin expression in different hematological malignancies and different myeloid cell lines. We investigated the expression of renin by RT-PCR in BM from patients with hematological malignancies (106 patients), in nine normal BM from healthy donors and in leukemic cell lines (K562, KU812, MEG-01, U-937 and HL60), as well in K562 cell line subjected to differentiation treatments. We have observed renin expression in cells from acute myeloid leukemia (AML), chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) cases. The highest frequency was observed in AML-non acute promyelocytic leukemia(APL) cases (47.2% of the cases). The disappearance of this expression was associated with the status of complete remission of AML. Renin is expressed in some myeloid human leukemia cell lines such as K562, KU812 and MEG-01. However, when K562 cells were treated with inducers of growth inhibition and/or differentiation, the expression did not disappear, indicating that renin expression is associated with a blastic phenotype rather than with cell proliferation. The obtained findings suggest that the renin expression could have a role on the disease development and could be used as an aberrant marker of leukemia.     

Rapamycin enhances dimethyl sulfoxide-mediated growth arrest in human myelogenous leukemia cells

Abstract Rapamycin and its derivatives have been proposed in the treatment of leukemia based on their cytostatic effects, but their possible role in differentiation therapy is less explored. The aim of the present study was to investigate the possible beneficial effects of the combination of rapamycin and dimethyl sulfoxide (DMSO) on growth arrest and differentiation of acute myelogenous leukemia (AML) cells. In myeloblastic HL-60, promyelocytic NB4, monocytic U937, immature KG-1 and erythro-megakaryocytic K562 cell lines, rapamycin alone had modest inhibitory effects, DMSO inhibited proliferation in a dose-dependent manner, and the combination of rapamycin and DMSO reduced the number of viable cells significantly more than either agent alone. In NB4 cells, rapamycin had no statistically significant effects on the DMSO-mediated increase in expression of CD11b, but increased apoptosis. These results demonstrate that rapamycin enhances DMSO-mediated growth arrest, and suggest that mTOR (mammalian target of rapamycin) inhibitors may have beneficial effects in differentiation therapy of AML.     

Effect of thimerosal in leukemia, in leukemic cell lines, and on normal hematopoiesis

Anti-thymocyte globulin (ATG), a horse antiserum to human thymus tissue, has been shown to induce granulocytic differentiation of the HL-60 human leukemia cell line. In this paper we describe the effect of ATG on leukemic blasts and its effect on other human leukemia cell lines in vitro. The in vitro differentiation effect of ATG was observed in blasts from two patients with leukemia and the human leukemia cell line K562. The differentiation effect of ATG was attributable to its preservative, thimerosal, separable from ATG by high pressure liquid chromatography or dialysis. Subsequent studies with thimerosal alone showed it to induce differentiation in leukemic blasts from three patients and the human leukemia cell lines U937, K562, and KG-1. The differentiation effect of thimerosal is blocked by a sulfhydryl-protective agent, dithiothreitol, suggesting that the mechanism of differentiation may be mediated via a sulfhydryl group-dependent process.     

miR-152在急性髓系白血病患者骨髓和细胞系中的表达及生物学功能的初步研究

目的:检测miR-152在急性髓系白血病(AML)患者骨髓和细胞系中的表达水平。初步研究miR-152在急性髓系白血病中的生物学功能。方法:收集急性髓系白血病患者40例,非恶性血液病对照组20例,提取骨髓有核细胞;培养U937、Kasumi-1、THP-1三种急性髓系白血病细胞系。RT-PCR检测miR-152的表达水平。分别对U937细胞系和Kasumi-1细胞系转染miR-152 mimics和inhibitor,CCK-8法检测U937细胞及Kasumi-1细胞增殖情况,Annexin V/PI流式实验检测细胞凋亡,流式细胞术检测细胞周期。结果:miR-152在急性髓系白血病患者骨髓及细胞系中较非恶性血液病对照组表达明显下降。U937细胞系转染miR-152 mimics后,增殖受抑制,细胞凋亡增加,细胞阻滞在G0-G1期;Kasumi-1细胞系转染miR-152 inhibitor后,增殖增加,细胞凋亡减少,G0-G1期细胞减少。结论:miR-152在急性髓系白血病患者骨髓及细胞系中的表达水平均较正常对照组显著降低。miR-152在急性髓系白血病中起抑癌基因的作用,过表达miR-152可以抑制细胞增殖,促进凋亡。     

Induction of erythroid differentiation of K562 human leukemic cells by herbimycin A, an inhibitor of tyrosine kinase activity

Herbimycin A, a benzoquinonoid ansamycin antibiotic, is found to reduce intracellular phosphorylation by tyrosine protein kinase. The human chronic myelogenous leukemia cell line K562 expresses a structurally altered c-abl protein with tyrosine kinase activity. When K562 cells are induced to undergo erythroid differentiation by hemin, reduction in the intracellular level of tyrosine phosphorylation occurs. In order to understand the relationship between induction of differentiation and reduction of tyrosine phosphorylation by the c-abl gene product, the effect that herbimycin A, a selective inhibitor of intracellular tyrosine kinase activity, exerts on the differentiation of K562 cells was examined. Reduction of tyrosine phosphorylation in K562 cells by herbimycin A was observed within 1 h. Noncytotoxic concentrations of herbimycin A induced erythroid differentiation of K562 cells but not of murine erythroleukemia 745A cells. The other human myeloid leukemia cell lines (HL-60, THP-1, and U937) tested were not induced to undergo cell differentiation by this antibiotic. Herbimycin A and the other well-known inducers such as hemin, butyric acid, Adriamycin, and 1-beta-D-arabinofuranosylcytosine had additive or more than additive effects on induction of erythroid differentiation of K562 cells. With respect to inhibition of cell growth, the sensitivity of K562 cells to herbimycin A was highest in the human leukemia cell lines we tested. Noncytotoxic concentrations of herbimycin enhanced the antiproliferative effect of Adriamycin or 1-beta-D-arabinofuranosylcytosine on K562 cells. Combination therapy with herbimycin A and its derivatives may be considered for use in the treatment of some types of leukemia where tyrosine kinase activities are implicated as determinants of the oncogenic state.     

miR -148b 在急性髓系白血病患者及细胞系中的表达与生物学功能

目的：检测 miR -148b 在急性髓系白血病患者和细胞系中的表达水平。初步研究 miR -148b 在急性髓系白血病中的生物学功能。方法：收集急性髓系白血病患者56例,非恶性血液病对照组20例,提取骨髓有核细胞;培养 U937、Kasumi -1、THP -1三种 AML 细胞系。RT - PCR 检测 miR -148b 的表达水平。分别对U937细胞系及 Kasumi -1细胞系转染 miR -148b mimics 和 inhibitor。CCK -8法检测 U937细胞及 Kasumi -1细胞增殖情况,Annexin V/ PI 流式检测细胞凋亡,流式细胞术检测细胞周期。结果：miR -148b 在急性髓系白血病患者及细胞系中较非恶性血液病对照组表达明显下降。U937细胞转染 miR -148b mimics 后,增殖受抑制,细胞凋亡增加,细胞阻滞在 G0- G1期;Kasumi -1细胞转染 miR -148b inhibitor 后,增殖增加,细胞凋亡减少,G0- G1期细胞减少。结论：miR -148b 在急性髓系白血病患者及细胞系中的表达水平均较正常对照显著降低。miR -148b 在急性髓系白血病中起抑癌基因的作用,过表达 miR -148b 可以抑制细胞增殖,促进凋亡。     

RNA interference is a functional pathway with therapeutic potential in human myeloid leukemia cell lines

BACKGROUND: RNA interference (RNAi) is a cellular pathway of gene silencing in a sequence-specific manner at the messenger RNA level. The basic mechanism behind RNAi is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence. In this study, we explored the effects of RNAi in reducing the target gene expression in human myeloid leukemia cell lines. METHODS: Four myeloid leukemia cell lines (HL-60, U937, THP-1, and K562) were transfected with dsRNA duplexes corresponding to the endogenous c-raf and bcl-2 genes and the gene expression inhibition was assessed. The effect of RNAi on cell differentiation was studied; the apoptosis induction and the sensitization of the leukemia cell lines to etoposide and daunorubicin were quantified by flowcytometric methods. RESULTS: Transfection of the myeloid leukemia cell lines with dsRNA corresponding to c-raf and bcl-2 genes decreased the expression of Raf-1 and Bcl-2 proteins. RNAi for c-raf gene blocked the appearance of the monocytic differentiation induced by treatment with TPA. Combined RNAi for c-raf and bcl-2 induced apoptosis in HL-60, U937, and THP-1 cells and increased chemosensitivity to etoposide and daunorubicin. CONCLUSIONS: RNAi is a functional pathway in human myeloid leukemia cell lines and combined RNAi of c-raf and bcl-2 genes may represent a novel approach to leukemia, providing a means to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in myeloid leukemia.     

Combined Inhibition of PI3K and mTOR Exerts Synergistic Antiproliferative Effect,but Diminishes Differentiative Properties of Rapamycin in Acute Myeloid Leukemia Cells

A novel strategy has been suggested to enhance rapamycin-based cancer therapy through combining mammalian target of rapamycin (mTOR)-inhibitors with an inhibitor of the phosphatydilinositol 3-kinase PI3K/Akt or mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway. However, recent study demonstrated the potentiating effect of rapamycin on all-trans-retinoic acid (ATRA)-mediated differentiation of acute myelogenous leukemia (AML) cells, prompting us to investigate the effects of longitudinal inhibition of PI3K/Akt/mTOR signaling pathway on both proliferation and differentiative capacity of AML. In NB4, HL-60, U937 and K562 cell lines, rapamycin exerted minimal antiproliferative effects, and combining PI3K inhibitor LY 294002 and rapamycin inhibited proliferation more than LY 294002 alone. Rapamycin potentiated differentiation of ATRA-treated NB4 cells, but the combination of rapamycin and LY 294002 inhibited the expression of CD11b in both ATRA- and phorbol myristate acetate (PMA)-stimulated cells more than PI3K inhibitor alone. These results demonstrate that, although the combination of PI3K inhibitor and rapamycin is more effective in inhibiting proliferation of AML, the concomitant inhibition of PI3K and mTOR by LY 294002 and rapamycin has more inhibitory effects on ATRA-mediated differentiation than the presence of PI3K-inhibitor alone, and diminishes positive effects of rapamycin on leukemia cell differentiation.     

PPARγ和RXRα配体联用对白血病细胞生长作用机制的研究

 目的　研究过氧化物酶体增生物活化受体（PPARγ）和维甲类X受体（RXRα）在人类白血病细胞（HL-60、K562、U937）中的表达及其对配体作用的反应。方法　采用MTT法检测细胞生长;半定量RT-PCR法检测PPARγ和RXRαmRNA的表达。结果　PPARγ和RXRα在HL-60细胞中有较强表达,在K562、U937细胞中表达较弱。PPARγ的配体土槿乙酸（PLAB）能显著抑制3株细胞的生长,PLAB与RXRα的配体9-顺式维甲酸（9-cisRA）联合作用时,与PLAB单独作用比较,对受体高表达的HL-60细胞抑制作用增强较明显（P＜0.05）,但对K562、U937的作用增强不明显（P＞0.05）。PLAB和9-cisRA分别上调HL-60细胞PPARγ和RXRα的表达,且联合作用组显著高于单独作用组（P＜0.05）。结论　在HL-60、K562、U937细胞中,HL-60细胞PPARγ和RXRα表达最强,这对配体发挥作用可能是必需的,配体发挥的抑制细胞生长的效应可能与其介导的信号途径有关。     

Beneficial effects of post-transfusional hepatitis in acute myelogenous leukemia may be mediated by lipopolysaccharides, tumor necrosis factor alpha and interferon gamma.

Post-transfusional hepatitis is often a complication in patients with acute myelogenous leukemia (AML) in whom survival is paradoxically prolonged. The etiology is unknown. In previous studies, we showed that impaired hepatic endotoxin (lipopolysaccharide, LPS) clearance in patients with acute viral hepatitis A, B, or C versus controls results in endotoxemia and tumor necrosis factor alpha (TNF-alpha) release. TNF-alpha mediates anti-proliferative and differentiating effects in AML cell lines. Interferon-gamma (IFN-gamma) released in acute viral hepatitis, acts in synergy with TNF-alpha. HL60, KG1, and U937 AML cells treated 3, 6, and 9 days with physiologically attainable TNF-alpha (10 U/ml), IFN-gamma (100 U/ml) and LPS (10 ng/ml) levels, have significantly diminished viability and cell growth versus controls. Treatment of HL60 AML cells with LPS/TNF-alpha/IFN-gamma also resulted in significantly increased monocytic pathway differentiation not seen with KG1 or U937 AML cells. HL60 AML cells treated with TNF-alpha/IFN-gamma for 6 days released endogenous TNF-alpha (1.57 U/10(6) cells) upon LPS stimulation compared to less than 0.01 U/10(6) cells in non-LPS-stimulated TNF-alpha/IFN-gamma-treated cells or untreated cells (p less than 0.0001). Untreated HL60 AML cells co-cultured with HL60 cells pretreated for 6 days with TNF-alpha/IFN-gamma and then subjected to LPS stimulation had significantly diminished cell growth compared to controls (p less than 0.0001). This effect could be reversed with anti-TNF-alpha antibody, supporting the concept that endogenous TNF-alpha release by LPS/TNF-alpha/IFN-gamma treated HL60 AML cells may act by paracrine means to suppress growth of other AML cells. The beneficial effects of post-transfusional hepatitis in AML patients may be mediated via LPS/TNF-alpha/IFN-gamma-induced AML cell growth suppression and/or terminal differentiation in which AML cells participate by releasing TNF-alpha after being acted upon by LPS/TNF-alpha/IFN-gamma. Endogenously released TNF-alpha might then act by autocrine/paracrine means to mediate further suppression and terminal differentiation.     

WT1(17AA+)剪接变异体在白血病中表达

目的：探讨不同白血病细胞株及不同亚型急性白血病患者原代骨髓细胞中WT1（17AA＋）剪接变异体的表达水平。方法：建立实时定量RT—PCR方法，检测K562、SHI、Jurkat、NB4、NB4／WT1A、NB4／WT1D等白血病细胞株及79例初诊急性白血病患者原代骨髓细胞中WT1基因和WT1（17AA＋）剪接变异体表达水平，并计算WT1（17AA＋）／WT1比值。结果：白血病细胞株K562、SHI、NB4、NB4／WT1A、NB4／WT1D中WT1基因表达水平较Jurkat、U937高2～3个对数级，且K562和NB4细胞株中WT1表达以WT1（17AA＋）剪接变异体优势表达。79例急性白血病患者原代骨髓细胞中WT1总体表达水平在0．03～34．17之间，中位数为1．02，明显高于23例对照非白血病患者（P〈0．001），其WT1（17AA＋）／WT1比值在0．30～0．93之间，中位数为0．62，WT1（17AA＋）剪接变异体表达在不同亚型急性白血病之间无统计学差异（F=0．152，P=0．979）。结论：高表达WT1的白血病细胞株及白血病患者骨髓细胞中WT1基因以WT1（17AA＋）剪接变异体优势表达，且WT1（17AA＋）剪接变异体表达在不同亚型急性白血病之间无明显统计学差别。     

FHL2基因表达改变对急性红白血病细胞生物学功能的影响

目的 探讨FHL2基因对人类急性红白血病细胞生物学功能的影响.方法 应用Western blot检测常见白血病细胞系（K562、HEK293T、U937、HL-60、Kasumi-1、SKNO-1、NB-4及Nalm-6）FHL2蛋白表达情况.采用RNA干扰技术设计并合成FHL2 mRNA特异性shRNA,构建pLKO.1-puro干扰载体,制备慢病毒并感染人类急性红白血病K562细胞;应用实时定量聚合酶链反应（PCR）方法检测干扰效率;采用四甲基偶氮唑蓝（MTT）法检测干扰FHL2对细胞增殖能力的影响;用流式细胞术分析细胞凋亡的变化.结果 FHL2在K562、Kasumi-1和Nalm-6等白血病细胞系中表达,尤其在K562细胞中高表达.Western blot检测显示干扰FHL2的K562细胞FHL2蛋白相对表达水平较转染阴性对照序列的K562细胞明显下降（相对表达水平分别为1、0.284,t=8.872,P=0.0004）,干扰K562细胞中FHL2基因表达能够抑制细胞的增殖,促进细胞的凋亡.结论 FHL2基因在多种白血病细胞系中表达升高.shRNA靶向干扰FHL2基因的表达可有效抑制K562细胞的增殖,促进其凋亡.     

Effect of X-irradiation dose rate on the clonagenic survival of human and experimental animal hematopoietic tumor cell lines: evidence for heterogeneity

It is a generally accepted principle of radiation biology that hematopoietic progenitor cells demonstrate dose rate independent killing by x-irradiation over the clinically relevant range for total body irradiation (TBI) (5-25 rad/min). To determine whether low dose rate (5 rad/min, or 20 rad/min) compared to conventional dose rate (200 rad/min) x-irradiation altered the clonagenic survival of leukemia and lymphoma cell lines, several permanent cell lines were studied. These included: bg/bg cl 1, mouse basophillic leukemia; LW12, [W/fu rat acute myelogenous leukemia (AML)]; and human cell lines: JY and Daudi (B-cell lymphomas); K45, (T-cell leukemia); K562, (erythroleukemia); HL60 and KG1 (monomyeloid leukemias), and U937 (human histiocytic/monocytic lymphoma). Dose rate independent killing was demonstrated at several plating densities with mouse and rat leukemia lines and all human leukemia lines tested except lines HL60 and U937. With HL60, increased plating density increased the D0 at each dose rate. This effect was not attributable to an increased plating efficiency. With line U937 there was a clear dose-rate effect with increase in D0 from 88 rad, n 4.6 at 200 rad/min, to D0 = 166, n 2.3 at 5 rad/min. The data demonstrate that some human hematopoietic tumor derived cell lines of myeloid/monocyte/macrophage lineage can exhibit atypical repair of irradiation damage in vitro. This repair may be enhanced by conditions relevant to clinical TBI including low irradiation dose-rate and cell to cell interactions by tumor cells in close proximity.     

GM-CSF incubation prior to treatment with cytarabine or doxorubicin enhances drug activity against AML cells in vitro: a model for leukemia chemotherapy

We studied the effect of preincubation with recombinant GM-CSF on the activity of cytarabine and doxorubicin against clonogenic acute myeloid leukemia cells (CFU-AML). Leukemia cells from seven persons with AML, three myeloid cell lines (HL60, KG1, K562) and two control cell lines (U937, MOLT3) were tested. Preincubation with GM-CSF (0.01-0.1 microgram/ml) increased DNA synthesis as measured by tritiated thymidine incorporation and intranuclear Ki67 expression in cells from six persons with AML and in HL60 cells. Leukemia cells preincubated with GM-CSF for 6-48 h were exposed to cytarabine (2-200 micrograms/ml) or doxorubicin (0.01-0.1 microgram/ml) for 3 h and CFU-AML assayed. This approach further reduced CFU-AML in samples from six persons with AML and in HL60 and KG1 cells compared to cells not preincubated with GM-CSF prior to drug treatment. In most instances, reduced CFU-AML correlated with GM-CSF induced DNA synthesis. These data suggest a possible strategy of GM-CSF pretreatment to increase anti-leukemia efficacy of chemotherapy in AML.     

Anti-Vascular Endothelial Growth Factor Effects of Sorafenib and Arsenic Trioxide in Acute Myeloid Leukemia Cell Lines

Acute myeloid leukemia (AML), is a clonal disorder caused by acquired somatic mutations and chromosomal rearrangements. According to some evidence, progression of hematolymphoid malignancies depends on the induction of new blood vessel formation under the influence of acute leukemia. Various factors are produced by cancer cells under hypoxic conditions to increase vascular formation. Among these, vascular endothelial growth factor (VEGF) plays a crucial role. Cytotoxicity and anticancer effects of arsenic trioxide (ATO) have been reported in many cancers. Sorafenib, known as an angiogenic inhibitor, decreases leukemic cell survival. The aim of this study was to indicate combination effects of ATO and sorafenib in two AML cell lines, KG-1 and U937. Effective doses was determined by MTT assay for both single and combination treatments. Percentages of apoptotic cells were evaluated by Annexin V FITC staining and mRNA levels of VEGF isoforms and receptor expression were investigated by Real-Time PCR. Our data show that sorafenib (5μM and 7μM in KG-1 and U937 cell lines respectively), ATO (1.618μM and 1μM in KG-1 and U937 cell lines respectively), and also their combination significantly increased the percentage of apoptotic cells. In addition the mRNA level of VEGF isoforms was downregulated in the U937 cell line while upregulated in KG-1 cells. Taken together, our results suggest that the VEGF autocrine loop may have an influence on AML development and progression and could be consider as a therapeutic target. The combination of sorafenib as a VEGF inhibitor with ATO synergistically inhibits cell proliferation and promotes apoptosis.