小干扰RNA逆转三氧化二砷耐药的白血病K562/AS2细胞Topo Ⅱ基因表达的实验研究

目的 研究小干扰RNA片段(shRNA)对三氧化二砷(ATO)耐药的白血病细胞株K562/AS2细胞的Topo Ⅱα、TopoⅡβ基因表达及其功能的影响.方法 设计并合成针对Topo Ⅱα和TopoⅡβ基因序列的shRNA各3对,在脂质体的介导下转染K562/AS2细胞;用荧光实时定量聚合酶链反应(PCR)分析Topo Ⅱα、TopoⅡβmRNA的表达水平;流式细胞术检测Topo Ⅱα、TopoⅡβ蛋白表达.结果 针对Topo Ⅱα-shRNA、TopoⅡβ的shRNA作用于K562/AS2细胞24 h后,Topo ⅡαmRNA水平和蛋白水平最大下调为(78.22±0.01)%、(31.17±1.27)%(P<0.05),TopoⅡβmRNA水平和蛋白水平最大下调为(57.36 ±0.01)%、(23.98 ± 1.22)%(P<0.05).结论 转染24 h后针对TopoⅡ的shRNA可抑制对ATO耐药的白血病细胞株K562/AS2细胞TopoⅡ基因的表达.     

低剂量地西他滨联合伊马替尼对K562细胞的增殖抑制作用

目的 研究低剂量地西他滨(DAC)联合伊马替尼(IM)对K562细胞株的增殖抑制作用及对bcr-abl表达的影响.方法 单药及两药联合后,通过四甲基偶氮唑蓝(MTT)法观察药物对K562细胞株的增殖抑制作用,流式细胞术检测药物对K562细胞株早期凋亡率及细胞周期,巢式反转录-聚合酶链反应(RT-PCR)半定量检测药物对K562细胞株bcr-abl mRNA表达.结果 DAC与IM单药对K562细胞的抑制作用呈浓度时间依赖性.两药联合用药抑制作用较单药组明显(F=43.947、165.580、321.193、296.101,均P<0.05),24、48、72 h各浓度组与对照组比较差异均有统计学意义(F=202.759、168.457、417.538,均P<0.05).DAC及IM单药作用药物对K562细胞株均使G,期细胞明显增多,IM0.2μmol/L作用于K562细胞株48 h可见6.7%早期凋亡细胞,IM 0.2 μmol/L联合DAC 4μmol/L早期凋亡细胞增加至8.4%.bcr-abl mRNA表达水平降低,DAC 4 μmol/L作用48 h后可降低K562细胞中bcr-abl mRNA表达(约14%),IM 0.2 μmol/L降低约40%,联合用药表达量明显降低(约60%).联合用药组与单药组比较差异有统计学意义(F=71.981,P<0.05).结论 DAC对K562细胞的增殖抑制作用与细胞周期阻滞、诱导凋亡及降低bcr-abl mRNA表达有关,两药联合可显著抑制K562细胞增殖.     

甲基化寡核苷酸灭活死亡相关蛋白激酶1基因对白血病K562细胞增殖的影响

目的 探讨互补甲基化寡核苷酸诱导灭活K562白血病细胞死亡相关蛋白激酶1基因(DAPK1)及对其增殖的影响.方法 应用Lipo2000将与DAPK1基因启动子序列互补的甲基化寡核苷酸转染进K562白血病细胞,分别应用甲基化特异性聚合酶链反应(MSP)和反转录PCR(RT-PCR)检测转染前后DAPK1基因启动子甲基化状态和mRNA表达改变.应用噻唑蓝(MTT)法检测转染前后细胞增殖变化.结果 正常组K562细胞的DAPK1基因启动子表现为未甲基化状态,可检测到相应mRNA表达;对照组寡核苷酸转染后,DAPK1基因启动子表现为未甲基化状态,mRNA表达和细胞增殖速度与正常组无明显差异;互补甲基化寡核苷酸转染后,DAPK1基因启动子呈甲基化状态,mRNA呈低表达状态,细胞增殖速度较正常组、甲基化对照寡核苷酸转染组显著增加.结论 互补甲基化寡核苷酸可诱导灭活K562白血病细胞DAPK1基因并抑制其mRNA表达,促进细胞增殖.     

Salivary cortisol and cardiovascular activity during stress in oppositional-defiant disorder boys and normal controls

The erythroleukemic cell line K562 can undergo further differentiation in erythroid or megakaryocytic lineage depending on the nature of the stimulus. Phorbol ester (PMA) stimulates megakaryocytic development whereas hemin promotes erythroid differentiation of these cells. We have examined the effect of PMA and hemin on the expression of the Kell blood group and CD10 antigens, two related proteins that belong to a family of membrane-bound neutral metalloendopeptidases. We show here that differentiation of K562 cells by PMA in the megakaryocytic lineage results in abolishment of Kell mRNA accumulation and protein expression and, in parallel, the induction of CD10 mRNA accumulation, protein expression, and enzymatic activity. Conversely, differentiation of these cells by hemin in the erythroid lineage is accompanied by an up-regulation of Kell mRNA and protein expression, with no changes in CD10 mRNA and protein expression. Thus, CD10 and Kell can be regarded as specific markers of the differentiation of K562 cells in the megakaryocytic and erythroid lineages, respectively.     

Retroviral vector-mediated transfer of the interferon-alpha gene in chronic myeloid leukemia cells

The transfer and expression of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. Interferon (IFN)-alpha may induce hematological remission in chronic myeloid leukemia (CML) patients, but only a small proportion of patients achieve a sustained, complete cytogenetic remission. We have investigated the possibility of transducing CML cells with the retroviral vector LIalpha2SN, which encodes the IFN-alpha2 gene. We first optimized the transduction efficiency using the CML-derived K562 cell line. A transduction efficiency of 50% and 85% after three and six infections, respectively, was obtained in K562 cells. We then expressed IFN-alpha2 in CML cells by transducing the latter with LIalpha2SN viral particles. The IFN-alpha secretion after three and six infections was 5,400 and 18,000 U/24 hours/10(6) cells for unselected K562 cells and 7,000 and 290 U/24 hours/10(6) cells for CML CD34+ cells at days 4 and 5. Moreover, the major histocompatibility complex class I antigens were overexpressed after infection with LIalpha2SN in both K562 and CML CD34+ cells. The proliferation (in liquid culture) and the cloning efficiency of these CML cells were significantly decreased after LIalpha2SN treatment. By contrast, the proliferation of cord blood CD34+ cells was not affected by transduction with LIalpha2SN. These results demonstrate the transduction efficiency of CML cells and suggest the possibility of CML cell immunotherapy with retroviral gene transfer of different cytokines such as IFN-alpha.     

Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation

The human erythroleukemia (K562) cell line undergoes megakaryocytic differentiation and cessation of proliferation when treated with phorbol myristate acetate (PMA). To investigate the role of individual protein kinase C (PKC) isotypes in these events, we have assessed PKC isotype expression during leukemic proliferation and PMA-induced differentiation. Immunoblot analysis using isotype-specific antibodies demonstrates that proliferating K562 cells express the alpha, beta II, and zeta PKC isotypes. PMA-induced differentiation and cytostasis lead to a decrease in beta II PKC and increases in alpha and zeta PKC levels. The role of the alpha and beta II PKC isotypes was further assessed in cells overexpressing these isotypes. K562 cells overexpressing human alpha PKC grew more slowly and were more sensitive to the cytostatic effects of PMA than control cells, whereas cells overexpressing beta II PKC were less sensitive to PMA. PMA-induced cytostasis is reversed upon removal of PMA. Resumption of proliferation is accompanied by reexpression of beta II PKC to near control levels, whereas alpha and zeta PKC levels remain elevated for several days after removal of PMA. Proliferation of PMA-withdrawn cells can be partially inhibited by antisense beta II PKC oligodeoxyribonucleotide. Growth inhibition is dose-dependent, specific for beta II PKC-directed antisense oligonucleotide, and associated with significant inhibition of beta II PKC levels indicating that beta II PKC is essential for K562 cell proliferation. Sodium butyrate, which unlike PMA induces megakaryocytic differentiation without cytostasis, causes increases in both alpha and beta II PKC levels. These data demonstrate that beta II PKC is required for K562 cell proliferation, whereas alpha PKC is involved in megakaryocytic differentiation.     

K562细胞硫氧还蛋白还原酶活力测定及其抑制剂体外抗白血病的初步研究

目的 探讨慢性粒细胞白血病(CML)细胞株K562中硫氧还蛋白还原酶(TrxR)的活力及其新型抑制剂乙烷硒啉(BBSKE)体外抗白血病作用.方法 应用胰岛素还原法检测K562细胞株及健康人骨髓单个核细胞中TrxR的活力.运用CCK-8法测定BBSKE对K562细胞的增殖抑制率.应用激光共聚焦显微镜、琼脂糖凝胶电泳以及Annexin V-FITC/PI双标记流式细胞术观察BBSKE的抗白血病作用.结果 K562细胞中TrxR的活性明显高于健康人骨髓单个核细胞,10 μmol/L BBSKE与K562细胞作用24 h,激光共聚焦显微镜可见典型的细胞凋亡表现,琼脂糖凝胶电泳后可见典型的DNA"梯"条带出现,流式细胞术检测凋亡率为(10.28±2.74)%;10 μmol/LBBSKE对CML患者原代细胞有诱导凋亡的作用,凋亡率为(5.70±0.48)%.结论 慢性粒细胞白血病细胞株K562中TrxR活力高于健康人骨髓单个核细胞,BBSKE有抑制TrxR活力、抑制K562细胞增殖和诱导凋亡的作用,是治疗CML潜在的有效药物.     

Abrogation of IL-3 requirements and stimulation of hematopoietic cell proliferation in vitro and in vivo by carboxylic acids

Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the beta-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced gamma globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observed in vivo in mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce gamma globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.     

Prospective study of mutant frequencies at the hprt and T-cell receptor gene loci in pediatric cancer patients during chemotherapy

With the use of three different hematopoietic cell lineages, the downregulation of telomerase activity was found to be a general response to the induction of differentiation. The decrease in telomerase activity occurred as early as 24 h when HL-60 and K562 cells were cultured in the presence of 1alpha, 25 dihydroxyvitamin D3 (VD3), all-trans-retinoic acid (ATRA) and hemin, and completely disappeared after 3 days. On the other hand, MEG-01 cells showed a marked inhibition of telomerase activity after 6 days of culture with 12-0-tetradecanoylphorbal 13-acetate (TPA). The analysis of telomeric DNA in the HL-60 cells and K562 cells demonstrated no detectable loss of telomeric DNA with cellular differentiation, with a loss of telomerase activity. The repression of telomerase is a common molecular event during leukemic cell differentiation.     

Enforced expression of Bcl-XS induces differentiation and sensitizes chronic myelogenous leukemia-blast crisis K562 cells to 1-beta-D-arabinofuranosylcytosine-mediated differentiation and apoptosis

Human chronic myelogenous leukemia-blast crisis K562 cells have been demonstrated to be relatively resistant to antileukemic drug-induced apoptosis. This has been attributed to the activity of p210bcr-abl tyrosine kinase present in the K562 cells, which is known to suppress drug-induced apoptosis. Recently, K562 cells have been shown to express the antiapoptosis Bcl-xL but not Bcl-2 proteins. To investigate the contribution of Bcl-xL toward resistance to drug-induced apoptosis, we created K562/Bcl-xS and K562/neo cells by electroporating the expression plasmids pSFFVneo-Bcl-xS and pSFFVneo, containing the bcl-xS and neomycin resistance genes, respectively, into K562 cells. K562/Bcl-xS but not K562/neo cells expressed the bcl-xS mRNA and p19Bcl-xS protein. In contrast, both cell types expressed equivalent levels of Bcl-xL, Bax, Bcl-2, Myc, retinoblastoma, p21cbor-abl, and p145abl proteins. A significant increase in the hemoglobin levels was observed in the K562/Bcl-xS compared with the K562/neo cells (P < 0.05). In addition, K562/Bcl-xS cells were significantly more sensitive than K562/neo cells to undergoing erythroid differentiation induced by low-dose 1-beta-D-arabinofuranosylcytosine (ara-C) and hexamethyl bisacetamide (P < 0.05), but not by all-trans-retinoic acid. Low-dose ara-C- or hexamethyl bisacetamide-induced differentiation was not associated with apoptosis of K562/Bcl-xS or K562/neo cells. Low-dose ara-C-induced erythroid differentiation was accompanied by conversion of the retinoblastoma protein to predominantly its underphosphorylated isoform as well as by down-regulation of Myc levels in K562/Bcl-xS and K562/neo cells. Importantly, exposure to high-dose ara-C (HIDAC; 100 microM ara-C for 4 h) caused internucleosomal DNA fragmentation and the morphological features of apoptosis in K562/Bcl-xS cells. These effects were modestly enhanced by cotreatment with HIDAC plus herbimycin A. In contrast, K562/neo cells were completely resistant to HIDAC- and herbimycin A-induced apoptosis. These results indicate that the expression of Bcl-xS induces erythroid differentiation and partially sensitizes chronic myelogenous leukemia-blast crisis-derived K562 cells to ara-C-induced differentiation and apoptosis.     

Expression of differentially phosphorylated Rb and mutant p53 proteins in myeloid leukemia cell lines

We studied the structure and expression of Rb and p53 genes in six myeloid leukemia cell lines (HL-60, KBM3, K562, KBM5, EM2, KBM7) in the light of the published reports that structural abnormalities of these genes are rarely seen in leukemic cells and also a recent finding that Rb gene expression can be regulated by the p53 protein. Except for HL-60 cells which have a truncated p53 gene, none of the other cell lines revealed any gross structural abnormalities in the Rb and p53 genes. KBM3, KBM5 and EM-2 expressed lower levels of Rb mRNA than HL-60, K562 and KBM7. The amount of Rb protein was lowest in KBM3 cells and in this and two other cell lines (KBM5, KBM7) Rb was markedly hypophosphorylated compared to the other three cell lines. HL-60 and K562 did not express p53 m-RNA, while the other four cell lines all expressed high levels of mutant p53 protein. Thus even in the absence of gross structural alterations, subtle abnormalities in the expression pattern of Rb and p53 genes occur in myeloid leukemia cells.     

Human hematopoietic progenitors express erythropoietin

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.     

Effect of HIV type 1 Tat protein on butyric acid-induced differentiation in a hematopoietic progenitor cell line

The trans-activator protein (Tat) of HIV-1 plays an important role in viral pathogenesis. Since Tat has been shown to alter expression of a number of host cellular genes, we have investigated the role of Tat in modulating gene expression and differentiation in hematopoietic progenitor cells. Tat protein was introduced in K562 cells, a human hematopoietic progenitor cell line, by either scrape-loading onto HeLa (HL)-tat cells or direct electroporation of an affinity-purified glutathione S-transferase (GST)-Tat fusion protein. Under these conditions, butyric acid-induced hemoglobin production in K562 cells was suppressed by 65 and 52%, respectively. However, coculturing with wild-type HeLa cells or electroporation with the control GST protein did not decrease hemoglobin production. To confirm the presence of bioactive Tat protein within K562 cells, the cells were transiently transfected with a pHIV/LTR-CAT prior to the introduction of Tat. A 30- to 40-fold induction in CAT gene expression was observed in the transfected K562 cells, which were either cocultured with HL-tat or were electroporated with GST-Tat. Simultaneous transient transfection of K562 cells with a TAR expression plasmid, to compete for the availability of Tat protein, significantly downregulated the HIV LTR trans-activation by Tat. In addition, overexpression of the TAR RNAs in K562 cells was able to downregulate the suppressive effect of Tat on butyric acid-induced differentiation. RT-PCR analysis of the total RNAs isolated from these cells demonstrated that Tat protein suppressed the butyric acid-induced gamma-globin gene expression by an average of 54% without affecting the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. These data indicate that the viral Tat protein plays a significant role in abrogating erythroid differentiation in K562 cells.     

Expression of the spr1 gene in cultured tracheal epithelial cells and its regulation by retinoids before and after confluence

The absence of vitamin A or vitamin A derivatives in culture media promotes squamous cell differentiation of tracheobronchial epithelial cells. This is especially true for the expression of a small proline-rich protein (20K; 98 amino acids) in pig trachea epithelial cells. Multigene families encode different small proline-rich proteins in different species, and these proteins are possible markers for squamous cell differentiation. 20K mRNA and 20K protein were detected in cells within 4 and 5 days in culture, respectively, when cells reached about 50% confluence, and expression increase 12-fold during cell proliferation until cells reached 100% confluence. Arotinoid (10(-9)M), a synthetic retinoid, essentially totally inhibited expression of 20K mRNA in proliferating tracheobronchial cells within 3 days of treatment while 20K protein levels were only decreased 4-fold after 5 days. However, if cells were exposed to arotinoid 3 days after reaching confluent growth, the levels of either 20K mRNA or 20K protein were unchanged. Cells exposed to arotinoid from the onset of culturing, and then removal of the retinoid from proliferating cells resulted in the expression of 20K mRNA and protein after 4 and 5 days as observed previously. 20K mRNA was not detected in cells that had been continuously exposed to arotinoid from the start of culture until 3 days post confluence, even 10 days following removal of arotinoid. Our results strongly suggest that the growth phase and state of cell differentiation greatly affect the response of these epithelial cells to vitamin A derivatives.     

IFN-gamma treatment increases insulin binding and MHC class I expression in erythroleukemia cells

We have investigated if interferon-gamma (IFN-gamma) treatment of human K562 tumor cells, which upregulates the expression of MHC class I antigens (MHC-I), simultaneously would influence insulin binding. Treatment of K562 cells with recombinant human IFN-gamma for 48 h caused a significant increase of insulin binding at 37 degrees C. Recombinant human tumor necrosis factor-alpha (TNF-alpha) alone had no effect but acted synergistically with IFN-gamma, leading to a two-fold increase of insulin binding. No change in affinity, number of binding sites or cell surface expression of insulin receptors (IR) after IFN-gamma treatment could be detected. The increased insulin binding observed at 37 degrees C was not seen at 4 degrees C, suggesting alteration of insulin internalization. The dose-response curve, as well as the time curve, for the increase in insulin binding after IFN-gamma treatment correlated with enhanced cell surface expression of MHC-I antigens. However, the correlation was not absolute. Our results show that IFN-gamma treatment alone or together with TNF-alpha, can alter the insulin binding to K562 cells without changing the expression or affinity of the IR. This correlates with the effect of IFN-gamma on MHC-I expression. These results support the findings that MHC-I molecules associate and interact with the IR at the cell surface.