红系KLF因子研究进展

β-珠蛋白基因簇是真核生物基因组中颇具代表性的模型,其表达具有红系组织专一性及发育时期特异性,这些特异性的产生与红系专一及通用的反式作用因子与相应的顺式作用元件之间相互作用有关。综述了反式作用因子中KLF家族中近年来新发现的三个因子EKLF、FKLF和FKLF-2,介绍了它们的结构、功能及它们与其他反式作用因子间的相互作用。     

KLFs对珠蛋白基因表达和红系分化的调控作用

Krüppel样因子(Krüppel-like factors, KLFs)是一组与真核基因转录调控密切相关的锌指蛋白.KLFs高度保守的羧基末端含3个串联的Cys2His2型锌指结构,用于结合GC盒和CACCC盒等DNA序列. 红细胞中特异表达的珠蛋白基因和许多红系调控因子中都含有CACCC盒.已有研究发现,多个KLFs通过结合CACCC盒参与调控珠蛋白基因表达和红系分化,例如,KLF1通过结合β-珠蛋白启动子和位点控制区(locus control region, LCR),促进β-珠蛋白的表达、γ-向β-珠蛋白基因的转换和红系分化;KLF2、KLF11和KLF13分别促进ε-和γ-珠蛋白基因的表达;KLF4促进α-和γ-珠蛋白基因的表达;KLF3和KLF8则抑制ε-和γ-珠蛋白基因的表达. 本文综述了KLFs调控珠蛋白基因表达和红系分化的研究进展.     

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.     

OAZ1基因诱导慢粒白血病K562细胞向成熟红系方向分化

近年来,鸟氨酸脱羧酶抗酶(OAZ)作为肿瘤治疗的潜在靶点备受关注.本文研究了OAZ1基因过表达对慢粒白血病K562细胞红系分化的作用.构建框移位点突变的OAZ1 过表达慢病毒载体pLVX-Neo-OAZ1-IRES-ZsGreen,包装病毒并感染K562细胞, Western 印迹验证其过表达效果.FACS检测细胞分化标志物CD71和GPA,结合联苯胺染色分析细胞红系分化情况.对比氯化高铁血红素(hemin)诱导组,实时RT-PCR检测与K562细胞红系分化、癌变的关键基因(GATA1、BCR/ABL、TGFβ)转录水平,对OAZ1 诱导分化的机制进行初步探索.结果表明,慢病毒过表达载体及K562细胞过表达体系构建成功.OAZ1过表达后细胞红系分化标志物CD71+/GPA+为(11.22±2.09)%,与对照组(4.07±1.04)%、空病毒组(1.79±2.36)%相比差异极显著(P<0.01);联苯胺蓝染阳性率为(14.037±0.083)%,与对照组、空病毒组比较,差异也极显著(P<0.01).定 量分析结果提示,相对于GATA1、BCR/ABL 基因mRNA转录水平的影响,OAZ1对TGFβ 基因的作用更为明显.为此推断,OAZ1基因可诱导白血病K562细胞向成熟红系方向分化,其作用机制可能与TGFβ信号转导通路相关.     

与抑制K562细胞向红系分化相关的Attractin基因的分离和初步鉴定

应用差异显示PCR（DDRT-PCR）方法分离野生型K562细胞株和能表达人β－珠蛋白基因的变种K562细胞株的4个差异cDNA片段,序列测定后,挑选差异最明显的片段dd1,经过RT-PCR、Northern 印迹鉴定确实为两株细胞间的差异片段,测序后经同源性分析等,发现其所对应的基因是吸引素（Attractin,GenBank注册号AF106861）。 根据其结构预测,Attractin有可能是K562 细胞表面一个黏附因子受体,与抑制K562细胞向红系分化有关。 Abstract:Differential display PCR method was used to isolate four differential display fragments between the wild type K562 cell line and the variant type K562 cell line that expressed the human β-globin gene.After sequencing,the most remarkable different fragment,named dd1,was selected for further study.The analysis of RT-PCR and Northern blot hybridization showed that dd1 was exactly the differentiation fragment between the two cell lines.The homology analysis indicated that dd1 was matched to Attractin (GeneBank registration No.AF106861).It might be an adhesion receptor related to inhibiting erythroid differentiation based on its structure.     

新的红细胞分化相关因子反义RNA特异抑制K562细胞中的GATA-1转录因子活性

以往报道了通过基因转染技术观察新的红细胞分化相关因子(EDRF1)反义RNA表达和EDRF1过表达对细胞增殖和分化的影响,结果表明,反义EDRF1表达载体转染后的K562细胞α-globin,γ-globin mRNA表达水平下降.本文利用基因芯片和真核基因转染技术观察了EDRF1对60种细胞因子及其受体表达水平的影响,发现EDRF1明显调节IL-6受体,GM-CSF受体,c-Jun/c-Fos,c-myc及c-kit(SCF受体)等基因的表达水平,并通过RNA点杂交进行验证,EDRF1对几个重要的细胞因子受体表达的调节可能是其执行功能的一个重要途径.Northern blot实验结果表明,GATA-1 mRNA在转染EDRF1反义表达载体后表达水平有所下调,随后的凝胶阻滞电泳实验(gel shift assay,EMSA)证明,与对照相比,EDRF1反义表达载体转染的K562细胞中,红系特异的转录因子GATA-1的活性受到明显的抑制,而另一个红系特异的转录因子NF-E2则没有明显的活性改变,对照NF-κB的转录活性也基本保持恒定.因此推测,K562细胞增殖和分化的调节很可能是通过直接影响红系特异的转录因子GATA-1与顺式序列相互作用的转录活性来实现的.     

红系NFE2反式作用因子

ＮＦＥ２是碱性亮氨酸拉链家族成员之一,由一个广泛表达的１８ｋＤ亚基和一个组织限制表达的４５ｋＤ亚基组成。ＮＦＥ２是生血组织特异的反式作用因子。它通过α和β珠蛋白基因簇的位点控制区吝节珠蛋白基因转录。而且对正常血小板的生成是必须的。     

过表达KLF4重组质粒的构建及其在白血病K562细胞中的表达

为了构建过表达锌指转录因子Krüppel样因子4(Krüppel-like factor 4,KLF4)基因的重组质粒pEGFP-KLF4,观察其在白血病K562细胞中的表达,以RT-PCR法扩增人KLF4基因,构建pEGFP-KLF4重组质粒;经酶切及测序鉴定后,将pEGFP-KLF4质粒电穿孔转染白血病K562细胞(K562/pEGFP-KLF4组),设pEGFP-C1空质粒转染K562细胞(K562/pEGFP-C1组)及空白K562细胞作为对照,荧光显微镜观察EGFP-KLF4融合蛋白的表达情况,同时用抗生素G418筛选阳性克隆并扩大培养建立稳定过表达KLF4基因的K562细胞株,随后用RT-PCR检测3组K562细胞中KLF4的m RNA表达水平。实验结果显示,pEGFP-KLF4重组质粒构建成功。该质粒转染K562细胞24 h后能观察到较高强度的绿色荧光;经G418筛选出的阳性克隆扩大培养后建立了稳定过表达KLF4基因的K562细胞株;与对照组相比,K562/pEGFP-KLF4细胞组KLF4的m RNA表达水平明显升高,其过表达率为65.71%(P0.05)。实验中构建的过表达KLF4基因的重组质粒pEGFP-KLF4能在K562细胞中高效表达,为进一步研究其在白血病中的作用奠定了基础。     

干细胞因子及其受体在红系分化中的作用

干细胞因子作为一种多能细胞因子,在临床上有较好的应用前景。随着CD34^ 细胞分离培养技术的完善,干细胞因子及其受体在红系增殖、分化中的重要作用正逐渐被发现并阐述清楚,对临床上相关疾病的治疗有重要的指导意义。本介绍了干系胞因子及其配体抑制红系祖细胞凋亡和促进红系祖细胞增殖和分化的功能,以及在临床上的应用前景。     

Role of ERK activation in growth and erythroid differentiation of K562 cells

Inhibition of signaling through Ras in BCR-ABL-positive pluripotent K562 cells leads to apoptosis and spontaneous differentiation. However, Ras-induced activation of the mitogen-activated protein kinase ERK has been suggested to play a critical role in either growth or differentiation in different model systems. We studied the role of ERK activation in the growth-promoting and anti-apoptotic effect of Ras and its involvement in hemin-induced nonterminal erythroid differentiation using the BCR-ABL-positive K562 cell line as a model. K562 cells were stably transfected with ERK1 or the dominant inhibitory mutant of ERK1 (ERK1-KR). Overexpression of ERK1-KR inhibited cell growth with an approximately fourfold increase in doubling time and induced apoptosis in K562 cells. Incubation with the MEK1 inhibitor UO126 inhibited cell growth and induced apoptosis in K562 cells in a dose-dependent manner as well. In the presence of exogenously added hemin, K562 cells differentiate into erythroblasts, as indicated by the production of large amounts of fetal hemoglobin. We examined the activation of MAP kinases during hemin-induced differentiation. The ERK1 and 2 activity increased within 2 h post hemin treatment and remained elevated for 24-48 h. During this time, fetal hemoglobin synthesis also increases from 0.8 to 10 pg/cell. There was no activation of JNK or p38 protein kinases. The hemin-induced accumulation of hemoglobin was inhibited in ERK1-KR overexpressing cells and was enhanced in the wild-type ERK1 transfectants. Our results suggest that ERK activation is involved in both growth and hemin-induced erythroid differentiation in the BCR-ABL-positive K562 cell line.     

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of a-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid pro     

Antisense EDRF1 gene inhibited GATA-1 transcription factor DNA-binding activity in K562 cells

Our previous studies showed that EDRF1 influenced expression of α-globin mRNA and synthesis of hemoglobin in K562 cells and modulated self-renewal of K562 cells. To illuminate the function of EDRF1 in K562 cells, sense and antisense EDRF1 constructs were prepared and transfected into K562 cells. By using microarray and dot blot assay, 60 cytokine receptors and some oncogenes sharing important functions in cell proliferation and differentiation were investigated. The results of this study demonstrated that IL-6 receptor, GM-CSF receptor, c-Jun/c-Fos, c-Myc and c-kit genes were regulated by antisense EDRF1 expression. The regulation was confirmed by RNA blot assay. GATA-1 mRNA expression was modulated by EDRF1 gene transfection. Electrophoretic mobility shift assay suggested that the DNA-binding activity of GATA-1 was remarkably inhibited in K562 cells expressing EDRF1 antisense gene. DNA binding activity of NF-E2 was at the same level as control experiment. Therefore EDRF1 may play a role in erythroid proliferation and differentiation by affecting the interaction between GATA-1 and its cis-elements.     

Fast apoptosis and erythroid differentiation induced by imatinib mesylate in JURL-MK1 cells

We compare the effects of Imatinib mesylate (Glivec) on chronic myeloid leukemia derived cell lines K562 and JURL-MK1. In both cell lines, the cell cycle arrests in G(1)/G(0) phase within 24 h after the addition of 1 microM Imatinib. This is followed by a decrease of Ki-67 expression and the induction of apoptosis. In JURL-MK1 cells, the apoptosis is faster in comparison with K562 cells: the caspase-3 activity reaches the peak value (20 to 30 fold of the control) after about 40 h and the apoptosis proceeds to its culmination point, the DNA fragmentation, within 48 h following 1 microM Imatinib addition. Unlike K562 cells, JURL-MK1 cells possess a probably functional p53 protein inducible by TPA (tetradecanoyl phorbol acetate) or UV-B irradiation. However, no increase in p53 expression was observed in Imatinib-treated JURL-MK1 cells indicating that the difference in the apoptosis rate between the two cell lines is not due to the lack of p53 in K562 cells. Imatinib also triggers erythroid differentiation both in JURL-MK1 and K562 cells. Glycophorin A expression occurred simultaneously with the apoptosis, even at the single cell level. In K562 cells, but not in JURL-MK1 cells, the differentiation process involved increased hemoglobin synthesis. However, during spontaneous evolution of JURL-MK1 cells in culture, the effects produced by Imatinib progressively changed from the fast apoptosis to the more complete erythroid differentiation. We suggest that the apoptosis and the erythroid differentiation are parallel effects of Imatinib and their relative contributions, kinetics and completeness are related to the differentiation stage of the treated cells.     

Effect of bacitracin on erythroid differentiation of MEL cells

Bacitracin, an antibiotic widely utilized in clinical and veterinary use, was tested on murine erythroleukemia (MEL) cells. Tests were performed to evaluate the capacity of the drug to interfere with erythroid differentiation. Cells were exposed to a single treatment in S phase at sublethal doses of bacitracin. Two responses were found depending on the drug concentration. At higher concentrations (25 g/ml and 250ng/ml) a reduction in number of differentiating cells was observed but the kinetics of the process remained unchanged. At lower concentrations (from 2.5 ng/ml to 2.5 fglml) a dramatic alteration of the dynamic of differentiation was found. These two responses are related to different activities of the DNA repair mechanisms. Higher doses of bacitracin stimulate repair while lower concentrations are not able to activate repair, as demonstrated by tests with hydroxyurea. The bacitracin-induced damage can be considered a stable genetic andlor epigenetic alteration, as demonstrated by the high frequency of mutant clones isolatedfrom low-dose treated cells. The suitability of MEL cells system in evaluating genotoxicity of drugs for veterinary use is underlined.Abbreviations MEL murine erythroleukemia - HU hydroxyurea     

MiR-543 regulates myoblast proliferation and differentiation of C2C12 cells by targeting KLF6

MicroRNA-543 (miR-543) has been found to play a suppressive role in various human cancers in many studies, whereas the specific functions of miR-543 in muscle development remain poorly understood. Here, we found that the expression of miR-543 was high in skeletal muscle and increased during the differentiation of C2C12 cells. Overexpression of miR-543 repressed C2C12 cell proliferation and promoted differentiation, while knockdown of miR-543 expression produced the opposite results. During myogenesis, we predicted and verified that Krüppel-like factor 6 (KLF6), a suppressor of multiple tumor cells, was a target gene of miR-543. Then, miR-543 was found to specifically target KLF6 and repress its expression. Besides this, knockdown of KLF6 promoted the differentiation but inhibited the proliferation of C2C12 cells. Si-KLF6 can rescue the influence of miR-543 inhibitor on C2C12 cell differentiation. Our results indicate a new regulatory mechanism of miR-543 on KLF6 expression and suggest the possibility of using the miR-543/KLF6 pathway as a potential target for studying myogenesis.     

Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells

Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.