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     

Initial function analysis of a novel erythroid differentiation related geneEDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

Initial function analysis of a novel erythroid differentiation related gene EDRF1

Erythroid differentiation depends on the establishment of specific patterns of gene expression. Hypersensitive site 2 (HS2, serving as a major enhancer of globin genes)-binding proteins may be involved in its natural open chromosomal environment formation. Previously we prepared monoclonal antibodies against HS2-binding nuclear proteins of terminal differentiated erythroid cells. By utilizing the monoclonal antibodies, we screened λ-gt11 human fetal liver cDNA expression library and obtained one cDNA clone, which was named erythroid differentiation related gene (EDRF1, Genbank accession number AF040247), encompassing an entire open reading frame. We investigated the expression pattern ofEDRF1 by RT-PCR technique. And a clue to the function ofEDRF1 has been found from confirmation of high levels ofEDRF1 mRNA in differentiated K562 and human fetal liver tissue. To illuminate the function ofEDRF1 in K562 cells, sense and antisenseEDRF1 constructs were prepared and transfected into K562 cells. α-globin mRNA was down-regulated and EpoR (erythropoietin receptor) mRNA expression was increased in antisense transfected cells. Cells transfected with sense construct grew more slowly than control cells suggested by [³H] thimidine incorporation experiments. Suppression of K562 proliferation was accompanied by increased spontaneous hemoglobin synthesis demonstrated by spectrometry. K562 cells transfected with sense construct exhibited reduced clongenicity compared with control cells in methycellulose culture. These data provided the evidence thatEDRF1 can influence globin expression and hemoglobin synthesis in K562 cells and modulated self-renewal in K562 cells.     

cDNA cloning and function analysis of two novel erythroid differentiation related genes

Our previous studies showed that some nuclear proteins that were expressed especially during terminal differentiation of erythroid cells might interact directly or indirectly with HS2 sequence to form the HS2-protein complexes and thus play an important role in the globin gene regulation and erythroid differentiation. Monoclonal antibodies against the nuclear proteins of terminal differentiated erythroid cells, including intermediate and late erythroblasts of human fetal liver and hemin induced K562 cells, were prepared by hybridoma technique. The monoclonal antibodies were used to screen λ-gtll human cDNA expression library of fetal liver in order to obtain the relevant cDNA clones. By the analysis of their cDNA clones and the identification of the proteins' functions, the regulation mechanism of the HS2 binding proteins might be better understood. Two cDNA clones (GenBank accession number AF040247 and AF040248 respectively) were obtained and one of them owns a full length and the other encodes a prote     

Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARγ) pathway

Evodiamine, a quinolone alkaloid, is one of the major bioactive compounds of Evodia rutaecarpa Bentham (Rutaceae). It exhibits excellent biological activities, especially the anticancer activity. This study aims to investigate the effect of evodiamine on the proliferation of leukemia cell line K562 and to explore the underlying mechanism. The effect of evodiamine on K562 cells proliferation was analyzed by trypan blue dye exclusion assay and MTT assay. The expression levels of peroxisome proliferators-activated receptor gamma (PPARγ), cyclin D1, and p21 were detected by western blot assay. The results demonstrated that evodiamine inhibited the proliferation and decreased the viability of K562 cells in a dose- and time-dependent manner. 2-Chloro-5-nitro-N-phenylbenzamide (GW9662) and/or PPARγ-siRNA pretreatment alleviated the cell growth suppression triggered by evodiamine. Meanwhile, evodiamine intervention elevated the expression of PPARγ in K562 cells, while pretreatment with GW9662 attenuated the enhanced upregulation of PPARγ expression induced by evodiamine. In addition, GW9662 and PPARγ-siRNA pretreatment also significantly attenuated the downregulation of the cell cycle control protein cyclin D1 and the upregulation of cyclin-dependent kinase inhibitor p21 induced by evodiamine. In conclusion, PPARγ signaling pathway may involve in the proliferation inhibition of evodiamine on K562 cells via inhibiting cylcin D1 and stimulating of p21.     

Antizyme1基因转染对K562细胞增殖与凋亡的影响

研究鸟氨酸脱羧酶抗酶蛋白对人红白血病K562细胞增殖、三氧化二砷（ As2O3）诱导凋亡时的影响。方法: 定点突变技术构建缺失frameshift位点的pEGFP-N1-AZ1-mutation重组表达载体。脂质体法转染K562细胞,通过G418筛选获得稳定表达antizyme1的K562pAZ1m细胞系。采用不同浓度的As2O3处理细胞,通过MTT法检测细胞增殖,流式细胞术分析细胞周期及凋亡变化。并通过RT-PCR方法检测antiyme1转染对cyclin D1和survivin基因表达的影响。结果：获得稳定表达antizyme1的K562-AZ1m细胞株后,其增殖能力明显减慢。CyclinD1基因表达降低,细胞主要停滞于G0/G1期。在 As2O3的诱导作用下,细胞凋亡增多,survivin基因表达降低。结论：AZ1基因能够抑制K562细胞增殖,通过对cyclinD1的负调控使细胞周期停滞于G0/G1期。并可能通过下调survivin表达来加强 As2O3对其的诱导凋亡作用     

X连锁凋亡抑制蛋白反义寡核苷酸逆转K562细胞及难治性癫痫大鼠对卡马西平和苯妥英钠耐药性

凋亡在癫痫发生机制中起重要作用,但其在难治性癫痫耐药机制中的作用尚不清楚．为研究X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein, XIAP)反义寡核苷酸对K562/Dox(阿霉素诱导)耐药细胞及难治性癫痫大鼠耐药性的影响,首先建立耐药的K562/Dox细胞株,比较XIAP在耐药细胞株和正常K562细胞株的表达情况,观察转染XIAP反义寡核苷酸后,线粒体膜电位变化以及对卡马西平和苯妥英钠耐药性的影响．另外,建立慢性杏仁核点燃癫痫模型,筛选出耐药组和药物敏感组,通过侧脑室注射XIAP反义寡核苷酸,对照组注射生理盐水．观察其对各组大鼠后放电阈值(after discharge threshold,ADT)、后放电时程(after discharge duration,ADD)等电生理指标的影响．结果发现,XIAP在K562/Dox耐药细胞上的表达明显高于正常K562细胞,XIAP反义寡核苷酸转染K562/Dox耐药细胞后,XIAP的表达明显下降．导致了K562/Dox细胞线粒体跨膜电位的下降,而且对苯妥英钠和卡马西平的耐药性明显下降,IC₅₀分别由(1 978.2 ± 90.3) mg/L和(1 875.6 ± 83.2) mg/L,降低到(1 123.5 ± 54.2) mg/L和(1 084.5 ± 60.6) mg/L,逆转倍数分别为1.76和1.73．同时动物实验发现,耐药组大鼠在给予XIAP反义寡核苷酸后,ADT明显高于对照组(P < 0.05), ADD时程也明显缩短．上述结果证明,XIAP在耐药的K562/Dox细胞株存在高表达,下调XIAP在K562/Dox细胞株表达可以改善K562/Dox对卡马西平和苯妥英钠的耐药性．而且下调XIAP表达可以协助AEDs改善耐药大鼠的电生理活动,提示XIAP参与了难治性癫痫的耐药．