BASIC STUDY ON GENE THERAPY FOR EWING'S SARCOMA・・・BY INTRODUCING ANTISENSE EWS/FLI-1 GENE・・・

摘要本研究在巨细胞病毒启动子作用下，将以反义方式克隆EWS/ fl -1全长C-DNA构建的EWS/ fl -1反义表达质粒转染到pCDNA表达载体中，制备了表达反义EWS/ fl -1转录本的稳定的尤文氏肉瘤细胞。用新霉素筛选得到10个克隆。在10个克隆中，我们用FLI-1抗体对3个克隆进行了免疫沉淀实验，用RT-PCR检测了EWS/FLI-1的反义m-RNA转录本，结果发现3个克隆中EWS/FLI-1蛋白的表达均下降。与仅转染pCDNA载体的亲本细胞相比，表达反义EWS/FLI-1转录本的克隆在裸鼠体内的生长速度较低，并且丧失了锚定独立生长和致瘤性。为了更好地了解ews / fli-1基因对尤文氏肉瘤致瘤性的作用机制，我们分析了pkc、plc、pld等参与信号转导的基因。WESTERN BLOTTING发现，在引入EWS/FLI-1反义蛋白的细胞中，plc - β 2、β 3、pkc - α、β 1、β 2减少，而plc - γ 1、δ 1、β 1、RhoA和CDC42不变。pma和PDGF刺激的PLD活性降低了50-65%，PDGF诱导的ip3生成也减少。然后合成针对ews / fli-1的核酶，在体外裂解ews / fli-1 m-rna，抑制ews / fli蛋白的生长和表达。这些数据提示，靶向ews / fli-1产物可能是治疗尤文氏肉瘤的有效途径。

IN THE PRESENT STUDY,WE MADE STABLE EWING'S SARCOMA CELLS EXPRESSING ANTISENSE EWS/FLI-1 TRANSCRIPTS BY TRANSFECTING THE ANTISENSE EWS/FLI-1 EXPRESSION PLASMID WHICH WAS CONSTRUCTED BY CLONING THE FULL LENGTH OF EWS/FLI-1 C-DNA IN ANTISENSE MANNER INTO pCDNA EXPRESSION VECTOR UNDER CYTOMEGALOVIRUS PROMOTER.BY SELECTION WITH NEOMYCIN,10 CLONES WERE OBTAINED.OUT OF THE 10 CLONES,WE STUDIED IMMUNOPRECIPITATION ASSAY WITH FLI-1 ANTIBODY IN 3 CLONES WHICH COULD BE DETECTED ANTISENSE EWS/FLI-1 m-RNA TRANSCRIPTS BY RT-PCR,AND WE FOUND THAT EWS/FLI-1 PROTEIN EXPRESSION DECREASED IN ALL OF THE 3 CLONES.COMPARED WITH THE PARENTAL CELLS WHICH WAS TRANSFECTED ONLY pCDNA VECTOR,THE CLONES EXPRESSING ANTISENSE EWS/FLI-1 TRANSCRIPTS SHOWED LOW GROWTH RATE AND LOSS OF ANCHORAGE INDEPENDENT GROWTH AND TUMORIGENICITY IN NUDE MICE.FOR THE BETTER UNDERSTANDING OF THE MECHANISM ON TUMORIGENICITY OF EWING'S SARCOMA BY EWS/FLI-1 GENE,WE ANALYZED SEVERAL GENES INVOLVED IN SIGNAL TRANSDUCTION SUCH AS PKC,PLC,PLD.BASED ON WESTERN BLOTTING,IN THE CELLS INTRODUCED ANTISENSE OF EWS/FLI-1, PLC-beta2, beta3, PKC-alpha, beta1, beta2DECREASED,WHILE PLC-gamma1, delta1, beta1, RhoA AND CDC42 WERE UNCHANGED.PLD ACTIVATION STIMULATED BY PMA AND PDGF WAS REDUCED BY 50-65% AND IP3 FORMATION BY PDGF ALSO DECREASED.THEN WE SYNPHESIZED RIBOZYMEZS AGAINST THE EWS/FLI-1, AND THEY CLEAVED THE EWS/FLI-1 M-RNA IN VITRO AND INHIBITED CELL GROWTH AND EXPRESSION OF THE EWS/FLI-I PROTEIN.THESE DATA SUGGEST THAT TARGETING THE EWS/FLI-1 PRODUCTS MAY BE USEFUL APPROACH FOR THE GENE THERAPY FOR EWING'S SARCOMA.