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

本研究将EWS/Fli-1 C-DNA全长反义克隆到巨细胞病毒载体pCDNA表达载体中，构建成反义EWS/Fli-1表达载体，经新霉素筛选，获得10个稳定表达反义EWS/Fli-1转录本的克隆。对其中3个经RT-PCR检测到反义EWS/Fli-1 m-RNA的克隆进行了免疫沉淀实验。与只转导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慢，失去了锚定非依赖性生长和致瘤性。为了更好地了解EWS/Fli-1基因对尤文肉瘤致瘤作用的机制，我们分析了几个参与信号转导的基因，如PKC、PLC、PLD、Western blotting，在细胞内引入了反义EWS/Fli-1、PLC-Beta2、Beta3、PKC-α、Beta1、Beta2DECASEDDelta1、β1、RhoA和CDC42为UNCHANGED。PMA和PDGF刺激的PLD活性降低50-65%，PDGF也抑制IP3的形成。我们合成了针对EWS/FLI-1的RIBOZYMEZS，它们在体外切割EWS/FLI-1 M-RNA，抑制细胞生长和EWS/FLI-I蛋白的表达。

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.