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

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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.