1996 Fiscal Year Final Research Report Summary
BASIC STUDY ON GENE THERAPY FOR EWING'S SARCOMA・・・BY INTRODUCING ANTISENSE EWS/FLI-1 GENE・・・
| Project/Area Number |
07671579
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Orthopaedic surgery
|
|---|
| Research Institution | DEPARTMENT OF ORTHOPAEDIC SURGERY,GIFU UNIVERSITY SCHOOL OF MEDICINE |
|---|
Principal Investigator |
OHNO Takatoshi GIFU UNIVERSITY SCHOOL OF MEDICINE,DEPARTMENT OF ORTHOPEDIC SURGERY,RESEARCH ASSOCIATE, 医学部, 助手 (60281052)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NISHIMOTO Yutaka GIFU UNIVERSITY SCHOOL OF MEDICINE,DEPARTMENT OF ORTHOPEDIC SURGERY,RESEARCH ASS, 医学部・附属病院, 助手 (20208234)
|
|---|
| Project Period (FY) |
1995 – 1996
|
| Keywords | EWING'S SARCOMA / EWS GENE / GENE THERAPY / ANTISENSE / RIBOZYME / SIGNAL TRANSDUCTION / CHIMERIC GENE |
|---|
| Research Abstract |
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.
|
