Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme.
In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.
Klein
D
D
Diabetes Research Institute, University of Miami School of Medicine, FL 33136, USA.
Ricordi
C
C
Pugliese
A
A
Pastori
R L
RL
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D005818:Genetic Engineering / Q000379:methods; D015316:Genetic Therapy / Q000379:methods*; D006085:Graft Survival; D007340:Insulinoma; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation / Q000276:immunology / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000235:genetics*; D012358:RNA, Transfer, Met / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000235:genetics* / Q000378:metabolism
D016337:RNA, Catalytic; D012358:RNA, Transfer, Met; D019014:fas Receptor