Inhibition of Fas-mediated apoptosis in mouse insulinoma βTC-3 cells via an anti-Fas ribozyme

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14 Scopus citations

Abstract

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 βTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic β cells, βTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-γ. 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-γ treatment, βTC-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 βTC-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 βTC-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 β cells, a feature that may significantly improve the survival of islet cell grafts.

Original languageEnglish (US)
Pages (from-to)1033-1045
Number of pages13
JournalHuman gene therapy
Volume11
Issue number7
DOIs
StatePublished - May 1 2000

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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