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

In this study we have designed and constructed an anti-Fas ribozyme and sho w that it can specifically cleave the Fas mRNA in vitro, Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into beta PTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expressi on. Like pancreatic beta cells, beta TC-3 cells do not constitutively expre ss Fas, but Fas expression can be induced with IL-1 and IFN-gamma, Transfec ted cells expressed an average of 5000 copies of anti-Fas ribozyme transcri pt per cell as assessed by reverse transcriptase-real-time PCR, After IL-1/ IFN-gamma treatment, beta TC-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 cel ls and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in beta TC-3 cells expressing the anti-Fas ribozy me correlated with resistance to Fas-mediated apoptosis as determined by th e number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the beta TC- S 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 s ignificantly improve the survival of islet cell grafts.