Type 1 and type 2 diabetes are characterized by progressive beta cell failure. Moreover, a substantial proportion of the transplanted islet mass fails to engraft due to death by apoptosis. The goal of this project is to explore novel molecular targets involved in the induction of beta cell death. To meet this objective, we developed an siRNA high content screen in MIN6 beta cell line. i. We optimized conditions to induce death by a cocktail of pro-inflammatory cytokines (TNF-a, IL-1-b and IFN-g) and optimize means to detect the effects of these cytokines on cell viability; ii. We optimized conditions for gene silencing by siRNA in Min6 cells; iii. We provided a ‘Proof of Principle’ that siRNAs directed towards selected known pro-apoptotic genes or cytokine receptors can inhibit death of Min6 cells; iv. We screened siRNA libraries constisting of ~4000 genes, having the potential to promote ß-cell death, in order to identify targets whose inhibition protects ß-cells from cytokine-induced death, or genes that further induce beta cell death by their inhibition. We screened the cells in an automated microscope to visually detect wells with high viability rates and we then applied two biochemical assays to detect caspase 3 activity and cell viability.
A few obvious genes were detected, the siRNAs of which inhibited cytomix-induced caspase-3 activity. These included caspase-3 itself as well as siRNAs to known pro-apoptotic genes such as PLK, Bid and FADD. These results attest as to the validity and reliability of the method applied. The pre-screen already revealed some unexpected genes that seems to be key players in cytokine-induced beta cell apoptosis. Validation of these findings is currently underway.
