Antisense oligonucleotide of clusterin mRNA induces apoptotic cell death and prevents adhesion of rat ASC-17D Sertoli cells

Clusterin has been known to play important roles in cell-cell and/or cell-s ubstratum interactions. Recently we reported the transient expression of cl usterin in pancreatic endocrine cells during the early developmental stages and suggested a role in aggregating the endocrine cells for islet formatio n. In the present study, we have investigated the involvement of clusterin in cell-substratum interaction by the inhibition of clusterin synthesis usi ng antisense oligonucleotide, The expression of clusterin was transiently i ncreased as early as 2-8 h after plating the ASC-17D Sertoli cells to the c ulture flask, which was the period of cell attachment. In addition, up-regu lation of clusterin mRNA was so much greater when the Sertoli cells were pl ated on the petri dish for the bacterial culture instead of in a animal cel l culture flask that therefore, the cells failed to attach to it. These fin dings suggested that interruption of cell to plate substratum interaction m ight lead to over-expression of clusterin from Sertoli cells to induce cell to cell aggregation or, perhaps, to re-establish attachment with the subst ratum. Transfection of ASC-17D Sertoli cells with a 20-base antisense oligo nucleotide against clusterin mRNA resulted in estracellular release of LDH and DNA fragmentation. Sertoli cell death by antisense oligonucleotide of c lusterin was sequence specific and dose dependent. Treatment of antisense o ligonucleotide induced a marked reduction of synthesis for clusterin protei n, but not for clusterin mRNA expression, suggesting the translational supp ression of clusterin by antisense oligonucleotide, Further, microscopic obs ervation showed that more noticeable cell death was induced by treating the antisense prior to plating the cells than by treating after cell attachmen t to the plate. From these results, we speculate that down-regulation of cl usterin expression in the anchorage-dependent Sertoli cells prevents them f rom attaching to the plate, and therefore induces cell death.