Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster

DNA fragmentation, a hallmark of apoptosis, is regulated by a specific nucl ease called caspase-activated DNase (CAD) and its inhibitor (ICAD). When ce ll lysates from Drosophila S2 cells were chemically denatured and the denat ured proteins were removed after dialysis, the supernatant inhibited Drosop hila CAD (dCAD). To identify the inhibitor, we tested recombinant DREP-1, w hich was previously identified using the Drosophila EST data base and found it also inhibited dCAD DNase. An antibody against DREP-1 inhibited the ICA D activity in the S2 cell extracts, confirming the identification of DREP-1 as a Drosophila homolog of ICAD (dICAD). The recombinant DREP-1/dICAD was cleaved at a specific site by human caspase 3 as well as by extracts prepar ed from S2 cells undergoing apoptosis. Biochemical fractionation and immuno precipitation of dICAD from S2 cell extracts indicated that dICAD is comple xed with dCAD in proliferating cells. The expression of the caspase-resista nt form of dICAD/DREP-1 in a Drosophila neuronal cell line prevented the ap optotic DNA fragmentation. Northern hybridization and the immunohistochemic al analyses revealed that the expression of the dICAD gene is developmental ly regulated.