Identification and Developmental Expression of Inhibitor of Caspase-activated DNase (ICAD) in Drosophila melanogaster

Abstract

DNA fragmentation, a hallmark of apoptosis, is regulated by a specific nuclease called caspase-activated DNase (CAD) and its inhibitor (ICAD). When cell lysates from Drosophila S2 cells were chemically denatured and the denatured proteins were removed after dialysis, the supernatant inhibited Drosophila CAD (dCAD). To identify the inhibitor, we tested recombinant DREP-1, which was previously identified using the Drosophila EST data base and found it also inhibited dCAD DNase. An antibody against DREP-1 inhibited the ICAD 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 prepared from S2 cells undergoing apoptosis. Biochemical fractionation and immunoprecipitation of dICAD from S2 cell extracts indicated that dICAD is complexed with dCAD in proliferating cells. The expression of the caspase-resistant form of dICAD/DREP-1 in a Drosophila neuronal cell line prevented the apoptotic DNA fragmentation. Northern hybridization and the immunohistochemical analyses revealed that the expression of the dICAD gene is developmentally regulated.

Degradation of chromosomal DNA during apoptosis

“…104 No specific function has been assigned to ICAD-S, and it is probably dispensable in most if not all species, as has been shown in Drosophila. 48 In this regard, results by Chen et al 49 indicating that rat and mouse tissues express only ICAD-S are difficult to explain. Human and mouse ICADs carry two caspase-recognition sites, at Asp-117 and 224, 30,44 while there is a single caspase recognition site in Drosophila ICAD at Asp-119.…”

“…On the other hand, the large-scale degradation of chromosomal DNA was observed in chicken DT40 cells lacking CAD, and Samejima et al 75 proposed that an ICAD-inhibitable non-CAD DNase causes this type of degradation. No caspase-dependent DNase activity was detected when the extracts from the thymocytes from CADnull mice were treated with caspase 3 (HN, and SN, unpublished results), and all the ICAD seems to be associated with CAD in Drosophila cells, 48 indicating that there are no additional ICAD-inhibitable DNases in mouse and Drosophila cells. Purification and characterization of the nuclease that causes the large-scale DNA degradation from chicken cells will be necessary to clarify this apparent controversy.…”

“…Splenocytes, thymocytes, and granulocytes from ICAD (DFF45)-null mice are resistant to DNA fragmentation upon exposure to various apoptotic stimuli such as staurosporine, etoposide, and actinomycin D. 47,76 Human cell lines (Jurkat lymphoma and IL-3-dependent TF-1 myeloid lymphoma) and the Drosophila BG-2 neuronal cell line that were transformed with the caspase-resistant ICAD, or thymocytes from the transgenic mice carrying the caspase-resistant ICAD gene, do not undergo DNA fragmentation after treatment with Fas ligand, factor deprivation, anticancer drugs, or kinase inhibitors. 48,50,73 Chicken DT40 cells that lack the CAD gene do not show fragmented DNA after treatment with etoposide. 75 Lymphocytes and hepatocytes from the CAD-null mice are also resistant to the apoptotic DNA fragmentation ( Figure 2, data not shown).…”

“…Human and mouse ICADs carry two caspase-recognition sites, at Asp-117 and 224, 30,44 while there is a single caspase recognition site in Drosophila ICAD at Asp-119. 48 Among members of the mammalian caspase family, caspases 3 and 7 cleave ICAD. 50,51 Mammalian ICAD must be cleaved at both sites to lose its CAD-inhibiting activity.…”

CIDE domains form functionally important higher-order assemblies for DNA fragmentation

“…S1A). In Drosophila, four DFF-related proteins (Drep1-4) are critical for apoptotic DNA fragmentation (10)(11)(12) (Fig. S1A), and Drep2 also acts as a unique synaptic protein important in learning and behavioral adaptation (13).…”