Caspases play an essential role in the execution of programmed cell death in metazoans. Although 14 caspases are known in mammals, only a few have been described in other organisms. Here we describe the identification and characterization of a Drosophila caspase, DRONC, that contains an amino terminal caspase recruitment domain. Ectopic expression of DRONC in cultured cells resulted in apoptosis, which was inhibited by the caspase inhibitors p35 and MIHA. DRONC exhibited a substrate specificity similar to mammalian caspase-2. DRONC is ubiquitously expressed in Drosophila embryos during early stages of development. In late third instar larvae, dronc mRNA is dramatically up-regulated in salivary glands and midgut before histolysis of these tissues. Exposure of salivary glands and midgut isolated from second instar larvae to ecdysone resulted in a massive increase in dronc mRNA levels. These results suggest that DRONC is an effector of steroid-mediated apoptosis during insect metamorphosis.

The release of cytochrome c from mitochondria is necessary for the formation of the Apaf-1 apoptosome and subsequent activation of caspase-9 in mammalian cells. However, the role of cytochrome c in caspase activation in Drosophila cells is not well understood. We demonstrate here that cytochrome c remains associated with mitochondria during apoptosis of Drosophila cells and that the initiator caspase DRONC and effector caspase DRICE are activated after various death stimuli without any significant release of cytochrome c in the cytosol. Ectopic expression of the proapoptotic Bcl-2 protein, DEBCL, also fails to show any cytochrome c release from mitochondria. A significant proportion of cellular DRONC and DRICE appears to localize near mitochondria, suggesting that an apoptosome may form in the vicinity of mitochondria in the absence of cytochrome c release. In vitro, DRONC was recruited to a >700-kD complex, similar to the mammalian apoptosome in cell extracts supplemented with cytochrome c and dATP. These results suggest that caspase activation in insects follows a more primitive mechanism that may be the precursor to the caspase activation pathways in mammals.

Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-2 ^−/− mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with E1A and Ras oncogenes, caspase-2 ^−/− MEFs grew significantly faster than caspase-2 ^+/+ MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse Eμ- Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-2 ^−/− mice. The caspase-2 ^−/− cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-2 ^−/− MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following γ-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein.

Dronc is a caspase recruitment domain-containingDrosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Here we show that ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99complex (reaper, hid, and grim) and enhanced by mutations in diap1. In contrast to previous reports, we show that the dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitorp35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Dark. Furthermore, extracts made from Dark homozygous mutant flies have reduced ability to process Dronc, showing that Dark is required for Dronc processing. Finally, using the RNA interference technique, we show that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death inDrosophila.