Programmed cell death is often triggered by the interaction of some cytokines with their cell surface receptors. Here, we report that ~/interferon (IFN-~/) induced in HeLa cells a type of cell death that had cytological characteristics of programmed cell death. In this system we have identified two novel genes whose expression was indispensable for the execution of this type of cell death. The rescue was based on positive growth selection of cells after transfection with antisense cDNA expression libraries. The antisense RNA-mediated inactivation of the two novel genes protected the cells from the IFN-~/-induced cell death but not from the cytostatic effects of the cytokine or from a necrotic type of cell death. One of those genes (DAP-1) is expressed as a single 2.4-kb mRNA that codes for a basic, proline-rich, 15-kD protein. The second is transcribed into a single 6.3-kb mRNA and codes for a unique 160-kD calmodulin-dependent serine/threonine kinase (DAP kinase) that carries eight ankyrin repeats. The expression levels of the two DAP proteins were selectively reduced by the corresponding antisense RNAs. Altogether, it is suggested that these two novel genes are candidates for positive mediators of programmed cell death that is induced by IFN-~/.

Kissil et al., 1995). Other laboratories adapted localized to the cytoskeleton, in association with the similar functional selection strategies and candidate genes microfilament system, and mapped a region within that mediate cell death induced by IL-3 deprivation or by the protein which is responsible for binding to the T-cell receptor activation were cloned including Requiem cytoskeleton. Several assays attributed a cell death (Gabig et al., 1994), ALG-2 and ALG-3 (Vito et al., 1996). function to the gene. Ectopic expression of wild-type Our method, called Technical Knock Out (TKO), was DAP-kinase induced the death of target cells, and the based on random inactivation of genes via introduction of killing property depended strictly on the status of the anti-sense cDNA expression libraries, prepared from a intrinsic kinase activity. Conversely, a catalytically mixture of non-treated and IFN-γ-treated cells. The genes inactive mutant that carried a lysine to alanine substituof interest were selected and cloned by virtue of the tion within the kinase domain, displayed dominantdefined phenotypic change-reduced susceptibility to the negative features and protected cells from interferoncytokine-induced cell death-which resulted from their γ-induced cell death. DAP-kinase is therefore a novel inactivation. HeLa cells, transfected with Epstein-Barr cytoskeletal-associated cell death serine/threonine virus (EBV)-based vectors carrying anti-sense cDNA kinase whose activation by Ca 2⍣ /calmodulin may be libraries, were subjected to positive selection of cells that linked to the biochemical mechanism underlying the survived in the continuous presence of IFN-γ. The rescued cytoskeletal alterations that occur during cell death.plasmids that were positively scored in a second round of Keywords: calcium/calmodulin/cytoskeleton/programmed transfection carried six non-overlapping groups of cDNA cell death/serine-threonine kinase fragments. Sequence analysis indicated that five of them corresponded to novel genes: DAPs 1-5 . The sixth gene was identical to a known aspartic protease, i.e. cathepsin D, the participation of which was

DAP kinase is a new type of calcium/calmodulin-dependent enzyme that phosphorylates serine/threonine residues on proteins. Its structure contains ankyrin repeats and the 'death' domain, and it is associated with the cell cytoskeleton. The gene encoding DAP kinase was initially isolated as a positive mediator of apoptosis induced by interferon-gamma, by using a strategy of functional cloning. We have now tested whether this gene has tumour-suppressive activity. We found that lung carcinoma clones, characterized by their highly aggressive metastatic behaviour and originating from two independent murine lung tumours, did not express DAP kinase, in contrast to their low-metastatic counterparts. Restoration of DAP kinase to physiological levels in high-metastatic Lewis carcinoma cells suppressed their ability to form lung metastases after intravenous injection into syngeneic mice, and delayed local tumour growth in a foreign 'microenvironment' Conversely, in vivo selection of rare lung lesions following injection into syngeneic mice of low-metastatic Lewis carcinoma cells or of DAP kinase transfectants, was associated with loss of DAP kinase expression. In situ TUNEL staining of tumour sections revealed that DAP kinase expression from the transgene raised the incidence of apoptosis in vivo. DAP-kinase transfectants also showed increased sensitivity in vitro to apoptotic stimuli, of the sort encountered by metastasizing cells at different stages of malignancy. We propose that loss of DAP kinase expression provides a unique mechanism that links suppression of apoptosis to metastasis.

A functional approach of gene cloning was applied to HeLa cells in an attempt to isolate positive mediators of programmed cell death. The approach was based on random inactivation of genes by transfections with antisense cDNA expression libraries, followed by the selection of cells that survived in the presence of the external apoptotic stimulus. An antisense cDNA fragment identical to human cathepsin D aspartic protease was rescued by this positive selection. The high cathepsin D antisense RNA levels protected the HeLa cells from interferon‐gamma‐ and Fas/APO‐1‐induced death. Pepstatin A, an inhibitor of cathepsin D, suppressed cell death in these systems and interfered with the TNF‐alpha‐induced programmed cell death of U937 cells as well. During cell death, expression of cathepsin D was elevated and processing of the protein was affected, which resulted in high steady‐state levels of an intermediate, proteolytically active, single chain form of this protease. Overexpression of cathepsin D by ectopic expression induced cell death in the absence of any external stimulus. Altogether, these results suggest that this well‐known endoprotease plays an active role in cytokine‐induced programmed cell death, thus adding cathepsin D to the growing list of proteases that function as positive mediators of apoptosis.

In this study we describe the identification and structure-function analysis of a novel death-associated protein (DAP) kinase-related protein, DRP-1. DRP-1 is a 42-kDa Ca