Louis Deiss

Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha.

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.

DAP-5, a novel homolog of eukaryotic translation initiation factor 4G isolated as a putative modulator of gamma interferon-induced programmed cell death.

A functional approach to gene cloning was applied to HeLa cells in an attempt to isolate cDNA fragments which convey resistance to gamma interferon (IFN-gamma)-induced programmed cell death. One of the rescued cDNAs, described in this work, was a fragment of a novel gene, named DAP-5. Analysis of a DAP-5 full-length cDNA clone revealed that it codes for a 97-kDa protein that is highly homologous to eukaryotic translation initiation factor 4G (eIF4G, also known as p220). According to its deduced amino acid sequence, this novel protein lacks the N-terminal region of eIF4G responsible for association with the cap binding protein eIF4E. The N-terminal part of DAP-5 has 39% identity and 63% similarity to the central region of mammalian p220. Its C-terminal part is less homologous to the corresponding region of p220, suggesting that it may possess unique functional properties. The rescued DAP-5 cDNA fragment which conveyed resistance to IFN-gamma-induced cell death was expressed from the vector in the sense orientation. Intriguingly, it comprised part of the coding region which corresponds to the less conserved C-terminal part of DAP-5 and directed the synthesis of a 28-kDa miniprotein. The miniprotein exerted a dual effect on HeLa cells. Low levels of expression protected the cells from IFN-gamma-induced programmed cell death, while high levels of expression were not compatible with continuous cell growth. The relevance of DAP-5 protein to possible changes in a cells translational machinery during programmed cell death and growth arrest is discussed.

Apoptosis and Cancer

Apoptosis, or Programmed Cell Death, is the activation of an inherent cellular suicide program that results in cell death [1]. There are two hallmarks in the death process. First, is a set of distinct morphologic changes such as membrane blebbing, cell shrinkage, and chromosomal condensation followed by chromosomal fragmentation. Second, is the rapid phagocytosis of the corpses of the dead cells, resulting in a limited local immune response. Recently it has been demonstrated that apoptosis plays a crucial role in cardiac damage and a variety of human diseases such as acute liver failure, Alzheimer’s disease, and cancer [2]. The realization that apoptosis constitutes a major mechanism of tumor suppression has dramatically advanced tumor biology by leading to a number of novel approaches for preventing, diagnosing, and treating cancer. The guiding principles for understanding and manipulating the apoptotic response have emerged from studies of both lower eukaryotes and mammalian model systems [3]. These principles, as well as strategies designed to harness the apoptotic response, are described in this chapter.