condense, and the organelles and plasma membrane retain their integrity in a process Kerr and his colleagues and Martin C. Raff Developmental Neurobiology Programme named apoptosis. The dead cells or their fragments are rapidly phagocytosed by neighboring cells or macro-MRC Laboratory for Molecular Cell Biology University College London phages before there is any leakage of the contents of the cells, and thus they do not induce an inflammatory London, WC1E 6BT United Kingdom response. Apoptotic cells in developing tissues are almost always inside other cells (Figures 1A-1C), suggesting that dying cells are usually phagocytosed before they display the morphological changes of apoptosis. Programmed cell death (PCD) occurs during the devel-

When the mammalian proto-oncogene bcl-2 is overexpressed it can protect various types of cells both from normal and from experimentally induced apoptosis, but the molecular mechanisms involved are unknown. Although the Bcl-2 protein is membrane-associated, its subcellular location is controversial: two studies have suggested that it is mainly associated with the nuclear envelope and endoplasmic reticulum, whereas another study has suggested that it is mainly located in the inner mitochondrial membrane. The latter study has suggested that Bcl-2 might protect cells from apoptosis by altering mitochondrial function and that mitochondria may be involved in apoptosis. Here we report that human mutant cell lines that lack mitochondrial DNA (mtDNA), and therefore do not have a functional respiratory chain, can still be induced to die by apoptosis, and that they can be protected from apoptosis by the overexpression of bcl-2, suggesting that neither apoptosis nor the protective effect of bcl-2 depends on mitochondrial respiration. We also show that the Bcl-2 protein in overexpressing cells is associated with the nuclear envelope and endoplasmic reticulum, as well as with mitochondria.

Programmed cell death (PCD) is a fundamental feature of animal cells, but the mechanism remains unknown. Similarly, the Bcl-2 oncoprotein can suppress PCD in a variety of cell types and circumstances, but it is not known how it does so. It has been suggested that PCD involves the generation of reactive oxygen species (ROS) and that Bcl-2 protects against PCD by inhibiting the generation or action of ROS. To determine whether ROS are required for PCD, we cultured cells in a near-anaerobic atmosphere where the generation of ROS would be expected not to occur, or at least to be greatly reduced. We find that these conditions inhibit PCD induced by ROS-generating agents but do not inhibit PCD induced by other means. Furthermore, we show that Bcl-2 can protect cells from PCD in these anaerobic conditions. These results suggest that ROS are not required for PCD, and that Bcl-2 protects against PCD in ways that do not depend on the inhibition of ROS production or activity.

The molecular basis of programmed cell death (PCD) is unknown. An important clue is provided by the Bcl‐2 protein, which can protect many cell types from PCD, although it is not known where or how it acts. Nuclear condensation, DNA fragmentation and a requirement for new RNA and protein synthesis are often considered hallmarks of PCD. We show here, however, that anucleate cytoplasts can undergo PCD and that Bcl‐2 and extracellular survival signals can protect them, indicating that, in some cases at least, the nucleus is not required for PCD or for Bcl‐2 or survival factor protection. We propose that PCD, like the cell cycle, is orchestrated by a cytoplasmic regulator that has multiple intracellular targets.

In situ killing of tumor cells using suicide gene transfer to generate death by a non-apoptotic pathway was associated with high immunogenicity and induction of heat shock protein (hsp) expression. In contrast, a syngeneic colorectal tumor line, CMT93, killed predominantly by apoptosis, showed low levels of hsp expression and less immunogenicity. When apoptosis was inhibited in CMT93 cells by overexpression of bcl-2, hsp was also induced. Furthermore, when cDNA encoding hsp70 was stably transfected into B16 and CMT93 cells, its expression significantly enhanced the immunogenicity of both tumors. Increased levels of hsp, induced by non-apoptotic cell killing, may provide an immunostimulatory signal in vivo which helps break tolerance to tumor antigens. These findings have important implications for the development of novel anti-cancer therapies aimed at promoting patients' immune responses to their own tumors.

The caspase recruitment domain (CARD) is a proteinbinding module that mediates the assembly of CARDcontaining proteins into apoptosis and NF-B signaling complexes. We report here that CARD protein 11 (CARD11) and CARD protein 14 (CARD14) are novel CARD-containing proteins that belong to the membraneassociated guanylate kinase (

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Abstract. In the presence of cycloheximide (CHX) to inhibit protein synthesis, a high concentration of staurosporine (STS) induces almost all cells in explant cultures of 8/8 types of newborn mouse organs and 3/3 types of adult mouse organs to die with the characteristic features of apoptosis. Eggs and blastomeres also die in this way when treated with STS and CHX, although they are less sensitive to this treatment than trophectoderm or inner cell mass cells whose sensitivity resembles that of other developing cells. Human red blood cells are exceptional in being completely resistant to treatment with STS and CHX. As (STS plus CHX)-induced cell deaths have been shown to display the characteristic features of programmed cell death (PCD), we conclude that all mammalian nucleated cells are capable of undergoing PCD and constitutively express all the proteins required to do so. It seems that the machinery for PCD is in place and ready to run, even though its activation often depends on new RNA and protein synthesis.