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.