John L. Farber

Primary cultures of adult rat hepatocytes were treated in the presence or absence of extracellular calcium with ten different membrane-active toxins. In all cases more than half the cells were killed in 1 to 6 hours in the presence but not in the absence of extracellular calcium. An effect of calcium on the primary mechanism of membrane injury by any of the agents cannot be implicated. Viability, as determined by trypan blue exclusion correlated well with other indices of viability such as plating efficiency and the hydrolysis of fluorescein diacetate. It is concluded that the cells are killed by processes that involve at least two steps. In each type of injury, disruption of the integrity of the plasma membrane by widely differing mechanisms is followed by a common functional consequence involving extracellular calcium, and most likely representing an influx of calcium across the damaged plasma membrane and down a steep concentration gradient. This later step represents, or at least initiates, a final common pathway for the toxic death of these cells.

Stably transfected Jurkat T cells were produced in which Bax expression is inducible by muristerone A. The cell death resulting from induction of the overexpression of Bax was prevented by inhibition of the mitochondrial permeability transition (MPT) with cyclosporin A (CyA) in combination with the phospholipase A2 inhibitor aristolochic acid (ArA). The caspase-3 inhibitor Z-Asp-Glu-Val aspartic acid fluoromethylketone (Z-DEVD-FMK) had no effect on the loss of viability. The MPT was measured as the CyA plus ArA-preventable loss of the mitochondrial membrane potential (DeltaPsim). The MPT was accompanied by the release of cytochrome c from the mitochondria, caspase-3 activation in the cytosol, cleavage of the nuclear enzyme poly(ADP-ribose)polymerase (PARP), and DNA fragmentation, all of which were inhibited by CyA plus ArA. Z-DEVD-FMK had no effect on the loss of DeltaPsim and the redistribution of cytochrome c but did prevent caspase-3 activation, PARP cleavage, and DNA fragmentation. It is concluded that Bax induces the MPT, a critical event in the loss of cell viability. In addition to the cell death, the MPT mediates other typical manifestations of apoptosis in this model, namely release of cytochrome c, caspase activation with PARP cleavage, and DNA fragmentation.

Current evidence suggests that O2 and H2O2 injure cells as a result of the generation of a more potent oxidizing species.In addition to O2 and H2O2 the third essential component of the complex that mediates the lethal cell injury is a cellular source of ferric iron.The hypothesis most consistent with all the available data suggests that O2 reduces a cellular source of ferric to ferrous iron, and the latter then reacts with H2O2 to produce a more potent oxidizing species, like the *OH or an equivalently reactive species.In turn, *OH initiates the peroxidative decomposition of the phospholipids of cellular membranes."OH also damages the inner mitochondrial membrane.Upon mitochondrial deenergization, a sequence of events is initiated that similarly leads to the loss of viability of the cell.DNA represents a third cellular target of *OH.Depending on the cell type, oxidative DNA dam- age can be coupled to cell killing through a mechanism related to the activation of poly (ADP-ribose) polymerase.-

Representative of Marshall Nirenberg's early work as part of the Biochemical Genetics group at NIH, and specifically Nirenberg's experiments involving gene expression in neurons, this article assesses the activities of marker enzymes in surface cultures of new born mouse brain cells, and in glial and nonbrain cell lines.