BIOCHEMICAL MECHANISMS IN TOXIC CELL DEATH

Cell killing is an important aspect of ischemic diseases such as myocardial infarction and stroke. In these diseases occlusion of an artery prevents or reduces blood supply to an organ, interrupting the oxygen supply to affected cells. When alternate energy sources are depleted the affected cells enter a state of irreversible injury and ultimately die. Reestablishing the blood supply before significant cell death occurs initiates rapid cell death rather than recovery. Several lines of evidence indicate that the agent in blood responsible for this phenomenon is Ca2+, which enters the cells down a steep electrochemical gradient; the energy-deprived cells are unable to pump the Ca2+ back out resulting in elevated intracellular Ca2+ which triggers cell death. Elevated intracellular Ca2+ concentrations can be modeled in cultured cells using the divalent cation ionophore A23187. Preliminary studies in this laboratory indicate that cell death triggered by elevated intracellular Ca2+ is an active process involving at least 3 identifiable biochemical steps, the first which appears to be a phospholipase. The aim of the proposed research is to understand the biochemistry of this process, i.e. to elucidate the steps involved and to characterize as far as practical the enzymes involved with each step. The long term goal of the research is to develop inhibitors of key steps in the process. These inhibitors could conceivably be developed into drugs to prevent cell death associated with ischemic diseases. The model system chosen for studying the biochemistry of cell death is a permanent cell line in culture making feasible mutant selection and other genetic approaches. Cell viability will be assessed by physical (e.g. trypan blue exclusion) and genetic (e.g. proliferation) criteria. Experimental approaches to elucidating the mechanism include (1) selection and characterization of mutant (variant) cell lines deficient in enzymes required for individual steps; (2) further characterization of the injured cells at the identified steps by biochemical criteria and response to pharmacological probes; and (3) characterization of the phospholipase activity associated with the first step.