MENADIONE AS A MODEL COMPOUND FOR CHARACTERIZING THE EFFECTS OF QUINONES ON ISOLATED MITOCHONDRIA FROM RAT LIVER AND HUMAN CELLS LINES USED IN THE NCI IN VITRO SCREEN

C.J. Glover, E.A. Sausville and R.L. Felsted
LDDRD, DTP, NCI-FCRDC, Frederick, MD 21702

Chemotherapeutic quinones have been shown to induce apoptosis in a number of human tumor cell lines. Bioactivation of these compounds to form reactive oxygen species (ROS) is required for cytotoxicity. These ROS and subsequent cellular redox changes are also implicated in apoptotic events involving the release of cytochrome c and the caspase cascade. Reactive oxygen species, especially H2O2, are also known to cause induction of the cyclosporin A-sensitive (and Ca+2 dependent) mitochondrial permeability transition (CyA-MPT), depolarization of the mitochondrial membrane, and release of stored calcium. A prototypical quinone, menadione, induces the CyA-MPT in isolated liver mitochondria and the subsequent swelling may cause rupture of the outer mitochondrial membrane and release of cytochrome c. We are using menadione as a model compound and mitochondria isolated from rat liver and from selected human cell lines used in the NCI in vitro screen to characterize and correlate drug effects on mitochondrial respiratory control ratios, induction of CyA-MPT, and outer membrane integrity. Hypothesized outer membrane disruption is being assessed by cytochrome oxidase assays in the presence of exogenously added cytochrome c (by spectrophotometry and O2 consumption) and by the release of adenylate kinase from the intermembrane space. These studies are designed to aid in characterizing the mechanism of action of quinone drugs identified by the NCI in vitro cancer cell screen.