The goal of our work is to elucidate the consequences of exposure of biological substrates to oxidative stress. In these studies, we investigate how oxidants may affect tumor progression, focusing on the two following questions: (1) how are tumor cells killed by oxidative stress? and (2) how can oxidants modulate tumor cell killing by anti-neoplastic drugs? Inherent in these studies is an investigation of cell death pathways. Cell death can occur through several different mechanisms which are distinguished by unique morphological and biochemical traits. The two most widely described forms of cell death are necrosis and apoptosis. Cells dying by apoptosis fragment into subcellular "apoptotic bodies" while cells dying by necrosis swell and then lyse. It is thought that death by apoptosis is physiologically advantageous because apoptotic cells can be phagocytosed by nearby cells such that the contents are degraded intracellularly. In contrast, death by necrosis is thought to promote an inflammatory response caused by the release of the intracellular contents. Most chemotherapeutic agents kill tumor cells by inducing apoptosis. Oxidants such as superoxide, hydrogen peroxide (H2O2), and the hydroxyl radical are generated under a variety of conditions in vivo such as during acute and chronic inflammation. Solid tumors are often infiltrated by inflammatory phagocytes which can generate oxidative stress within the tumor tissue. Treatment of cells in vitro with H2O2 causes DNA strand breaks, oxidation of lipids and proteins, activation of poly(ADP)-ribosylation, and depletion of cellular energy stores. We have found that in the presence of H2O2, human B lymphoma cells are unable to undergo apoptosis. This was established using the chemotherapy drug VP-16 to induce apoptosis in Burkitt's lymphoma cell lines and measuring markers of apoptotic death, including cell morphology (fluorescence microscopy), annexin V binding (FACS analysis), induction of an oligonucleosomal endonuclease (agarose gel electrophoresis), and caspase activation (enzyme assays and Western blot immunoassays). When cells are treated with VP-16 in the presence of 75-100 ?M H2O2, all of these biochemical hallmarks of apoptosis are inhibited and the cells undergo non-apoptotic cell death, similar to the death observed when they are treated with H2O2 alone. The mechanism whereby H2O2 inhibits apoptosis is by depleting the cells of ATP. The effects of H2O2 can be overcome by inhibitors of poly(ADP)-ribosylation, which preserve cellular ATP levels, and can be mimicked by agents such as oligomycin which inhibit ATP synthesis. Overall, our data show that H2O2 can manipulate cell death pathways, diverting the cell away from apoptosis. The main physiological significance of these findings will be found in whether oxidative stress interferes with chemotherapy-induced tumor cell death and clearance in vivo. Experiments are currently underway to address this question.