Oxidants and Cell Death

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Summary: We are investigating how inflammatory oxidants such as hydrogen peroxide (H2O2) kill tumor cells and how they may affect tumor cell recognition and elimination by the immune system. Most chemotherapeutic agents kill tumor cells by inducing apoptosis. Solid tumors are often infiltrated by inflammatory phagocytes which can generate oxidative stress within the tumor tissue. Previously, we found that in the presence of H2O2, human Burkitt's lymphoma (BL) cells are unable to undergo apoptosis in response to cancer chemotherapy drugs and die instead by a form of necrosis. One of the most important consequences of the interaction between H2O2 and the chemotherapy drugs is that the cells do not become phagocytosed by co-cultured macrophages until after their membranes have lysed. This can lead to an undesirable inflammatory reaction which can further complicate tumor cell depletion. Studies carried out during the past year were aimed at identifying the molecular mechanism whereby H2O2 inhibits uptake of dying tumor cells by macrophages. Shortly after the induction of apoptosis, dying cells are targeted for phagocytosis via the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. We found that H2O2 inhibits phagocytosis of BL cells even when they express PS on the exofacial surface of the plasma membrane. H2O2 treatment does not inhibit protein (annexin) binding to PS, nor does it modify the spatial distribution of PS on the apoptotic cell membrane (as determined by confocal microscopy). These results indicate that PS is necessary, but is not sufficient for recognition and uptake of apoptotic cells by macrophages. Further, they suggest that H2O2 acts by modifying a separate, as yet unidentified phagocytic marker on the surface of apoptotic cells. In a separate series of studies, we sought to determine whether the mechanism of cell killing by H2O2 is typical of all oxidants generated by inflammatory cells. Previously, we found that the primary mode of death induced in human B lymphoma cells by H2O2 is non-apoptotic. In more recent studies, we examined the mechanism of cell killing by the two other oxidants known to be generated by activated phagocytes. Apoptosis and necrosis were assessed by nuclear morphology, caspase activity, and PS flipping. We found that superoxide generated by xanthine oxidase kills cells entirely through formation of H2O2 such that the cells die primarily by pyknosis/necrosis. In contrast, HOCl kills the cells by apoptosis. The difference between H2O2 and HOCl appears to lie in their effects on ATP levels; H2O2 causes intracellular ATP to drop below 25% of control levels whereas ATP is maintained above 50% of control after HOCl treatment. When ATP levels are maintained by inhibiting poly(ADP)ribose polymerase activity, H2O2 also kills the cells by apoptosis. The molecular targets for these oxidants are being sought.