We are investigating how inflammatory oxidants such as hydrogen peroxide (H2O2) kill tumor cells and how they may influence 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 to the dying cells, 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 and at identifying cofactors for the phagocytic process. 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. Recently, we discovered that phagocytosis of apoptotic lymphoma cells requires the presence of protein S, a serum protein that regulates the activity of activated protein C. H2O2 inhibits the protein S-stimulated 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. We found that superoxide kills cells entirely through formation of H2O2 such that the cells die primarily by pyknosis/necrosis. In contrast, HOCl kills the cells by apoptosis which is mediated by formation of aminoacyl chloramines in the growth medium. The molecular targets for these oxidants are being sought so that we may identify additional mechanisms of controlling cell death.
