Oxidants and Cell Death

总结：我们正在研究炎症氧化剂如过氧化氢（H2 O2）如何杀死肿瘤细胞，以及它们如何影响免疫系统对肿瘤细胞的识别和消除。 大多数化疗药物通过诱导细胞凋亡来杀死肿瘤细胞。 实体瘤通常被炎性吞噬细胞浸润，炎性吞噬细胞可在肿瘤组织内产生氧化应激。 以前，我们发现，在H2 O2的存在下，人伯基特淋巴瘤（BL）细胞不能进行细胞凋亡，以响应癌症化疗药物，而死亡，而不是一种形式的坏死。 H2 O2和化疗药物之间相互作用的最重要的结果之一是细胞不会被共培养的巨噬细胞吞噬，直到它们的膜溶解之后。 这可能导致对垂死细胞的不良炎症反应，这可能进一步使肿瘤细胞耗竭复杂化。 在过去一年中进行的研究旨在确定H2 O2抑制巨噬细胞摄取垂死肿瘤细胞的分子机制，并确定吞噬过程的辅助因子。 在诱导细胞凋亡后不久，通过磷脂酰丝氨酸（PS）从质膜的内小叶易位到外小叶，垂死细胞被靶向吞噬。 最近，我们发现凋亡淋巴瘤细胞的吞噬作用需要蛋白S的存在，蛋白S是一种调节活化蛋白C活性的血清蛋白。 H2 O2抑制蛋白S刺激的BL细胞的吞噬作用，即使当他们表达PS的外表面上的质膜。 H2 O2处理不抑制蛋白质（膜联蛋白）结合到PS，也不修改PS在凋亡细胞膜上的空间分布（通过共聚焦显微镜测定）。 这些结果表明，PS是必要的，但不足以识别和摄取凋亡细胞的巨噬细胞。 此外，他们认为H2 O2通过修饰凋亡细胞表面上的一个单独的、尚未鉴定的吞噬标志物起作用。 在另一系列研究中，我们试图确定H2 O2杀死细胞的机制是否是炎症细胞产生的所有氧化剂的典型机制。 以前，我们发现过氧化氢诱导的人B淋巴瘤细胞死亡的主要模式是非凋亡。 在最近的研究中，我们研究了由活化的吞噬细胞产生的另外两种氧化剂杀死细胞的机制。 我们发现超氧化物完全通过形成H2 O2杀死细胞，使得细胞主要通过固缩/坏死死亡。 相反，HOCl通过细胞凋亡杀死细胞，细胞凋亡是由生长培养基中氨酰氯胺的形成介导的。 正在寻找这些氧化剂的分子靶点，以便我们可以确定控制细胞死亡的其他机制。

Summary: 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.