Mechanisms and mediators of apoptosis : with special reference to hematological disorders

Abstract: Life and death are inextricably entwined. Naturally occurring cell death or apoptosis is essential for the of homeostasis and serves to remove extraneous or dangerous cells in a swift and unobtrusive manner. work has demonstrated that such cell death is genetically regulated and transpires according to evolutionarily conserved pathways. The skewing of the delicate balance between life and death may impinge on numerous processes, and the modulation of apoptosis for therapeutic purposes can therefore be envisioned. The present attempts to provide an overview of contemporary apoptosis research, with particular emphasis on the mediators and mechanisms of apoptosis at the molecular level, and the consequences of defective or inadvertent apoptosis forhuman disease. An important mediator of apoptosis in the immune system is Fas, a member of the death receptor family. In a series of studies, we mapped the epitope targeted by agonistic and antagonistic anti-Fas antibodies to a linear region which was shown, by homology-based modeling, to correspond to a hairpin loop on the surface of the Fas molecule. In a subsequent study, we attempted to delineate the role of reactive oxygen species (ROS) in the regulation of the cysteine proteases known as caspases. Caspases were activated in human neutrophils undergoing spontaneous and Fas-triggered apoptosis, and the degree of activation was enhanced in the presence of diphenylene iodonium, an inhibitor of the NADPH oxidase. Moreover, in neutrophils triggered to undergo a respiratory burst, caspase activation was completely abrogated, suggesting that ROS serve to suppress caspases in these cells. Neutrophils were also shown to possess two distinct mechanisms of externalization of phoshatidyiserine (PS). an important marker for phagocytosis. Neutrophils from patients afflicted with chronic granulomatous disease (CGD), a genetic disorder with defective generation of ROS and hence severe and protracted bacterial infections, were shown to lack the oxidant-dependent pathway of PS exposure, while the caspase-dependent pathway remained intact. We then tested whether externalization of PS is a universal event during apoptosis. A panel of tumor cell lines of hematopoietic origin were triggered to undergo apoptosis and characteristic nuclear changes with concomitant caspase activation were demonstrated to occur in all cells. However, only some cell types yielded detectable PS exposure, and this was shown to correlate with a pronounced drop in the mitochondrial transmembrane potential. The caspase-dependent cleavage of Bcl-2 in etoposide-treated myeloid leukemic cells was also demonstrated, and evidence was provided that this is a relatively early event in the apoptotic cascade, which occurs predominantly in the mitochondrial compartment of the dying cell. Finally, we turned to the rare and fatal childhood disorder termed familial hemophagocytic lymphohistiocytosis (FHL), which is characterized by a non-malignant accumulation and multivisceral infiltration of activated T lymphocytes and histiocytes, in an attempt to assess putative defects in these patients. Neutrophils and lymphocytes from FHL patients were found to undergo etoposide- and Fas-triggered apoptosis in vitro in a manner comparable to cells obtained from healthy controls. Specifically, PS externalization, caspase activation and subsequent cleavage of Bcl-2 proceded unabated in all patients tested. However, several FHL patients displayed a low or absent spontaneous activation of caspase-3-like enzymes it activated lymphocytes, as well as elevated serum levels of soluble Fas ligand.