The goal of the proposed research is to define the role of programmed cell death in chemotherapy drug action, so as to identify biochemical and metabolic factors that predispose or protect cells from endonuclease activation after DNA damage. programmed cell death, or apoptosis, is distinguishable from necrosis by morphologic and biochemical features, and typically occurs during fetal development, endocrine atrophy, and as a consequence of cell mediated immunity. The hallmark of apoptosis is chromatin cleavage into oligonucleosomal fragments by an endogenous Ca++ /Mg++dependent endonuclease. Recent evidence suggests that certain chemotherapy drugs at damage the DNA of interphase cells may trigger programmed cell death, and preliminary experiments using 2-chloro-2'-deoxyadenosine (CdA) support such a mechanism in chronic lymphocytic leukemia (CLL) cells. However, little is known about the requirements for Ca++ flux, macromolecular synthesis, or ADP-ribosylation in target cells induced to undergo apoptosis by DNA-damaging chemotherapeutic drugs. The Research Plan focuses on two target cell model systems to study programmed cell death in chemotherapy: (i) CLL and hairy cell leukemia as models of malignant, nondividing cells, and (ii) normal blood monocytes, a model of non-dividing cells with phagocytic and secretory functions contribute to the pathogenesis of autoimmune disorders. Target CLL and hairy cell leukemia cells and monocytes will be exposed in vitro to CdA and to other chemotherapy agents known to cause DNA damage. Time-course measurements will be made of DNA strand breaks and of oligonucleosomal DNA fragmentation, the latter by methods of fragment release and by visualization after electrophoresis. Perturbations in NAD metabolism will also be measured, and specific modulation of Ca++ flux, macromolecular synthesis, and ADP-ribosylation will be performed to determine critical biochemical pathways that govern the transition from DNA strand breaks to programmed cell death. CdA is in clinical trial at this institute for the treatments of chronic lymphoid malignancies and for rheumatoid arthritis. The research plan presents a unique opportunity to complement in vitro biochemical analyses with flow cytometric studies to detect apoptosis in the same target cell populations during CdA treatment in vivo. In addition to the clinical relevance of this combined approach, an understanding of biochemical mechanisms in programmed cell death should provide an important basis for novel strategies for anticancer and immunosuppressive therapy.
