CRYSTALLOGRAPHIC AND BIOCHEMICAL ANALYSIS OF AN INHIBITOR OF APOPTOSIS, BCL-2. Jason W. O'Neill and Kam Zhang, Division of Basic Sciences, Fred Hutchingson Cancer Research Center, Seattle, WA

Programmed cell death (apoptosis) plays an important role in animal development and homeostasis of cell numbers. The primary regulator of human apoptosis is Bcl-2, a novel cellular gene which can function to block most modes of apoptosis. Elevated levels of Bcl-2 protein are strongly correlated with tumorgenesis and is found deregulated in a variety of cancers, including; colorectal (90%), breast (70%), and lymphoma (65%). Bcl-2 may function to inhibit apoptosis through effects on antioxidant protection pathways or by modulating the apoptotic effects of Ca²⁺ release from ER. The regulation of Bcl-2 is thought to occur through phosphorylation and/or heterodimer formation with related promoters of apoptosis, such as Bax. To pursue Bcl-2's function and regulational control: a) We have expressed human Bcl-2 in E. coli using a pET-His-tag expression vector. Bcl-2 has been successfully purified to high concentrations and is found as a homodimer as measured by dynamic light scattering. Currently we are undergoing crystallization trials. b) We also look at the effects of phosphorylation to Bcl-2's function, and how the addition of Bax influences Bcl-2's function using an in vitro apoptotic assay. Combining the 3D-structure with the biochemical data will be critical in unraveling Bcl-2's biological mechanism and for its regulation during apoptosis. Furthermore, this knowledge will be extremely useful in structure based design of inhibitors of Bcl-2, which could lead to potent therapeutics for cancers and a broad range of other malignancies.