Greater than 85% percent of human follicular B cell lymphomas exhibit a t(14;18) chromosomal translocation, which results in elevated expression of the Bcl-2 oncoprotein. Bcl-2 has been shown to block apoptosis in a variety of hematopoietic and neuronal cell types and may contribute to follicular lymphomas by this type of mechanism. Attempts to devise therapies that are based on blocking Bcl-2 function, have been hindered by the fact that little is known about the molecular mechanism of Bcl-2 action. Our broad, long-term objective is to define the mechanism of Bcl- 2 action, by characterizing the interactions of Bcl-2 with other cellular proteins. In preliminary studies, we have cloned two cDNAs which code for novel proteins, BAP1 and BAP2, that bind to Bcl-2 in vitro. BAP1 and BAP2 are derived from distinct, yet related, genes, and define a novel family of Bcl-2-associated proteins. Forced expression of the BAP2 protein in an interleukin-3 (IL-3)-dependent cell line resulted in accelerated apoptosis following IL-3 withdrawal. Thus, the BAP proteins likely play important roles in cellular apoptosis.
The Specific Aims of this proposal are: 1) to characterize expression of the BAP1 and BAP2 genes in normal murine tissues and defined hematopoietic cell lines; 2) to investigate physical interactions between Bcl-2 and the BAP1 and BAP2 proteins in defined hematopoietic cell lines; 3) to investigate the capabilities of BAP1 and BAP2 to block Bcl-2 function; and 4) to investigate physical interactions between the BAP1 and BAP2 proteins and other members of the Bcl-2 protein family, including Bax, Bcl-XL, Bcl-XS, and Mcl-1. These studies may provide a basis for novel therapies aimed at blocking Bcl-2 function in vivo. Expression of the BAP1 and BAP2 proteins in normal murine tissues and defined hematopoietic cell lines will be studied by immunohistochemistry and Western blotting using polyclonal antisera generated against the recombinant proteins. Northern blotting and in situ hybridization will be used to study mRNA expression. To investigate potential intracellular protein-protein interactions, we will coexpress Bcl-2 with BAP1 or BAP2 in IL-3-dependent cell lines, and determine whether Bcl-2 can be specifically coimmunoprecipitated with BAP1 or BAP2. In parallel, we will investigate the effects of forced BAP1 or BAP2 expression on the ability of Bcl-2 to suppress apoptosis following IL-3 withdrawal. Investigations of interactions between the BAP proteins and other members of the Bcl-2 protein family will be performed in vitro, using purified recombinant proteins.
Showing the most recent 10 out of 11 publications
