Research Project
6722795
DESCRIPTION (provided by applicant): Our broad aim is to better understand the roles of surface IgM and IgD (slgM and slgD respectively) receptors in B lymphomas and normal cells. Using anti-mu and anti-delta antibodies to crosslink and activate slgM and slgD receptors respectively, we established that anti-mu and not anti-delta induced growth arrest and apoptosis in B lymphoma cells. Although anti-mu, and anti-delta both stimulate c-Myc, only anti-mu, ultimately ablates c-Myc protein expression and mediates a large induction of p27Kip1 protein. We also demonstrated that c-Myc protein expression is controlled through the ITAM and we recently established PI-3K regulation as the mediator of anti-mu, and anti-delta action. Therefore, our first aim is to explore the role of ITAMS and accessory BCR proteins in PI-3K recruitment and signaling. This will be performed by using CD8-Ig-alpha fusion protein constructs which contain ITAM and non-ITAM mutations first using immunohistochemical then molecular methods. We will also directly test our hypothesis that aborted apoptotic signaling via the slgD receptor results from its failure to co-localize negative regulators which could include adapter proteins, kinase(s), phosphatase(s) or even possibly ITIM-containing co-receptors. Our second aim is to directly establish the role(s) of PI-3K pathway proteins in anti-mu driven growth arrest and apoptosis. We will subclone active and dominant negative PI-3K modulators and effector cDNAs into HIV-TAT fusion vectors. These fusion proteins will be transduced into lymphoma cell lines and primary cells to determine their roles and the hierarchy of their influence on slgM or slgD receptor signaling. Our third aim is to further explore and contrast positive and negative signaling in BCR induced apoptosis. We recently observed that anti-mu massively stimulates PTEN protein expression, whereas anti-delta does not. Therefore, I will establish the mechanisms that regulate PTEN protein expression following BCR engagement and determine its role in BCR mediated outcomes. Transduction of dominant negative PTEN should render lymphoma cells resistant to anti-mu mediated growth arrest whereas transduction of active PTEN should mimic or augment the anti-mu (apoptotic) and anti-delta (pro-apoptotic) responses. Our last aim is to compare and contrast selected gene expression with slgM and slgD receptor signaling. Using a novel gene expression technique, RAGE, we recently observed that anti-mu, specifically modulates a number of cDNAs. We will perform kinetic analysis of the expression of their corresponding proteins and determine their role(s) in the anti-mu effect. Together, these studies will delineate proteins and signaling events, modules and programs initiated by the surface IgM or surface IgD receptors to effect survival, growth arrest and/or apoptosis in B-lymphoma cells.
