BTK mediated apoptosis, a possible mechanism for failure to generate high titer retroviral producer clones.

Abstract

It has been shown previously that mutations in the cytoplasmic protein kinase, Bruton's tyrosine kinase (BTK) lead to X-linked agammaglobulinemia, an inherited primary immunodeficiency, thus making it a potential candidate for gene therapy. Producer cell lines using retroviral BTK constructs were generated and retroviral titers determined. Southern blot analysis was performed to check for pro-viral integrity in the respective clones. Furthermore, cotransfection of green fluorescent protein (GFP) with BTK expression plasmids was used in HeLa cells to establish and characterize the role of BTK in apoptosis. Following the attempt to generate retroviral producer clones by conventional methods, we observed that the BTK gene is deleted from neomycin-resistant high titer clones. We show that BTK mediated apoptosis in GP#E86 and HeLa cells. Furthermore, membrane targeting and kinase activity are required for this effect. In addition, BTK induced apoptosis could be inhibited by using a specific inhibitor for p38 mitogen-activated protein kinase (MAPK), SB203580. Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product.