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

Background It has been shown previously that mutations in the cytoplasmic p rotein kinase, Bruton's tyrosine kinase (BTK) lead to X-linked agammaglobul inemia, an inherited primary immunodeficiency, thus making it a potential c andidate for gene therapy. Methods Producer cell lines using retroviral BTK constructs were generated and retroviral titers determined. Southern blot analysis was performed to c heck for pro-viral integrity in the respective clones. Furthermore, cotrans fection of green fluorescent protein (GFP) with BTK expression plasmids was used in HeLa cells to establish and characterize the role of BTK in apopto sis. Results Following the attempt to generate retroviral producer clones by con ventional methods, we observed that the BTK gene is deleted from neomycin-r esistant high titer clones. We show that BTK mediated apoptosis in GP#E86 a nd HeLa cells. Furthermore, membrane targeting and kinase activity are requ ired for this effect. In addition, BTK induced apoptosis could be inhibited by using a specific inhibitor for p38 mitogen-activated protein kinase (MA PK), SB203580. Conclusion Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product. Copyri ght (C) 2000 John Wiley & Sons, Ltd.