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 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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
Failure to generate retroviral producer clones may be caused by the induction of apoptosis mediated by the therapeutic gene product.
Islam
T C
TC
Center for BioTechnology, Department of Biosciences, Karolinska Institute, NOVUM, Huddinge, Sweden. tahmina.islam@csb.ki.se
Brandén
L J
LJ
Kohn
D B
DB
Islam
K B
KB
Smith
C I
CI
D000077329:Agammaglobulinaemia Tyrosine Kinase; D017209:Apoptosis / Q000235:genetics*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012190:Retroviridae / Q000201:enzymology / Q000235:genetics*; D048051:p38 Mitogen-Activated Protein Kinases
D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020558:GTP Phosphohydrolases