EXPRESSION OF DNA-DEPENDENT PROTEIN-KINASE HOLOENZYME UPON INDUCTION OF LYMPHOCYTE DIFFERENTIATION AND V(D)J RECOMBINATION

Murine preB lymphocytes grow in tissue culture in the presence of stro mal cells and interleukin 7 (IL-7), and can be induced to differentiat e to surface-immunoglobulin-positive B cells in vitro by withdrawal of IL-7. Upon differentiation, proliferation ceases, and upregulation of Rag-1 and Rag-2 expression, and induction of V(D)J immunoglobulin-gen e rearrangements occur. DNA-dependent protein kinase (DNA-PK) is requi red for effective V(D)J recombination and repair of DNA double-strand breaks. The holoenzyme comprises a catalytic subunit (DNA-PKcs) and th e Ku heterodimer (Ku70/Ku80). We have analyzed expression of Ku70, Ku8 0 and DNA-PKcs upon induction of differentiation in preB cells derived from wild-type, severe combined immunodeficiency (SCID) and Rag-2(-/- ) mice. Protein levels of Ku80 and Ku70 moderately decrease after indu ction in all three cell types. A distinct polypeptide that crossreacts with anti-Ku Ig appears in the cytoplasm of wild-type and Rag-2(-/-) cells, but not of SCID cells. In mouse preB cells, Ku70 and Ku80 are p resent in the nuclei and cytoplasm before and after onset of different iation. In vivo, Ku70 is predominantly expressed in V(D)J-recombinatio n-active, early-preB and CD4/CD(-)thymocyte cell populations. Upon dif ferentiation, protein levels of DNA-PKcs are unaltered. DNA-PK activit y, which is not detectable in SCID cells, increases in wild-type and R ag-2(-/-) cells more than twofold shortly after induction of different iation, then falls back to about 50% of starting levels.