A Eriksson1, R Lewensohn, A Nilsson. 1. Department of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Hospital, Stockholm, Sweden.
Abstract
BACKGROUND: DNA-PK is a serine/threonine protein kinase that has been shown to play an important role in V(D)J recombination, Ig switching and DNA double strand break (DSB) repair. We have investigated DNA-PK in whole cell extracts from B and T lymphocytes, monocytes and granulocytes and the effects of phorbolmyristate acetate (PMA) and Concanavalin A (Con A) on DNA-PK in B and T lymphocytes. MATERIALS AND METHODS: Lymphocytes were separated from human peripheral blood using the E-rosette technique and DNA-PK was analysed using Western blot, DNA-PK kinase activity assay and flow cytometry. RESULTS: The levels of DNA-PK activity and content varied among the leukocytes. PMA and Con A stimulation consistently increased both DNA-PK activity and content in B lymphocytes as cells entered the S phase. Up-regulation could normally not be seen in T lymphocytes, although cells entered the S phase following PMA and Con A stimulation. CONCLUSION: Mitogenic stimulation up-regulates DNA-PK activity and protein expression in B lymphocytes. T lymphocytes show interindividual differences in DNA-PK. Regulation of DNA-PK is not strictly cell cycle dependent for all cell types.
BACKGROUND:DNA-PK is a serine/threonine protein kinase that has been shown to play an important role in V(D)J recombination, Ig switching and DNA double strand break (DSB) repair. We have investigated DNA-PK in whole cell extracts from B and T lymphocytes, monocytes and granulocytes and the effects of phorbolmyristate acetate (PMA) and Concanavalin A (Con A) on DNA-PK in B and T lymphocytes. MATERIALS AND METHODS: Lymphocytes were separated from human peripheral blood using the E-rosette technique and DNA-PK was analysed using Western blot, DNA-PK kinase activity assay and flow cytometry. RESULTS: The levels of DNA-PK activity and content varied among the leukocytes. PMA and Con A stimulation consistently increased both DNA-PK activity and content in B lymphocytes as cells entered the S phase. Up-regulation could normally not be seen in T lymphocytes, although cells entered the S phase following PMA and Con A stimulation. CONCLUSION: Mitogenic stimulation up-regulates DNA-PK activity and protein expression in B lymphocytes. T lymphocytes show interindividual differences in DNA-PK. Regulation of DNA-PK is not strictly cell cycle dependent for all cell types.