The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase.
scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.
Kachnic
L A
LA
Laboratory of Molecular and Cellular Radiation Biology, Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
Wu
B
B
Wunsch
H
H
Mekeel
K L
KL
DeFrank
J S
JS
Tang
W
W
Powell
S N
SN
D000818:Animals; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004249:DNA Damage; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004622:Embryo, Mammalian / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism / Q000528:radiation effects; D005434:Flow Cytometry; D007259:Infrared Rays; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases