The response of proliferating cell nuclear antigen to ionizing radiation in human lymphoblastoid cell lines is dependent on p53.
Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and epsilon involved in DNA replication and nucleotide excision repair. There are two intranuclear fractions: a detergent-extractable, soluble fraction and a tightly DNA-bound fraction. To function, PCNA forms a trimeric sliding clamp which is loaded onto DNA. To better understand the role of the p53/p21 pathway in the regulation of PCNA after irradiation, we studied three closely related human lymphoblastoid cell lines, WTK1, TK6 and TK6E6, an HPV16 E6-transfected line, that differ in p53 status, radiosensitivity and susceptibility to radiation-induced apoptosis. Time-dependent changes in PCNA levels were measured in the different nuclear fractions by Western blot analysis after protein crosslinking. The results were compared to those for human diploid fibroblasts studied under different growth conditions. There was no change in total cellular levels of PCNA after irradiation, consistent with predominantly post-translational regulation. Changes in intranuclear distribution and complex formation occurred in a p53/p21-dependent manner. The loading of PCNA onto DNA was increased in cells with low p21 levels. A disruption of PCNA trimers was observed in exponentially growing p53+ cells in the soluble fraction. Thus the p53/p21 signal transduction pathway appears to play a significant role in the regulation of the response of PCNA to radiation.
Wenz
F
F
Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Azzam
E I
EI
Little
J B
JB
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000502:physiology; D004249:DNA Damage; D019281:Dimerization; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000528:radiation effects*; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D018809:Proliferating Cell Nuclear Antigen; D016159:Tumor Suppressor Protein p53