P53-independent PML-p21 Pathway In DNA Damage Response

Objective: As a key transcriptional factor in DNA damage response, p53 can directly position in PML nuclear bodies, which activates the cycle regulation protein p21.And PML-p53-p21 pathway participates in multiple cellular fundamental processes, including the cell cycle progression, apoptosis, gene transcription and DNA damage repair. This experiment will research and expound the p53-independent functional relationship between PML and p21 inγ-irradiation induced DNA damage responses.Knocked down PML expression in p53-null cells to figure out whether p53 is necessary for PML aroused p21 induction,and the effects on cell cycle, proliferation and DNA damage repair in DNA damage response.Methods: Knockdown or over-expression PML , treating with CHX and MG132 in p53-null tumor cells to observe the change trend of p21 in protein level in H1299 and HCT-116 cells.RT-PCR and Dual Luciferase Reporter Gene Assay in transcriptional level. MTT reduction assay,M phase synchronization,Flow cytometric analysis and Immunofluorescence to analysis inγ-irradiation induced DNA damage responses after PML Knockdown in H1299 cells.Examining interactions of p21 with anther factors( Ubquitin,PCNA ) in p53-independent PML-p21 pathway by Co-IP and Immunoprecipitation.Results: Knockdown PML by specific siRNA resulted in down-regulation of p21 in p53-null H1299 tumor cells. Similar results were identified in p53-null HCT-116 (p53^-/-) cells, indicating PML is physiologically involved in p21 maintenance in un-stressed p53 deficiency cells. The half-life of p21 was significantly decreased ,but Ubiquitinylation of p21 was not changed in PML siRNA transfected H1299 cell. Over-expression PML-I and PML-IV can not up-regulate p21 protein expression in H1299 cell.γ-irradiation induced PML and p21 up-regulation, p21 protein expression and mRNA was inhibited by PML knockdown. PML knockdown did not significantly affect p53-null H1299 and HCT-116 (p53^-/-) cells proliferation.Deficiency in PML results in a decreased S phase and G2/M-phase checkpoint, suggest that PML plays an important role in the activation of S and G2/M DNA damage checkpoints after irradiation. p21 over-expression rectified the repair deficiency in PML knockdown H1299 cells PML contributes to maintain the basic expression and the stability of p21. p53-independent PML-p21 pathway plays an important role in cell cycle progression, proliferation and DSBs repair inγ-irradiation induced DNA damage responses.