The P53-inducible Gene3(PIG3) Parcitipated In Ionizing Radiation-induced DNA Damage Response Through Regulating DNA-PKcs

The p53-inducible gene3, PIG3, is one of p53induced genes before onset ofapoptosis. PIG3is homologous to NADPH oxidoreductase TED2in plants andzeta-Crystallin in mammalian cells. Its oxidoreductase activity has also been reportedand involved in the reactive oxidore specie (ROS) generation. PIG3repression willreduce ROS and repress apoptosis. In addition, PIG3mediates cells death induced byGlutathione Peroxidase3. It is considered as a proapotosis marker. However, PIG3recently has been reported be induced by ionizing radiation and play an important rolein DNA damage response induced by UV irradiation or treatment with radiomimeticdrug neocarzinostatin (NCS). But the mechanism how PIG3participates in DNAdamage response is poorly unknown.DNA-dependent kinase catalytic subunit (DNA-PKcs) together with ATM andATR, belong to Phosphatidylinositol-3-kinase-like kinase (PIKK) family. As a Thr/Serkinase DNA-PKcs phosphorylate a series of downstream substrates, and centers onNHEJ pathway, V(D)J recombination and the stability of chromosome. Recently,DNA-PKcs was shown to participate in DNA damage response and regulation of cellcycle checkpoint.In the present study, small interference RNA (siRNA) and lentivirus-mediatedshRNA expression strategies were used to knock down PIG3transiently or stably andcell phenotypes post ionizing radiation were analyzed, in order to figure out themechanism PIG3regulates DNA damage response. We acquired some results asfollow:1. PIG3increased post ionizing radiation. Irradiated with6,8Gy induced PIG3notably in A549lung cancer cell line, and PIG3was induced in a dose dependentmanner in AHH1cells. These results indicate the participation of PIG3in DNAdamage response.2. The cell lines stable silenced or overexpressed PIG3have been constructed.And pulse field gel electrophoresis assay shows PIG3knockdown impact DSB repair. In addition, the phosphorylation of γ-H2AX, chk1, chk2and kap-1down-regulatedpost IR, accompied by a prolonged G2/M arrest. These findings support theinvolvement of PIG3in DNA damage response.3. A double block approach using thirdmine (TdR) was performed tosynchronize cells at G1phase, and cell cycle procession post G1block released waschecked. PIG3-depleted cells showed a prolonged G2/M phase, and a high level ofcyclin B1and p53protein. Cyclin B1is notable high at2-4hours post G1block butcome to comparable between PIG3-depleted and control cells at8-12hours post G1block release when cells enter G2/M phase. p53was induced during G2/M phase andpeaked at12hours post release. High level of p53could repress the expression ofcyclin B1and its activity, lead to G2arrest and take more time for cell proliferation.Consist with this, the growth curve shows a decreased proliferation capability inPIG3-depleted cells. In addition, PIG3knockdown also make cells sensitive toPaclitaxel.4. DNA-PKcs is down regulated in PIG3-depleted cells accompanied with ATMdecrease. PIG3doesn’t influent the mRNA level of DNA-PKcs or its promoteractivity. In ATM deficient cell line ATS4and AT5BIVA, knockdown PIG3could alsorepress DNA-PKcs, indicates PIG3regulates DNA-PKcs in an ATM independentmanner.5. A compensatory feedback of increased mRNA expression of DNA-PKcs wasformed in PIG3-depleted cells after a few passages or cell cycles of subculture, whichled the recovery of the DNA-PKcs protein level and the consequent recoveredefficiency of the DNA damage response. DNA-PKcs decreased at10-12day postPIG3depletion and then recovered in the next few days. This phenomenon appears inmany cell lines with different kinetics.6. The increases of cyclin B1in PIG3knockdown cells is due to the repressionof DNA-PKcs. Knocking down or inhibit DNA-PKcs activity led cyclin B1accumulated in cells and exit delay from mitosis. The degradation of cyclin B1decreased post DNA-PKcs knockdown or inhibition, because the ubiquitination defect.Proteasome inhibitor MG-132abolished the increase of cyclin B1, and one subunit ofAPC/C APC3is down regulated in DNA-PKcs-depleted cells. In addition, DNA-PKcsinteracts with another APC/C subunit APC2.Our work reveals a novel mechanism through which PIG3contributes to theDNA damage response. Inactivation of PIG3leads temporarily to the attenuation of DNA-PKcs, which in turn causes a depression of ATM and a defect in DNA damageresponse and abnormal cell cycle procession. After a certain cell cycles or passages ofsubculture, there followed a feedback of compensatory increased transcription activityof DNA-PKcs, which resulted in recovery of the cellular DNA-PKcs level and ATM,and consequently the DNA damage response activities. We also showed that PIG3regulates cell cycle procession through DNA-PKcs dependent cyclin B1degradation.DNA-PKcs interact with APC2and regulate APC3level, finally result in cyclin B1ubiquitination decreased and stabilize cyclin B1. These results provide a new insightinto PIG3’s role in DNA damage signaling and the regulation network of the cellularDNA-PKcs homeostatic balance, and demonstrated a novel function of PIG3regulating cell cycle through DNA-PKcs-cyclin B1pathway.