Study On The Correlation Between The P53Phosphorylation And Nasopharyngeal Carcinoma Radio-sensitivity

Objective:Non radio-sensitive cell line was established basing on the HNE2cell line and named HNE2-NRC. In order to find the relationship between the phosphorylation of P53and the therapeutic response in NPC radiotherapy, the differences of P53phosphorylation expression status between the HNE2and HNE2-NRC was assessed and the phosphorylation level of P53Ser376locus was measured by NPC tissue microarray.Methods:To establish the HNE2-NRC cell line we exposed the HNE2cell line in the X-ray of semi-lethal dose or increasing dose. Before and after exposure we observed the changes in cell morphology, cell proliferation, cell apoptosis and cell cycle in HNE2-NRC and HNE2respectively. The change in cell morphology can be observed in inverted microscope and the cell clone formation rate and growth curve can be obtained with clone formation assay. We identified the inhibition of the cell proliferation after exposure by MTS assay. To assess the differences in cell cycle between HNE2-NRC and HNE2we adopted flow cytometry. We monitored the differences in the status of P53phosphorylation using Western Blotting and detected the phosphorylation level of P53Ser376locus in NPC tissue microarray by IHCResults:The results of the clone formation assay showed that the ability of clone formation in HNE2-NRC is stronger than the ability of the HNE2and the SER is0.796less than1. In MTS test we observed the growth inhibition in HNE2is more obvious than the HNE2-NRC by radiation and the differences is statistically significant in6h,2d,6d and8d respectively. The differences in cell cycles between these two cell lines were detected in flow cytometry and the cell proportion in G2/M phase of HNE2and HNE2-NRC are23.88%and10.84%respectively. The increase in the cell proportion in G0/G1phase of HNE2-NRC can also be detected. The results of Western Blot showed that the phosphorylation levels in P53, P53Ser15, Thr81, Thr55and Ser389are higher in HNE2-NRC than HNE2and the phosphorylation levels in Ser6, Ser9, Ser315and Ser376are lower in HNE2-NRC than HNE2. IHC results of NPC tissue microarray indicate that there is a decreasing tendency in the phosphorylation level of P53Ser376among the patients whose survival span is less than5years. Conclusions:1.Our study established the non radio-sensitive cell line HNE2-NRC and this cell line possessed the radio-resistant growth character in contrast to HNE2.2.The P53Ser15, Thr55phosphorylation levels increased in the HNE2-NRC and the dephosphorylation of P53Ser315and Ser376can be found in the HNE2-NRC. This phenomenon suggests that there is a relationship between the phosphorylation spectrum of P53associated loci and the NPC radio-resistance.3.At the histological microarray level there is a relationship between the phosphorylation level of P53Ser376and the prognosis of NPC patients who received the radiotherapy. This phenomenon suggests the P53phosphorylation spectrum can be used to predict the radio-sensitivity.