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The Effects and Mechanisms of P53Regulated Cell Autophagy in DNA Radiation Damage Response

Author: LiJunYing
Tutor: ZhouPingKun
School: Anhui Medical University,
Course: Biochemistry and Molecular Biology
Keywords: p53 p62 SQSTM1 G2 M arrest DNA damage repair cell apoptosis
CLC: Q75
Type: Master's thesis
Year: 2013
Downloads: 5
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Abstract


Objective:p53is one of the most extensively studied tumor suppressor, it is also a highly potent transcription factor, DNA radiation damage can induce p53stabilized leading to changes in the expression of p53‐responsive genes. The biological outcome inducing this pathway can be either growth arrest and apoptosis or senescence to maintain the integrity of the genome or to delete the damaged cells. So, p53is a pivotal tumour suppressor and a mainstay of our body’s natural anticancer defence. This article could provide some useful information for further study on the mechanisms of tumorigenesis and its progression, and also could contribute to the discovery of antitumor agents.Methods:Cell survival of HCT116p53+/+ and HCT116p53-/- were determined by colony formation assay. Cell cycle and protein expression level were respectively analyzed by flow cytometry and western blotting. To study the role of p53in cell cycle progression, the cell cycle synchronization of HCT116p53+/+ and HCT116p53-/- was achieved by thymidine double‐blocking. P53stable knockdown and control A549cell lines were constructed with lentivirus, and identified by Western blotting. The transcriptional regulation of p53to p62/SQSTM1was detected by luciferase reporter gene assay, using Annexin V‐FITC/PI double staining combined with flow cytometry detect cell apoptosis. Immunofluorescence and western blotting was used to detect the expression of autophagy protein p62/SQSTM1, autophagic vacuoles were observed by transmission electron microscopy.Results:1．P53enhanced cell survival after ionizing radiation, through colony formation assay and found that HCT116p53+/+ cell survival rate was significantly higher than HCT116p53-/- cells after ionizing radiation.2．After ionizing radiation, the G2/M arrest of HCT116p53-/- cells was earlier and longer than HCT116p53+/+ cell.3．The G2/M phase of HCT116p53-/- cells was longer than HCT116p53+/+ cell after cell cycle synchronization.4．Add1uM doxorubicin to cells, we found that the S phase of HCT116p53-/- cells was longer than HCT116p53+/+ cell.5．Western blotting results show that p53stable knockdown cell lines were successfully constructed. Through luciferase reporter gene assay, we found that4Gy irradiation can strongly activate the transcription of p62, and the transcriptional activity doubled at2h after irradiation. In addition, in p53+/+ cells it slowly increased after4h, decreased at24h. While in p53-/- cells, it declined for a while and increased rapidly until24h.6．Through flow cytometric analysis, we found that in control cells the G2/M arrest was occurred at8h after irradiation, while in p53knockdown cells, the G2/M arrest was occurred at4h. After serum starvation, the G2/M block was postponed apparently, and the difference between p53knockdown and control cells was reduced. 7．The cell apoptosis results show that the apoptosis rate of p53knockdown cells was significantly lower than the control cells, the apoptosis rate of serum hunger cells were significantly lower than the control cells, while3‐MA treatment groups have the highest cell apoptosis rates.Conclusion:1．p53enhanced cell survival after ionizing radiation.2．P53may inhibit the activation of G2/M checkpoint, and p53may play an important role in the transition from G2to M phase or the exit of M phase cells.3．After DNA damage induced by irradiation, p53inhibited autophagy, and the inhibition was likely achieved by inhibiting the transcription of p62.4．Promote autophagy can promote cell survival after ionizing radiation, while inhibit autophagy will Increase radiation sensitivity, this radiation sensitizing effect is likely achieved through the inhibition of cell cycle arrest.

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