The Mechanism Of Irradiation-induced Egr-1/p300-mediated Cathepsin L Activation By Mutant P53

Part ?Objective:To study the significance of p53 gene mutation for irradiation-induced transcription of Cathepsin L,and to explain the relationship between Egr-1/p300 and mutated p53.To investigate that Egr-1/p300 is an important target in the process of irradiation-induced transcription of Cathepsin L,and to confirm that Cathepsin L is an important participator of mutant p53 gain of function.Methods:Different tumor cell lines were treated by 10 Gy dose of irradiation.Western blot was subjected to detect the effect of irradiation on different tumor cell lines protein level.Cathepsin L,Egr-1 and p300 antibody were applied in western blot assays.Luciferase reporter gene assay was used to check the effect of irradiation on Cathepsin L promoter p53 binding region activity in different tumor cell lines.ChIP assay was used to detect the binding status of p53 or p300 on Cathepsin L promoter after irradiation treatment.Si-RNAs anti-p300 or Egr-1 were used to knockdown the p300 or Egr-1 gene expression in tumor cells.After p300 knockdown,ChIP assay was used to detect the binding status of mutant p53 on Cathepsin L promoter after irradiation treatment.Western blot was used to check the protein level of Cathepsin L in anti-p300/Egr-1 si-RNA-treated tumor cells after irradiation.Luciferase reporter gene assay was used to check the effect of anti-p300/Egr-1 si-RNA on Cathepsin L promoter p53 binding region activity in anti-p300/Egr-1 si-RNA-treated tumor cells after irradiation.Colon cancer cell line HT-29(mutant type p53)and RKO(wild type p53)were used to establish nude mice models subcutaneously,grouped into control group and irradiation group.Western blot and Immunohistochemistry assays were performed to detect the expression level of Cathepsin L and Egr-1 in subcutaneous tumors.Clinical colon and breast cancer tissue were collected to study the expression relationship between Cathepsin L and Egr-1/p300.P53 gene was sequenced for gene mutation analysis.Immunohistochemistry assays were performed to detect the expression status of Cathepsin L,Egr-1 and p300 in wild-type or mutant p53 clinical colon cancer samples.Results:After irradiation treatment,western blot assay results showed that the expression level of Cathepsin L,Egr-1 and p300 were up-regulated in p53-mutated tumor cell lines U251,HT-29 and MDA-MB-468 but not in p53 wild tumor cell lines U87,RKO or MCF-7.Luciferase reporter gene assay results demonstrated that irradiation induced the activity of p53 binding region on Cathepsin L promoter.Si-RNAs anti-p300 or Egr-1 significantly abolished the increase of Cathepsin L expression induced by irradiation.And ChIP assay results showed that the irradiation-activated transcription of Cathepsin L was also p300 dependent.Additionally,in the HT-29 and RKO expriments in vivo,analyse on tumor volume change showed that the therapy efficiency of HT-29 cells irradiation group was significantly worse than RKO cells irradiation group comparing with control group.Western blot and Immunohistochemistry assays results showed the expression level of Cathepsin L,Egr-1 and p300 were much higher in irradiation group of HT-29 cells comparing with control group.However,there was no significant change in RKO cell subcutaneous tumors between irradiation and control group.Cathepsin L and Egr-1 were both high expressed in clinical colon and breast cancer tissue comparing with normal tissue.P53 gene mutation analysis showed 2 of clinical colon samples are p53-273 mutation.Immunohistochemistry assays showed Cathepsin L,Egr-1 and p300 expression levels were higher in p53-273-mutated colon samples than in normal tissue,and nuclear translocation of Cathepsin L was observed in p53-273-mutated tumor tissue but not in p53 wild-type tissue.P300 expression was detected higher only in breast cancer tissue than in normal tissue.Conclusions:Mutant p53 could regulate the transcription of Cathepsin L via Egr-1 and p300 under the treatment of irradiation.Part ?Objective:To study the irradiation-induced transcription mechanism of Cathepsin L by mutant p53 via methylation on the promoter of Cathepsin L,and to confirm the role of epigenetics in transcriptional regulation of Cathepsin L.Methods:After irradiation treatment,western blot assay was used to detected the expression level of DNA methyltransferase protein(Dnmtl,Dnmt3a and Dnmt3b),and the total DNA of tumor cells was extracted and methylation status of Cathepsin L promoter was checked by methylation specific PCR(MSP)assays,and CpG islands of p53 binding site upstream on Cathepsin L promoter were checked by bisulfite sequencing PCR(BSP)assays.Results:western blot assay showed irradiation induced the expression increase of DNA methyltransferase protein in p53-mutated cell lines.Methylation specific PCR(MSP)assays showed that irradiation induced methylation of upstream of p53 binding site on Cathepsin L promoter in p53-mutated tumor cell lines.Bisulfite sequencing PCR(BSP)assays showed the effect of irradiation on methylation of p53 binding site upstream on Cathepsin L promoter was not very remarkable.The methylation level of U251 cells was raised by irradiation from 58%to 65%,and HT-29 was increased from 75%to 80%.Conclusions:The methylation level of Cathepsin L promoter in Cathepsin L low-expressing cell lines was correlated with the methylation level of Cathepsin L promoter.Irradiation raised the methylation level of Cathepsin L promoter in p53-mutated tumor cells,and thus the irradiation-induced transcriptional activation of Cathepsin L in p53-mutated tumor cells was not through the methylation of p53 binding region upstream on Cathepsin L promoter.