Roles Of Nucleolin In The Changes Of P53 Induced By Irradiation

Nucleolin(i.e., C23), one of proteinum of entoblasts, possesses specific biological effects such as regulating ribosomal biosynthesis and maturation, cell proliferation,cell growth,embryogenesis,cytocinesis, and so on. Otherwise Nucleolin prosesses the characteristics of shuttling between cytosol and nucleus.If cells stimulated, Nucleolin can shift from nucelus to cytosol. It is reported that Nucleolin could regulate the expression of p53 by competitively binding with RPL26 to the 5'UTR of p53 gene. When DNA damaged RPL26 preferentially binds to 5'UTR of p53 and consequently increases the transcription of p53, induces cell G1 arrest. In this study, the changes of p53 and nucleolin in MCF7 cells were detected after the treatment of irradiation (IR). RNAi is also used to knock down the expression of p53 to observe the possible feedback regulation of p53 to Nucleolin. At last, the binding activity of nucleolin to p53 was detected by protein supershift experiment. The results will be helpful to approach regulatory mechanism of p53.1. The time course changes of Nucleolin and p53 after IR with 4 Gy X-rays1.1 Time course of changes of total Nucleolin and p53 proteins in MCF7 cells after IRThe Western blot was applied to detect the changes of total Nucleolin and p53 proteins in MCF7 cells at 2 h～48 h intervals after IR with 4 Gy. Results showed that total Nucleolin and p53 proteins increased with times as compared with sham-irradiated groups.1.2 Time course changes of Nucleolin and p53 proteins in cytoplasma of MCF7 cells after IRThe Western blot was applied to detect the changes of Nucleolin and p53 proteins in cytoplasma of MCF7 cells at 2 h～48 h intervals after IR with 4 Gy. Results showed that both increased at 2 h, 4 h, 8 h and 16 h and began to recover after 24 h.1.3 Time course changes of Nucleolin and p53 neucleoprotein in MCF7 cells after IRThe Western blot was applied to detect the changes of Nucleolin and p53 neucleoprotein in MCF7 cells at 2 h～48h intervals after IR with 4 Gy. Results showed that Nucleolin and p53 neucleoprotein decreased at 2 h, 4 h, 8 h and 16 h and began to recover after 24 h.2. Dose effect changes of Nucleolin and p53 protein in MCF7 cells 4h after IR with different doses of X-rays2.1 Dose effect changes of Nucleolin and p53 total protein in MCF7 cells 4h after IRThe Western blot was applied to detect the changes of Nucleolin and p53 total protein in MCF7 cells at 4h after IR with doses of 0.5 Gy～4 Gy. Results showed that the Nucleolin and p53 total protein in 4 Gy groups increased as compared with sham-irradiated groups.2.2 Dose effect changes of Nucleolin and p53 proteins in cytoplasma of MCF7 cells 4h after IR The Western blot was applied to detect the changes of Nucleolin and p53 proteins in cytoplasma of MCF7 cells at 4h after IR with doses of 0.5 Gy～4 Gy. Results showed thatboth of them increased in 0.5Gy and 4 Gy groups as compared with sham-irradiated groups.2.3 Dose effect of changes of Nucleolin and p53 neucleoprotein in MCF7 cells 4h after IRThe Western blot was applied to detect the changes of Nucleolin and p53 neucleoprotein in MCF7 cells at 4h after IR with doses of 0.5 Gy～4 Gy. Results showed that the Nucleolin and p53 neucleoprotein increased with the increase of dose and reach the peak in 4 Gy groups.3. The expression of nucleolin in MCF7-p53-/- cell model The RNAi and the vector of Super-P53-RNAi was applied to interact the expression of p53 in MCF7 cells to establish the MCF7-p53-/- model.3.1 Time course changes of total Nucleolin protein in MCF7-p53-/- cells after IR with 4 Gy X-raysThe Western blot was applied to detect the changes of total Nucleolin protein in MCF7-p53-/- cells at 2 h～48h after IR with 4 Gy. Results showed that total protein decreased at 4 h to 48 h, compared with sham-irradiated groups.3.1.1 Time course changes of Nucleolin protein in cytoplasma of MCF7-p53-/- cells after IR with 4 Gy X-raysThe Western blot was applied to detect the changes of Nucleolin protein in cytoplasma of MCF7-p53-/- cells at 2 h～24 h after IR with 4 Gy. Results showed that Nucleolin protein in cytoplasma increased at 4 h to 24 h, compared with sham-irradiated groups. 3.1.2 Time course changes of Nucleolin neucleoprotein in MCF7-p53-/- cells after IR with 4 Gy X-raysThe Western blot was applied to detect the changes of Nucleolin neucleoprotein in MCF7-p53-/- cells at 2 h～24 h after IR with 4 Gy. Results showed that Nucleolin neucleoprotein decreased at 4 h to 24 h, compared with sham-irradiated groups.3.2 Dose effect changes of total Nucleolin protein in MCF7-p53-/- cells 4h after IRThe Western blot was applied to detect the changes of total Nucleolin protein in MCF7-p53-/- cells at 4 h after IR with doses of 0.5 Gy～4 Gy. Results showed that the total Nucleolin protein decreased in a dose-dependent manner, and reached the peak in 4 Gy groups as compared with sham-irradiated groups.3.2.1 Dose effect changes of Nucleolin protein in cytoplasma of MCF7-p53-/- cells 4h after IRThe Western blot was applied to detect the changes of Nucleolin protein in cytoplasma of MCF7-p53-/- cells at 4 h after IR with doses of 2 Gy～8 Gy. Results showed that Nucleolin increased in a dose-dependent manner, and reached the peak in 8 Gy groups as compared with sham-irradiated groups.3.2.2 Dose effect changes of Nucleolin neucleoprotein in MCF7-p53-/- cells 4h after IRThe Western blot was applied to detect the changes of Nucleolin neucleoprotein in MCF7-p53-/- cells at 4h after IR with doses of 2 Gy～8 Gy. Results showed that the neucleoprotein decreased in a dose-dependent manner, and reached the peak in 8 Gy groups as compared with sham-irradiated groups. 4. The expression of Nucleolin and p53 proteins after nuclear export inhibition with Leptomycin B 4.1 Time course changes of proteins in cytoplasma of MCF7 cells after IRLeptomycin B was used to inhibit the shuttle of nucleoprotein to cytoplasma and Western blot was applied to detect the changes of proteins after IR with 4 Gy X rays. Results showed that Nucleolin increase at 4 h and 8 h, recover at 16 h. p53 increase at 8 h.then recover at 16 h.4.2 Time course changes of neucleoproteins in MCF7 cells after IRLeptomycin B was used to inhibit the shuttle of nucleoprotein to cytoplasma and Western blot was applied to detect the changes of nucleoproteins in MCF7 cells after 4 Gy X rays. Results showed that Nucleolin and p53 nucleoprotein increased as compared with blank control. While after IR the expressions of Nucleolin and p53 nucleoprotein decrease beginning from 4 h and reached the peak at 8 h, then recovered from 16 h as compared with sham-irrad group treated only by Leptomycin B.4.3 Dose effect changes of proteins in cytoplasma of MCF7 cells 4h after IRLeptomycin B was used to inhibit the shuttle of nucleoprotein to cytoplasma and Western blot was applied to detect the changes of proteins in cytoplasma of MCF7 cells 4 h after IR with different doses of X-rays . Results showed that Nucleolin and p53 both increased in a dose-dependent manner.4.4 Dose effect changes of neucleoproteins in MCF7 cells 4h after IRLeptomycin B was used to inhibit the shuttle of nucleoprotein to cytoplasma and Western blot was applied to detect the changes of neucleoproteins in MCF7 cells 4 h after IRwith different doses of X-rays. Results showed that Nucleolin nucleoprotein increased with the increase of dose, while p53 only increased in 2 Gy groups .then recover.5. The Changes of in DNA binding activity of Nucleolin in MCF7 cells after IR with 4 Gy X-rays5.1 Time course Changes of DNA binding activity of NucleolinThe protein supershift was applied to detect the DNA binding activity of Nucleolin neucleoprotein in MCF7 cells at 2 h-48 h after IR with 4 Gy. Results showed that the DNA binding activity decrease at 2 h,4 h,8 h and 16 h, and reached the peak in 4 h groups as compared with sham-irradiated groups.5.2 Dose effect changes of DNA binding activity of NucleolinThe protein supershift was applied to detect the DNA binding activity of Nucleolin neucleoprotein in MCF7 cells 4 h after IR with different doses of 0.5 Gy～4 Gy. Results showed that the DNA binding activity decrease at 4 h,compared with sham-irradiated groups.This study will provide the possible evidence for the regulary roles of Nucleolin in the changes of p53 induced by IR...