Wang B S, Zhao H W, Qiao L X, Shan J Q, Hou Q S, Chen D X, Guo H L
Department of General Surgery, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Cancer Hospital and Institute, Jinan 250117, China.
Department of Medical Records Management, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Cancer Hospital and Institute, Jinan 250117, China.
Zhonghua Zhong Liu Za Zhi. 2017 May 23;39(5):332-338. doi: 10.3760/cma.j.issn.0253-3766.2017.05.003.
To investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53. The wild-type p53 was ectopically expressed in HCT116-p53(-/-) (endogenous Δ40p53 expression), HCT116-p53(+ /+) (wild-type p53) and H1299 (p53-null) cells by adenoviral delivery, while Δ40p53 plasmid were transfected into these cells to overexpress Δ40p53. The levels of Δ40p53 and p53 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR. The expression of related proteins was deter mined by Western blotting. The interaction of p53 and Δ40p53 was observed by co-immunoprecipitation assay. Calcein-AM/propidium iodide (PI) staining and flow cytometry were used to detect the apoptotic rate of tested cells in each group. HCT116-p53(-/-) cells expressed endogenous Δ40p53 isoform. Neither transcription nor protein expression of wild-type p53 was interfered by the increased expression of Δ40p53. Full length p53 and Δ40p53 could bind to each other. Calcein-AM/PI staining showed that the apoptotic rates of H1299-Control, HCT116-p53(-/-) -Control, H1299+ p53, HCT116-p53(-/-)+ p53, H1299+ oxaliplatin (Oxa), HCT116-p53(-/-)+ Oxa, H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups were (2.50±0.47)%, (2.40±0.32)%, (5.20±0.58)%, (4.10±0.18)%, (22.40±1.73)%, (19.30±1.11)%, (29.90±1.15)% and (39.30±2.26)%, respectively. It was statistically significant between H1299+ p53+ Oxa and HCT116-p53(-/-)+ p53+ Oxa groups (=3.721, =0.0205). Moreover, the apoptotic rates of H1299-Control, H1299+ Δ40p53, H1299+ p53, H1299+ p53+ Δ40p53, H1299+ Oxa, H1299+ Δ40p53+ Oxa, H1299+ p53+ Oxa and H1299+ p53+ Δ40p53+ Oxa groups were (2.60±0.35)%, (2.20±0.17)%, (4.80±0.49)%, (4.90±1.10)%, (20.30±1.10)%, (19.60±1.45)%, (27.90±1.39)%, (35.20±1.43)%, respectively. Furthermore, flow cytometry assay showed that the apoptotic rates of above cells were (2.70±0.32)%, (2.20±0.24)%, (4.60±0.48)%, (3.90±0.67)%, (19.30±1.11)%, (17.70±0.66)%, (28.30±2.76)% and (37.50±1.51)%, respectively. H1299+ p53+ Δ40p53+ Oxa cells showed higher cell apoptosis than H1299+ p53+ Oxa cells (=2.930, =0.042). Δ40p53 isoform can bind to full-length p53, and enhance its pro-apoptotic function in tumor cells.
为研究p53的一种缺失N端的可变剪接异构体Δ40p53对p53促凋亡功能的影响。通过腺病毒载体将野生型p53在HCT116 - p53(-/-)（内源性表达Δ40p53）、HCT116 - p53(+/+)（野生型p53）和H1299（p53基因缺失）细胞中异位表达，同时将Δ40p53质粒转染至这些细胞以过表达Δ40p53。通过逆转录 - 聚合酶链反应（RT - PCR）和定量PCR检测Δ40p53和p53 mRNA的水平。通过蛋白质印迹法测定相关蛋白的表达。通过免疫共沉淀试验观察p53与Δ40p53的相互作用。采用钙黄绿素 - AM/碘化丙啶（PI）染色和流式细胞术检测每组受试细胞的凋亡率。HCT116 - p53(-/-)细胞表达内源性Δ40p53异构体。野生型p53的转录和蛋白表达均未受到Δ40p53表达增加的干扰。全长p53和Δ40p53能够相互结合。钙黄绿素 - AM/PI染色显示，H1299 - 对照、HCT116 - p53(-/-) - 对照、H1299 + p53、HCT116 - p53(-/-)+ p53、H1299 + 奥沙利铂（Oxa）、HCT116 - p53(-/-)+ Oxa、H1299 + p53 + Oxa和HCT116 - p53(-/-)+ p53 + Oxa组的凋亡率分别为(2.50±0.47)%、(2.40±0.32)%、(5.20±0.58)%、(4.10±0.18)%、(22.40±1.73)%、(19.30±1.11)%、(29.90±1.15)%和(39.30±2.26)%。H1299 + p53 + Oxa组与HCT116 - p53(-/-)+ p53 + Oxa组之间差异具有统计学意义（=3.721，=0.0205）。此外，H1299 - 对照、H1299 + Δ40p53、H1299 + p53、H1299 + p53 + Δ40p53、H1299 + Oxa、H1299 + Δ40p53 + Oxa、H1299 + p53 + Oxa和H1299 + p53 + Δ40p53 + Oxa组的凋亡率分别为(2.60±0.35)%、(2.20±0.17)%、(4.80±0.49)%、(4.90±1.10)%、(20.30±1.10)%、(19.60±1.45)%、(27.90±1.39)%、(35.20±1.43)%。此外，流式细胞术检测显示上述细胞的凋亡率分别为(2.70±0.32)%、(2.20±0.24)%、(4.60±0.48)%、(3.90±0.67)%、(19.30±1.11)%、(17.70±0.66)%、(28.30±2.76)%和(37.50±1.51)%。H1299 + p53 + Δ40p53 + Oxa细胞比H1299 + p53 + Oxa细胞表现出更高的细胞凋亡率（=2.930，=0.042）。Δ40p53异构体能够与全长p53结合，并增强其在肿瘤细胞中的促凋亡功能。
