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. 2017 May 23;39(5):332-338.
doi: 10.3760/cma.j.issn.0253-3766.2017.05.003.

[Effect of Δ40p53 isoform on enhancing the pro-apoptotic function of p53 in tumor cells]

[Article in Chinese]
B S Wang  1 , H W Zhao  2 , L X Qiao  3 , J Q Shan  1 , Q S Hou  1 , D X Chen  3 , H L Guo  1
Affiliations

[Effect of Δ40p53 isoform on enhancing the pro-apoptotic function of p53 in tumor cells]

[Article in Chinese]
B S Wang et al. Zhonghua Zhong Liu Za Zhi. .

Abstract
in English, Chinese

Objective: To investigate the effect of Δ40p53, an alternative spliced isoform of p53 lacking the N-ter minus, on the pro-apoptotic function of p53. Methods: 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. Results: 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 (t=3.721, P=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 (t=2.930, P=0.042). Conclusion: Δ40p53 isoform can bind to full-length p53, and enhance its pro-apoptotic function in tumor cells.

目的： 探讨p53亚体Δ40p53对p53促肿瘤细胞凋亡作用的影响。 方法： 体外培养HCT116－p53(－/－)结肠癌细胞系(内源性表达Δ40p53)、HCT116－p53(＋/＋)结肠癌细胞系(内源性表达野生型p53)和H1299肺癌细胞系(p53缺失)，感染p53腺病毒使p53过表达，转染Δ40p53质粒使Δ40p53过表达。逆转录聚合酶链反应(RT－PCR)检测Δ40p53和p53 mRNA，实时荧光定量PCR检测Δ40p53对p53转录水平的影响，Western blot检测相关蛋白的表达，免疫共沉淀检测Δ40p53与p53相互作用关系，Calcein－AM/PI染色和流式细胞术检测细胞凋亡水平。 结果： Δ40p53在HCT116－p53(－/－)细胞中能够稳定转录并表达。实时荧光定量PCR和Western blot检测显示，Δ40p53对p53的转录及表达水平无影响。免疫共沉淀检测显示，Δ40p53与p53能相互结合。Calcein－AM/PI染色显示，H1299－Control组、HCT116－p53(－/－)－Control组、H1299＋p53组、HCT116－p53(－/－)＋p53组、H1299＋奥沙利铂(Oxa)组、HCT116－p53(－/－)＋Oxa组、H1299＋p53＋Oxa组和HCT116－p53(－/－)＋p53＋Oxa组的细胞凋亡率分别为(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组的细胞凋亡率差异有统计学意义(t＝3.721，P＝0.021)。Calcein－AM/PI染色显示，H1299－Control组、H1299＋Δ40p53组、H1299＋p53组、H1299＋p53＋ Δ40p53组、H1299＋Oxa组、H1299＋Δ40p53＋Oxa组、H1299＋p53＋Oxa组和H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率分别为(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)%和(35.20±1.43)%，H1299＋p53＋Oxa组与H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率差异有统计学意义(t＝3.629，P＝0.022)。流式细胞术检测显示，H1299－Control组、H1299＋Δ40p53组、H1299＋p53组、H1299＋p53＋Δ40p53组、H1299＋Oxa组、H1299＋Δ40p53＋Oxa组、H1299＋p53＋Oxa组和H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率分别为(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＋Oxa组与H1299＋p53＋Δ40p53＋Oxa组的细胞凋亡率差异有统计学意义(t＝2.930，P＝0.042)。 结论： Δ40p53与p53可相互结合，并能够增强p53的促肿瘤细胞凋亡作用。.

Keywords: Apoptosis; Neoplasms; p53; Δ40p53.

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