野生型p53基因对白血病细胞系K562中心体过度复制的抑制作用。

Tian Wen-Jun, Feng Wen-Li, Wang Hong-Bin, Huang Shi-Feng, Cao Wei-Xi, Huang Zong-Gan

Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Chongqing University of Medical Sciences, Chongqing, PR China.

Ai Zheng. 2009 Feb;28(2):122-6. Epub 2009 Feb 23.

BACKGROUND AND OBJECTIVE

Mutation and deletion of the p53 gene in tumor cells is one of the major reasons for aneuploid development and genomic instability. Abnormal centrosomes exist in chronic myelogenous leukemia patients at different stages; furthermore, the degree of abnormality is associated with the clinical stage and more severe in the blast crisis stage. This study was to establish the leukemia cell line K562 with the exogenous wild-type p53 (wt-p53) gene, and to explore the effect of the p53 gene on centrosomes in K562 cells.

METHODS

The recombinant adenoviruses carrying the wt-p53 gene (Ad5wtp53), the mutant p53 gene (Ad5mtp53) and the green fluorescent protein gene (Ad5GFP) were amplified respectively in HEK293 cells, and co-infected with cation polybrene into K562 cells respectively; uninfected K562 cells were used as blank control. The infection efficiency was analyzed by flow cytometry. P53 expression was detected by Western blot. Centrosomes were counted under the laser confocal microscope after indirect immunofluorescence staining. The expression of Gadd45a (growth arrest and DNA damage), BubR1 (Bub 1 related) and Aurora A was detected by western blot.

RESULTS

K562 cell line with exogenous wt-p53 gene was established. The infection efficiencies of three groups were over 60%, and P53 sustained expression for 72 h. The percentage of cells with amplified centrosomes (more than 2/cell) in Ad5wtp53 group was decreased to (0.38 +/- 0.02)%, lower than that of blank control group (p < 0.05). Meanwhile, the protein levels of Gadd45a and BubR1 in Ad5wtp53 group were upregulated by 93% and 88% of blank control (p < 0.05), respectively, and the protein level of Aurora A was downregulated by 56% of blank control (p < 0.05).

CONCLUSIONS

P53 protein is sustained to express in K562 cells after being infected by Ad5wtp53. wt-p53 can suppress excessive replication of centrosomes that may contribute to the upregulation of Gadd45a and BubR1 protein expression as well as the downregulation of Aurora A protein expression.

背景与目的

肿瘤细胞中p53基因的突变和缺失是异倍体发育和基因组不稳定的主要原因之一。慢性粒细胞白血病患者在不同阶段均存在中心体异常；此外，异常程度与临床分期相关，在急变期更为严重。本研究旨在构建携带外源性野生型p53（wt-p53）基因的白血病细胞系K562，并探讨p53基因对K562细胞中心体的影响。

方法

分别在HEK293细胞中扩增携带wt-p53基因的重组腺病毒（Ad5wtp53）、突变型p53基因的重组腺病毒（Ad5mtp53）和绿色荧光蛋白基因的重组腺病毒（Ad5GFP），并分别与阳离子多聚赖氨酸共感染K562细胞；未感染的K562细胞作为空白对照。采用流式细胞术分析感染效率。通过蛋白质免疫印迹法检测p53表达。间接免疫荧光染色后，在激光共聚焦显微镜下计数中心体。通过蛋白质免疫印迹法检测生长停滞和DNA损伤诱导蛋白（Gadd45a）、Bub1相关蛋白（BubR1）和极光激酶A（Aurora A）的表达。

结果

成功构建携带外源性wt-p53基因的K562细胞系。三组的感染效率均超过60%，且p53持续表达72小时。Ad5wtp53组中中心体扩增（每个细胞超过2个）的细胞百分比降至（0.38±0.02）%，低于空白对照组（p<0.05）。同时，Ad5wtp53组中Gadd45a和BubR1的蛋白水平分别比空白对照组上调93%和88%（p<0.05），而Aurora A的蛋白水平比空白对照组下调56%（p<0.05）。