The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria.
The mechanisms of death of an erythroleukemic cell line by p53: involvement of the microtubule and mitochondria.
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DOI：
10.3109/10428199909093740
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发表时间：
1999-03
影响因子：
2.6
通讯作者：
Mitsuo V. Kato
中科院分区：
文献类型：
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作者：
Mitsuo V. Kato
建立了只表达温度敏感性突变型p53基因（密码子135处的Ala至瓦尔取代）的小鼠红白血病细胞系（1-2-3）。这些细胞在32 ℃培养时表现出典型的凋亡特征。在此过程中，p53恢复了野生型p53的功能，p21（waf 1/cip 1/sdi 1）、cyclin G1和gadd 45基因的表达增加。而mdm 2、bcl-2、bax、fas和fasl基因的表达无明显变化，提示存在其他与凋亡相关的基因。采用mRNA差异显示法筛选p53上调表达基因。其中一个上调基因被鉴定为延伸因子1 α（EF-1 α）基因。EF-1 α也是一种微管切断蛋白。在温度变化后，细胞产生了与用长春新碱（一种影响微管的药物）处理的细胞相似的形态和α-微管蛋白的定位。与p53上调EF-1 α相关的微管断裂可能是细胞死亡的原因。另一方面，尽管1-2-3细胞在凋亡早期表现出cyclin G1基因的显著增加，但cyclin G1的功能尚不清楚。酵母双杂交系统用于鉴定细胞周期蛋白G1相关蛋白。一种是细胞色素c（Cyt c）氧化酶亚基II（COXII）。从表达高水平细胞周期蛋白G1的人骨肉瘤细胞系的提取物中免疫共沉淀细胞周期蛋白G1和COXII。考克斯的活性也增加了温度的变化，在这个细胞系。考克斯活性的变化模式密切反映了细胞周期蛋白G1基因的表达。细胞周期蛋白G1和COXII在体内相互物理关联，并且通过与p53上调的细胞周期蛋白G1结合而激活COXII可能与细胞凋亡相关。这两个新的途径，p53-EF-1 α-微管-切断（-细胞骨架的扭曲）和p53-细胞周期蛋白G1-COXII（-CytC，ATP-半胱天冬酶-3激活），可能合作，以诱导该细胞系的凋亡。
A murine erythroleukemic cell line (1-2-3) which expresses only the temperature-sensitive mutant p53 gene (Ala-to-Val substitution at codon 135) was established. These cells showed typical characteristics of apoptosis, when they were cultured at 32 degrees C. In this process, p53 recovered the wild-type p53 function and the expression of the p21 (waf1/cip1/sdi1), cyclin G1 and gadd45 genes was increased. However, no significant changes were detected in the expression of the mdm2, bcl-2, bax, fas and fasl genes, suggesting the existence of other genes associated with apoptosis. Genes up-regulated by p53 were screened by the mRNA differential display method. One of the up-regulated genes was identified as the elongation factor 1 alpha (EF-1 alpha) gene. EF-1 alpha is also a microtubule-severing protein. Upon the temperature-shift, the cells developed the morphology and the localization of alpha-tubulin similar to those of the cells treated with vincristine, a drug that affects microtubules. The microtubule-severing associated with up-regulation of EF-1 alpha by p53 may be a cause of the cell death. On the other hand, the function of cyclin G1 is not so clear despite the fact that 1-2-3 cells showed a significant increase of the cyclin G1 gene during the early stage of apoptosis. The yeast two-hybrid system was used to identify cyclin G1-associated proteins. One is a cytochrome c (Cyt c) oxidase subunit II (COXII). Cyclin G1 and COXII were co-immunoprecipitated from an extract of human osteosarcoma cell line that expressed high levels of cyclin G1. COX activity was also increased by temperature-shift in this cell line. The pattern of changes in COX activity was closely reflected by the expression of the cyclin G1 gene. Cyclin G1 and COXII associate physically with each other in vivo and that activation of COXII by binding to cyclin G1 upregulated by p53 may be associated with apoptosis. These two new pathways, p53-EF-1 alpha-microtubule-severing (-distortion of cytoskeleton) and p53-cyclin G1-COXII (-CytC, ATP-caspase-3 activation), may cooperate to induce apoptosis in this cell line.