Gao C Y, Zelenka P S
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Exp Cell Res. 1995 Aug;219(2):612-8. doi: 10.1006/excr.1995.1271.
The present study examines whether cyclin B may be involved in apoptosis of neuronally differentiated PC12 cells following withdrawal of NGF. Cyclin B mRNA increased approximately 10-fold 4 days after NGF withdrawal, as indicated by competitive RT/PCR. Sequencing of the PCR product confirmed that it was derived from cyclin B mRNA. Cyclin B protein increased in parallel with cyclin B mRNA, as shown by immunoblotting. Immunoprecipitation with anti-cyclin B antibody demonstrated that cyclin B was associated with H1K activity, which reached a maximum 5 days after NGF withdrawal. When proteins immunoprecipitated with anti-cyclin B antibody were immunoblotted with anti-PSTAIR antibody, a protein with apparent molecular weight of 34 kDa was detected. This protein was identified as p34cdc2 on the basis of immunoreactivity with antibody against the C-terminal portion of mouse p34cdc2. Since cyclin B/p34cdc2 complexes are known to catalyze chromosomal condensation and nuclear envelope breakdown during mitosis, these results suggest that cyclin B/p34cdc2 may play some role in the nuclear changes accompanying apoptosis of PC12 cells.
本研究检测了细胞周期蛋白B是否参与了神经生长因子（NGF）撤除后神经分化型PC12细胞的凋亡过程。竞争性逆转录聚合酶链反应（RT/PCR）结果显示，NGF撤除4天后，细胞周期蛋白B的信使核糖核酸（mRNA）增加了约10倍。对聚合酶链反应产物进行测序证实其来源于细胞周期蛋白B的mRNA。免疫印迹结果表明，细胞周期蛋白B的蛋白质水平与mRNA水平平行升高。用抗细胞周期蛋白B抗体进行免疫沉淀显示，细胞周期蛋白B与H1K活性相关，该活性在NGF撤除5天后达到最大值。当用抗细胞周期蛋白B抗体免疫沉淀的蛋白质用抗PSTAIR抗体进行免疫印迹时，检测到一种表观分子量为34 kDa的蛋白质。基于与抗小鼠p34cdc2 C末端部分抗体的免疫反应性，该蛋白质被鉴定为p34cdc2。由于已知细胞周期蛋白B/p34cdc2复合物在有丝分裂过程中催化染色体凝聚和核膜破裂，这些结果表明细胞周期蛋白B/p34cdc2可能在PC12细胞凋亡伴随的核变化中发挥某种作用。
