Gao Sizhi, Scott Robert E
Department of Pathology, University of Tennessee Health Science Center, Memphis TN 38163, Tennessee, USA.
J Cell Physiol. 2002 Nov;193(2):199-207. doi: 10.1002/jcp.10163.
Mitotic cells show a tenfold increase in immunoreactive P2P-R protein. During mitosis, the distribution of P2P-R protein also changes from a primary nucleolar localization in interphase cells to the periphery of chromosome in mitotic cells. These findings suggest that P2P-R might serve a functional role in mitosis. To test this possibility, human Saos2 cells were stably transfected with P2P-R DNA constructs and the biological effects of P2P-R overexpression were evaluated. Overexpression of near full-length P2P-R was found to have paradoxical effects on the relationship between proliferation and mitosis in the nine Saos2 cell clones that were studied. A significant repression in the population doubling rates was observed in all nine clones even though a significant increase in the frequency of easily detached cells with a mitotic morphology was apparent. Flow cytometric analysis confirmed that greater than two thirds of the cells with a mitotic morphology had a 4n DNA content. Confocal microscopy further established that 85% of the mitotic cell population had prometaphase characteristics suggesting that P2P-R overexpression restricts mitotic progression at prometaphase. Many cells with a mitotic morphology also showed signs of apoptosis with prominent cell surface blebs. Confocal microscopy confirmed that 25-40% of such mitotic cells were apoptotic with chromosomal abnormalities and cell surface blebbing. In association with mitotic apoptosis, P2P-R protein appears to dissociate from the periphery of chromosomes and localize in the cytoplasm and in cell surface blebs. The presence of P2P-R in cell surface blebs was confirmed by analysis of highly enriched populations of apoptotic cell surface blebs wherein Western blotting documented the presence of 250 kDa P2P-R. These results therefore suggest that P2P-R overexpression promotes both prometaphase arrest in mitosis and mitotic apoptosis.
有丝分裂细胞中免疫反应性P2P-R蛋白增加了十倍。在有丝分裂过程中，P2P-R蛋白的分布也从间期细胞中的主要核仁定位转变为有丝分裂细胞中染色体的周边。这些发现表明P2P-R可能在有丝分裂中发挥功能作用。为了验证这种可能性，将P2P-R DNA构建体稳定转染到人Saos2细胞中，并评估P2P-R过表达的生物学效应。在所研究的九个Saos2细胞克隆中，发现近全长P2P-R的过表达对增殖与有丝分裂之间的关系具有矛盾的影响。尽管具有有丝分裂形态的易于脱离细胞的频率明显增加，但在所有九个克隆中均观察到群体倍增率的显著抑制。流式细胞术分析证实，超过三分之二具有有丝分裂形态的细胞具有4n DNA含量。共聚焦显微镜进一步证实，85%的有丝分裂细胞群体具有前中期特征，表明P2P-R过表达在前中期限制有丝分裂进程。许多具有有丝分裂形态的细胞也显示出凋亡迹象，细胞表面有明显的泡状突起。共聚焦显微镜证实，25%-40%的此类有丝分裂细胞凋亡，伴有染色体异常和细胞表面泡状突起。与有丝分裂凋亡相关，P2P-R蛋白似乎从染色体周边解离并定位在细胞质和细胞表面泡状突起中。通过对凋亡细胞表面泡状突起的高度富集群体进行分析，证实了P2P-R在细胞表面泡状突起中的存在，其中蛋白质印迹法记录了250 kDa P2P-R的存在。因此，这些结果表明P2P-R过表达促进有丝分裂前中期停滞和有丝分裂凋亡。
