P2P-R protein overexpression restricts mitotic progression at prometaphase and promotes mitotic apoptosis.
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.
Gao
Sizhi
S
Department of Pathology, University of Tennessee Health Science Center, Memphis TN 38163, Tennessee, USA.
Scott
Robert E
RE
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D017209:Apoptosis; D002352:Carrier Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002466:Cell Nucleolus / Q000378:metabolism; D002869:Chromosome Aberrations; D002999:Clone Cells; D006801:Humans; D007399:Interphase; D008677:Metaphase; D051379:Mice; D008938:Mitosis; D011003:Ploidies; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C104647:PRKRA protein, human; D016601:RNA-Binding Proteins; C491290:Rbbp6 protein, mouse