P2P‐R protein overexpression restricts mitotic progression at prometaphase and promotes mitotic apoptosis

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Abstract
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.