Abstract
Ex-vivo expansion of human HSC prior to bone marrow transplantation is still an unrealized goal that could greatly extend the usefulness of this mainstay strategy for treating numerous human hematologic diseases. The safety of this process for potential use in humans depends in large part on the maintenance of karyotypic stability of HSC during expansion, a lack of which could contribute to serious, even fatal, complications such as cancer, and could also contribute to engraftment failure. The spindle checkpoint and its linkage to apoptosis initiation is one of the most important cellular processes that helps maintain chromosomal stability in rapidly proliferating cell populations by removing aneuploid and karyotypically abnormal cells via activation of cell death programs. Detailed understanding of the molecular regulation of this vital cell cycle checkpoint is important to maximize safety of in-vitro HSC expansion techniques. It is widely accepted that mammalian cells enter the next G1-phase with 4N DNA after slippage from prolonged drug-induced mitotic block caused by activation of the transient spindle checkpoint that it is from this state that polyploid/aneuploid cells initiate apoptosis. However, definitive biochemical evidence for G1 is scarce or unconvincing; in part because of methods of protein extraction required for immunoblot analysis that cannot take into account the cell cycle heterogeneity of cell cultures. We used single-cell-intracellular-flow-cytometric analysis to define important factors determining cell fate after mitotic slippage. Results from human and mouse embryonic stem cells that reenter polyploid cell cycles are compared to human somatic hematopoietic cells that die after MS. We now report for the first time that phosphorylation status of pRb, p53, CDK1, and cyclin B1 levels are important for cell fate/apoptosis decision in mitotic-slippage cells, which occurs in a unique, intervening, non-G1, tetraploid subphase. Hyperphosphorylated Rb was extremely abundant in mitotic-slippage cells, a cell signaling event usually associated with early G1-S phase transition. P53 was phosphorylated at sites known to be associated with apoptosis regulation. Cyclin A and B1 were undetectable in mitotic slippage cells; yet, CDK1 was phosphorylated at sites typically associated with its activation. Evidence is also presented raising the possibility of cyclin B1-independent CDK1 activity in mitotic-slippage cells. These findings challenge the current models of spindle checkpoint-apoptosis linkages. Our new model could have important implications for methods to maintain karyotypic stability during ex-vivo HSC expansion.
The conserved AAA-ATPase PCH-2 TRIP13 regulates spindle checkpoint strength
