The effects of p55Cdc/Cdc20 on apoptosis and G1/S transition in myeloid cells.

摘要

p55Cdc/Cdc20 is a member of a family of proteins involved in protein degradation during the cell cycle. This family of proteins is characterized by the presence of WD repeats, which are involved in protein interactions, and by their ability to activate the anaphase-promoting complex (APC). The APC participates at the spindle assembly checkpoint, a requirement of the cell cycle that ensures duplicated chromosomes attached to the mitotic spindle have properly lined up during metaphase before separation during anaphase. The p55Cdc/Cdc20 protein serves to link APC substrates with the APC for degradation.; Previous studies have focused on the role of p55Cdc/Cdc20 during metaphase-anaphase transition. We studied other biological functions of this protein using a cell culture system with 32Dcl3 cells, a murine myeloid cell line. We overexpressed p55Cdc/Cdc20 using an inducible metallothionein promoter. Our studies have shown that increased p55Cdc/Cdc20 expression leads to an increased rate of apoptosis in 32Dcl3 cells deprived of serum and growth factor.; We have also shown that 32Dcl3 cells overexpressing p55Cdc/Cdc20 exhibit an earlier transition from G1 to S phase of the cell cycle. The levels of various proteins such as cyclin D1, cyclin E, cdk2, and cdc2 were not affected by p55Cdc/Cdc20 overexpression. The level of p27Kip1, a cyclin-dependent kinase inhibitor, appeared to decrease with p55Cdc/Cdc20 overexpression although this may be an effect of the earlier G1/S transition. The activity of cdk2 increased in cells overexpressing p55Cdc/Cdc20 compared to control cells.; We have commenced in vivo studies of p55cdc/Cdc20 in mice using knockout and transgenic approaches. Mapping of the genomic region around p55Cdc/Cdc20 has been done in preparation for knockout targeting vector construction. Additionally, a transgenic mouse model with p55Cdc/Cdc20 cDNA and myeloid-specific h-MRP8 promoter was created and studied. We have demonstrated that five lines of C57BL/6 mice successfully incorporated the transgene. (Abstract shortened by UMI.)