Abstract
Introduction
We have previously proposed that Cdh1 is a tumor suppressor by maintaining genomic stability. We also found Cdh1 downregulated in several tumor cell lines including AML (Oncogene 2008; 27:907-17). Heterozygous Cdh1 knockout mice develop epithelial tumors, myelodysplasia and plasma cell dyscrasias (Nat. Cell Biol. 2008;10:802-11). By analyzing primary AML samples from bone marrow (BM) or peripheral blood (PB) we detected downregulation of Cdh1 in the vast majority of samples when compared to normal CD34+ HSCs. Progression through the cell cycle is tightly regulated by different cyclin-dependent kinases (Cdks) and their activating cyclin subunits. Stage-specific proteolysis of cyclins and other cell cycle regulators is important for transition to the next cell cycle phase. The anaphase-promoting complex/cyclosome (APC/C) is an E3-ubiquitin ligase that controls mitosis and G1 through degradation of these proteins. Through its activating subunits Cdh1 and Cdc20 the APC/C ensures substrate-specifity. While Cdc20 regulates progression through mitosis, Cdh1 is activated in late mitosis to coordinate accurate entry into S-phase. Thereby, the APC/C is crucial for maintaining genomic stability during the cell cycle. Suppression of APC/C-Cdh1 can lead to unscheduled cyclin expression and Cdk activity, which can cause cell cycle defects leading to the accumulation of DNA alterations and further to malignant transformations. However, the exact nature of the origin of genomic instability upon downregulation of Cdh1 is unclear.
Methods
To investigate stability of cyclins in Cdh1-knockdown (kd) cells, origin loading and start of replication, cells were released from a mitotic block and samples were taken every 2 h until S-phase entry for FACS and immunoblotting. For live-cell imaging cells were seeded 24 h before imaging in chambered coverslips, after which progression through the cell cycle was analyzed by automated microscopy.
Results
Characterization of a Cdh1-kd revealed strong stabilization of the substrates cyclin A/B leading to diminished loading of mini-chromosome maintenance (MCM) proteins on replication origins in G1. Stabilization of cyclin A/B and unscheduled Cdk1/2 activity may cause the observed premature entry into S-phase, while the reduced loading of MCMs in G1 could be responsible for the prolonged replication in S-phase seen in Cdh1-kd cells. Accordingly, treatment with the Cdk1 inhibitor RO-3306 restored reduced MCM loading. Polo-like kinase 1 (Plk1) was stabilized in Cdh1-kd cells, which may cause bypass of the Cdc14B-Cdh1-Plk1 dependent DNA damage checkpoint. Indeed, potential replication stress in Cdh1-kd cells did not lead to G2/M arrest, but was enforced by inhibition of the Cdh1 substrate Plk1. Underreplicated DNA and replication intermediates in mitosis may be the reason for increased genomic instability, namely lagging chromosomes, anaphase bridges and micronuclei in Cdh1-kd cells detected by live-cell imaging. In addition, aberrant cytokinesis and the development of polyploid cells generated by misseparation of chromosomes during mitosis were enhanced in Cdh1-kd cells. Finally, monitoring of 53BP1, a DNA-repair marker, in living cells showed amplified DNA-damage through increased double-strand breaks in Cdh1-kd cells.
Conclusions
Downregulation of the tumor suppressor APC/C-Cdh1 leads to deregulation of DNA-replication by stabilizing cyclin A and B in G1 and reduced loading of replication origins with MCM proteins resulting in the accumulation of enhanced genomic instability and DNA damage.
Disclosures:
No relevant conflicts of interest to declare.
Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells