The cell cycle is driven by oscillating cyclin-dependent kinase (CDK) activities. It is primarily controlled by temporal phosphorylation of CDKs and degradation of cyclins. While cyclin B1-CDK1 is known to be the major component of mitosis promoting factor, the essential functions of cyclin A2-CDKs remain to be deciphered. Our group has recently defined the role of cyclin A2 in activating cyclin B1-CDK1 through the WEE1 axis....[ Read more ]

The cell cycle is driven by oscillating cyclin-dependent kinase (CDK) activities. It is primarily controlled by temporal phosphorylation of CDKs and degradation of cyclins. While cyclin B1-CDK1 is known to be the major component of mitosis promoting factor, the essential functions of cyclin A2-CDKs remain to be deciphered. Our group has recently defined the role of cyclin A2 in activating cyclin B1-CDK1 through the WEE1 axis.

As most of the functional studies of cyclin A2 employ RNA interference techniques, a method was developed to conveniently validate the knockdown phenotypes. I have devised a vector, called pKAR1, that could express short hairpin RNA (shRNA) and the respective rescue cDNA together. This system was particularly useful in the case of cyclin A2, as protracted knockdown of cyclin A2 led to apoptosis. Using the pKAR1 system, I have validated the specificity of cyclin A2 shRNA and successfully generated stable cell lines that displayed conditional depletion of cyclin A2.

Together with geminin, cyclin A2 is proposed to play a major role in inhibiting DNA re-replication. However, I found that no significant re-replication occurred when cyclin A2 was depleted. A small extent of re-replication occurred when both cyclin A2 and cyclin B1 were depleted together. In contrast, chemical inhibition of CDK1 induced significant re-replication up to 16N. Surprisingly, knockdown of cyclin A2 abolished the DNA re-replication induced by CDK1 inhibition. Since APC/C is proposed to be a master regulator of DNA replication, an APC/C activity reporter system was generated to monitor the APC/C activity in living cells. I found that the APC/C activity continued to oscillate after CDK1 inhibition. The activation of APC/C was preceded by the activation of CDK2; and inhibition of cyclin A2-CDK2 prevented APC/C activation. On the other hand, interruption of APC/C activation by either over-expressing its inhibitor EMI1 or by depleting its adaptor protein CDH1 prevented re-replication. Taking together, these results show that cyclin A2-CDK2 is essential to DNA re-replication, most probably through the activation of APC/C.

Prolonged depletion of cyclin A2 also induced a loss of viability. This cell death was observed after transfection of both shRNA as well as siRNA against cyclin A2, and was validated by the pKAR1 vector system, indicating the specificity of the effect. I found that this cell death was caspases-dependent and could be rescued by microtubule-disrupting agents. Apoptosis triggered by specific chemical inhibitors of CDK2, but not CDK1, was also suppressed by microtubule-disrupting agents. These data suggest that knockdown of cyclin A2 induces apoptosis via a microtubule network-mediated mechanism.