Abstract
BackgroundCDK-inhibitors can diminish transcriptional levels of cell cycle-related cyclins through the inhibition of E2F family members and CDK7 and 9. Cyclin A1, an E2F-independent cyclin, is strongly upregulated under genotoxic conditions and functionally was shown to increase NHEJ activity. Cyclin A1 outcompetes with cyclin A2 for CDK2 binding, possibly redirecting its activity towards DNA repair. To see if we could therapeutically block this switch, we analyzed the effects of the CDK-inhibitor R-Roscovitine on the expression levels of cyclin A1 under genotoxic stress and observed subsequent DNA damage and repair mechanisms.ResultsWe found that R-Roscovitine alone was unable to alter cyclin A1 transcriptional levels, however it was able to reduce protein expression through a proteosome-dependent mechanism. When combined with DNA damaging agents, R-Roscovitine was able to prevent the DNA damage-induced upregulation of cyclin A1 on a transcriptional and post-transcriptional level. This, moreover resulted in a significant decrease in non-homologous end-joining (NHEJ) paired with an increase in DNA DSBs and overall DNA damage over time. Furthermore, microarray analysis demonstrated that R-Roscovitine affected DNA repair mechanisms in a more global fashion.ConclusionsOur data reveal a new mechanism of action for R-Roscovitine on DNA repair through the inhibition of the molecular switch between cyclin A family members under genotoxic conditions resulting in reduced NHEJ capability.
Highlights
Cyclin-dependent kinases (CDK)-inhibitors can diminish transcriptional levels of cell cycle-related cyclins through the inhibition of E2F family members and CDK7 and 9
Since one of the most relevant effects of CDK inhibitors is the downregulation of cell cycle related cyclins, we investigated if the inhibition of DNA repair mechanisms by Roscovitine may occur through the modulation of the expression levels of cyclin A family members
We found that cyclin A1 upregulation is dose dependent with a plateau that is reached around 2.5 μM (IC90)
Summary
CDK-inhibitors can diminish transcriptional levels of cell cycle-related cyclins through the inhibition of E2F family members and CDK7 and 9. To see if we could therapeutically block this switch, we analyzed the effects of the CDK-inhibitor R-Roscovitine on the expression levels of cyclin A1 under genotoxic stress and observed subsequent DNA damage and repair mechanisms. In preclinical studies Roscovitine has shown significant in vitro and in vivo antitumor activity on a wide panel of human cancers and is currently in phase II clinical trials[5]. It has become evident that, CDK-inhibitors, such as Roscovitine, may curb the activity of DNA repair machinery[6,7], becoming an attractive candidate for therapeutic association with either radiation therapy[8,9] or genotoxic agent-based chemotherapy[10].
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