The Functional Link Between CDK1 and Retinoic Acid Receptor γ (RARγ) in Response to Treatment with All-Trans Retinoic Acid

Abstract 2485

Alterations in cell cycle pathways and retinoic acid signaling are implicated in the pathogenesis of leukemia and treatment response. The retinoic acid receptors (RARα, RARβ and RARγ) mediate the differentiation, cell cycle arrest and cellular response to all-trans retinoic acid (ATRA). However, it is largely unknown whether the cyclin dependent kinases (CDK1 and CDK2) mediate the ATRA effects, also whether and how CDK1 and CDK2 are functional linked to RARs. Therefore in the present study, we examined the function of CDKs and their regulation in relationship to the RAR α, β and γ.

The use of siRNA mediated knockdown of CDK1 in human leukemic U-937 cells affected cell cycle distribution, resulting in a decreased fraction of G0/G1 cells which coincided with the reduced expression of p27 protein. Treatment of U-937 si-CDK1 cells with ATRA demonstrated reduced terminal differentiation and decreased cell cycle arrest at G0/G1 phases in comparison to U-937 siRNA control cells. We observed a reduced responsiveness to ATRA treatment due to the loss of CDK1 function, and this was accompanied with the alterations in the expression of RARγ and the increased phosphorylation of Akt. However, loss of CDK2 function showed no effects on ATRA-mediated growth arrest and differentiation in U-937 cells.

We also found that nuclear CDK1 was actively phosphorylated and was rapidly degraded upon the induction of ATRA, in contrast to its cytoplasmic counterpart in human leukemic U-937 cells. When examining the functional link between RARs and the regulation of CDKs in response to ATRA, we found that RARβ and RARγ, but not RARα, participated in ATRA-mediated protein degradation of CDK1 and CDK2. Moreover, we compared the effects of ATRA on ubiquitin-mediated protein degradation in the F9 teratocarcinoma cells and in F9 cells harboring targeted deletions of the RARα, RARβ and RARγ genes. ATRA induced ubiquitin conjugates showed striking changes in patterns in cells lacking RARγ. This indicates that the blockage in protein degradation of CDK1 may be due to the loss of RARγ function. Among the RAR isoforms, RARγ co-localized with CDK1 predominantly in the nuclear bodies. There was no detectable complex formation between RARβ and CDK1. This suggests that the phosphorylation and degradation of the nuclear CDK1 and its interaction with RARγ may be important events in mediating ATRA-induced effects on cell cycle and differentiation.

Our study has revealed a novel mechanism by which CDK1 cooperates with RARγ to mediate the effects of all-trans retinoic acid.