Research Article Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells Ade Kallas, Martin Pook, Annika Trei, and Toivo Maimets Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia Correspondence should be addressed to Ade Kallas; ade.kallas@ut.ee Received 11 June 2014; Revised 10 October 2014; Accepted 14 October 2014; Published 17 November 2014 Academic Editor: Pavel Hozak Copyright © 2014 Ade Kallas et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the efects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter fow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no signifcant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells diferences in ectodermal, endodermal, and mesodermal lineages were found. Te results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in diferentiation of hES cells. 1. Introduction Cyclin-dependent kinases (CDKs) regulate cell cycle progres- sion and RNA transcription in diferent cell types. CDKs form complexes that infuence several upstream and downstream pathways regulating cell cycle, cell proliferation, and apopto- sis. Since alterations in cell cycle progression occur in several malignancies, inhibition of CDKs is regarded as a promising target for cancer treatment. Among the CDKs responsible for cell cycle progression CDK2 is an inherently fexible protein [1] with many conformations needed for interactions with various ligands. CDK2 regulates cell cycle progression by forming (a) cyclin E-CDK2 complexes at the boundary of G1 to S transition and (b) cyclin A-CDK2 complexes for orderly S phase progression and G2 to M phase transition. Te inhibition of CDK2 has therefore been an attractive, albeit complicated, task. Using structural-drug design several small molecules and peptides have been developed to target ATP binding subsites or other important binding sites needed for active confrmation of CDK2. Creating highly selective CDK2 compounds is a challenge due to the identity of ATP binding subsites within CDK1, CDK2, and CDK3 molecules; CDK2 also possesses 92% and 80% sequence identity in CDK5 and CDK6 molecules, respectively (RCSB Protein Data Bank code: 1b38). In order to afect CDK2 binding to a specifc ligand it would be important therefore to optimize interactions between CDK2 inhibitors and CDK2 residues. Various specifc CDK2 inhibitors have been shown to be efective in inducing apoptosis and reducing proliferation of various cancer cells [2]. In normal cells an induced cell cycle arrest has been shown to be reversible [3, 4]. Te properties of CDK2 inhibitors to afect cell cycles are however not completely understood. Only a weak G1 arrest has been observed in CDK2−/− MEFs [5, 6] or afer siRNA ablation in established tumor cell lines [7]. An arrest of the cell cycle in the G1 phase has however been detected in cells that have been synchronized and released from a nocodazole- induced mitotic block [8]. Additionally the CDK2 inhibitor Hindawi Publishing Corporation International Journal of Cell Biology Volume 2014, Article ID 280638, 16 pages http://dx.doi.org/10.1155/2014/280638