As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription CDKs are attractive targets for creating cancer cell treatments. 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 significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal endodermal and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells. 1 Introduction Cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription in different cell types. CDKs form complexes that influence several upstream and downstream pathways regulating cell cycle cell proliferation and apoptosis. Since alterations in cell cycle progression occur in several malignancies Z-LEHD-FMK inhibition of CDKs is regarded as a promising target for cancer treatment. Among the CDKs responsible for cell cycle progression CDK2 is an inherently flexible protein [1] with many conformations needed for interactions with various ligands. CDK2 regulates cell cycle progression by forming (a) cyclin Z-LEHD-FMK 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. The 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 confirmation of CDK2. Z-LEHD-FMK 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 affect CDK2 binding to a specific ligand it would be important therefore to optimize interactions between CDK2 inhibitors and CDK2 residues. Various specific CDK2 inhibitors have been shown to be effective 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 The properties of CDK2 inhibitors to affect cell cycles are however not completely understood. Only a weak G1 arrest has been observed in CDK2?/? MEFs [5 6 or after 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 flavopiridol was more cytotoxic to transformed cells when treated within the S phase [9]. Cells in certain cell cycle phases are thus likely more sensitive to CDK2 inhibition. Some cancer cells however possess resistance to CDK2 inhibition as shown by a unique upregulation of CDK2 target proteins and preexisting cellular polyploidy in cancer cells [10]. Among CDK2 inhibitors those with purine-based structures (NU6140 and its derivatives) have shown higher specificity to inhibit CDK2 interaction with cyclin A compared to other interactions (CDK1/cyclin B CDK4/cyclin D CDK5/p25 and CDK7/cyclin H) [11 12 NU6140 induces apoptosis in HeLa cervical carcinoma cells arrests cells in the G2/M phase and reduces Pgf cell survival both by itself and in combination with paclitaxel [13]. In epithelial cells however NU6140 has no effect on apoptosis [14]. Exactly how NU6140 affects the cell cycle in carcinoma-derived cells and whether the effect is reversible have remained unclear. Several specific features of human Z-LEHD-FMK embryonic stem (hES) cells are of special interest in studying the effect of CDK2 inhibition. First hES cells are characterized by both unlimited proliferative potential and pluripotency providing them with the capacity to differentiate into all three cell lineages-ectoderm endoderm Z-LEHD-FMK and mesoderm [15-17]. The capacity to differentiate provides an opportunity to investigate whether CDK2 inhibition could alter the differentiation potential of these cells. Second hES cells possess a unique cell cycle profile with an abbreviated G1 phase and long S phase [18]. Third a recent study on phosphoproteome of hES cells during differentiation revealed that CDK2 and Cdc2 activities were central in promoting pluripotency and self-renewal.