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,Annika Trei,Martin Pook,Toivo Maimets

doi:10.1155/2014/280638

Abstract

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 effects 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 flow 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 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.

Highlights

Cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription in different cell types
We found that both human embryonic stem (hES) and hEC cells were sensitive in suppressing the expression of NANOG and OCT4 with nocodazole or NU6140, whereas the effect on SOX2 expression was more pronounced after nocodazole treatment
The differences we found in commitment of cells into endodermal, ectodermal, and mesodermal lineages between untreated hES cells and those treated with NU6140 indicates that inhibition of CDK2 with

Summary

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, 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 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 been an attractive, albeit complicated, task. Using structural-drug design several small molecules and peptides have been developed to target

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