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Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells.

Kallas A, Pook M, Trei A, Maimets T - Int J Cell Biol (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.

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.

No MeSH data available.


Related in: MedlinePlus

NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.
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Related In: Results  -  Collection


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fig1: NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.

Mentions: Cell permeabilisation, fixation, staining, and data acquisition for all samples were done on the same day. For more accurate analysis we used sequential selection of cell populations based on size and granularity. Only single cells were selected for detecting cells in different cell cycle phases and only these cells were used to analyse for the expression of various parameters (see Figures 1–6), allowing the minimisation of nonspecific signals.


Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells.

Kallas A, Pook M, Trei A, Maimets T - Int J Cell Biol (2014)

NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4248398&req=5

fig1: NU6140 treatment compared to nocodazole treatment affects differently the expression of pluripotency markers in hES cells. (a) Flow cytometric analysis of the expression of pluripotency markers NANOG, OCT4, and SOX2 in hES cells treated with nocodazole, NU6140, and DMSO. Fixed and permeabilised cells were stained with anti-NANOG (PE), anti-OCT4 (Alexa Fluor 647), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI. For analysis cellular debris and doublets were excluded. (b) Correlation between expression of CDK2 with NANOG and SOX2. Fixed and permeabilised cells were stained with anti-CDK2 (Alexa Fluor 488 conjugate), anti-NANOG (PE), and anti-SOX2 (PerCp Cy5.5 conjugate) antibodies and with DAPI.
Mentions: Cell permeabilisation, fixation, staining, and data acquisition for all samples were done on the same day. For more accurate analysis we used sequential selection of cell populations based on size and granularity. Only single cells were selected for detecting cells in different cell cycle phases and only these cells were used to analyse for the expression of various parameters (see Figures 1–6), allowing the minimisation of nonspecific signals.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.

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.

No MeSH data available.


Related in: MedlinePlus