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Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

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Gene expression signature of conditionally reprogrammed HFKs.A, Heatmap of the top 30 up- and down-regulated genes based on the changes in the ratio of Y/F, J2/F and J2Y/F. B, Gene expression signatures related to the effects of Y, J2 or J2+Y. A list of changes in gene expression is in Table C in S1 File.
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pone.0116755.g002: Gene expression signature of conditionally reprogrammed HFKs.A, Heatmap of the top 30 up- and down-regulated genes based on the changes in the ratio of Y/F, J2/F and J2Y/F. B, Gene expression signatures related to the effects of Y, J2 or J2+Y. A list of changes in gene expression is in Table C in S1 File.

Mentions: To obtain a global picture of gene transcription resulting from conditional reprogramming, gene expression analysis of freshly isolated HFKs grown for two days with or without J2 cells and Y was evaluated. Gene expression analysis revealed that treatment with Y differentially regulated 293 genes, whereas, J2 cells modulated 215 genes, and the combination of J2 cells and Y differentially affected 495 genes (Table C in S1 File). A heatmap of the expression of 30 genes from each of the three comparisons that demonstrated the greatest degree of positive and negative regulation indicated unique as well as cooperative effects between J2 cells and Y on HFKs (Fig. 2A). The distinct effects of Y and J2 cells were achieved by different sets of genes associated with increased proliferation and reduced differentiation and other processes (Fig. 2B). A list of the changes in gene expression is shown in Table C in S1 File.


Multifactorial analysis of conditional reprogramming of human keratinocytes.

Ligaba SB, Khurana A, Graham G, Krawczyk E, Jablonski S, Petricoin EF, Glazer RI, Upadhyay G - PLoS ONE (2015)

Gene expression signature of conditionally reprogrammed HFKs.A, Heatmap of the top 30 up- and down-regulated genes based on the changes in the ratio of Y/F, J2/F and J2Y/F. B, Gene expression signatures related to the effects of Y, J2 or J2+Y. A list of changes in gene expression is in Table C in S1 File.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4340869&req=5

pone.0116755.g002: Gene expression signature of conditionally reprogrammed HFKs.A, Heatmap of the top 30 up- and down-regulated genes based on the changes in the ratio of Y/F, J2/F and J2Y/F. B, Gene expression signatures related to the effects of Y, J2 or J2+Y. A list of changes in gene expression is in Table C in S1 File.
Mentions: To obtain a global picture of gene transcription resulting from conditional reprogramming, gene expression analysis of freshly isolated HFKs grown for two days with or without J2 cells and Y was evaluated. Gene expression analysis revealed that treatment with Y differentially regulated 293 genes, whereas, J2 cells modulated 215 genes, and the combination of J2 cells and Y differentially affected 495 genes (Table C in S1 File). A heatmap of the expression of 30 genes from each of the three comparisons that demonstrated the greatest degree of positive and negative regulation indicated unique as well as cooperative effects between J2 cells and Y on HFKs (Fig. 2A). The distinct effects of Y and J2 cells were achieved by different sets of genes associated with increased proliferation and reduced differentiation and other processes (Fig. 2B). A list of the changes in gene expression is shown in Table C in S1 File.

Bottom Line: Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming.To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening.These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Georgetown University, Washington, DC, 20007, United States of America.

ABSTRACT
Co-culture of human primary epithelial cells with irradiated 3T3 fibroblast feeder cells (J2 cells) and the Rho kinase inhibitor Y-27632 (Y) allows for the unrestricted growth of cells of epithelial origin by the process termed conditional reprogramming. To better understand the nature of the signaling processes associated with conditionally reprogrammed cells, the effect of the two critical components of the co-culture conditions, J2 cells and Y, on the growth of human foreskin keratinocytes (HFKs) was evaluated by gene expression profiling, reverse-phase protein arrays and siRNA screening. J2 cells and Y acted cooperatively to down-regulate differentiation, and upregulate proliferation and cell adhesion, including increased pT308Akt and pERK, and reduced TGF-β pathway signaling. These findings establish a mechanistic basis for the unlimited growth potential of human epithelial cells that will be invaluable to assess the effect of genetic changes in pathologic tissues and their response to therapeutic agents.

Show MeSH
Related in: MedlinePlus