scholarly journals Identification of Novel Keratinocyte Differentiation Modulating Compounds by High-Throughput Screening

2006 ◽  
Vol 11 (8) ◽  
pp. 977-984 ◽  
Author(s):  
Masaru Honma ◽  
Mark Stubbs ◽  
Ian Collins ◽  
Paul Workman ◽  
Wynne Aherne ◽  
...  
Keyword(s):  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Histone Deacetylase Inhibitors ◽  
Terminal Differentiation ◽  
Mek Inhibitor ◽  
Keratinocyte Differentiation ◽  
Immunofluorescent Staining ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes

The authors have designed high-throughput screens to identify compounds that promote or inhibit terminal differentiation of primary human epidermal keratinocytes. Eleven known inhibitors of signaling pathways and approximately 4000 compounds of diverse structure were screened using an In-Cell Western system based on immunofluorescent staining of the terminal differentiation marker, involucrin. Staurosporine, a nonspecific protein kinase C inhibitor, and H89, a protein kinase A inhibitor, promoted expression of involucrin. Conversely, U0126, a MEK inhibitor, and SAHA or SBHA, 2 histone deacetylase inhibitors, reduced the expression of involucrin during calcium-induced stratification. In addition, the authors found 1 novel compound that induced keratinocyte differentiation and 2 novel compounds that were inhibitory to calcium-induced differentiation. The differentiation-inducing compound also inhibited growth of a human squamous cell carcinoma line by stimulating both differentiation and apoptosis. Because the compound affected the tumor cells at a lower concentration than primary keratinocytes, it may have potential as an antitumor therapy.

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

1996 ◽  
Vol 109 (13) ◽  
pp. 3013-3023 ◽  
Author(s):  
A.J. Zhu ◽  
F.M. Watt
Keyword(s):  
Cell Adhesion ◽  
Terminal Differentiation ◽  
Intercellular Adhesion ◽  
Dominant Negative ◽  
Epidermal Keratinocytes ◽  
Dominant Negative Mutant ◽  
Beta Catenin ◽  
Human Epidermal Keratinocytes ◽  
Negative Mutant ◽  
E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

scholarly journals TRTH-28. HIGH THROUGHPUT SCREENING OF NOVEL HISTONE DEACETYLASE INHIBITORS FOR EPIGENETIC THERAPY OF PRIMARY BRAIN TUMORS

2017 ◽  
Vol 19 (suppl_4) ◽  
pp. iv57-iv58
Author(s):  
Viktoria Marquardt ◽  
David Pauck ◽  
Finn K. Hansen ◽  
Daniel Picard ◽  
Jörg Felsberg ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Histone Deacetylase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Histone Deacetylase Inhibitors ◽  
Epigenetic Therapy ◽  
Primary Brain Tumors

A robust and quantitative assay platform for multiplexed, high throughput screening of protein kinase inhibitors

2016 ◽  
Vol 52 (81) ◽  
pp. 12112-12115 ◽  
Author(s):  
Jieon Lee ◽  
Il-Soo Park ◽  
Ginam Park ◽  
Kyukwang Cho ◽  
Hee-Sung Park ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Protein Kinase ◽  
High Throughput ◽  
High Throughput Screening ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Protein Kinase Activity ◽  
Activity Assay ◽  
Kinase Activity Assay

We present a new platform for multiplexed protein kinase activity assay using TiO2decorated graphene oxide (GO), which is applicable to high throughput inhibitor screening.

scholarly journals β1 Integrins Regulate Keratinocyte Adhesion and Differentiation by Distinct Mechanisms

2000 ◽  
Vol 11 (2) ◽  
pp. 453-466 ◽  
Author(s):  
Laurence Levy ◽  
Simon Broad ◽  
Dagmar Diekmann ◽  
Richard D. Evans ◽  
Fiona M. Watt
Keyword(s):  
Point Mutations ◽  
Terminal Differentiation ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Proximal Part ◽  
Epidermal Keratinocytes ◽  
Β1 Integrin ◽  
Primary Human Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Β1 Integrins

In keratinocytes, the β1 integrins mediate adhesion to the extracellular matrix and also regulate the initiation of terminal differentiation. To explore the relationship between these functions, we stably infected primary human epidermal keratinocytes and an undifferentiated squamous cell carcinoma line, SCC4, with retroviruses encoding wild-type and mutant chick β1 integrin subunits. We examined the ability of adhesion-blocking chick β1-specific antibodies to inhibit suspension-induced terminal differentiation of primary human keratinocytes and the ability of the chick β1 subunit to promote spontaneous differentiation of SCC4. A D154A point mutant clustered in focal adhesions but was inactive in the differentiation assays, showing that differentiation regulation required a functional ligand-binding domain. The signal transduced by β1 integrins in normal keratinocytes was “do not differentiate” (transduced by ligand-occupied receptors) as opposed to “do differentiate” (transduced by unoccupied receptors), and the signal depended on the absolute number, rather than on the proportion, of occupied receptors. Single and double point mutations in cyto-2 and -3, the NPXY motifs, prevented focal adhesion targeting without inhibiting differentiation control. However, deletions in the proximal part of the cytoplasmic domain, affecting cyto-1, abolished the differentiation-regulatory ability of the β1 subunit. We conclude that distinct signaling pathways are involved in β1 integrin–mediated adhesion and differentiation control in keratinocytes.

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