Ethanol affects cell signalling. A variety of cellular functions are regulated by these mechanisms. In this project, the aim is to investigate a newly discovered effect of ethanol on cell tyrosine kinase. Thy hypothesis is that """"""""ethanol modulates cell tyrosine kinase and the consequential response."""""""" A-431 cells, a human epidermoid cell, have an abundance of tyrosine kinase and peridermal growth factor (EGF) receptors. EGF receptor is a tyrosine kinase (EGF-RTK). Based on preliminary results, A-431 cells will be used as a model to initiate this project. Subsequently, other systems will be tested. The following two objectives will test this hypothesis. Objective I: To determine and characterized the effects of ethanol on tyrosine kinase and its cellular substrates. [A] Membranes of A-431 cells will be used to study the effect of ethanol, as well as other aliphatic alcohols, on basal and EGF stimulated tyrosine kinase using a synthetic 13 amino acid peptide substrate for its assay. Subsequently, intact A-431 cells, labelled with [32P], will be treated with ethanol and then challenged with EGF.[32P] Protein phosphorylation, [32P] phosphoamino acids, and autophosphorylation of EGF-R will be characterized. As a functional response, ion transport will be monitored in these studies and correlated with the effect of ethanol in tyrosine kinase. [B] Effects of ethanol on cell signalling components (e.g., PLC-gamma, pp60, GAP, PI-3 kinase), which are targets of tyrosine kinase will be determined using specific antibodies. Objective II. To determine the effects of long-term exposure to ethanol on cell tyrosine kinase. [A] A-431 cells will be exposed continuously to ethanol for periods ranging from 2 hrs to 1 week and their membrane tyrosine kinase and phosphorylation of tyrosine kinase substrates (seeI) will be monitored systematically and compared with control incubations. [B] Other cell models will be used to provide further evidence for the ethanol effect on tyrosine kinase. Primary cultures and other cell of brain (NG 108), liver (HEP-1 or ARL), and kidney (OK-1, or MDCK) origin will be particularly selected since these organs are profoundly affected by alcohol ingestion. This exploratory grant will provide firm evidence for ethanol affects on cell tyrosine kinase and on some of its substrates. It functional co- relationship to ion transport will emerge. Prevalence of this effect in different models will also be known. The proposal offers a new dimension into the mechanism of action of ethanol at cell signalling level. Besides it role in signal transduction, tyrosine kinase is also emerging as an essential component in cell proliferation, growth and development. These studies will therefore have an impact on our assessment of the long term detrimental effects of ethanol where these processes are affected.
| Reddy, M A; Shukla, S D (1996) Potentiation of mitogen-activated protein kinase by ethanol in embryonic liver cells. Biochem Pharmacol 51:661-8 |
