Long-term objectives-To test the hypothesis that reactive species derived from the cellular metabolism of chemicals of environmental significance-mainly quinonoid compounds-[a] subvert normal metabolic controls, thereby eliciting toxic responses (e.g., stimulation of abnormal growth) and/or [b] potentiate normal regulatory activities (e.g., mitogenic activities in tissue repair). This hypothesis is further detailed by the implication that the reactive species produced through cellular metabolism of quinonoid compounds stimulate a membrane-associated, tyrosine-specific protein phosphorylation and inositol phospholipid phosphorylation cascade that is closely associated with growth factor receptor(s). This results in a stimulation or potentiation of cell proliferation. Since these growth factor receptor systems exist in many different mammalian tissues, such effects-initially triggered by oxidants derived from the redox transitions of quinones-will be pleiotropic.
Specific aims -The aims of this research are directed towards an understanding of the molecular mechanism inherent in quinone- stimulated cell proliferation mediated by phosphorylation of tyrosine- and phosphatidylinositol kinases.
These aims represent a systematic and comprehensive approach to gather such information: [1] to elucidate the identities and biochemical properties of the quinone- sensitive tyrosine protein kinase(s) and phosphatidylinositol kinase(s) and the possibility that they are components of a protein phosphorylation cascade. [2] To study the growth-promoting effects of the oxygen free radical-generating systems (quinone redox transitions) in the vascular endothelial cell culture following the sequence of events that are known to be associated with growth factor receptor actions. [3] To evaluate the physicochemical properties of quinones, which are essential to their reactivity in terms of potentiation of kinase autophosphorylation. [4] To test selective quinonoid compounds that are known constituents of environmental pollutants in their growth-promoting effects and compare with those of model quinonoids used so far in our studies and in Specific Aims No.3. Experimental designs and methods-The specific aims [1-2] will be carried out by experiments designed to [a] separate and identify both kinases; [b] elucidate the potential relationship between them, and [c] pinpoint the site of interaction between quinones and growth factor(s) leading to potentiation of cell growth.
The specific aims [3-4] involve experiments designed to establish those physico-chemical properties of quinones essential for stimulation of enzyme modification and promotion of cell growth. The biological preparations utilized for the above purposes will be mainly rat liver plasma membranes and cultured rabbit aortic endothelial cells and cell membranes. Separation and identification techniques to be used include: Sephacryl S-300 chromatography, immunoprecipitation using antiphosphotyrosine antibody agarose, cellulose acetate electrophoresis, and protein and lipid phosphorylation. Spectro-and fluorometric methods along with HPLC methods with different detection modes will be used to measure enzyme activities, active oxygen species, redox status of glutathione, quinones, and quinone thioether derivatives.
| Chen, Y; Chan, T M (1993) Orthovanadate and 2,3-dimethoxy-1,4-naphthoquinone augment growth factor-induced cell proliferation and c-fos gene expression in 3T3-L1 cells. Arch Biochem Biophys 305:9-16 |
