This project seeks to gain an understanding of some of the mechanisms involved in growth control in 'normal' cultured mammalian cells and to describe some of the metabolic and regulatory consequences of oncogene expression in cultured cells. Tumor cells differ from normal cells in the regulation of cell proliferation and cell differentiation; much recent evidence suggests that these differences stem from inappropriate expression of specific DNA sequences or oncogenes. The gene products of a substantial number of these oncogenes have been identified and characterized but there is considerable uncertainty about the function of these proteins in cells. Since the loss of growth control associated with oncogene expression reflects an alteration of the normal mechanisms of growth regulation it is important to examine those mechanisms in a normal, untransformed cell as a control for the transformed cell. In the work proposed, we intend to contribute to an understanding of this subject by applying NMR to study the correlates of a transition from quiescence to growth elicited by mitogenic stimulation of mouse fibroblast 3T3 cells. The parameters which have been reported to be affected by such stimuli include among others; an increase in ion fluxes, an increase in intracellular pH and an increase in the intracellular content of a number of cations, most significantly Na+, K+ and Ca++. These parameters are accessible to study with NMR spectroscopy which being a noninvasive and nondestructive technique offers very significant advantages over other techniques utilized in the study of these phenomena. Thus far we have developed methodologies which allow us to obtain unique information on intracellular pH, and while we further pursue such studies, we intend to refine and elaborate methodologies to observe and study the other parameters mentioned above. With these new methodologies and with reference set of data on the untransformed 3T3 cell line, we intend to extend our studies to cell lines derived from the 3T3 cells by transformation with specific oncogenes such as src and ras and possibly to other cell lines such as those derived from tumors.
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