We wish to test the hypothesis that turnover of phosphatidylinositol (PI) is an important regulator of terminal differentiation and acts in part by controlling the expression of the proto-oncogene, c-myc and of a gene coding for an immunologically-related protein, p48. Friend erythroleukemia cells and epidermal keratinocytes will be used as model systems. We have observed a rapid decrease in diacylglycerol (DG), a second-messenger product of PI turnover, during Friend cell differentiation. The decrease precedes a fall in c-myc mRNA and in myc-related proteins. We also showed that synthetic DGs inhibit differentiation. Myc expression in undifferentiating cells is cell-cycle independent, but after the initial decrease is re-established under cell cycle control. We will determine whether DG production also converts from a constitutive to a cell-cycle dependent mode during differentiation and whether addition of phorbol esters or DGs blocks the initial decrease in myc expression. Pulse-labeling will be used to determine the mechanism by which DG decreases during induction. Protein kinase C distribution between cytosol and membranes will provide a measure of the effect of changes in DG. To directly determine whether kinase C can activate myc transcription we will attempt to activate expression by phorbol ester addition to permeabilized cells. To determine if the decrease in myc is necessary for differentiation, we will co-transfect cells with myc and neo genes and select for G418 resistance. Cloned cells will be tested for resistance to differentiation. Finally, the relation of tyrosyl kinase activity to myc expression will be analysed using vanadate to inhibit phosphotyrosyl phosphatases. Vanadate is a potent inhibitor of Friend cell differentiation. Total cell and p42 phosphotyrosine levels will be determined along with measurements of DG, myc RNA and S6 kinase activities. Keratinocytes will be used to establish whether the changes during Friend cell differentiation can be generalized to other, unrelated cell-types. These experiments should help to define the mechanisms by which a reversal of the transformed phenotype can be induced.
