The goal of our research is to understand the modulation of signal transduction mechanisms during the expression of a well-defined oncogene. Our previous finding that ras transformation of a kidney cell line (MDCK cells) leads to loss of glucogan sensitivity, raises a possible role of ras product in hormone signal transduction. From our studies and others, it has been shown that ras transformation not only affects cyclic AMP-mediated signal, it also elevates phosphoinositol level and abolishes certain functions of EGF. In order to examine the causal relationship between the expression of ras gene and the various changes of signal transduction, a new approach has been taken to allow more precise control of the production of p21. We have obtained a transfection NIH-3T3 cell line containing MMTV LTR ras oncogene. Our preliminary results show that in the presence of dexamethasone in culture, when p21 is produced, there is a concomitant decrease in EGF binding of the transfected 3T3 cells, while the changes in cyclic AMP-mediated hormone response is less pronounced. The ras- -transformed cells are known to have an elevated level of diacylglycerol, whether the loss of EGF binding is a consequence of activation of protein kinase C is being studied. This finding is consistent with our previous observation that in a ras- transformed 3T3 cell line, EGF binding is markedly reduced. Since NIH-3T3 cells are relatively undifferentiated, it is desirable to examine the effect of inducible ras gene in a more differentiated cell line. We have obtained plasmids, containing glucocorticoid- inducible normal and viral ras, from others. MDCK cells have now been transfected with these plasmids. We are currently selecting clones expressing the most highly inducible ras gene. Once these transfected clones are identified, we will attempt to examine the temporal relationship between the expression of ras product and the reduction of EGF binding, the elevation of phosphoinositol and the changes in hormone response.
