The cell biology of ras-induced transformation: Insights from studies utilizing an inducible hybrid oncogene system

The c-Ha-ras oncogene has been implicated as a causative agent in the development of tumors in humans as well as mice. The molecular nature of the ras-induced tumorigenic process remains unclear, however. To address this question directly we have constructed a cell line which carries a zinc-inducible metallothionein-ras hybrid oncogene, transformant 212. Upon exposure to zinc for 24-48 hr, 212 cells assume a highly transformed morphology, concomitant with the induction of ras-expression. Natural killer cells constitute a subpopulation of lymphoid effector cells which have for a long time been hypothesized to be involved in the earliest stages of antitumor surveillance. Central to this hypothesis is the prediction that NK sensitivity arises during cellular transformation. By carrying out cytotoxicity assays against the 212 transformant, we showed that, indeed, increased sensitivity to NK-mediated lysis correlated with expression of the ras oncogene, which is consistent with the above hypothesis. We then addressed the question of the biochemical mechanism of ras-induced transformation. Owing to their similarity to G proteins, regulatory elements interposed between cell-surface receptors and their effector enzymes, it has been postulated that p21, the ras oncogene protein, mediates its transforming effects by constitutive activation of proliferative signal transduction pathways. We studied the effect of ras expression on the regulation of adenylate cyclase (A.C.), key enzyme of one such major pathway. We found that ras expression correlated with a dampening of responsiveness of A.C. to several stimuli, including hormones such as isoproterenol and other agents such as GMP-PNP, forskolin and fluoride-ion. Accumulation of cAMP as measured by RIA in intact cells, as basal or in response to stimulation of A.C. activity with forskolin, was also decreased (~10-fold) with ras expression. Because the regulation of calcium, another important second messenger is dependent, in part, upon cAMP and GTP-binding proteins, we investigated the possible influence of ras expression on the intracellular concentration of calcium. Steady-state intracellular free Ca²⁺ concentration, as measured by fluorimetry, was indeed increased by approximately 50-125% in association with ras expression. Finally, we studied the possible influence of p21ras on protein kinase C (PKC), which is a key enzyme in the important signal transduction pathway of phosphatidylinositol lipid turnover. We assessed PKC activity directly, in a cell-free system, by measuring the ability of the enzyme to transfer radiolabelled phosphate from γ-³²P-ATP to histone, an exogenous substrate. Utilizing this system, we observed that induction of rats in 212 transformants lead to translocation of PKC activity from cytosol to particulate fraction with a concomitant decrease of particulate as well as cytosolic PKC activity. Quantitation of PKC by immunoblotting revealed a decrease in the 80k band which corresponds to PKC. These findings are consistent with the hypothesis that p21ras mediates activation of PKC which leads to down-regulation of the enzyme, possibly as a result of an increased rate of degradation. The possible ramification of these findings for the cell physiology of ras-expressing cells are discussed.