A CARDIOTONIC STEROID BUFALIN-INDUCED DIFFERENTIATION OF THP-1 CELLS - INVOLVEMENT OF NA-ATPASE INHIBITION IN THE EARLY CHANGES IN PROTOONCOGENE EXPRESSION(, K+)

Human monocytic leukemia THP-1 cells were induced to differentiate int o macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with s imilar kinetics to ouabain, a specific inhibitor of the enzyme. This i nduction of differentiation was characterized by loss of proliferation , cell adherence, increased ability to reduce Nitro Blue tetrazolium ( NBT), and increased expression of interleukin 1 beta (IL-1 beta). Duri ng this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar chan ges in protooncogene expression and induced phenotypic markers of diff erentiated cells at concentrations comparable to bufalin. The 12-O-tet radecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhi bitor H7 failed to inhibit bufalin-mediated c-fos induction further su pports the theory that the signal transduction machinery caused by buf alin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was r eplaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ mediu m. These findings indicate that an increased intracellular Na+ concent ration resulting from the Na+, K+-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.