Protein Kinase C as the Receptor for the Phorbol Ester Tumor Promoters: Sixth Rhoads Memorial Award Lecture1

The focus of my research has been on understanding the initial events in phorbol ester action. The phorbol esters are natural products derived from Croton tiglium, the source of croton oil, and from other plants of the family Euphorbiaceae (1). The phorbol esters initially became the object of intense research interest on the basis of their potent activity as mouse skin tumor promoters (2). The behavior of tumor promoters has been characterized in detail in the mouse skin system. Briefly, tumor promoters are compounds which by themselves are not carcinogenic or indeed mutagenic but which if administered chronically after exposure of an animal to a subeffective dose of a carcinogen are then able to lead to the rapid appearance of skin tumors. Whereas the action of the carcinogen is irreversible, as shown by the fact that one can delay the promotion treatment by up to 1 year after application of the carcinogen, referred to as an initiator, the action of the tumor promoters is reversible. Thus, if the chronic tumor promoter treatments precede rather than follow the treatment with the initiator, tumors do not arise. Although tumor promoting activity provided the initial mo tivation for studying the mechanism of the phorbol esters, as people began to examine the effects of the phorbol esters in different in vitro systems, it turned out that the phorbol esters had profound effects on a wide variety of biological systems (35). These effects included (a) induction or inhibition of differ entiation, (b) the partial mimicry in normal cells of the trans formed phenotype, (c) stimulation of secretory responses in a variety of cell types such as platelets and neutrophils, (d) modulation of many membrane activities, and (e) generation of active oxygen species. The range of these effects suggested early on that the phorbol esters might be functioning through stim ulation of some central regulatory pathway within cells. Two indirect arguments had suggested that the phorbol esters might bind to specific receptors: (a) the compounds had ex traordinarily high potency, being active at nanomolar concen trations; (h) small structural changes in the molecule led to marked differences in activity. For example, elimination of the hydroxyl group at position 20 of phorbol led to complete loss of activity, and methylation of the hydroxyl group at position 4 led to a several hundred-fold loss in potency. Initial efforts to demonstrate specific phorbol ester binding to cells were frus trated, however, because investigators used the biologically most active phorbol ester, phorbol 12-myristate 13-acetate. The myristate side chain makes the molecule highly lipophilic, and specific binding is therefore obscured by very high nonspecific uptake. On the basis of structure-activity analysis which we had carried out in vitro together with published data concerning the lipophilicity of different phorbol esters as a function of biolog-