Although the signal transduction pathways for promoting agents discussed above are generally applicable, it is useful to consider the more specific molecular mechanisms and pathways of the most commonly employed promoting agents in experimental systems. These are tetrade- canoylphorbolacetate (TPA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), promoting agents effective in a variety of tissues, and phenobarbital (PB), a promoting agent effective in only a few tissues.
The receptor for TPA was shown to be a cytosolic serine/threonine protein kinase termed protein kinase C (Leach et al., 1983; Ashendel et al., 1983). Pathways activated by the TPA interaction with protein kinase C in its membrane-bound form are outlined in Figure 7.10 (Kazanietz and Blumberg, 1996). Diacylglycerol, the normal endogenous activator of protein kinase C, may be produced by the action of phospholipases after being activated either from a transmembrane growth factor receptor or a multiple transmembrane protein receptor (Figure 7.6). TPA is a more effective ligand activator than the endogenous diacylglycerol, thus causing the enhancement of the various pathways noted that involve the activation of another protein kinase, Raf-1 (B-raf), and the subsequent cascade of various events ending with the phosphorylation. This ultimately results in the changes in phosphorylation levels of a dimeric transcription factor, AP-1, usually consisting of a heterodimer comprising one molecule each of the proto-oncogenes c-jun and c-fos. This heterodimer interacts with a DNA sequence, termed the TPA-responsive element (TRE), whose sequence is TGA(G/C)TCA (Kazanietz and Blumberg, 1996). The regulatory re- gions of many genes possess AP-1 sites thus resulting in the numerous effects of TPA on cells (Chapter 14, Table 14.9).
The other signal transduction pathway activated by TPA is that involving the nuclear transcription factor NFκB. This protein is normally maintained in the cytoplasm in a complex with another protein, IkB, which prevents its entrance into the nucleus by formation of the complex. Phosphorylation of IFκB by specific protein kinases (cf. Karin, 1999) that themselves may be phosphorylated by protein kinase C causes the degradation of IFκB by the ubiquitin pathway, freeing the NFκB protein for entrance into the nucleus and interaction at its specific sequence motif, GGG(A/G)NNYYCC (Miyamoto and Verma, 1995). NFκB may also be acti- vated by alteration in intracellular thiols, presumably involving oxidative stress (cf. Keyse,1997). Just as with AP-1, which is also affected by oxidative stress, the target genes for NFκB comprise an extensive list, a number of which are concerned with the immune system (Schreck et al., 1997).
It is of interest that although TPA activates protein kinase C, chronic administration of the promoting agent causes a downregulation of this gene in mouse epidermis and in preneoplastic papillomas (Hansen et al., 1990). The activation of protein kinase C appears also to phosphory- late the epidermal growth factor receptor at serine residues (Davis and Czech, 1984), which may be related to the demonstration that the epidermal growth factor receptor binding to EGF is dra- matically inhibited by TPA. This inhibition is reversed in the absence of the promoting agent (Magun et al., 1980). While these seemingly discordant results may be explained by a variety of mechanisms, it is clear that TPA induces cell replication in the epidermis, the overriding factor in its promoting action.
Another model promoting agent to be considered is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an effective promoter in a variety of tissues but primarily in liver and skin in the rodent (Pitot et al., 1980; Poland et al., 1982). TCDD is an environmental contaminant that is both a by- product of a variety of chemical syntheses and a product of various types of combustion (Hites,1990; Bumb et al., 1980). TCDD is extremely toxic (cf. Lilienfeld and Gallo, 1989; Pohjanvirta and Tuomisto, 1994) and is likely the most effective promoting agent on a per-molecule basis that has been described (Pitot et al., 1980). Virtually all of the toxicities of TCDD (Pohjanvirta and Tuomisto, 1994) are mediated through the interaction of TCDD with the Ah receptor as first described by Poland (cf. Poland and Knutson, 1982). The signal transduction pathway ef- fecting the toxicity and promoting activity of TCDD has now been established to some degree
Figure 7.10 Pathways of protein kinase C–activated signal transduction. Interaction of TPA with the membrane-associated protein kinase C leads to an enhancement of its catalytic domain (C), which in turn may phosphorylate several different substrates as noted in the figure. This leads to a phosphorylation cas- cade with ultimate transcriptional activation of phorbol ester-inducible genes. Transcriptional activation is mediated by two independent routes, the ras/raf-1 and the IkB/NF-κB pathways. Abbreviations: R, regula- tory domain of PKC; C, catalytic domain of PKC; PLC, phospholipase C; GRB-2, growth receptor–bound protein 2 (an adapter between the growth receptor tyrosine kinase and SOS through its SH2 domain; SOS (son of sevenless), a guanine-nucleotide dissociation protein that releases GDP from RAS and allows GTP to bind and activate RAS; MAP kinase, mitogen-activated protein kinase; NF-κB, nuclear factor κB; IκB, inhibitory subunit of nuclear factor κB. (Adapted from Kazanietz and Blumberg, 1996, with permission of the authors and publisher.)
(Figure 7.11). As shown in the figure, TCDD interacts with the Ah receptor in the cytoplasm, the receptor being maintained in a native form through interaction with heat-shock proteins or chap- erones (Henry and Gasiewicz, 1993; Whitlock, 1993). Interaction of the receptor with the ligand (TCDD) in the cytoplasm leads to a dissociation of the chaperone-Ah receptor complex and its binding to another somewhat related protein, the Ah receptor nuclear translocator protein (Arnt) (Reyes et al., 1992). This complex is then translocated into the nucleus, where it interacts with a specific core nucleotide sequence, T_GCGTG (Whitlock, 1993). Just as with TPA, this complex transcriptionally activates a large number of different genes, including many of the phase I xeno- biotic metabolizing genes such as members of the cytochrome P450 family and a variety of oth- ers (DeVito and Birnbaum, 1994; Grassman et al., 1998). Like TPA, TCDD administration in vivo, albeit at doses considerably in excess of those used for tumor promotion, downregulated both the epidermal growth factor receptor (Sewall et al., 1995) and protein kinase C (Bombick et al., 1985). In addition, in cell culture, TCDD inhibited intercellular communication, like many other tumor promoting agents (Chapter 14) (De Haan et al., 1994). Like TPA, TCDD induces DNA synthesis and cell replication in preneoplastic foci but has very little if any effect on the nonfocal hepatocytes in such livers (Buchmann et al., 1994). This finding is in concert with that of its similar selective effect in inhibiting apoptosis of cells of altered hepatic foci without signif- icantly altering the apoptotic index of the nonfocal liver (Stinchcombe et al., 1995). A potential mechanistic explanation of this latter finding is the demonstration that TCDD also suppresses apoptosis in rat hepatocytes treated in vitro with an apoptogenic dose of ultraviolet light. TCDD administration resulted in a dose-dependent increase in the phosphorylation of the p53 tumor suppressor protein (Wörner and Schrenk, 1998). This suggests that phosphorylation of key sites on the p53 protein prevents its action as a mediator of apoptosis (Bates and Vousden, 1999).