[CANCER RESEARCH 49, 5907-5912, November 1, 1989] Independent Mechanisms for Tumor Promoters Phénobarbitaland 12-0-Tetradecanoylphorbol-13-acetate in Reduction of Epidermal Growth Factor Binding by Rat Hepatocytes1 Sharon A. Meyer, T. Avery Gibbs, and Randy L. Jirtle2 Division of Radiation Oncology, Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710 ABSTRACT Primary cultures of hepatocytes derived from adult Fischer 344 rats were used to test for effects of the liver tumor promoter phénobarbital on several components of the epidermal growth factor (EGF) receptor signal transduction pathway. Phénobarbitalhad no effect on the binding of 12SI-labeledEGF by its hepatocyte receptor at 4°Cor on EGF-induced receptor down-regulation. However, pretreatment of hepatocytes with phénobarbital (3 mivi)at 37°C caused inhibition of subsequent I25l-labeled EGF binding. This response temporally resembled that of hepatocytes to 12-O-tetradecanoylphorbol-13-acetate (TPA) in that maximal inhibition occurred after l h of pretreatment but was reversed after longer pretreat- ment times. The inhibitory effects of phénobarbitaland TPA on EGF binding were additive, suggesting that distinct mechanisms mediated the responses to these two tumor promoters. In addition, treatment with TPA, but not phénobarbital,caused a redistribution of the activity of Ca2*/phospholipid-dependent protein kinase C. In untreated and pheno- barbital-treated hepatocytes, 20% of protein kinase C activity was iso lated with a membranous fraction, while 75% of the activity was mem brane associated in TPA-treated hepatocytes. These results demonstrate that phénobarbital, like TPA and other tumor promoters, can modulate the EGF receptor system but suggest that it does so without directly competing with EGF for binding to its receptor or by activating protein kinase C. INTRODUCTION Formation of putative preneoplastic lesions during the pro motional phase of multistep carcinogenesis involves the expres sion of a growth advantage by initiated cells relative to normal cells (1). Thus, one focus of tumor promotion research has been directed towards describing the effects of tumor promoters on mitogenic responses of initiated and normal cells to growth factors and on the signal transduction pathways mediating their effects. EGF3 is a mitogen for several types of cells (2) and the pathway mediating EGF-induced mitogenesis has been shown to be a target of tumor promoter action. The most well-docu mented effects are those of the phorbol ester class of skin tumor promoters, e.g., TPA, on the EGF receptor. A rapid, transient decrease in EGF binding of several types of cultured cells is generally observed (3-5) and results from phosphorylation of the EGF receptor as a consequence of TPA activation of Ca2"1"/ phospholipid-dependent protein kinase C (6-8). A similar re sponse to other structurally distinct classes of skin tumor pro moters has also been described (9-11), although one of these, palytoxin, mediates its effects on the EGF receptor independent of protein kinase C (11). Experimental carcinogenesis of rodent liver, like that of skin, is a multistep process, and in this system phénobarbitalis a Received 2/20/89; revised 7/27/89; accepted 8/4/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work was supported by USPHS Grants CA25951 and CA40172. 2To whom requests for reprints should be addressed, at Department of Radiology, Box 3433, Duke University Medical Center, Durham, NC 27710. 'The abbreviations used are: EGF. epidermal growth factor; TPA, 12-O- tetradecanoylphorbol-13-acetate; MEM, minimal essential medium: EGTA, eth- ylene glycol bis(f)-aminoethyl ether)-/V,/V,yv',JV'-tetraacetic acid. strong tumor promoter (1, 12, 13). EGF is a potent mitogen for rat hepatocytes (14) and phénobarbital, added directly to the medium of cultured rat hepatocytes, biphasically alters EGF-dependent mitogenesis. Stimulation has been observed at phénobarbitalconcentrations of 1 mM or less, while inhibition is observed at higher concentrations (15, 16). These observa tions suggest that phénobarbitalmodulates some component(s) of the EGF-induced mitogenic pathway in hepatocytes. By analogy to the effect of skin tumor promoters, the hepatocyte EGF receptor may therefore be a potential site of phénobarbital action. Also, a recent report (17) describing phénobarbital inhibition of protein kinase C activity suggests that phénobar bital could possibly affect EGF binding by modifying protein kinase C-dependent receptor phosphorylation. The purpose of the studies described here was to test whether phénobarbitaladded to the medium of cultured rat hepatocytes affected early events involved in EGF interaction with its recep tor. We describe results demonstrating that phénobarbitaldid not directly compete with EGF for binding to its receptor or alter EGF-induced receptor down-regulation but that pretreat ment (37°C)with phénobarbital did cause a rapid, transient decrease in EGF binding at the hepatocyte surface. Although this response was temporally similar to that induced by TPA, further evidence is presented suggesting that the reduction of hepatocyte EGF receptors by phénobarbital occurred in the absence of protein kinase C activation. MATERIALS AND METHODS Materials. Female Fischer 344 rats (—175g)were from Charles River Laboratories (Wilmington, MA). MEM (Earle's salts) plus nonessential amino acids was from GIBCO (Grand Island, NY). Collagenase type I and receptor grade EGF were purchased from Worthington Biochem- icals (Freehold, NJ) and Collaborative Research (Bedford, MA), re spectively. Amersham (Arlington Heights, IL) was the source of 125I- labeled EGF and [-y-"P]ATP was from NEN-Dupont (Boston, MA). Sodium phénobarbitalwas from Mallinckrodt (Paris, KY). Leupeptin was purchased from United States Biochemical Corp. (Cleveland, OH). Sigma (St. Louis, MO) was the source of bovine serum albumin, bovine pancreas insulin, histone (type III-S), soybean trypsin inhibitor (type I- S), phosphatidylserine, TPA, and ff-phorbol. Hepatocyte Isolation and Culture. Hepatocytes were isolated by in situ collagenase perfusion, plated (10s cells/cm2) on collagenase-coated tissue culture plastic, and cultured (37°C, 5% CO2) as described previ ously (18). Culture medium was MEM with nonessential amino acids supplemented with pyruvate (1 mM), aspartate (0.2 mM), serine (0.2 mM), insulin (0.1 //M), and gentamicin (50 ¿ig/ml).Fresh medium or medium containing phénobarbital,TPA, 0-phorbol, or dimethyl sulf- oxide was applied by replacing the plating medium after allowing 2 h for hepatocyte attachment, and drug treatments were continued at 37°C for the times specified. Stock solutions of sodium phénobarbital(10 mM), made on the day of each experiment, were prepared in culture medium and the pH was readjusted to 7.4 with HC1. TPA and ß- phorbol were added to the culture medium from 1000-fold stock solutions in dimethyl sulfoxide. EGF-induced down-regulation of the EGF receptor was induced by culturing hepatocytes for 16 h with 10 fig/ml EGF. Medium containing unlabeled EGF was then removed and 5907 on March 18, 2016. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from