Liver Fatty Acid-Binding Protein
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Proc. Nati. Acad. Sci. USA Vol. 91, pp. 848-852, February 1994 Cell Biology Liver fatty acid-binding protein: Specific mediator of the mitogenesis induced by two classes of carcinogenic peroxisome proliferators (fatty acds/liver nens/t d htoma cels) SHAHID H. KHAN AND SAM SOROF* Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 Communicated by Sidney Weinhouse, October 12, 1993 (receivedfor review August 5, 1993) ABSTRACT Peroxisome proliferators (PP) are a diverse nected the protein to mitogenesis of rat hepatocytes. Thus, group of chemicals that Induce dramatic increases in peroxi- L-FABP expression is markedly elevated in the cytoplasm of somes in rodent hepatocyles, followed by hypertrophy, hepa- hepatocytes during mitosis in normal and regenerating rat tomegaly, alterations in lipid metabolism, mit s, and livers (13-15) and throughout the cell cycle in hyperplastic finally hepatocarclnomas. Termed mongenotoxic ros, hepatocytes and hepatocarcinomas produced in rats by in- they do not interact with DNA, are not mut c in bacterial gestion of the genotoxic liver carcinogens 2-acetylaminoflu- assays, and fail to elicit many ofthe phenotypes a id with orene (N-2-fluorenylacetamide) and aminoazo dyes (13, 16). classic genotoic carcinogens. We report here that the mito- In addition, L-FABP binds many ligands of potential impor- genesis induced by the major PP class, the amphipatic car- tance in the modulation of cell multiplication, including four boxylates, and by the tetrazole-substituted acetophenones spe- types of liver carcinogens-i.e., genotoxic metabolites of cifically requires liver fatty acid-binding protein (L-FABP) in both 2-acetylaminofluorene and aminoazo dyes during hepa- cultured rat hepatoma cells transected with the sense cDNA of tocarcinogenesis (17, 18) and two classes ofnongenotoxic PP L-FABP, in contrast to L-FABP-nonexpressing cells trans- carcinogens in vitro (19-22)-the essential fatty acids linoleic fected with it antlsense cDNA. The mitogenic actions of acid and arachidonic acid (8-12), prostaglandin E1 (23), L-FABP were protein-specific, inasmuch as no other protein in growth modulatory hydroxy and hydroperoxy metabolites of the cels could act like L-FABP. L-FADP was arachidonic acid (24), growth inhibitory prostaglandins (25), previously shown not only (i) to interact covalently with mitogenic lysophosphatidic acids (26), lysophosphatidylcho- mntabolites of the two genotoxic carcinogens 2-acetylamino- lines (27), antiproliferative and anticancer selenium (28), fluorene and dyes during liver carcinogenesis, but retinyl palmitate (29), and heme (30). also (ii) to bind noncowalently the two classes ofPP in vitro with PP differ in their abilities to elicit peroxisomal responses aVWditles that correlate with their abilities to elicit peroxisomal (e.g., elevated peroxisomal 3-oxidation of fatty acids), car- enzymatic responses, and (iii) together with unsaturated fatty cinogenicities in rodents, and avidities for L-FABP in vitro. acds, eseilly linoleic acid, to promote multiplication of the Among the synthetic PP, the largest group consists of the transfected hepatoma cells in culture. The convergence of the amphipathic carboxylates, such as Wy-14,643, bezafibrate, two types of genotoxic carcinogens with the two classes of PP ciprofibrate, nafenopin, fenofibrate, and clofibric acid (see nongenotoic carcinogens, and also with unsa fatty formulas in Fig. 1). Fenofibrate, an ester, is thought to be acids, at L-FABP actions in inducing mitogen allows the hydrolyzed to the amphipathic carboxylic acid in cells. These following hypothesis. During tumor promotion of carcinogen- compounds are strong to moderate PP and hepatocarcino- esis in vivo, these groups of genotoxic and nougenotoic car- gens in rats and mice (4-7). Brandes et al. (19) initially cinogens act on the normal process by which L4FABP, func- reported that bezafibrate binds to L-FABP. Eacho and co- tionig as a specific receptor ofunt ed fatty acids or their workers (21, 22) then found a correlation between the differ- metabolites, promotes hepatocyte proliferation. ent avidities of both amphipathic carboxylates and four closely similar tetrazole-substituted acetophenones in bind- Induced cell proliferation (mitogenesis) is increasingly ing to L-FABP in vitro and their diverse abilities to induce a thought to play a key role in carcinogenesis in animals and peroxisomal enzymatic response. Among the amphipathic humans (1-3). The actions of the peroxisome proliferators carboxylates, the strong carcinogen Wy-14,643 was the most (PP) have been revealing. This diverse group of chemicals potent PP and most avid binder to L-FABP, followed in induces dramatic increases in peroxisomes in rodent hepa- decreasing rank order by bezafibrate, ciprofibrate, and tocytes, followed by hypertrophy, hepatomegaly, alterations clofibric acid. Among the acetophenones, the rank order of in lipid metabolism, mitogenesis, and finally hepatocarcino- both L-FABP binding and peroxisomal response was mas. Termed nongenotoxic carcinogens, they do not interact LY189585 2 LY171883 >> LY213768 2 LY163443 (see with DNA, are not mutagenic in bacterial assays, and fail to in elicit many phenotypes associated with classic genotoxic formulas Fig. 2). LY171883, which is a leukotriene D4 carcinogens. Considerable interest surrounds the unknown antagonist, increases the incidence of hepatocellular carci- intracellular mediators and the mechanisms of the induced nomas in female B6C3 F1 mice (31). mitogenesis and carcinogenesis (1-7). Whether the interactions ofthe many ligands with L-FABP Liver fatty acid-binding protein (L-FABP), a cytoplasmic actually modulate the mitogenesis of hepatocytes has been 14-kDa protein previously termed Z protein, is convention- unknown. Therefore, we had derived stable cell lines (32) ally considered an intracellular carrier of fatty acids in containing the sense and antisense cDNAs of rat L-FABP in hepatocytes (8-12). Multiple associations have also con- Abbreviations: PP, peroxisome proliferator(s); L-FABP, liver fatty acid-binding protein. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed at: Institute for payment. This article must therefore be hereby marked "advertisement" Cancer Research, Fox Chase Cancer Center, 7701 Burholme Av- in accordance with 18 U.S.C. §1734 solely to indicate this fact. enue, Philadelphia, PA 19111. 848 Downloaded by guest on October 5, 2021 Cell Biology: Kahn and Sorof Proc. Natl. Acad. Sci. USA 91 (1994) 849 a subline ofrat hepatoma HTC cells that do not endogenously However, only the L-FABP-expressing cells were induced express L-FABP RNA or its protein. The cells transfected by amphipathic carboxylate and tetrazole-substituted ace- with the L-FABP sense cDNA express the recombinant tophenone PP to proliferate additionally. Throughout the L-FABP RNA and protein, whereas the cells transfected with study, changes in cell numbers from day 1 to day 7 were the antisense cDNA do not. This technique provides a compared in the presence vs. the absence of PP in L-FABP- zero-background homologous cell model of L-FABP expres- expressing and L-FABP-nonexpressing cell cultures. In all sion suitable for controlled studies ofthe intracellular roles of cases, the PP increased the number of L-FABP-expressing L-FABP. With this system, unsaturated fatty acids, espe- cells, whereas the number of L-FABP-nonexpressing cells cially linoleic acid, were found to cooperate with L-FABP in was virtually unaffected or relatively reduced. Fig. 3 shows promoting DNA synthesis and cell multiplication (32-34). the increases in cell numbers over day 1-day 7 from the This cell model has now been applied to a study of the role combination of background multiplication of cell lines and of L-FABP in PP-induced mitogenesis. We report here that PP-induced mitogenesis. In contrast, Figs. 1, 2, and 4 deal the mitogenesis induced by the major PP class, the amphi- only with the latter-i.e., the mitogenesis induced by the PP pathic carboxylates, and by the tetrazole-substituted ace- and its dependence on L-FABP. The methods of computa- tophenones specifically requires L-FABP in the rat hepatoma tions of both types of cell multiplication are indicated in the cells transfected with the sense cDNA of L-FABP. The figure legends. convergence of actions of certain genotoxic carcinogens, Mitogenesis Induced by Amphipathic Carboxylates. Induc- these nongenotoxic carcinogens, and fatty acids at L-FABP tion of mitogenesis by the amphipathic carboxylic PP re- in induction of mitogenesis implies that, in these cases, both quired L-FABP expression. Wy-14,643, the most potent types of carcinogens act on the normal process by which inducer of peroxisomal (3-oxidation (4-7, 21), the most avid L-FABP promotes the multiplication of hepatocytes. binder to L-FABP in vitro (21), and a strong liver carcinogen (4-7), was the best inducer ofmitogenesis ofall examined PP. Further, the cell proliferation was most attributable to MATERIALS AND METHODS L-FABP presence. Fig. 1A shows that Wy-14,643 at 10-7 M Cells. Rat hepatoma HTC-R3T2 cells that do not contain brought about the greatest elevation in number oftransfected L-FABP mRNA or protein had been transfected with sense L-FABP-expressing cells; matching treatment of the and antisense L-FABP cDNA (32); expression of the trans- L-FABP-nonexpressing cells elicited virtually no change or fected L-FABP cDNA was under the promoter control of the was inhibitory. Daily measurements for 7 days revealed that long terminal repeat of Rous sarcoma virus. The cloned after a 3-day lag, Wy-14,643 steadily increased only the transfected cell lines were routinely passaged in RPMI 1640 number of L-FABP-expressing cells (Fig. 4). medium/10%o calf serum (both from GIBCO)/sodium bicar- Likewise, in decreasing rank order, nafenopin, clofibric bonate (2 g/liter)/penicillin (50 units/ml)/streptomycin sul- acid, and fenofibrate stimulated mitogenesis of the L-FABP- fate (50 p.g/ml)/kanamycin sulfate (100 pg/ml) at 37°C in a expressing cells, whereas the L-FABP-nonexpressing cells humidified atmosphere of 5% CO2.