Proc. Natl. Acad. Sci. USA Vol. 77, No. 3, pp. 1551-1555, March 1980 Cell Biology Control of differentiation of a mammary cell line by lipids (domes/tumor promoters/fatty acids/lysolecithins) RENATO DULBECCO*, MAURO BOLOGNAt, AND MICHAEL UNGER The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, California 92037 Contributed by Renato Dulbecco, December 17, 1979 ABSTRACT A rat mammary cell line (LA7) undergoes profound effect on cultured cells of various types (13, 14). These spontaneous differentiation into domes due to production of effects include stimulation of growth (15), suppression of several specific inducers by the cells. Some of these inducers may be kinds of differentiation and induction of some other lipids, and we show that lipids regulate this differentiation as (16-24), both inducers and inhibitors. One inhibitor is the tumor pro- kinds of differentiation (25-27). TPA also induces a phenotype moter tetradecanoyl-13 phorbol 12-acetate. The inducers are similar to that of transformed cells (28-30), with a reduced saturated fatty acids of two groups: butyric acid and acids with contact inhibition of growth (31), increased production of chain lengths from C13 to C16, especially myristic acid (C14). plasminogen activator (32-35), increased phospholipid turnover Other inducers are myristoyl and palmitoyl lysolecithins, (36-38), decrease of LETS protein (39), and induction of myristic acid methyl ester, and two cationic detergents with a polyamine synthesis (40). TPA may alter the function of the cell tetradecenyl chain. We propose that the lipids with a C14-CI6 plasma membrane, as its alkyl chain affect differentiation by recognizing specific re- suggested by ability to inhibit the ceptors through their alkyl chains and that the effects obtained binding of epidermal growth factor to its receptors at the cell depend on the head groups. These lipids may be physiological surface (41, 42). The phorbol ester appears to act without prior regulators in the mammary gland. metabolism (43) although its metabolism by the cells can be detected (44). A rat mammary cell line, Rama 25, derived from a dimethyl- The correlation between some of the chemical features of the benzanthracene-induced carcinoma (1), and some of its sublines lipids that are dome inducers and those that are inhibitors of give rise to differentiation-like events in vitro. In an irreversible dome formation raises the question of whether certain lipids event, the Rama 25 cells generate fusiform cells, probably re- physiologically play a role in regulating the functions and dif- lated to myoepithelial cells (1). The LA7 subline undergoes ferentiation of mammary gland cells. reversible changes characterized by the formation of micro- structures, designated as domes and ridges (2). This article will MATERIALS AND METHODS deal with factors controlling the appearance of domes. Chemicals were purchased from Sigma, with the following Trypsinized LA7 cells are uncommitted; whether they de- exceptions: Nonidet P-40 was from Particle Data Laboratories velop domes or ridges depends on the culture medium. Domes (Elmhurst, IL); surfactant OPPE 100 (Triton X-100) was from form spontaneously due to the action of endogenous inducers Atlas (San Diego, CA); sodium deoxycholate was from Cal- (3); medium conditioned by LA7 cells, presumably containing biochem; and sodium dodecyl sulfate was from BDH Chemicals these inducers, promotes dome formation in test cultures (3). (Poole, England). Domes are also in Fatty acids longer than C4 were dissolved in induced LA7 cells by inducers of erythroid ethanol; phorbol, phorbol esters, and myristic acid methyl ester differentiation in proerythroid cells such as dimethyl sulfoxide were dissolved in acetone; other substances were (Me2SO) (4). These agents also induce domes in other epithelial dissolved in cell water. In the cultures, the solvent never exceeded a concen- lines, including the Rama 25 line (5-7). tration of 0.2% (vol/vol). All substances and solutions were kept The ability of butyric acid to act as a dome inducer suggested at -20'C. that fatty acids might be the endogenous inducers present in conditioned The LA7 cell line is a clonal derivative of Rama 25 (1) that medium because fatty acids are synthesized by is able to form spontaneous domes in abundance (2). Cells were normal mammary cells and some of them affect the growth of grown in reinforced Dulbecco's mammary cells in culture (8). Participation of fatty acids in the modified Eagle's medium induction of supplemented with 10% (vol/vol) calf serum and 50 ng each spontaneous domes in LA7 cultures is supported of insulin and hydrocortisone per ml. For transfer, cells were by the observation that dome-inducing factors can be extracted detached in trypsin/EDTA. by ether from conditioned medium at pH 2 (unpublished ob- Drop cultures used for the assays were started with 0.1 ml of servations). medium The observations reported in this article show that several containing 7.5 X 104 trypsinized cells deposited as a long-chain saturated fatty acids (C13-CW6), especially myristic drop in the middle of a 35-mm plastic dish. About 6 hr later, acid (C14), are strong after the cells had attached, 2 ml of medium (modified Eagle's dome inducers and that other alkyl medium with 10% calf serum and 50 ng of hydrocortisone per compounds with similar chain lengths are also dome inducers, ml) was to some very potent. In contrast, a phorbol ester that also has a added the dish. Twenty-four hours later each culture saturated C14 acyl chain (tetradecanoyl-13 phorbol 12-acetate, formed a confluent disc of cells in the center of the dish. These or TPA) is a potent inhibitor of dome formation. This common cultures were used for assays. Drop cultures were used because chemical feature of dome inducers and dome inhibitors may they have a high ratio of medium to cells and, therefore, have interesting implications because TPA is a tumor promoter changes in the medium due to exhaustion of components or in two-stage carcinogenesis of mouse skin (9-12) and has a Abbreviations: Me2SO, dimethyl sulfoxide; TPA, tetradecanoyl-13 phorbol 12-acetate. The publication costs of this article were defrayed in part by page * Also Department of Pathology and Medicine, University of California charge payment. This article must therefore be hereby marked "ad- at San Diego, La Jolla, CA 92093. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t On leave of absence from the Department of Pathology, University this fact. of L'Aquila Medical School, L'Aquila, Italy. 1551 Downloaded by guest on September 25, 2021 1552 Cell Biology: Dulbecco et al. Proc. Natl. Acad. Sci. USA 77 (1980) addition of cell-produced substances occur slowly. In these cultures the cell density decreases from the center to the free edge. Hence, spontaneous domes, which are highly dependent on cell density, form in the center of the cultures whereas those induced by exogenous inducers, such as Me2SO, being less de- E - pendent on cell density, form more or less evenly throughout 0 0.6 the culture (3). Because drop cultures are confluent when they 0.4 are used, domes can develop without any further cell multi- 1- __ _ _ _ _ _ plication. Therefore most assays were carried out in serum-free 0.2 ' modified Eagle's medium containing 50 ng of hydrocortisone per ml. 0!. The dome-inducing power of substances added to the culture 0 1 2 3 4 5 medium was assayed by counting the total number of domes TPA, ng/ml per culture. If the background of spontaneous domes was high, FIG. 1. Proportion of domes formed with various concentrations domes were counted in peripheral microscopic fields (i.e., with of TPA in the medium. TPA was added to the assay medium at time 0, and the proportion of domes formed, relative to those formed in the outlines tangent to the free edge of the culture), which are absence of TPA, was measured 2 days later. 0, Endogenous domes. generally free of spontaneous domes. Within the same experi- Domes induced by: v, conditioned medium; 0, 11.5 ,uM myristic acid; ment the two measurements gave essentially identical results, &, 50 AM myristic acid; 0, 250 mM Me2SO. but when the background was high, the statistical significance of the differences was higher with the latter procedure. Domes added on the first day (data not shown). These differences in were counted once or twice daily by using an inverted micro- the inhibitory effect of TPA may be related to the strength of scope under phase contrast with a magnification of lOOX. When the inducers owing to the type or the concentration of each the activities of substances were compared, the assay cultures inducer. were all simultaneously fixed with a neutral formaldehyde The dome-inhibiting effect of TPA was compared to that of solution, containing 3.5% (vol/vol) formaldehyde, before the i3-phorbol and phorbol dibutyrate, which are not tumor pro- domes were counted. This procedure avoided variation due to moters (Fig. 3). Only TPA inhibited the appearance of domes; temperature or pH changes produced by repeated opening of the other two compounds were inactive in the same concen- the incubator. We counted only domes in which the cell layer tration range. had clearly become elevated (i.e., out of focus when the main Induction of Domes by Fatty Acids and Related Com- cell layer was in focus). Incipient domes (45) were not pounds. Most of the saturated fatty acids, between propionic counted. (C3) and lignoceric (C24), have been tested by addition to the Conditioned medium was prepared by adding modified assay medium of drop cultures at concentrations below the Eagle's medium containing 50 ng of hydrocortisone per ml to critical micellar concentration (46).