Carcinogenesis vol.13 no.2 pp. 199-203, 1992

Regulation of gap junctional communication in Syrian hamster embryo cells by retinoic acid and 12-0-tetradecanoylphorbol-13-acetate

Edgar Rivedal and Tore Sanner Laboratory for Environmental and Occupational Cancer, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo 3, Norway

Introduction Epidemiological studies have shown that intake of retinoids as well as their precursor /3-carotene may reduce the risk of cancer (1-3). This has been supported by experimental studies where vitamin A analogs have been shown to protect against the induction of benign as well as malignant tumors (4-6). Thus, retinoids are interesting compounds for use in cancer prevention, and are presently undergoing widespread clinical trials as cancer chemopreventive agents (7—9). Some studies on the effect of retinoids on carcinogenesis have, however, given apparently contradictory results. Retinoids have been found to increase the frequency of cancer in some studies on experimental animals (10-15). In tumor promotion studies, retinoids both inhibit (5) and enhance (15,16) the induction of •Abbreviations: SHE, Syrian hamster embryo; TPA, 12-0-tetradecanoylphorbol-13-acetate; EGF, epidermal growth factor, DMEM, Dulbecco's modified EagJe's medium; FBS, fetal bovine serum; B[a]P, benzo[a]pyrene; CE, cloning efficiency; PKC, protein kinase C. © Oxford University Press

Materials and methods Chemicals Retinoic acid, retinol, cholera toxin, A23187, 12-Cketradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF) and Lucifer Yellow were purchased from Sigma Co., St Louis, MO. [I23I]EGF and [3H]arachidonic acid (60-90 Ci/mmol) was from Amersham, UK. All chemicals were of the highest purity available. Cell cultures The cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) at 37°C in a 10% CO? atmosphere. Stock cultures were kept frozen in liquid nitrogen. Primary cell cultures from Syrian hamster (Wright, Chelmsford, Essex, UK) embryos were prepared at 14 days of gestation as described by Pienta et aL (32). BPNi is a non-tumorigenic hamster embryo cell line derived from primary hamster embryo cells exposed to 0.4 /»M benzo[a]pyrene (B[a]P) and 19 iiM nickel sulfate for 4 days (33). The cells have escaped senescence and have been grown for - 75 passages. The cells have normal colony morphology, but are characterized by very high sensitivity to the induction of criss-crossed transformation-like morphology and block in intercellular communication by tumorpromoting phorbol esters (34). Taxtcity The toxkity was determined as effect on the colony-forming ability of SHE and BPNi cells. Two hundred SHE cells or 80 BPNi cells were seeded 1 day after 6 x 104 X-irradiated (5000 rad) SHE feeder cells. The cells were exposed to the compounds from the next day, and after a total of 8 days of growth the colonies were fixed with methanol and stained with Giemsa (10% v/v in water). Cloning efficiency (CE) is expressed as number of surviving colonies as a percentage of control. Cell—cell communication Intercellular communication was measured as spreading of micro-injected Lucifer Yellow (10% w/v in 0.33 M lithium chloride) to neighboring cells in a monolayer of BPNi or primary SHE cells. A Narishige (Tokyo, Japan) micromanipulator

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Retinoids enhance the frequency of Syrian hamster embryo (SHE) cell colonies with transformed morphology in a similar way to tumor-promoting phorbol esters. The present study shows that retinoids are also potent inhibitors of gap junctional intercellular communication in SHE cells at noncytotoxic concentrations. This is in apparent contrast to the results observed in transformation systems using the mouse cell lines C3H10T1/2 and BALB/c 3T3, where retinoids have been found to reduce the induction of transformation, and also to enhance gap junctional cell communication. Retinoids are thus potent modulators of transformation and cell communication in three transformation systems. For all three cell types, enhancement of communication by retinoids is related to reduced transformation, and inhibition of communication to enhanced induction of transformation. Communication in the SHE cells is completely blocked following 1 h exposure to 30 /xM retinoic acid, while concentrations of 0.3—15 ^M results in a gradual downregulation of communication during 1 - 5 h exposure. Removal of retinoic acid results in complete restoration of communication to control values within a few hours. Primary SHE cells and the cell line BPNi show similar sensitivity for inhibition of communication after exposure to retinoic acid, while BPNi cells are far more sensitive to inhibition of communication by 12-O-tetradecanoylphorbol-13-acetate (TPA) than primary SHE cells. Retinoic acid does not induce inhibition of epidermal growth factor binding, potentiate adenylate cyclase activation or enhance arachidonic acid release, as does TPA, suggesting different mechanisms of action.

tumors under different conditions, and have been found to bring about bom antagonistic and similar biochemical alterations as observed for phorbol ester tumor promoters (17—21). Tumor promoters of different chemical classes are potent inhibitors of intercellular communication. This effect has been related to their tumor-promoting properties (reviewed in 22,23). In some studies the exposure to retinoids has resulted in enhancement of gap junctional communication (24,25), while other studies report potent inhibition of cell communication (26-29). The results with systems using the mouse cell lines BALB/c 3T3 and C3H10T1/2 are of special interest. The retinoids were potent inhibitors of carcinogen-induced focus transformation, and did, in accordance with the proposed involvement of inhibition of gap junctional communication in cell transformation, enhance communication in these cells (24,25,30). We have previously shown that in contrast to the finding in die mouse cell line transformation systems, retinoids behave similarly to phorbol ester tumor promoters in the cell transformation assay using Syrian hamster embryo (SHE*) cells (31). In these cells retinoids both enhance and induce by themselves a high frequency of morphologically transformed colonies. In the present study we show that in accordance with their effect on cell transformation, retinoids inhibit gap junctional communication in hamster embryo cells in a similar way to phorbol esters, though probably through a different mechanism.

E.Rivedal and T.Sanner mounted on a Nikon Diaphot microscope was used. The cells were exposed as indicated, and the test compound was present during dye transfer. The number of dye-coupled cells was counted 10 min following injection using fluorescence equipment attached to the same microscope. Effect on EGF binding Cells were seeded in 24 well plates and grown to confluence. The cells were then exposed to the chemicals indicated in 1 ml DMEM with 10% FBS for 2 h before 0.05 (iCi (137 ^Ci//ig) [I2SI]EGF was added to each well. After incubation for 30 min at 37°C, the plates were put on ice for 5 min prior to washing with ice-cold Hank's balanced salt solution with 1 mg/ml BSA. The cells were dissolved in 0.5 ml, 0.5 M NaOH and bound [12iI]EGF was counted in a gamma counter. Background binding was determined by adding 200 times excess cold EGF. cAMP measurements Cells were grown to confluence in 10 cm dishes and exposed to the chemicals indicated for 4 h. cAMP was extracted with acidic ethanol (1 ml 1 M HC1 in 100 ml ethanol), dried using a vacuum centrifuge (Savant), resuspended in 300 ^1 buffer (50 mM Tris-HCl, pH 7.5, 4 mM EDTA), and measured using a R1A kit from Amersham.

Results Effect on cell—cell communication Retinoic acid strongly inhibits communication between hamster embryo fibroblasts. Figure 1(A) shows data from exposure of the cell line BPNi to /ra/ts-retinoic acid at concentrations from 0.3 to 30 fiM for periods between 30 min and 5 h. Exposure to 30 /iM retinoic acid results in an almost instant block in cell communication. At concentrations between 3 and 15 /iM, the effect on cell communication increases gradually with increasing exposure period. The effect on communication is reversible, i.e. removal of the retinoic acid following 1 h exposure to 30 fiM results in an almost complete reoccurrence of communication following 2 h incubation with growth medium (data not shown). Other retinoids such as fra/w-retinol and fra/w-retinal were tested and showed similar potency to retinoic acid in inhibition of cell communication in these cells (data not shown). The inhibition of intercellular communication takes place at retinoid concentrations where there are no effects on the colony-

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Fig. 2. (A) Effect of TPA on cell-cell communication in BPNi and SHE cells. Cells were exposed to different concentration of TPA for 5 h. (B) Effect of retinoic acid on cell-cell communication in BPNi and SHE cells. Cells were exposed to different concentration of retinoic acid for 1 or 5 h. Bars: SEM, n = 15-30.

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Fig. 1. (A) Effect of fra/is-retinoic acid on cell-cell communication in BPNi cells. Confluent monolayers of BPNi cells were exposed to different concentrations of retinoic acid for periods of between 30 min and 5 h. Lucifer Yellow was then micro-injected into single cells and the spreading of the fluorescent dye to the neighboring cells was measured 10 min later. The figure shows the number of dye-coupled cells as a percentage of control for the different exposure regimes. 100% = 5 0 - 6 0 communicating cells. Bars: SEM, n = 15-30. (B) Toxicity of mmj-retinoic acid in SHE and BPNi cells. Cloning efficiency is the number of surviving colonies as a percentage of seeded target cells. Bars: SD.

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.01 .1 1 10 100 CONCENTRATION iiM) Fig. 3. Binding of [I251]EGF to BPNi cells after 2 h exposure to different concentrations of retinoic acid or TPA. Bars: SD.

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Arachidonic acid metabolite release assay BPNi cells were seeded in 24 well plates at a density of 2 x 104 cells/well in 0.5 ml DMEM with 10% FBS. The following day, cultures were labelled overnight with 0.5 fiCi/ml [3H]arachidonic acid (60-90 Ci/mmol). The medium was then removed and cultures were washed three times with medium containing FBS. Cells were treated with various reagents for 2 h. Aliquots of supernatant medium were then removed and radioactivity counted in Optifluor scintillation fluid (Packard).

forming ability. This is a very sensitive way of assessing toxic effects. Figure 1(B) shows that rram-retinoic acid has no effect on colony formation of BPNi or primary SHE cells at concentrations of 30 nM or less. The cell line BPNi is very sensitive to TPA-induced transformed colony morphology (33,34). Close to 100% of the colonies may show transformed morphology following exposure to TPA. In concurrence with this, the cell line BPNi appears to be far more sensitive than primary SHE cells with regard to inhibition of communication by TPA (Figure 2A). While gap junctional communication in BPNi cells is almost completely blocked at concentrations > 10 nM TPA, the level of communication in the primary SHE cells is decreased by