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Advances in Brief [CANCER RESEARCH 56. 483—489.February 1. 1996J Advances in Brief Inhibition of Tumor Promoter-induced Transformation by Retinoids That Transrepress AP-1 without Transactivating Retinoic Acid Response Element Jian-Jian Li,1 Zigang Dong, Marcia I. Dawson, and Nancy H. Colburn Cell Biology Section. Laboratory of Viral @arcinogenesis, National Cancer Institute, Frederick, Maryland 21702-1201 Ii-.!. L, N. H. C'.J: Hormel Institute, University of Minnesota, Austin, Minnesota 55912 Ii DI: and SRI, Menlo Park California 94025 [M. I. D.J Abstract Retinoids are a group of natural and synthetic vitamin A analogues that are considered to be potential antitumor agents. Retinoids inhibit Both retinoic acid (RA) treatment and dominant-negativec-Jun mutant tumor cell growth and induce differentiation of certain malignant cells expression effectively inhibit phorbol ester-induced AP-1 activity and (7, 8). The retinoid RA proved to be effective in inhibiting papilboma induced neoplastic transformation in mouse epidermal JB6 cells. How ever, both reagents also target non-AP-1 molecules in addition. Because formation in mouse skin (9, 10) and transformation of mouse JB6 cells liganded retinoic acid receptors interact with and transactivate RA re (11), and inducing differentiation of keratinocytes (12, 13). Recent spouse elements (RAREs) on DNA, as well as interact with Jun protein to results indicate that RA can also cause a phenotypic reversion of the block AP-1 activity, the question arises as to which of these two activities TPA-transformed JB6 cell line RT1O1 (14). The biological effects of retinoids is responsible for antitumor-promoting activity. To address produced by RA are believed to be mediated by the RARs in the cell this question we generated JB6 promotion-sensitive (P@')celllines that are nucleus. The RARs belong to a large superfamily of ligand-inducible stably transfected with a construct containing the collagenase promoter transcription factors that include the steroid, vitamin D, and thyroid bearing one AP-1.binding site that drives a luciferase reporter gene. The hormone receptors (15—17).The retinoid receptors consist of two stable collagenase-luciferase-transfected cell lines showed 1.5—3.5-fold en classes, RARs (cx, j3, @y)andRXRs (a, (3, ‘y)(18—20).Both RAR-a hanced AP-1 activity when treated with 12-O-tetradecanoyl-phorbel-13- and RAR-y are expressed, but RAR-@3is not detected in JB6 cells acetate (TPA). Up to 90% of TPA-induced AP-1 activity was blocked by (21). RAR-y was implicated as the receptor that may mediate the retinoids SR11238, SR11302, or trans-RA, but not by retinoid SR11235. of theseretinoids,onlyRAand SR11235wereableto transactivate inhibitory function of RA and synthetic retinoids on tumor promoter RARE-dependent gene expression. Transrepression ofTPA-induced AP-1 induced transformation in JB6 cells (21). However, because RA both and transactivation of RARE by RA, SR11238, and SR11302 were con transactivates RARE-dependent gene expression and transrepresses @ centration dependent at 1O@0 10'6 Mretinoid. When tested for activity AP-1-dependent expression, the possibility that the antipromoting in inhibiting tumor promoter-induced transformation in JB6 P―cells,the activity is mediated through RARE transactivation rather than through retinoids specific for AP-1 transrepression were inhibitory, whereas AP-1 transrepression has not been excluded. Several new synthetic SR11235, which only activated RARE, showed little effect. We thus con retinoids have been shown to selectively inhibit AP-l transactivation; elude that the AP-1-blocking activity ofretinoids is likely to be responsible others have been shown to selectively induce RARE transactivation for the antitumor-promoting activity. This result, together with the ob (22, 23). Only the AP-1 transrepressors suppressed the growth of lung servation that dominant-negative Jun blocks transformation, argues for a and breast cancer cells (23). These transcription factor-selective reti requirement of induced AP-1 in the tumor promoter-inducedtransforma tion process. noids have not, however, been reported to act during tumor promotion or progression. In the present study, we investigated the effects of Introduction these selective retinoids on TPA-induced AP-l transactivation and cell transformation in JB6 promotion-sensitive cells stably transfected The JB6 family of mouse epidermal clonal genetic variants that are with an AP-l reporter. We show that retinoids that transrepress AP-1, transformation promotion sensitive (P@) or resistant (P) provides a but not retinoids that preferentially transactivate RARE, blocked suitable in vitro model for studying critical gene regulation events that TPA-induced cell transformation. occur during carcinogenesis (1, 2). AP-1 transactivation by tumor promoters TPA2 or EGF appears to be causally related to transforma Materials and Methods tion in the JB6 model. c-Jun expression and AP-1 transactivation are induced by TPA or EGF in promotion-sensitive but not in promotion Cell Culture and Reagents.JB6 mouseepidermalcell lines (Cl4l) were resistant cells (3, 4); TPA-induced cell transformation is blocked by grown at 36°C in EMEM supplemented with glutamine, gentamicin, and 4% FBS. Cells were cultured 24 h (80% confluency) before switching to 2% serum AP-l inhibiting RA and the steroid fluocinolone acetonide, or by in EMEM with TPA with or without retinoid. FBS was from BioWhittaker expression of a dominant-negative c-Jun (5), which sequesters endog (Walkersville, MD); TPA was from Chemicals for Cancer Research (Edina, enous Jun and Fos in transcriptionally inactive complexes (6). MN), and DMSO was from Pierce (Rockford, IL), LipofectAMlNE was from Life Technologies (Grand Island, NY), and [a-32P]dCFP was from Amersham Received 11/7/95; accepted 12/14/95. (Arlington Heights, IL). Luciferase assay substrate was from Promega (Mad The costs of publication of this article were defrayed in part by the payment of page ison, WI). RA was from Sigma Chemical Co. (St. Louis, MO), and the charges. This article must therefore be hereby marked advertisement in accordance with synthetic retinoids were the same as reported (23). 18 U.S.C. Section 1734 solely to indicate this fact. I To whom requests for reprints should be addressed, at Cell Biology Section, Labo Plasmids. A sequence of the collagenase promoter region [—73to +67 ratory of Viral Carcinogenesis, National Cancer Institute, P.O. Box B, Building 560, containing one AP-l-binding site (TGAGTCA)] was excised from a collagen Room 21—27,Frederick, MD 21702-1201. Phone: (301) 846—1333;Fax: (301) 846—1909. ase-AP-l CAT construct as reported (14) and then inserted into the luciferase 2 The abbreviations used are: TPA, l2-O-tetradecanoylphorbol-l3-acetate; EGF, epi reporter vector p012-basic (Promega) to make an AP-l luciferase reporter dermal growth factor; CMV, cytomegalovirus; EMEM, Eagle's MEM; FBS, fetal bovine serum; RA, trans-retinoic acid; RARE, retinoic acid response element; RAR, retinoic acid construct. CMV-neo-selecting plasmid (pBKCMV) was obtained from Invitro receptor; RXR, retinoid X receptor. gen (San Diego, CA). pMexMTX-TAM67 dominant-negative c-Jun plasmid 483 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. INHIBITION OF AP-1 AND CELL TRANSFORMATIONBY RETINOIDS A 4 C) 0 3 a- 2 Fig. 1. Stable presence of luciferase gene and AP-1-luciferase expression in clonal collagenase luciferase transfectants. A, stable expression of AP-l lucif 1 erase reporter gene. JB6 P@ cells (clone Cl41) were cotransfected with colla genase AP-l luciferase construct and the marker plasmid CMV-neo by using LipofectAMINE as mentioned in “Materialsand Methods.―After transfection, clonal lines were selected in medium containing G4l8 (250—500 p.g/ml). At passages as labeled, cell lysates were made, and luciferase activity was meas tired. Values shown are absolute luminometer readings with the entire set of 0 10 20 clones measured separately at each passage indicated. •and @,cell lines transfected with CMV-Neo plasmid only. The cell lines labeled 7—24(R, 7; 0, PassageNumber 8; 4, II; 0, 13; @,19;X, 24) were clonal lines cotransfected with collagenase AP-l and CMV-Neo plasmids and selected in 0418. B, presence of introduced luciferase reporter gene in cloned JB6 cell lines. Genomic DNA of each cell line was digested with the restriction enzymes HindIlI/AvaI, size-separated by 0.8% B 12345678 gel electrophoresis, and transferred onto a Zetabind filter. The filter was hy bridized with 32P-labeled luciferase eDNA probes and visualized on X-ray film. Lane I, control (Con. ) cDNA (collagenase-luciferase plasmid); Lane 2, parental JB6 C14l (P'); Lanes 3 and 4, control cell lines transfected with CMV-neo plasmid only (V1. V2); Lanes 5—8,cell lines cotransfected with collagenase AP-l luciferase reporter plasmid and the CMV-neo plasmid (Col-l 1, 13, 19, and 24. Col-l 1 showed two sites of reporter plasmid incorporation, and Col-13 lost the reporter gene during cell passaging, which was indicated as the decreased luciferase activity during cell passage and lack of genomic incorporation of luciferase gene detected by Southem blot; Col-19 was subsequently selected for further experiments. @—Luciferase @ _, ___ Luciferase Con.P―V1 V2 11 13 19 24 (24) was provided by Dr. M. Birrer (National Cancer Institute, Bethesda, MD). negative c-Jun (TAM67) expression on AP-l transactivation, the collagenase The @3RAREtk-luciferasereporter plasmid containing a (3RARE, 5'-gatc AP-l luciferase reporter cell line (Col-l9) was cotransfected for 12 h with cgctagcAAG GOT TCA CCG AAA GTF CAC TCG CATa-3' driven by a TAM-67 plasmid or control vector (CMV-neo) with LipofectAMlNE. Cells herpes simplex virus thymidine kinase gene promoter (25), was provided by were then cultured in 2% EMEM with or without TPA or EGF for 20—24h, Dr. K. Ozato (NIH, Bethesda, MD). Plasmid containing the (3-galactosidase and luciferase activity was measured as indicated above.
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