Advances in Brief

[CANCER RESEARCH 56. 483â€”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 (15â€”17).The retinoid receptors consist of two stable collagenase-luciferase-transfected cell lines showed 1.5â€”3.5-fold en classes, RARs (cx, j3, @y)andRXRs (a, (3, â€˜y)(18â€”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 Pâ€•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 36Â°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 [â€”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 21â€”27,Frederick, MD 21702-1201. Phone: (301) 846â€”1333;Fax: (301) 846â€”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

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 â€œMaterialsand Methods.â€•After transfection, clonal lines were selected in medium containing G4l8 (250â€”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. â€¢and @,cell lines transfected with CMV-Neo plasmid only. The cell lines labeled 7â€”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 5â€”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.

@â€”Luciferase

@ _, ___ Luciferase

Con.Pâ€•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 20â€”24h, Dr. K. Ozato (NIH, Bethesda, MD). Plasmid containing the (3-galactosidase and luciferase activity was measured as indicated above. Results were cx driven by a CMV promoter was obtained from CLONTECH Laboratories, Inc. pressed as percentage of the luciferase activity of cells transfected with control Transfection and Luciferase Assay of AP-1 and RARE-dependent vector and treated with TPA. Transactivation. For studying retinoid effects on gene transactivation in JB6 Generation ofStable AP-1 Reporter-transfected Cell Lines. To measure cells, JB6 Cl41 cells were cotransfected for 4â€”6hby the calcium phosphate the effects of synthetic retinoids on TPA-induced AP-l transactivation and cell method using 10 @xgRARE luciferase reporter plasmid DNA and 2 @xg transformation, JB6 cell lines stably expressing the collagenase AP-l lucifer fJ-galactosidasereporter plasmid. Alternatively, cotransfection was performed ase reporter were generated by using LipofectAMINE reagent (26). Cells were using LipofectAMINE reagent (26) with 2 @xgRAREluciferase reporter cultured in 60-mm dishes for 24 h (60â€”80% confluency) and cotransfected plasimid and 1 @xgf3-galactosidaseplasmids. Cells were cultured in 2% FBS with the coblagenaseluciferase reporter vector (5 @xg),aCMV-neo containing for 12â€”24h.The cell lysate was mixed with the luciferase assay reagent vector (0.5 ,.sg),and 5 @tlofLipofectAMINE. Cells were transfected for 16 h, (Promega), and luciferase activity was measured by a luminometer (Monobight diluted with 2 ml EMEM containing 4% FBS, and incubated for 24â€”72h.The 2010, Analytical Luminescence Laboratory, San Diego, CA) and normalized transfected cells were trypsinized with 0.033% trypsin and 0.125 mist EDTA, by f3-galactosidaseacitvity. For analysis of the inhibitory effect of dominant resuspended in 4% FBS EMEM, and plated at three concentrations onto 484

INHIBITION OF AP-1 AND CELL TRANSFORMATIONBY RETINOIDS

Table 1 Similar transformation response to TPA in P@ parental cells and in cell lines Results stably transfected with AP-1 luciferase reporter gene

RelativeCell Transfection and Stable Expression of an AP-1 Luciferase Re frequencyC14llineTPAâ€•transformation porter Gene in JB6 P@ Cells. To characterize the relationship be p@â€”0.00V1â€• tween anti-AP-l activity and transformation inhibition by different retinoids, we stably transfected JB6 P@ cells (C141) with the coblag - 0.00 + 1.17 enase-buciferase reporter gene (containing one AP-1-binding site in V2@'+ - 0.00 the promoter region). Southern analysis indicated that the reporter 0.72Col-7â€” +1.00 gene was incorporated into the genomic DNA (>2 copies) in the

0.65Col-8â€” +0.00 stable transfectants (Fig. 1). Basal AP-l activity was detected in the cbonal reporter gene-transfected cell lines (such as clones 7, 8, 11, 19, +0.00 0.58Col-llâ€” and 24; Fig. 1 and data not shown). These stably transfected JB6 cell 0.82Col-13â€” â€”0.00 lines were useful for the present retinoid experiments because a constant basal AP-l activity was detected that provided the sensitivity 1.10Col-19â€” +0.00 needed for reproducible detection of TPA-induced AP-1 transactiva 0.95Col-24â€” +0.00 tion. In addition, cellular trauma during a transient transfection proc

+1.00 1.07 a TPA concentration: 3 ng/ml (5 nM)of 0.33% soft agar in EMEM. b â€˜@â€˜@andV2 were vector control cell lines. A C The transformation frequency of 1.00 corresponds to 310 colonies per lO@ cells (two experiments; the variation was less than 10%).

100-mm tissue culture dishes, then incubated at 36Â°C for 14 days with G4l8 (250 @xg/ml).Clonalcell lineswerering-isolatedandpassagedin 12-welltissue culture dishes with EMEM containing 500 @xg/ml0418.Stable AP-l reporter transfected cell lines were screened by assay of luciferase activity and by Southern blotting. The basal luciferase activity in established cell lines was DNA Binding tested every three passages, and cell lines with constant expression of lucif (GelShift) erase activity for more than 15passages were selected for further study. Stably transfected cells were cultured in 0418-free EMEM for at least 2 passages before each experiment. Southern Hybridization. Isolation of genomic DNA and Southern hybrid ization were performed according to established methods (27â€”29).Briefly, 10 ,.ag genomic DNA from each AP-l reporter-transfected clonal line were digested with restriction endonucleases Hindill and AvaI and were separated by 0.8% gel electrophoresis. The DNA was denatured and transferred onto a Zetabind filter (CUNO, Inc., Meriden, CT). The filter was hybridized with the luciferase gene HindIIlJAvaI DNA fragments that were labeled with [32P]dCTP using a Rediprime labeling kit (Amersham). Prehybridization, hybridization, and blocking were performed in the presence of 6X SSC at 42Â°C.(1X SSC TPAtreatment(hours) 0 6 12 24 48 contains 0.15 MNaC1plus 0.015 Msodium citrate, pH 7.0.) Selective hybrid ization of luciferase sequences was achieved by washing the filters in 0. 1X SSC/0.l% SDS at 65Â°C.Thehybridization blot was visualized by exposing to X-ray film at 70Â°C. B 3 Nuclear Protein Analysis. Gel shift assays were used to detect AP-l binding activity after exposure to the tumor promoter TPA for different times. An AP-I-binding sequence from the human collagenase promoter region, I 5'-AGCATGAGTCAGACACCTCTGGC-3' [human (h)-col AP-l, position @2 @ â€”73 to â€”54 (30)], was synthesized and labeled with [32PJdCTP using the AP-1Transactivation @2 Klenow fragment (Life Science Co., Gaithersburg, MD). Three @xgofnuclear protein extracted from cells exposed to TPA for indicated time intervals were added to the DNA-binding buffer, which contained 5 X l0@cpm 32P-labeled @ oligonucleotide probe, 1.5 @xgpoly(dIdC), and 3 @xgBSA. The reaction / mixture was incubated on ice for 10 mm, followed by incubation at room temperature for 20 mm. The DNA-protein complexes were resolved in a 6% nondenaturing acrylamide gel. The gel was dried and exposed to X-ray film at â€”70Â°Covernight. Anchorage-independent Growth Assay. Retinoid inhibition of TPA-in 0 6 12 24 48 duced cell transformation was investigated in the parental JB6 Cl41 or AP-l reporter stably transfected Cl41 cells. Exponentially growing cells were sus TPA Treatment (hour) pended in 1.5 ml 0.33% agar EMEM over 7 ml 0.5% agar medium containing Fig. 2. TPA-induced AP-l transactivation and DNA-binding activity in collagenase 5 nM TPA with or without retinoids (l0 â€˜Â°tolO_6 M). The cultures were luciferase stably transfected cell line. A, collagenase-luciferase reporter-transfected clone maintained in a 36Â°C incubator for 14 days, and anchorage-independent Col-19 was exposed to TPA (10 ng/ml) for the time intervals indicated. Three @agof colonies greater than 8 cells were scored by a computerized image analyzer. nuclear protein purified as in â€œMaterialsand Methodsâ€•were incubated with 32P-labeled The efficiency of retinoid inhibition of TPA-induced cell transformation is AP-1 oligonucleotide for gel shift assay. B, cell lysates of duplicate dishes from the same TPA experiments were used for assays of AP-l transactivation as measured by luciferase presented as a percentage of the transformation frequency when the cells were activity. Columns, average value for 6 dishes; bars, SEM. The result of gel shift shown is treated with TPA only. one of two duplicate experiments. 485

1.2

C 1.0 0 (5 > U 0.8 (5 (I) Fig. 3. Dominant-negative c-Jun transfection inhib C its both TPA- and EGF-induced AP-1 transactivation (5 in the reporter cells. The Col-19 JB6 reporter cell line 0.6 was transfected with the dominant-negative c-Jun 1â€¢@ plasmid TAM67 or control vector (CMV-neo) 24 h before exposure to TPA (10 ng/ml) or EGF (10 ng/mI) for 20 h. Data were normalized to the value of control 0) 0.4 vector transfection with TPA treatment and expressed > as the mean Â±SEM(bars) of 9 independent cell wells (5 from 3 experiments. 0) 0.2

0.0 TPA EGF Vector TAM-67

ess was eliminated as a source of variability. To be sure that the stable reporter transfectants retain the characteristics of the parental original tumor promoter sensitivity in the reporter gene transfectants JB6 p@ cells with respect to tumor promoter-induced AP-1IDNA was not altered by the process of gene transfection and selection, the binding and AP-l activation kinetics, as well as inhibition of AP-l transformation response to TPA was measured using anchorage inde activity by dominant-negative c-Jun. pendence as the end point (2, 31). All 6 of the reporter gene-trans Transcription Factor Specificity of Retinoids. To ascertain the fected cell lines that were tested retained sensitivity to the TPA specificity of retinoids with respect to RARE transactivation and AP-l induced neoplastic transformation that characterizes the parental P@ transrepression in the JB6 model, the activation of RARE-luciferase C14l cells. Individual clones varied in the magnitude of TPA-induced was measured in transiently transfected JB6 Cl41 cells (Fig. 4A), and transformation (Table 1), with values ranging from 0.58 to 1.17 the repression of TPA-induced AP-1 activity was measured in the relative to the parental Cl 41 response, and a relative value of 0.95 for AP-l reporter expressing Col-l9 cells (Fig. 4B). Transrepression of Col-l9, the clone chosen for further study. The choice of Col-19 was TPA-induced AP-l activity occurred in response to RA and to syn based on its similarity to parental cells for TPA-induced transforma thetic retinoids SR11238 and SR1 1302 in a concentration range of tion response and for AP- 1 transactivation in transient transfection 10@â€˜Â°tol0_6 M (Fig. 4B). However unlike RA, SR1 1238 and assays (5). SR113O2 did not show RARE transactivation (Fig. 4A). The magni Comparison of AP-l-dependent Response in JB6 Cells Stably tude of AP-l transrepression by SR1 1238 or SRll302 was similar to or Transiently Transfected with AP-1-inducible Promoter-driven that by RA (78% inhibition at lO_6 M). In contrast, the retinoid Luciferase Reporter. Both API transactivation and DNA-binding SR1 1235 produced a much lower AP-l transrepression (20%) that activity were induced by TPA in the reporter transfectants. Assay of was not concentration dependent but was able to transactivate the nuclear extract binding to a collagenase promoter oligonucleotide RARE in a dose-dependent manner, exhibiting a 2.9-fold response at showed that the AP-l-binding activity was elevated as early as 30 mm lO_6 M, a reponse that is 81% of the maximal RARE activation after TPA addition (data not shown) and increased during the course (3.6-fold) attained with RA (Fig. 4A). No RARE activation was of TPA treatment until 24 h (Fig. 14). The AP-l-luciferase transac detected after treatment with SR11238 or SR1 1302. Thus, in mouse tivation, represented as luciferase activity after TPA treatment (Fig. JB6 cells, SR11238 and 11302 are specific for transrepression of 2B), paralleled the increase of AP- 1 DNA binding from 6 to 24 h, with AP-l, whereas SR11235 shows relative specificity for RARE trans the highest values at 24 h. This result was in agreement with those of activation. another experiment, which used the same JB6 clone 41 parental cell Specificity of Retinoids in Blocking TPA-induced Neoplastic line and a transient reporter transfection method (data not shown and Transformation. The concentrations of RA and synthetic retinoids Ref. 5). Because dominant-negative mutant c-Jun has been shown to found to be active for RARE or AP- 1 regulation were used to assess inhibit TPA-induced AP-l activity in JB6 cells (5) and to block tumor the inhibitory effect on TPA-induced JB6 cell transformation. Fig. 5 formation by mouse epidermal carcinoma cells (32), we transiently shows the anchorage-independent transformation-inhibitory activity transfected a stable reporter clonal line (Col-l9) with a dominant of retinoids in the collagenase luciferase reporter Col-19 cells. negative c-Jun construct (TAM67), which contains a mutant c-Jun SR11238, SR11302, and RA, which showed AP-l transrepression recombinant cDNA deleted in the transactivation domain (lacking (Fig. 4, A and B), inhibited TPA-induced transformation dose depen amino acids 3â€”122)driven by a metallothioneine promoter (24). Fig. dently to about 10% of the TPA-only controls. Retinoid SR1 1235 3 showsthatTPA-orEGF-inducedAP-ltransactivationwasblocked showed a small dose-independent inhibition of cell transformation in the TAM67 transiently transfected Col-l9 cells with greater inhi (20% transformation inhibition in the concentration range l0 â€˜Â°to bition in the TPA-treated cells than in the EGF-treated cells. The basal l0@ M) of a magnitude similar to that for TPA-induced AP-l tran AP-l transactivation showed no significant inhibition by TAM67 srepression (Fig. 4B). Thus, AP-l transrepression, but not RARE transfection when compared with vector control (Fig. 3). Thus, the transactivation, by retinoids appears to be causally related to their 486

A but not RARE transactivation. Another retinoid (SR11235) transacti â€”0-â€” 235 vated RARE-dependent gene expression but produced little inhibition ..â€”.-.-238 of either AP-l transactivation or neoplastic transformation by TPA. ...-fJ---, 302 This finding provides new evidence that further implicates AP-1 but 3 now excludes RARE as a reqiurement in antipromotion by retinoids. Previous reports have demonstrated the activity of AP-l-transrepress

0 ing but not RARE-transactivating retinoids in blocking maintenance of tumor phenotype as assessed by anchorage-independent growth of lii tumor cells (22). The present study is, to our knowledge, the first to

2 demonstrate such specificity in the process of carcinogenesis. This 0) observation lends additional independent support to the previous lines > 0) of evidence implicating AP-1 , rather than certain other coregulated 0) transcription factors, in tumor promoter-induced transformation. Ret inoids, glucocorticoids, and dominant-negative Jun all inhibit tumor promoter-induced transformation (5). Because the only apparent corn mon target of these three pleiotropic agents is Jun protein (17, 33â€”35), the promotion-relevant target is likely to be a Jun-containing or Jun-dependent transcription factor. These earlier studies argued against but did not exclude a robe for RAR- or glucocorticoid receptor 0 -10 -9 -8 -7 dependent gene transcription in antipromotion as modeled by JB6 Retinoid Concentration (log M) cells. The present studies suggest that a requirement for RARE in antipromotion by retinoids can be excluded. They also suggest that B retimoids free of specific side effects associated with RARE activation might be valuable for chemoprevention. 2.4 An advantage of the present experimental approach is that we used a stably transfected JB6 P@ cell line that showed cellular responses to 2.2 a tumor promoter that were very similar to those seen with transiently transfected parental JB6 P@ cells (1, 2, 31). A constant basal level of 2.0 AP-l transactivation was detected for many passages in the AP-1 U ,( reporter transfectants, thus circumventing the variables linked to tran 1.8 sient transfection, such as transfection efficiency, internal controls, 4 and cytotoxic effects of the transfection process. Moreover, stably 0) 1.6 â€”0â€” 235 integrated reporter genes are likely to model gene regulation in 0) â€”..-â€” 238 @ 0) 302 chromatin better than the naked DNA of transiently transfected plas ---*.. RA 1.4

1.2

C 0 1.0 0 4' solvent control 0)

5-E 0 0) 0 -10 .9 .8 -7 .6 C 0) 5- Retinoid Concentration (log M) I. â€¢0 Fig. 4. Retinoids RA, SR 11238 (238), and 11302 (302) transrepressed AP-l , whereas 0) RA and SR11235 (235) transactivated RARE in JB6 cells. A, Cl4l cells were transiently 0 .5 transfected with a RARE luciferase reporter construct as described in â€œMaterialsand C Methodsâ€•for 4â€”6h and then exposed to retinoids at various concentrations as indicated for 16â€”20h. Retinoid-induced luciferase transactivation was determined by assaying for a. luciferase activity. Data were presented as the fold induction compared to the value of Iâ€” luciferase activity in C141 cells transiently transfected with RARE luciferase reporter 0 without treatment by retinoids. B, collagenese-luciferase stably transfected Col-19 cell C line was exposed to TPA (10 ng/ml) with retinoids in the dose range as indicated. AP-l 0 transactivation was determined by measuring luciferase activity and presented as fraction .t of the value of TPA-only-treated cells. The DMSO (O.Ol%)-treated cells served as a solvent control as marked. Points, mean of 6 independent cell dishes from two experi C ments (A) or 8 independent dishes from 4 experiments (B); bars, SEM.

inhibitory effect on TPA-induced transformation. Only the retinoids that blocked AP-l transactivation showed a high efficiency in anti promotion. Retlnold Concentration (log M) Fig. 5. AP-l-transrepression-specific retinoids inhibit TPA-induced anchorage-inde Discussion pendent transformation. Collagenase-luciferase reporter cell line (Col-19) was exposed to TPA (3 ng/ml, 5 nat) with retinoids RA, SR11235 (235), SR11238 (238), or SR11302 In the present study, we have shown that two synthetic retinoids (302) at concentrations from l0 â€˜Â°tol0_6 M in 0.33% soft agar. Anchorage-independent colonies were scored after 14 days incubation at 36Â°Cand 100% humidity. Data are (SRi 1238 and SR1 1302) blocked TPA-induced neoplastic transfor presented as the fractions of the transformation frequency with TPA treatment only. mation by a mechanism that appears to involve AP-l transrepression Points, mean of three experiments; bars, SEM. 487

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. INHIBITION OF AP-1 AND CELL TRANSFORMATION BY RETINOIDS raids. These AP- 1 reporter P@ cells should serve as useful tools for References identifying new antipromoting retinoids, as well as other pharmaco I. Colbum, N. H., Former, B. F., Nelson, K. A., and Yuspa. S. H. Tumour promoter logical or dietary chemprevention agents that act via AP-l transre induces anchorage independence irreversibly. Nature (Land.). 281: 589â€”591, 1979. presson. 2. Colburn, N. H., Wendel, E., and Srinivas, L. Responses of prenoplastic epidermal cells to tumor promoters and growth factors: use of promoter-resistant varients for It has been well documented that the RAR and RXR classes of mechanism studies. J. Cell. Biochem., 18: 261â€”270,1982. nuclear receptors and their a, @3,and â€˜yisoforms exhibit distinct 3. Bernstein, L. R., and Colbum, N. H. AP1/jun function is differentially induced in patterns of expression in cells (18, 36). RAR-a and RAR-y are promotion sentitive and resistant JB6 cells. Science (Washington DC), 244: 566â€”569, 1989. expressed in mouse epidermis (37), whereas only low levels of 4. Bernstein, L. R., Ferris, D. K., Colbum, N. H., and Sobel, M. E. A family of MAP RAR-@3expression were detected in adult human (38) and mouse skin kinase related proteins interacts in sis'o with the AP-l transcription factor. J. Biol. Chem., 269: 9401â€”9404, 1994. (37). RARs bind both all-trans-RA and 9-cis-RA, whereas RXRs bind 5. Dong. Z., Birrer, M. J., Watts, R. G., Matrisian, L. M., and Colburn, N. H. Blocking only 9-cis-RA (39â€”41). Reports have indicated that both RARs and of tumor promoter-induced AP-l activity inhibits induced transformation in JB6 RXRs can act as ligand-dependent transrepressors of AP- 1 (c-Jun/c- mouse epidermal cells. Proc. NatI. Acad. Sci. USA, 91: 609â€”613,1994. 6. Brown, P. H., Chen, T. K., and Birrer, M. J. Mechanism of action of a dominant Fos) activity and the AP-l complex, in turn, can inhibit transactivation negative mutant of c-Jun. Oncogene, 9: 791â€”799,1994. by RARs and RXRs (33, 35). SRl 1238 and SRi 1302, which bind 7. Jetten, A. M., and Goldfarb, R. H. Action of epidermal growth factor and retinoids on RARs, inhibited AP-l transactivation and the proliferative growth of anchorage-dependent and -independent growth of nontransformed rat kidney cells. Cancer Res., 43: 2094â€”2099,1983. several tumor lines but did not induce differentiation in embryonal 8. Bollag, W., and Holdener, E. E. Retinoids in cancer prevention and therapy. Ann. carcinoma F9 cells (23). In contrast, 5Rll235, an RXR-a-selective Oncol., 3: 513â€”526,1992. ligand, had little effect on AP-l transactivation (23). Our results also 9. Verma, A. K., and Boutwell, R. K. Vitamin A acid (retinoic acid), a potent inhibitor or 12-O-tetradecanoyl-phorbol-13-acetate-induced omithine decarboxylase activity in show some inhibitory effect of 5R11235 (dose independent) on TPA mouse epidemis. Cancer Res., 37: 2196â€”2201, 1977. induced cell transformation (10 â€˜Â°tol0@ M). The magnitude of 10. Verma, A. K., Shapas, B. G., Rice, H. M., and Boutwell, R. K. Correlation of the inhibition by retinoids of tumor promoter-induced mouse epidermal ornithine decar inhibition by SRl 1235 was small compared to that of AP-l-specific boxylase activity and of tumor promotion. Cancer Res., 39: 419â€”425, 1979. SRi 1238 or SR1 1302 but is consistent with its low inhibitory effect 11. Colburn, N. H., Ozanne, S., Lichti, U., Ben, T., Yuspa. S. H., Wendel, E., Jardini, E., on AP-l transactivation. Our data further indicate that the inhibitory and Abruzzo, 0. Retinoids inhibits promoter-dependent preneoplastic progression in mouse epidermal cell lines. Ann. NY Acad. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1996 American Association for Cancer Research. Inhibition of Tumor Promoter-induced Transformation by Retinoids That Transrepress AP-1 without Transactivating Retinoic Acid Response Element

Jian-Jian Li, Zigang Dong, Marcia I. Dawson, et al.

Cancer Res 1996;56:483-489.

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