Vol. 1, 619–630, June 2003 Molecular Cancer Research 619 Identification and Characterization of Retinoic Acid Receptor B2 Target Genes in F9 Teratocarcinoma Cells Yong Zhuang,1 Teresa N. Faria,1,* Pierre Chambon,2 and Lorraine J. Gudas1 1Pharmacology Department, Weill Medical College of Cornell University, New York, NY, and 2Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS/INSERM/ULP, Colle`ge de France, CU de Strasbourg, France Abstract Introduction Retinoids, a group of natural and synthetic analogues of Retinoids, a group of natural and synthetic analogues of vitamin A (retinol), modulate the differentiation of many vitamin A, exert profound effects on many biological cell types. Retinoids are also used for the prevention and processes, such as vertebrate embryonic development (1) treatment of cancer. The actions of retinoids are and cell growth and differentiation (2). Vitamin A insuffi- generally mediated by the retinoic acid receptors (RARs ciency during pregnancy results in the death of the fetus, as A, B, and ;) and the retinoid X receptors (RXRs A, B, well as congenital malformations affecting the eyes and ocular and ;). One of the RARs, RARB, is expressed at reduced tissues, myocardium, respiratory, urogenital, and circulatory À/À levels in many human carcinomas, and F9 RARB2 cells systems (1). Vitamin A deficiency in experimental animals is do not growth arrest in response to RA. To determine if also associated with a higher incidence of some types of RARB2 regulates the expression of a unique set of genes, cancer, and with increased susceptibility to chemical carci- through the use of subtractive hybridization and DNA nogens (3). Experimental models of carcinogenesis have array analysis we have identified and characterized demonstrated the efficacy of pharmacological administration À/À genes that are differentially expressed in F9 RARB2 of retinoids in preventing the development of cancers of the teratocarcinoma cells. These genes, which encode skin, oral cavity, lung, mammary gland, prostate, bladder, transcription factors, cell surface signal transduction liver, and pancreas in animals exposed to carcinogenic agents molecules, and metabolic enzymes, include c-myc, (3). Clinical trials have indicated that retinoids may be useful FOG1, GATA6, glutamate dehydrogenase, glutathione for prevention of cancers of the upper aerodigestive tract, skin, S-transferase homologue (p28), Foxq1, Hic5, Meis1a, breast, and ovaries (4). In addition, retinoids have been Dab2, midkine, and the PDGF-a receptor. These genes successfully used in the treatment of acute promyelocytic are regulated specifically by RARB2 in F9 wild-type (Wt) leukemia (APL) (5, 6), and other cancers (for review, see cells as indicated by their expression profiles in F9 Refs. 7 and 8). À/À À/À RARB2 cells as compared to F9 Wt, RARA ,or The biological effects of retinoids are primarily mediated RAR;À/À cells, and their responsiveness to specific by two classes of nuclear retinoid receptors: retinoic acid retinoid receptor agonists. The basal expression levels receptors (RARs) and retinoid X receptors (RXRs) (9, 10). of some of these genes, such as c-myc, are higher in the RARs and RXRs are members of the nuclear receptor À/À F9 RARB2 cells than in F9 Wt in the absence of superfamily that also include estrogen, androgen, thyroid exogenous retinoids, suggesting that RARB2 can inhibit hormone, vitamin D, PPAR, and orphan receptors. The gene expression in the absence of a ligand. The RARB2 retinoid receptors are encoded by six distinct genes: RARa, target genes are transcriptionally activated by retinol, as RARb, RARc, RXRa, RXRb, and RXRc. Each of these well as RA, in F9 Wt cells. Because the lack of RARB2 receptors includes several isoforms formed by different alters both the control of proliferation and differentiation splicing and usage of alternative promoters (9, 10). All-trans in F9 cells, the genes that we have characterized may retinoic acid (RA) binds and activates RARs, and 9-cis- mediate key effects of RA, via RARB2, on these retinoic acid (9-cis RA) binds and activates both RARs and processes. RXRs (11). Retinoid receptors activate transcription in a ligand-dependent manner by binding to retinoic acid response elements (RAREs) located in the promoter regions or enhancers of target genes. RAREs generally consist of two directly repeated half-sites of the consensus sequence Received 2/18/03; revised 5/9/03; accepted 5/9/03. AGGTCA spaced by 2 or 5 bp (DR2 and DR5 elements) The costs of publication of this article were defrayed in part by the payment of (12). Recently, various coactivators for the nuclear receptors page charges. This article must therefore be hereby marked advertisement in have been identified, including CBP/p300, SRC-1, TIF-2/ accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant support: NIH grant R01 CA43796 to L.J.G.; a research grant from the Grip1, and ACTR/RAC3/p/CIP. They are recruited by ligand- American Institute for Cancer Research; and in part by a Cancer Pharmacology activated receptors and enhance transcription (13, 14). Many Training Grant (CA62948-08) (Y.Z.). of these coactivators possess acetyltransferase activity that can Requests for reprints: Lorraine J. Gudas, Room E409, Pharmacology Depart- ment, Weill Medical College of Cornell University, 1300 York Avenue, New modify histones and may increase promoter access to DNA- York, NY 10021. Phone: (212) 746-6250; Fax: (212) 746-8858. E-mail: binding proteins and the transcriptional machinery (15–18). [email protected] *Current address: Bristol-Myers Squibb Company, New Brunswick, NJ 08903. The strong conservation of each RAR across vertebrates Copyright D 2003 American Association for Cancer Research. suggests that each receptor performs unique functions (19). In Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. 620 RARh2 Target Genes our laboratory, we generated F9 teratocarcinoma cells with identified include transcription factors, protein tyrosine mutations in both alleles of RARa, RARb2, or RARc by kinases, homeobox proteins, and oncoproteins. We have homologous recombination and showed that different RARs initiated the further characterization of eleven target genes in have different functions (20–23). Mice homozygous for this study. RARa,RARh,orRARg have also been generated to understand the function of each receptor. Mutant mice lacking Results RARa, RARh , RARh, or RARg are viable but exhibit 2 Identification of RARb Target Genes in F9 Cells abnormalities in several tissues (24–27). For example, 2 We hypothesized that RARh , which is induced by RA in RARhÀ/À mice exhibit greatly reduced hippocampal CA1 2 many cell types, mediated the growth inhibitory actions of RA long-term potentiation (LTP) and long-term depression (LTD) in F9 cells. To test this hypothesis, an F9 teratocarcinoma cell (28), activities which are likely to play a role in learning line that contained targeted disruptions in both alleles of the and memory. RARh gene was generated by homologous recombination. We The RARb gene has four isoforms: h , h , h , and h . The h 2 1 2 3 4 2 showed that the F9 Wt and RARh+/À heterozygous lines could isoform is the most abundant RARh isoform and the major RA 2 undergo RA-induced growth arrest, but that RA did not cause inducible form (29). An RARE that mediates RA-induced growth arrest in the F9 RARhÀ/À cells (21). While these data RARh gene expression in many different cell types was 2 2 confirmed our hypothesis, more information about the target identified in the promoter region (30, 31). The RARh RARE 2 genes regulated by RARh was then required to understand the consists of two direct repeats of the core motif sequence 2 downstream effects of RARh . We anticipated that RARh AGGTCA separated by five nucleotides (31, 32). Activation of 2 2 positively regulated the transcription of some of its target genes, the RARh promoter is mediated by RAR/RXR heterodimers 2 and negatively regulated other target genes. (33). A thyroid hormone receptor/RXR heterodimer can also h h h To isolate RAR 2 target genes, F9 Wt cells and F9 bind strongly to the RARE and activates the RAR 2 promoter À/À RARh cells were treated with 1 AM all-trans RA for 24 h. in response to RXR ligands (34). RARh exhibits a pattern of 2 Subtractive hybridization and microarray analysis were then expression during development and in the mature organism (35) employed. By comparing the gene expression patterns between which is different from those of the other RARs. This suggests F9 Wt cells and the F9 RARhÀ/À cells, we identified that RARh performs specific functions. 2 approximately 80 genes from subtractive hybridization and RARh is not expressed, or is expressed at low levels, in a 300 target genes from the microarray analyses (see ‘‘Materials number of malignant tumors, including lung carcinoma, and Methods’’). Approximately one-half of the putative target squamous cell carcinoma of the head and neck, breast cancer genes were expressed at higher levels in RA-treated F9 Wt cells cell lines, and esophageal carcinoma (36–40). RARh is as compared to F9 RARhÀ/À cells, while the remaining genes encoded by a gene located on the short arm of chromosome 2 were expressed at higher levels in RA-treated RARhÀ/À cells 3 (p24) (41). In breast cancer, loss of heterozygosity (LOH) 2 than in F9 Wt. has frequently been detected at chromosome 3p22–25 (42). All of these findings support the concept that the specific loss of RARh expression may be an important event in Target Genes Identified by Subtractive Hybridization tumorigenesis. We examined 20 of the genes isolated by subtractive Recent studies have indicated that a decrease in RARh hybridization by Northern analysis of F9 Wt versus F9 À/À expression results in resistance to the growth inhibitory RARh2 cells and found that 7 out of 20 target genes were actions of retinoids.