Identification and Characterization of Retinoic Acid Receptor B2 Target Genes in F9 Teratocarcinoma Cells

Vol. 1, 619–630, June 2003 Molecular Cancer Research 619

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 / 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 / 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). / / RARB2 cells as compared to F9 Wt, RARA ,or The biological effects of retinoids are primarily mediated RAR;/ 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 / 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

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 RARh/ 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+/ 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 RARh/ 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 / 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 RARh/ 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 RARh/ 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 RARh/ 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 / expression results in resistance to the growth inhibitory RARh2 cells and found that 7 out of 20 target genes were actions of retinoids. Indeed, transfection of RARh into reproducibly altered in the Northern blot analysis. We RARh-negative cervical, breast, and lung cancer cells sequenced these seven target genes and identified them using increased responsiveness to growth inhibition and induction NCBI Blast Programs. Among them, six are known genes of apoptosis by retinoids (43–47), and the restoration of (Table 1) and one is an uncharacterized gene. The six known RARh expression in RARh-negative lung cancer cell lines genes are Dab2 (p96 form), glutamate dehydrogenase, Foxq1, inhibited tumorigenicity in nude mice (48, 49). Adminis- Meis1a, midkine, and glutathione S-transferase homologue tration of 13-cis RA to patients with premalignant oral (p28). To determine whether or not these genes were specific lesions can restore the expression of RARh, as well as RARh2 targets, the regulation of these genes by RA was also reverse the lesions (50). Conversely, the loss of both alleles examined by Northern blot analysis in F9 RARa/ and / of the RARb2 in an F9 teratocarcinoma line, generated in this RARg lines, generated by homologous recombination in laboratory by homologous recombination, resulted in the loss our laboratory (22, 23). We also examined RARh2 mRNA of growth inhibition by RA (21). expression. As expected, RARh2 mRNA was not expressed in / We hypothesized that each RAR regulates a specific subset F9 RARh2 cells and its expression was stimulated by RA in of target genes. Thus, to understand the functions of the F9 Wt, RARa/, and RARg/ cell lines (Fig. 1). transcription factor RARh2, the target genes specifically Dab2 (disabled-2), one of the two mammalian orthologues regulated by RARh2 must be identified and analyzed. Very of the Drosophila Disabled gene, is thought to be a tumor few RARh target genes have been identified to date. By suppressor in ovarian cancer since its expression is lost or comparing RA-treated F9 wild-type (Wt) teratocarcinoma cells greatly reduced in 85% of breast and ovarian cancers (51). We / and F9 RARh2 cells, we identified some of the RARh2 show here that Dab2 mRNA expression was up-regulated by target genes through the use of subtractive hybridization and RA in F9 Wt cells, while Dab2 mRNA levels were not affected / DNA expression microarray techniques. The target genes by RA in F9 RARh2 cells (Table 1 and Fig. 1, C and D).

/ Table 1. Summary of Genes Differentially Expressed in RA-Treated F9 Wt and F9 RARB2 Cells

GenBank Identity GenBank Method of Identification Regulation by RA RARh2 Accession No. in Wild-Type F9 Cells Specific

Myelocytomatosis oncogene (c-myc) L00039 Microarray # Y Friend of GATA-1 (FOG) AF006492 Microarray " Y GATA-binding protein 6 U51335 Microarray " Y Glutamate dehydrogenase (GLUD) X57024 Subtractive hybridization " Y Glutathione S-transferase homologue (p28) MMU80819 Subtractive hybridization NC Y Foxq1 (Hfh-1L) AF010405 Microarray and subtractive hybridization " N Hic-5 L22485 Microarray NC Y Meis1a U33629 Subtractive hybridization " Y Mitogen-responsive phosphoprotein p96 (Dab2) U18869 Microarray and subtractive hybridization " N midkine M35833 Subtractive hybridization " Y PDGF-a receptor M57683 Microarray " N

Note: NC, no change; Y, yes; N, no.

Furthermore, Dab2 basal mRNA levels were about 10-fold Glutamate dehydrogenase (GDH) is a mitochondrial enzyme / lower in the F9 RARh2 cells as compared to Wt. This that catalyzes the oxidative deamination of glutamate to a- + + indicates that in the absence of exogenous RA, RARh2 can ketoglutarate using NAD or NADP as a cofactor (56). In the positively regulate the Dab2 gene. Dab2 mRNA expression in brain, besides its function in metabolism, GDH influences F9 RARg/ cells was also low, both before and after RA glutamate neurotransmitter availability (57). The basal GDH / treatment, as compared to Wt cells (Fig. 1, C and D), indicating mRNA level was lower in F9 RARh2 cells than in F9 Wt that RARg may also modulate its expression. cells (data not shown). RA treatment caused an induction of Foxq1 (formerly Hfh-1L) belongs to the family of the FOX glutamate dehydrogenase mRNA expression in F9 Wt cells, but / (Forkhead box) transcription factors (previously called HNF-3/ not in F9 RARh2 cells (Table 1). forkhead transcription factors) (52). RA strongly induced Foxq1 Finally, glutathione S-transferase homologue (p28) is a small / expression in F9 Wt cells, but not in F9 RARh2 cells. For stress response protein which belongs to a family of GST-like instance, Foxq1 mRNA was induced by 5.6- and 7.8-fold after (glutathione S-transferase) proteins (58). p28 expression was not 48 and 72 h RA treatment in F9 Wt cells. By contrast, in F9 affected by RA at either 24 or 48 h in Wt, RARa/, or RARg/ / / RARh2 cells, after 48 and 72 h, Foxq1 mRNA was only cells, and its expression was 4-fold lower in F9 RARh2 cells slightly induced (Fig. 1, A and B; Table 1). However, Foxq1 compared to the other three cell lines (Fig. 1, C and D). Thus, p28 appeared not to be a specific RARh2 target. Foxq1 mRNA exhibits the properties of an RARh2 specific target gene. expression in F9 RARg/ cells was also low, both before and after RA treatment, as compared to Wt cells (Fig. 1, A and B), indicating that RARg may also modulate its expression. Target Genes Identified by Microarray Analyses Meis1a, a member of a family of genes encoding the TALE We used gene microarray analyses to identify RARh2 target (three-amino-acid loop extension) subset of homeodomain- genes, and obtained EST clones for the genes showing the / containing proteins (53), acts as a cofactor of certain Hox largest fold changes between F9 Wt cells and F9 RARh2 proteins and may play a major role in normal hematopoiesis cells. Seven out of the seven target genes from microarray (54). Meis1a mRNA was expressed in F9 Wt, RARa/, and analysis were confirmed by semi-quantitative RT-PCR or RARg/ cell lines in the absence of RA. In contrast, Meis1a Northern analysis, indicating that the microarray analysis was / mRNA is barely detectable in the untreated F9 RARh2 cells, generally reliable. These genes are listed in Table 1. Dab2 and indicating that the basal expression level of Meis1a mRNA in F9 Foxq1 were also identified by subtractive hybridization and / / Wt, RARa , and RARg cells may be regulated by RARh2 were discussed above. in the absence of exogenously added ligand. After 24 h of RA c-myc is a proto-oncogene that is activated in various animal treatment, the Meis1a mRNA level was increased by 3-, 3-, and and human tumors. It is a transcription factor that plays a 2.5-fold in the F9 Wt, RARa/, and RARg/ cell lines, pivotal role in many biological functions including cell growth / respectively, but it was not changed in the F9 RARh2 cells and differentiation (59, 60). c-myc mRNA in untreated / (Fig. 1, A and B). Meis1a mRNAwas only slightly induced in the RARh2 cells was expressed at a 3.4-fold higher level than / / / F9 RARh2 cells, even after 48 or 72 h RA treatment, indicating in untreated F9 Wt, RARa , or RARg cells. Furthermore, / that Meis1a is a specific RARh2 target gene in these cells. c-myc mRNA expression in F9 RARh2 cells is 4.5-fold Midkine is a member of a family of heparin-binding growth higher than in F9 Wt cells after 6 h of RA treatment (Fig. 1, A factors and was originally isolated as a retinoic acid-responsive and B). After 24 h of RA treatment, the expression of c-myc gene (55). midkine mRNA was increased by RA in F9 Wt, mRNA is reduced by 3.7-, 4-, and 4.9-fold in F9 Wt cells, RARa/, and RARg/ cells, and after 48 h, RA increased RARa/, and RARg/ cells, respectively, while in the / midkine mRNA levels by 5.5-, 4-, and 5-fold, respectively. In RARh2 cells, the c-myc mRNA level is still higher than in contrast, RA led to a slight reduction in the midkine mRNA Wt cells. These data suggest that RARh2 suppresses c-myc / level in F9 RARh2 cells (Fig. 1, C and D). Thus, midkine mRNA expression in F9 Wt cells even in the absence of displays the features of a RARh2 specific target gene. exogenously added RA.

FOG1 is a member of the FOG (for Friend of GATA) family of multitype zinc finger proteins that interact with GATA and play essential roles in development (61). FOG1 is expressed in erythroid cells and megakaryocytes, liver, and testis (61). GATA6 is a member of the GATA family of transcription factors which regulate gene expression during development (62). GATA6, together with GATA4 and GATA5, is involved in the formation of the extra-embryonic and embryonic endoderm, as well as the cardiogenic mesoderm (63). FOG1 and GATA6 mRNAs were increased by RA in the F9 Wt, RARa/, and RARg/ cell lines, but were not increased by RA or increased / to a lesser extent in the F9 RARh2 cells (Fig. 1A). Thus, FOG1 and GATA6 are RARh2 specific target genes. Hic5 is a focal adhesion protein that is very similar to paxillin (64). Studies have suggested that Hic5 is involved in the negative regulation of cell growth, since overexpression of Hic5 results in cell growth inhibition (65). Like p28,the expression of Hic5 is not affected by RA treatment, but basal Hic5 mRNA expression is much higher in F9 Wt cells than in / F9 RARh2 cells (Table 1). FIGURE 2. Northern blot analysis of the effects of actinomycin D on PDGF-a receptor (PDGFaR) is one of the two receptors for / RARh2 target gene expression. F9 Wt cells and F9 RARh2 cells were platelet-derived growth factor (PDGF); it has been suggested grown for 12 h in the absence () or presence (+) of 1 AM RA. At 12 h, RNA that the PDGFaR plays a role in embryonic development (66). was isolated or 2 Ag/ml transcriptional inhibitor actinomycin D (ACT D) was added to the medium and RNA was isolated after 6 h. After size It was previously shown that PDGFaR expression is up- fractionation on the gel, the RNA was transferred to nylon and hybrid- regulated during RA-induced F9 cell differentiation (67). Our ization to different cDNAs for the target genes was performed. This results show that the PDGFaR mRNA level is increased by RA experiment was performed twice with similar results; one experiment / is shown. in F9 Wt cells, but not in F9 RARh2 cells (Table 1). PDGFaR mRNA levels are also lower in F9 RARa/ and F9 / h/ RARg cells, indicating that multiple RARs can regulate the seen after 18 h of RA treatment in F9 RAR 2 cells was expression of this gene. greatly reduced after 6 h of actinomycin D treatment, which is consistent with the previous finding that the half-life of c-myc

Target Genes are Transcriptionally Regulated by RARb2 transcripts in F9 cells is about 40 min (70). To determine if the target genes were transcriptionally regulated by RARh2, we inhibited transcription by using RARb Agonists, in the Presence of a RXR Agonist, actinomycin D, an inhibitor of RNA polymerase II (68). F9 Wt Stimulate the Expression of Target Genes in F9 Wt but / / cells and F9 RARh2 cells were first treated for 12 h with or not in F9 RARb2 Cells without 1 AM RA. At 12 h, RNA was isolated from some dishes To study further the function of RARh2 in the regulation / of cells, while actinomycin D (2 Ag/ml) (69) was added to other of its target genes, F9 Wt and RARh2 cells were treated dishes of cells to inhibit RNA transcription. RNA was isolated for 48 h with 1 AM RA or RARh selective agonists (100 nM after an additional 6 h. The RNA samples were analyzed by BMS185411 or 1 AM CD2314), and/or a pan-RXR agonist Northern blot analysis (Fig. 2). Consistent with results shown in (1 AM BMS188649 [BMS649]) (Fig. 3). Neither BMS185411 Fig. 1, RA induced target gene expression in F9 Wt cells, but nor CD2314, the RARh selective agonists, alone could / not in the F9 RARh2 cells. It is also worth noting that the induce the expression of any target genes in F9 Wt cells. stimulatory effect of RA can be seen as early at 12 h. Treatment Similarly, BMS649 (pan-RXR) alone had no effect on the of Wt cells with actinomycin D alone resulted in a decrease in expression of target genes. However, BMS649 (pan-RXR) target gene mRNA. Furthermore, RA induction of target gene and the RARh selective agonists (BMS185411 or CD2314) mRNA was completely blocked by treatment with actinomycin synergistically induced the expression of all RARh2 target / D, indicating that ongoing transcription is required for RA- genes in F9 Wt cells, but not in RARh2 F9 cells. Thus, induced expression of target genes. The c-myc mRNA level RXR-RARh2 heterodimers appear to play a major role in

/ / FIGURE 1. Northern blot analysis of the clones obtained from microarray analysis and subtractive hybridization analysis. A. F9 Wt cells, RARh2 , a , / and g F9 cells were treated with 1 AM RA for different times and total RNA was extracted. After size fractionation on the gel, the RNA was transferred to nylon and hybridized to different cDNAs for the target genes. h-Actin was used as an RNA loading control. This experiment was repeated three to five times with different RNA preparations and very similar results were obtained. A representative experiment is shown. B. Quantitative analysis of the effect of 1 AM RA on the mRNA levels of some target genes. C. Northern blot analysis of three clones obtained from subtractive hybridization analysis. This experiment was repeated three to five times with different RNA preparations and very similar results were obtained. One representative experiment is shown. D. Quantitative analysis of the effect of 1 AM RA on the mRNA levels of the target genes. Quantitative analyses in panels B and D were performed using NIH Image 1.62 or Image Quant 1.2. The mRNA levels are normalized to h-actin mRNA and are expressed relative to the value derived from Wt cells not treated with RA. Columns, means from three to five experiments; bars, SE. Y-axes, arbitrary density units.

/ FIGURE 3. Expression of RARh2 target genes is increased by a RARh-specific agonist and pan-RXR agonist. A. F9 Wt cells and RARh2 cells were treated for 48 h with 1 AM RA, 1 AM or 100 nM RARh-specific agonist (BMS185411 or CD2314), 1 AM pan-RXR agonist (BMS188649), or different combinations of drugs. Total RNA was then extracted and size fractionated on the gel. The RNA was transferred onto nylon and hybridized to different cDNAs for the target genes. The experiments were repeated with different RNA preparations. h-Actin is used as an RNA loading control. One representative experiment is shown here. B. Quantitation of the effects of different agonists on the mRNA levels of the target genes in F9 Wt cells. The mRNA levels are normalized to h-actin mRNA and are expressed relative to the value derived from Wt cells not treated with RA. Columns, means from three experiments; bars, SE. Y-axes, arbitrary units.

/ mediating the induction of the expression of RARh2 target F9 RARh2 cells were treated with RACT, or different genes. This is consistent with the conclusion that RXR-RAR combinations of RARh agonist and/or RXR agonist in the heterodimers are the functional units transducing the retinoid presence of CT (dibutyryl cAMP and theophylline) for 48 h. signal (9). The RNA samples were analyzed by Northern blot analysis (Fig. 4). As expected, CT alone did not have an effect on Expression of RARb2 Target Genes Is Induced in F9 Wt expression of target genes (72). Like RA, RACT induced the / Cells Treated With RACT, but not in F9 RARb2 Cells expression of target genes in F9 Wt cells. Addition of the pan- RACT (retinoic acid, dibutyryl cAMP, and theophylline) RXR agonist BMS649 decreased the Foxq1, FOG1, and treatment causes F9 Wt cells to differentiate into parietal Meis1a expression induced by RACT in F9 Wt cells, but endoderm, while RA alone results in primitive endoderm (71). increased the GATA6 expression induced by RACT. In the We examined the regulation of the expression of some of the presence of CT, BMS649 (pan-RXR) and the RARh selective RARh2 target genes in F9 cells treated with RACT. F9 Wt or agonists (BMS185411 or CD2314) induced the expression of

/ / FIGURE 4. Expression of RARh2 target genes in F9 Wt cells and RARh2 cells treated with RACT. F9 Wt cells and F9 RARh2 cells were treated for 48 h with 1 AM RA, 100 nM (CD2314)or1AM (BMS185411) RARh-specific agonist, 1 AM pan-RXR agonist, or different combinations in the presence of CT [dibutyryl cAMP (250 AM) and theophylline (250 AM)]. Total RNA (10 Ag) was then extracted and size fractionated on the gel. The RNA was transferred onto nylon and hybridized to different cDNAs for the target genes. The experiments were performed two times with different RNA preparations. h-Actin is used as an RNA loading control. Quantitation of some of the mRNAs is shown below the figure.

all RARh2 target genes in F9 Wt cells. In addition, retinol (Rol; and human tumors (76). c-Myc is a transcription factor which vitamin A) induced the expression of all of the RARh2 target regulates many biological processes, including the cell cycle and / genes in F9 Wt cells. In contrast, in F9 RARh2 cells, cell differentiation (77). The c-Myc protein can regulate some expression of FOG1, Foxq1, GATA6, and Meis1a mRNA was cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors, very low with various treatments (Fig. 4). which are cell cycle regulators (78). Deregulated c-myc We also studied the regulation of expression of the Hoxa1 expression often results in increased cyclin E and cyclin A gene, a gene we have studied previously (23, 69, 73, 74). expression (79, 80). In addition, expression of c-myc can Unlike the RARh2 target genes, a similar pattern of Hoxa1 decrease p27 levels and interfere with its function (81). / mRNA expression was observed in F9 Wt and F9 RARh2 Our data indicate that expression of c-myc mRNA is repressed cells (Fig. 4). Hoxa1 mRNA expression was induced by RACT by RARh2 in F9 Wt cells, even in the absence of exogenous RA, / in both F9 Wt and the RARh2 cells. Furthermore, Hoxa1 and that c-myc mRNA is expressed at a much higher level in / mRNA expression was not significantly increased by the RXR untreated and 24 h RA treated F9 RARh2 than F9 Wt cells agonist (BMS649) and RARh selective agonists (BMS185411 (Fig. 1). We conclude that in F9 Wt cells, a low level of RARh2 or CD2314) in the presence of CT (Fig. 4). Rol only slightly expression (without exogenous RA addition but in the presence induced Hoxa1 mRNA expression in F9 Wt cells; thus, Rol of low levels of Rol from serum) inhibits c-myc mRNA treatment was more effective in inducing several of the RARh2 expression. The reduction of c-myc mRNA by RA in F9 Wt target genes, especially GATA6 and Foxq1, than in inducing cells has been observed in several studies, and down-regulation Hoxa1 mRNA, consistent with our prior genetic data that the of c-myc mRNA by RA is associated with RA-induced growth Hoxa1 gene is primarily transcriptionally regulated by RARg arrest of F9 cells (82). The increased level of c-myc mRNA in / and not RARh2 in F9 cells (23). RARh2 cells may play a role in preventing these cells from undergoing growth arrest on RA treatment. Since changes in the c-myc gene or its expression are associated with approximately Discussion one-seventh of U.S. cancer deaths (76), experiments will be In this study, using subtractive hybridization and microarray carried out to determine if the loss of RARh2 expression in h analyses, we identified some of the RAR 2 targets in F9 cells. tumors causes the overexpression of c-myc transcripts and h To our knowledge, this is the first time that specific RAR 2 concomitant abnormal regulation of tumor cell growth. target genes have been identified and confirmed by a RA has been shown to regulate Dab2 expression during h/ / / comparison of Wt, RAR 2 , RARa , and RARg cell embryonic development (83). Moreover, Dab2 has been lines. Most of the target genes are regulated by RA, though our suggested to be a major mediator of RA cell growth inhibition. data suggest that some genes, such as Dab2, Hic5, and p28, are Transient transfection of Dab2 was sufficient to uncouple h also regulated by RAR 2 in the absence of exogenously added MAPK activation and c-fos expression, and was associated h RA. The RAR 2 target genes are also induced by Rol (Fig. 4) with growth inhibition in F9 cells (84). Since we found Dab2 to to a greater extent than a primary RA target gene such as be a RARh2 target in this study, RARh2 could mediate the Hoxa1, which is not an RARh target (23). As we have shown growth inhibitory effects of RA by up-regulating Dab2 previously that Rol is not metabolized to RA in F9 cells (75), a expression. Studies of GATA6-deficient mouse embryos Rol metabolite, 4-oxoRol, is likely to be involved in the suggest that Dab2 expression is regulated by GATA6 (85). regulation of these target genes in F9 cells. We have not yet Since we have shown that GATA6 is also a RARh2 specific h determined the number of primary versus secondary RAR 2 target, Dab2 may be an indirect target of RARh2 and may be target genes, as this requires much more extensive character- regulated by RARh2 through GATA6. The lack of a known ization of the promoters of the genes. The target genes RARE in the Dab2 promoter (86) supports this suggestion. The identified in this study encode a wide range of proteins that observation that the expression of Dab2 mRNA is eliminated in regulate a variety of biological processes. This suggests that, in 85–95% of breast and ovarian tumors suggests that it is a tumor addition to the regulation of cell proliferation, the transcription suppressor gene (51). Indeed, overexpression of Dab2 reduced h factor RAR 2 participates in the regulation of many biological the tumorigenicity of carcinoma cells (87). Conversely, the processes via the transcription of multiple target genes. reduction of RARh2 levels in human breast tumors (88) could h We found that some target genes are RAR 2 specific result in the down-regulation of Dab2, which in turn could alter h/ (Table 1) in that the F9 RAR 2 cells exhibited reduced or no the control of cell proliferation. expression of these target genes, but the F9 RARa/ and F9 RARg/ cell lines exhibited expression levels similar to those in F9 Wt cells. These data provide evidence that each RAR Target Genes and Development isoform has distinct functions. Several targets identified in this study provide insight into the roles of RARh2 in development. Meis1a is a member of the Target Genes and Cell Growth and Cancer homeobox gene family of transcription factors and has been Consistent with our data showing that the induction of suggested to play a role in peripheral neural system develop- RARh2 is necessary for the growth arrest of F9 cells, two of the ment. Correct expression of Meis1a is essential for correct target genes we identified, c-myc and Dab2, from this study patterning in the embryonic peripheral nervous system (89). have been shown to play major roles in cell growth and Our finding that Meis1a is a target of RARh2 is consistent with proliferation. The c-myc proto-oncogene, the cellular homologue the finding that RARh is expressed and plays a role in the of the retroviral v-myc oncogene, is activated in various animal developing nervous system (90).

FOG1 is the founding member of the FOG family of prehybridized, and hybridized at 42jC in 50% (v/v) formamide, proteins that interact with GATA transcription factors and 5 SSC, 50 mM NaH2PO4-Na2HPO4 (pH 7.4), 5 mM EDTA, modulate their activity (61). FOG1 knockout mice die at and 0.1 mg/ml salmon sperm DNA. cDNA probes of target embryonic day 11.5 of gestation because of arrested erythroid genes were labeled with [32P]dCTP using a random primer cell maturation and complete failure of megakaryopoiesis (91). labeling kit (Roche, Indianapolis, IN). cDNA inserts for target As FOG1 was shown to be a target of RARh2 in these F9 cells, genes were either from the F9 cDNA library described below or RARh2 may play a role in megakaryopoiesis. were commercially available EST clones. EST clones for The GATA6 transcription factor is involved in the formation GATA6 (AI894084), PDGF-a receptor (BE630572), and FOG1 of the extra-embryonic and embryonic endoderm, as well as in (AW763708) were obtained from Incyte (Palo Alto, CA). EST cardiogenic mesoderm (92). GATA6 is essential for lung clones for c-myc (BG243402) and Hic5 (BF536993) were from epithelial differentiation in the mouse embryo (63). Our result ATCC. Northern blots were quantitated by phosphorimager that GATA6 is a RARh2 specific target suggests that RARh has analysis, and the software NIH Image 1.62 or Image Quant 1.2. a role in the lung epithelial cell differentiation. Consistent with this, RARh is expressed in the epithelia of the lungs (93). Subtractive Hybridization In the midgestation period, midkine is expressed in the A PCR-Select cDNA Subtraction Kit (Clontech, Palo Alto, endoderm-derived lung epithelium and the adjacent mesen- CA) was used to carry out subtractive hybridization analysis. F9 / chyme, which suggests a key role for midkine in the regulation Wt teratocarcinoma cells and the F9 RARh2 line were treated h of lung development (94). Interestingly, RAR is also with 1 AM all-trans RA for 24 h and total RNA was extracted. expressed in the endoderm-derived lung epithelium and the After reverse transcription, cDNAs from the two cell lines were adjacent mesenchyme at the same stage of embryogenesis. In subjected to subtractive hybridization according to the manu- the embryo, PDGF-A is expressed primarily by epithelial cells, facturer’s protocol. Products from the secondary PCR reactions whereas the PDGF-a receptor is expressed primarily in were radiolabeled with [a-32P]dCTP and hybridized to duplicate mesenchymal cells. This ligand-receptor pair is thought to be filters containing phage clones from a cDNA library in a Lambda important to epithelial-mesenchymal signaling in the lung ZAP vector; this cDNA library was synthesized from F9 Wt cells during development (95). These findings, together with our treated with RA for 72 h.1 After autoradiography, the filter h / results that midkine and the PDGF-a receptor are RAR 2 patterns of F9 Wt cells and of F9 RARh2 cells were compared target genes, suggest that RARh plays a role in epithelial- for each spot. Differentially hybridized clones were picked and mesenchymal signaling during lung development. the inserts were subjected to Northern blot analysis. Foxq1 is expressed throughout embryonic development of the mouse (52). Although its function remains unclear, it is DNA Microarray Analysis likely to play a crucial role during embryogenesis, given its Total cellular RNA was extracted from F9 Wt teratocarci- similarity to other family members. Another family member, / noma cells and the F9 RARh2 cells, both of which were Foxp2, is involved in the development of speech (96). treated with 1 AM all-trans RA for 24 h. Microarray analysis h Further characterization of the RAR 2 target genes identi- was carried out according to the Affymetrix Genechip fied in this study will be helpful in increasing our understanding expression analysis technical manual. Briefly, total RNA from h of the functions of RAR 2 in a variety of biological processes. both cell lines was reverse transcribed into cDNA. After Future studies should also provide important insights into the second-strand synthesis, cDNAs were then in vitro transcribed h molecular mechanisms by which the silencing of the RAR 2 into cRNA with biotinylated ribonucleotides (Enzo Diagnostics, gene leads to cancer. Farmingdale, NY). cRNA (20 Ag) was fragmented by heating at 94jC for 35 min. A cocktail containing fragmented cRNA, Materials and Methods control oligonucleotide B2, control cRNA (Biotin B, Biotin C, Materials Biotin D, and Cre), and herring sperm DNA was hybridized to The RARh agonist BMS185411 and the pan-RXR agonist microarray chips (MG-U74Av2) for 16 h at 45jC. After BMS188649 are from Bristol-Myers Squibb (Buffalo, NY). The washing and staining, the distribution of fluorescent material on RARh agonist CD2314 is from CIRD Galderma (Sophia the array was measured using a laser scanner. The resultant Antipolis, Valbonne, France). Actinomycin D was purchased image was processed with MAS (Microarray Suite) software from Sigma Chemical Co. (St. Louis, MO) and used at a (Affymetrix, Santa Clara, CA). The microarray experiments concentration (2 Ag/ml) shown to inhibit RNA synthesis (69). were repeated three times with different RNA preparations. Only genes which exhibited different expression patterns / between the F9 Wt and F9 RARh2 cells in three independent Cell Culture, RNA Extraction, and Northern Blot Analysis / / / array experiments were further analyzed. F9 Wt, RARh2 , RARa , and RARg cells were maintained in DME supplemented with 10% bovine calf serum Microarray Data Analysis and 2 mM glutamine. Total cellular RNA was extracted using The Microarray Core of the Weill Medical College of RNA-STAT 60 (Tel-Test, Friendswood, TX) following the Cornell performed data preparation and data analysis. manufacturer’s protocol. Northern blot analysis was performed as described previously (97). Briefly, total RNA (10 Ag) was resolved on 1% agarose gels containing 5% formaldehyde and RNA was transferred to nylon filters. Nylon filters were 1Thompson and Gudas, unpublished data.

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Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2003 American Association for Cancer Research. Identification and Characterization of Retinoic Acid Receptor β2 Target Genes in F9 Teratocarcinoma Cells1 1 NIH grant R01 CA43796 to L.J.G.; a research grant from the American Institute for Cancer Research; and in part by a Cancer Pharmacology Training Grant (CA62948-08) (Y.Z.).

Yong Zhuang, Teresa N. Faria, Pierre Chambon, et al.

Mol Cancer Res 2003;1:619-630.

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