RARbeta2 Promoter Research Articles

Cooperative Action of 1alpha,25-Dihydroxyvitamin D3 and Retinoic Acid in NB4 Acute Promyelocytic Leukemia Cell Differentiation Is Transcriptionally Controlled.

All-trans-retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we have shown that in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA-transcriptional activity through an RARbeta promoter. By EMSA and ChIP assays, we further demonstrate that while both VDR and RAR are bound to the RARbeta promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA-transcriptional activity in leukemic cells suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation.

Cooperative action of 1α,25-dihydroxyvitamin D3 and retinoic acid in NB4 acute promyelocytic leukemia cell differentiation is transcriptionally controlled

All-trans-retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we show that, in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA transcriptional activity through an RARbeta promoter. By EMSA and ChIP assays, we further demonstrate that, while both VDR and RAR are bound to the RARbeta promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter, while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA transcriptional activity in leukemic cells, suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells, the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation.

Hyperacetylation enhances the growth-inhibitory effect of all-trans retinoic acid by the restoration of retinoic acid receptor β expression in head and neck squamous carcinoma (HNSCC) cells

The chemotherapeutic effects of all-trans-retinoic acid (atRA) are mediated by the retinoic acid receptor beta (RARbeta), but RARbeta expression is reduced in a number of head and neck carcinoma (HNSCC) cells which causes resistance to RA treatment in half the patients with HNSCC. The possible mechanism for the reduced RARbeta expression has been suggested as the methylation of the CpG islands adjacent to the RA response elements (RARE) in the RARbeta promoter and the loss of histone acetylation. The suppressed RARbeta expression can be reactivated by a demethylating agent (5-aza-2'-deoxycytidine, 5-AzaC) or a histone deacetylase inhibitor (trichostatin A, TSA). Therefore, we sought to determine if the restoration of RARbeta activity, or a combination of these drugs, could restore the sensitivity to RA in RARbeta-negative HNSCC cells with an epigenetically methylated RARbeta promoter region. SqCC/Y1 cells resistant to atRA showed methylated and unmethylated forms in the RARbeta promoter region. RARbeta expression of these cells was restored by 5-AzaC or TSA treatment. Also, treatment with TSA and atRA combined synergistically increased the growth-inhibitory effect and highly induced the transcriptional activation of the RARbeta promoter compared to atRA treatment in HNSCC cells. Additionally, TSA alone and the combination 5-AzaC and TSA increased lysine-9 (Lys-9) acetylation and Lys-4 methylation of the first exon at the RARbeta gene, while decreasing the methylation of Lys-9 in the HNSCC cells.

PARP-1 Determines Specificity in a Retinoid Signaling Pathway via Direct Modulation of Mediator

We show that PARP-1 is indispensable to retinoic acid receptor (RAR)-mediated transcription from the RARbeta2 promoter in a highly purified, reconstituted transcription system and that RA-inducible expression of all RARbeta isoforms is abrogated in PARP-1(-/-) cells in vivo. Importantly, PARP-1 activity was independent of its catalytic domain. PARP-1 directly interacts with RAR and Mediator. Chromatin immunoprecipitation experiments confirmed the presence of PARP-1 and Mediator on RAR-responsive promoters in vivo. Importantly, Mediator was inactive (Cdk8+) under basal conditions but was activated (Cdk8-) upon induction. However, in PARP-1(-/-) cells, Mediator was retained in its inactive state (Cdk8+) upon induction consistent with the absence of gene expression. PARP-1 became dispensable for ligand-dependent transcription in a chromatin reconstituted transcription assay when Mediator was devoid of the Cdk8 module (CRSP). PARP-1 appears to function as a specificity factor regulating the RA-induced switch of Mediator from the inactive (Cdk8+) to the active (Cdk8-) state in RAR-dependent transcription.

In vivo imaging of retinoic acid receptor β2 transcriptional activation by the histone deacetylase inhibitor MS‐275 in retinoid‐resistant prostate cancer cells

In retinoid resistant epithelial tumors, the lack of retinoic acid receptor beta2 (RARbeta2) expression due to epigenetic silencing impairs the activation of retinoid target genes including RARbeta2, and has been associated with the development of cancer. In this study we developed a strategy to monitor the re-activation of RARbeta2 by chromatin remodeling agents combined with retinoids in real time, and to correlate the RARbeta2 re-activation with anti-tumor activity. We selected the RARbeta2-negative retinoid resistant human prostate carcinoma cell line PC3 and stably transfected it with a luciferase expression vector under the control of a functional segment of RARbeta2 promoter (pGL2-RARbeta2-PC3). Then, we used the bioluminescence technology to monitor the reporter gene expression in real time both in vitro and in vivo following combination treatment with the histone deacetylase inhibitor MS-275 and 13-cis retinoic acid (CRA). Based on the effective dose for the RARbeta2 re-activation, we tested the anti-tumor activity of this drug combination. Following combination treatment with MS-275 and CRA, we observed endogenous RARbeta2 re-expression, acetylation at the RARbeta2 promoter level, and synergistic activation of the luciferase reporter gene by real time imaging both in vitro and in vivo. Combination treatment with MS-275 and CRA restored retinoid sensitivity in human prostate carcinoma cell lines, and had a greater inhibitory effect on tumor cell growth than single agents in vitro and in vivo. This study provides evidence that HDAC inhibitors restore retinoid sensitivity in prostate cancer cells, and in vivo real time imaging of RARbeta2 activation may represent a useful tool to study the pharmacodynamics of combination therapy with HDAC inhibitors and retinoids.

Promoter methylation of retinoic acid receptor beta 2 and the development of second primary lung cancers in non-small-cell lung cancer.

To investigate whether the promoter hypermethylation of retinoic acid receptor beta 2 (RARbeta2) is associated with the development of second primary lung cancers (SPLCs) differentially according to smoking status in primary non-small-cell lung cancer (NSCLC). We retrospectively analyzed the relationship between RARbeta2 methylation and the SPLC development in a total of 342 NSCLCs. The methylation status of RARbeta2 was determined by using methylation-specific polymerase chain reaction. The difference in the time to SPLC development was analyzed by using the log-rank test and the Cox proportional hazards model. The median follow-up was 4.1 years. SPLCs developed in 19 (5.6%) of the 342 NSCLCs, and overall incidence rate of SPLC development was 1.54 per 100 patient-years. SPLCs did not occur in 39 patients who had not smoked. After controlling for possible confounding factors, the hazard of failure for former smokers with RARbeta2 hypermethylation was about 2.87 (95% CI, 0.92 to 13.64; P =.08) times higher compared to those without RARbeta2 methylation. However, for current smokers, hypermethylation of the RARbeta2 was found to have a protective effect against the SPLC development (hazard ratio = 0.23; 95% CI, 0.11 to 0.87; P =.03). Hypermethylation of RARbeta2 promoter had a differential effect on the development of SPLCs in NSCLC, and this was dependent on smoking status. Our study suggests that a combination of retinoids and/or a demethylating agent may be effective in the prevention of SPLCs in never-smokers and former smokers with NSCLC.

Hypermethylation of the retinoic acid receptor-beta(2) gene in head and neck carcinogenesis.

Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation. RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.

β-Carotene and β-Apo-14′-Carotenoic Acid Prevent the Reduction of Retinoic Acid Receptor β in Benzo[a]pyrene-Treated Normal Human Bronchial Epithelial Cells

Low-dose beta-carotene (BC) supplementation, such as would be provided by daily consumption of approximately 5-9 servings of fruits and vegetables, has no apparent detrimental effects, but rather appears to have a protective effect against cigarette smoke-induced lung lesions in ferrets. In the present study, we investigated the effects of BC, beta-apo-14'-carotenoic acid (14'CA), or benzo[a]pyrene (BP; a primary lung carcinogen from cigarette smoke) treatments, either alone or in combination, on cell growth and expression of the retinoic acid receptor (RAR) of normal human bronchial epithelial (NHBE) cells. We found that both BC and 14'CA inhibited the growth of NHBE cells (P < 0.05) with or without BP. The level of RARbeta, a tumor suppressor, but not RARalpha or RARgamma, was reduced by 50% in the NHBE cells treated with BP. However, treatment with either BC or 14'CA significantly induced the expression of RARbeta in the NHBE cells, and prevented the reduction of RARbeta by BP. Furthermore, 14'CA transactivated the RARbeta promoter primarily via its conversion to retinoic acid (RA). In the presence of 3-mercaptopropionic acid, an inhibitor of fatty acid oxidation, both RA formation and transactivation activity from 14'CA were decreased. These observations indicate that the growth inhibitory effects of BC and beta-apo-carotenoic acid are through their conversion to RA and upregulation of RARbeta.

Downregulation of retinoic acid receptor-beta(2) expression is linked to aberrant methylation in esophageal squamous cell carcinoma cell lines.

To study the role of hypermethylation in the loss of retinoic acid receptor beta(2) (RARbeta(2)) in esophageal squamous cell carcinoma (ESCC). The role of hypermethylation in RARbeta(2) gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP), and its methylation status was compared with RARbeta(2) mRNA expression by RT-PCR. The MSP results were confirmed by bisulfite sequencing of RARbeta(2) promoter regions. Methylation was detected in 4 of the 6 cell lines, and the expression of RARbeta(2) was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARbeta(2) was restored in one RARbeta(2) -downregulated cell line with the partial demethylation of promoter region of RARbeta(2) after 5-aza-2'-deoxycytidine (5-aza-dc) treatment. The methylation of the 5' region may play an important role in the downregulation of RARbeta(2) in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARbeta(2) expression in ESCC cell lines. This study may have clinical applications for treatment and prevention of ESCC.

Carcinogen exposure differentially modulates RAR-beta promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis.

The retinoic acid receptor beta (RAR-beta) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-beta has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-beta gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), RAR-beta was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-beta methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-beta is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.

The Coactivator p/CIP/SRC-3 Facilitates Retinoic Acid Receptor Signaling via Recruitment of GCN5

p/CIP/SRC-3 is a member of a family of steroid receptor coactivators/nuclear receptor coactivators (SRC/NCoA) proteins that mediate the transcriptional effects of nuclear hormone receptors (NRs). Using deletion analysis we have mapped the location of two distinct activation domains in p/CIP (AD1 and AD2) capable of activating transcription in mammalian cells when fused to the Gal4-DNA binding domain. In addition to AD1 being coincident with the interaction domain for CBP, we demonstrate a novel in vivo interaction between the AD1 and GCN5. Overexpression of a Gal4-AD1 fusion protein in yeast leads to growth arrest that is relieved by mutation of genes encoding components of the SAGA complex including GCN5, ADA3, and SPT7. In addition, the AD1 of p/CIP and the ADA3 gene are shown to be essential for retinoic acid receptor alpha-dependent transcription in yeast. Transient transfection assays in mammalian cells indicate that GCN5 cooperates with p/CIP as a coactivator of RAR alpha-dependent transcription. Down-regulation of GCN5 using small interfering RNA in mammalian cells indicates that the AD1 domain and the RAR beta promoter activity are dependent, in part, on GCN5. Mutational analysis of AD1 has identified two helical motifs that are required for interactions with GCN5 and CBP. Taken together, these results support a model by which p/CIP functions as a ligand-dependent adapter, through specific protein-protein interactions with AD1, to recruit members from at least two distinct families of acetyltransferase proteins to NRs.

Response to histone deacetylase inhibition of novel PML/RARα mutants detected in retinoic acid–resistant APL cells

Resistance to all-trans retinoic acid (ATRA) remains a clinical problem in the treatment of acute promyelocytic leukemia (APL) and provides a model for the development of novel therapies. Molecular alterations in the ligand-binding domain (LBD) of the PML/RARalpha fusion gene that characterizes APL constitute one mechanism of acquired resistance to ATRA. We identified missense mutations in PML/RARalpha from an additional ATRA-resistant patient at relapse and in a novel ATRA-resistant cell line, NB4-MRA1. These cause altered binding to ligand and transcriptional coregulators, leading to a dominant-negative block of transcription. These mutations are in regions of the LBD that appear to be mutational hot spots occurring repeatedly in ATRA-resistant APL patient cells. We evaluated whether histone deacetylase (HDAC) inhibition could overcome the effects of these mutations on ATRA-induced gene expression. Cotreatment with ATRA and TSA restored RARbeta gene expression in NB4-MRA1 cells, whose PML/RARalpha mutation is in helix 12 of the LBD, but not in an APL cell line harboring the patient-derived PML/RARalpha mutation, which was between helix 5 and 6. Furthermore, ATRA combined with TSA increases histone 4 acetylation on the RARbeta promoter only in NB4-MRA1 cells. Consistent with these results, the combined treatment induces differentiation of NB4-MRA1 only. Thus, the ability of an HDAC inhibitor to restore ATRA sensitivity in resistant cells may depend on their specific molecular defects. The variety of PML/RARalpha mutations arising in ATRA-resistant patients begins to explain how APL patients in relapse may differ in response to transcription therapy with HDAC inhibitors.

Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas.

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma. CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test). However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma.

Nicotine modulates the effects of retinoids on growth inhibition and RAR beta expression in lung cancer cells.

Epidemiological and animal studies have demonstrated that vitamin A and its natural and synthetic derivatives, retinoids, are effective agents in preventing the development of tobacco-associated cancers. Unfortunately, clinical trials of retinoids on cigarette smokers have shown lack of efficacy in preventing lung cancer. In our study, we investigated the effect of nicotine on the anti-cancer activity of all trans-retinoic acid (trans-RA) in human lung cancer cells. Our results demonstrated that nicotine could abrogate the growth inhibitory effect of trans-RA by suppressing its ability to induce the expression of RA receptor beta (RAR beta), a tumor suppressor. The inhibitory effect of nicotine was accompanied with induction of orphan receptor TR3. Inhibition of TR3 expression by overexpression of TR3 anti-sense RNA in H460 lung cancer cells strongly prevented the suppressive effect of nicotine on trans-RA activity. Treatment with nicotine or the cotransfection of TR3 expression vector inhibited the induction of RAR beta promoter activity by trans-RA in transient transfection assays. The inhibition of RAR beta promoter activity was due to the interaction of TR3 with orphan receptor COUP-TF, resulting in inhibition of COUP-TF DNA binding and transactivation on the RAR beta promoter. Furthermore, we found that nicotine failed to suppress the effect of a retinoid X receptor (RXR)-selective retinoid SR11237 on inducing both growth inhibition and RAR beta promoter activity, due to the ability of SR11237 to activate the RAR beta promoter through the RXR/TR3 heterodimer. Together, our results demonstrate that nicotine suppresses the growth inhibitory effects of trans-RA by inhibiting RAR beta expression through its induction of TR3 expression and suggest that RXR-selective retinoids may be more effective than classical retinoids for preventing and treating tobacco-associated cancers.

Phosphorylation of histone H3 is functionally linked to retinoic acid receptor beta promoter activation.

Ligand-dependent transcriptional activation of retinoic acid receptors (RARs) is a multistep process culminating in the formation of a multimeric co-activator complex on regulated promoters. Several co-activator complexes harbor an acetyl transferase activity, which is required for retinoid-induced transcription of reporter genes. Using murine P19 embryonal carcinoma cells, we examined the relationship between histone post-translational modifications and activation of the endogenous RARbeta2 promoter, which is under the control of a canonical retinoic acid response element and rapidly induced upon retinoid treatment. While histones H3 and H4 were constitutively acetylated at this promoter, retinoid agonists induced a rapid phosphorylation at Ser10 of histone H3. A retinoid antagonist, whose activity was independent of co-repressor binding to RAR, could oppose this agonist-induced H3 phosphorylation. Since such post-translational modifications were not observed at several other promoters, we conclude that histone H3 phosphorylation may be a molecular signature of the activated, retinoid-controlled mRARbeta2 gene promoter.

Role of coactivators and corepressors in the induction of the RARbeta gene in human colon cancer cells.

We previously reported that the retinoic acid (RA) insensitivity of RARbeta induction is a general feature of human colon cancer cells (Biochem. Pharmacol., 59: 485-496, 2000). In the present investigation, we analyzed potential transcriptional defects associated with the expression of the RARbeta gene in colon cancer cells. Transfection of reporter constructs containing the RARbeta gene promoter as well as truncated fragments of the promoter showed a significant induction of reporter activity by RA treatment in RA-sensitive HCT-15 cells, but not in RA-resistant DLD-1 cells. The results suggest that the transcriptional defect of RARbeta expression may not be due to the presence of a specific cis-element in the RARbeta promoter. Next we examined whether coactivators and core-pressors of nuclear receptors were involved in the RA sensitivity of colon cancer cells. Transfection of coactivators such as CREB binding protein (CBP) and p300 up-regulated the RA-responsive element present in the RARbeta promoter (betaRARE) in DLD-1 cells up to the level in HCT-15, while coexpression of the nuclear receptor corepressor (NCoR) suppressed the betaRARE activity in HCT-15 cells. The expression level of CBP protein was consistently higher in HCT-15, while that of NCoR and Sin3A was higher in DLD-1 cells. Treatment with the histone deacetvlase inhibitor trichostatin A (TSA) increased both basal and RA-induced betaRARE activity in DLD-1, indicating that histone deacetylase is involved in the regulation of RARbeta gene expression. Taken together, our results show that differential function of coactivators and corepressors may determine the level of RARbeta induction that may mediate retinoid action in colon cancer cells.

Nerve growth factor activates the RARbeta2 promoter by a Ras-dependent mechanism.

Nerve growth factor (NGF) and retinoic acid (RA) exert important actions on PC12 cells. We have previously shown that incubation with NGF induces retinoic acid receptor beta (RARbeta) binding to a hormone response element in PC12 cells. In this study we show that NGF increases RARbeta protein levels by enhancing basal RARbeta2 promoter activity, and potentiates stimulation by RA in transient transfection assays. The effect of RA is mediated by a RA response element (RARE) located at -37/-53 and mutation of this element abolishes activation by the retinoid, as well as cooperation with NGF. However, the action of NGF is independent of the RARE and is mediated by sequences overlapping the TATA box and the INR comprising nucleotides -59 to +14. NGF produces a strong decrease in some of the complexes that bind to the INR. These results suggest that the RARbeta2 gene could be in a basal repressed state and NGF could increase RARbeta2 transcription by inducing the release of some inhibitory factors from the INR. Functional Ras is required for RARbeta2 promoter activation by NGF because expression of oncogenic Ras increases promoter activity and a dominant inhibitory Ras mutant blocks the effect of NGF. Oncogenic Raf also mimics the effect of NGF on the promoter. Other ligands of tyrosine kinase receptors that stimulate Ras also cause RARbeta2 promoter activation and act cooperatively with RA. These results indicate the existence of cross-coupling of the Ras-Raf signal transduction pathway with retinoid receptor pathways which could increase sensitivity to RA and be important for PC12 cell function.

Constitutive Activation of Retinoic Acid Receptor β2 Promoter by Orphan Nuclear Receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.

Evidence of epigenetic changes affecting the chromatin state of the retinoic acid receptor beta2 promoter in breast cancer cells.

Retinoic acid (RA)-resistance in breast cancer cells has been associated with irreversible loss of retinoic acid receptor beta, RARbeta, gene expression. Search of the causes affecting RARbeta gene activity has been oriented at identifying possible differences either at the level of one of the RARbeta promoters, RARbeta2, or at regulatory factors. We hypothesized that loss of RARbeta2 activity occurs as a result of multiple factors, including epigenetic modifications, which can pattern RARbeta2 chromatin state. Using methylation-specific PCR, we found hypermethylation at RARbeta2 in a significant proportion of both breast cancer cell lines and primary breast tumors. Treatment of cells with a methylated RARbeta2 promoter, by means of the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR), led to demethylation within RARbeta2 and expression of RARbeta indicating that DNA methylation is at least one factor, contributing to RARbeta inactivity. However, identically methylated promoters can differentially respond to RA, suggesting that RARbeta2 activity may be associated to different repressive chromatin states. This supposition is supported by the finding that the more stable repressive RARbeta2 state in the RA-resistant MDA-MB-231 cell line can be alleviated by the HDAC inhibitor, trichostatin A (TSA), with restoration of RA-induced RARbeta transcription. Thus, chromatin-remodeling drugs might provide a strategy to restore RARbeta activity, and help to overcome the hurdle of RA-resistance in breast cancer.

Synergistic effects of 8-Cl-cAMP and retinoic acids in the inhibition of growth and induction of apoptosis in ovarian cancer cells: induction of retinoic acid receptor beta.

Both cAMP and retinoids play a role in cell differentiation and the control of cell growth. A site-selective cAMP analog, 8-Cl-cAMP and retinoic acid synergistically inhibit growth and induce apoptosis in certain cancer cells. In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective even at doses that are toxic to the host. The objective of our present study was to examine the mechanism(s) of synergistic effects of retinoic acid (9-cis, 13-cis or all-trans RA) and 8-Cl-cAMP on apoptosis in human ovarian cancer NIH: OVCAR-3 and OVCAR-8 cells. RA induced growth inhibition and apoptosis in OVCAR-3 and OVCAR-8 cells. 8-Cl-cAMP acted synergistically with RA in inducing and activating retinoic acid receptor beta (RARbeta) which correlates with growth inhibition and apoptosis in both cell types. In addition, induction of apoptosis by RA plus 8-Cl-cAMP requires caspase-3 activation followed by cleavage of anti-poly(ADP-ribose) polymerase. Furthermore, mutations in CRE-related motif within the RARbeta promoter resulted in loss of both transcriptional activation of RARbeta and synergy between RA and 8-Cl-cAMP. RARbeta expression appears to be associated with induction of apoptosis. Introduction of the RARbeta gene into OVCAR-3 cells resulted in gain of RA sensitivity. Loss of RARbeta expression, therefore, may contribute to the tumorigenicity of human ovarian cancer cells. Thus, combined treatment with RA and 8-Cl-cAMP may provide an effective means for inducing RARbeta expression leading to apoptosis in ovarian cancer cells.