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Background: Hand eczema is the most common occupational skin disease. The etiology is multifactorial. Systemic alitretinoin, a pan-retinoic receptor agonist, has proven efficacy in the treatment of recalcitrant chronic hand eczema; however, its precise mechanism of action in hand eczema is not fully understood., Aims: Assessment of the level of expression of retinoid receptors (RAR and RXR) in the skin of patients with hand eczema in an attempt to explain their possible role in the pathogenesis of the disease., Methods: Thirty patients with hand eczema and 30 age- and sex-matched healthy controls were included. Full clinical examination was done, and tissue levels of retinoic acid receptor (RAR) and retinoid x receptor (RXR) were measured by quantitative real-time PCR (qRT-PCR)., Results: The levels of RAR and RXR expression were significantly downregulated in the patient group compared to the control group; (P < 0.001) for both. In addition, there was a statistically significant negative correlation between Osnabrück Hand Eczema Severity Index (OHSI) and the levels of RAR and RXR expression (P < 0.001)., Conclusion: Deficient retinoid receptor expression has a primary role in the pathogenesis, clinical phenotype, and severity of hand eczema and sheds light on the mechanism of action of retinoids in the treatment of chronic hand eczema., (© 2020 The International Society of Dermatology.)

Of numerous genes regulated by retinoic acid (RA), CYP26A1 is the most inducible gene by RA. In this study, we have used a shortened construct form, E4, of the CYP26A1 gene promoter, in a promoter-less vector with either luciferase or red fluorescent protein (RFP) as the reporter gene and have tested its responses to retinoids in transfected HepG2 and HEK293T cells. The promoter responded linearly to a wide concentration range of RA in cells cotransfected with retinoic acid receptors. It also responded quantitatively to retinol and other retinoids. An isolated clonal line of HEK293T cells permanently transfected with the promoter driving the expression of RFP responded to both RA and retinol, and the responses could be measured by fluorescence microscopy and flow cytometry. The promoter was used to assess the retinoid activity of 3 novel synthetic retinoid analogues, as well as of the intact serum samples of rats. Among the synthetic retinoid analogues tested, EC23 is more potent than RA at lower concentrations and was more stable than RA. The retinoid activities could be measured in control rat serum samples and were increased in the serum of RA-treated rats. This system offers a biologically-based alternative to mass-based retinoid analysis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hypothyroidism has been linked to infertility, but the mechanisms underlying infertility-related hypothyroidism have yet to be fully elucidated. Therefore, in this study, effects of hypothyroidism on expression of the proteins related to thyroid hormone function in the uterus, which were thought to play a role implantation, including thyroid hormone receptor (TR), thyroid stimulating hormone receptor (TSHR), retinoic acid receptor (RAR) and extracellular kinase (ERK) were identified. Pregnant female rats were rendered hypothyroid by giving methimazole (MMI), orally. Following hypothyroid induction, rats were grouped into control (non-treated) and received subcutaneous thyroxine at 20, 40, and 80 μg/kg/day for five consecutive days. At Day 6, which is the day of implantation (GD 6), rats were sacrificed and the number of embryo implantation site in the uterus was calculated. Then, uterine horns were harvested and expression of the above proteins and their mRNAs were identified by Western blotting and real-time PCR, respectively. In non-treated hypothyroid pregnant rats, the number of embryo implantation sites decreased as compared to euthyroid and hypothyroid rats receiving thyroxine treatment. Similarly, expression of TRα-1, TRβ-1, TSHR, ERK1/2 and RAR proteins and mRNA in the uterus of non-treated hypothyroid rats also decreased (P < 0.05 when compared to euthyroid and thyroxine-treated hypothyroid rats). In conclusion, downregulated expression of the thyroid hormone related proteins in the uterus at the day of implantation might result in infertility as reported in hypothyroid condition., (© 2019 International Federation for Cell Biology.)

Scope: Vitamin A (VA) is an essential nutrient for the development of the brain. We previously found that children with autism spectrum disorder (ASD) have a significant rate of VA deficiency (VAD). In the current study, we aim to determine whether VAD is a risk factor for the generation of autistic-like behaviors via the transcription factor retinoic acid receptor beta (RARβ)-regulated cluster of differentiation 38 (CD38)-oxytocin (OXT) axis., Methods and Results: Gestational VAD or VA supplementation (VAS) rat models are established, and the autistic-like behaviors in the offspring rats are investigated. The different expression levels of RARβ and CD38 in hypothalamic tissue and serum retinol and OXT concentration are tested. Primary cultured rat hypothalamic neurons are treated with all-trans retinoic acid (atRA), and recombinant adenoviruses carrying the rat RARβ (AdRARβ) or RNA interference virus RARβ-siRNA (siRARβ) are used to infect neurons to change RARβ signal. Western blotting, chromatin immunoprecipitation (ChIP), and intracellular Ca 2+ detections are used to investigate the primary regulatory mechanism of RARβ in the CD38-OXT signaling pathway. We found that gestational VAD increases autistic-like behaviors and decreases the expression levels of hypothalamic RARβ and CD38 and serum OXT levels in the offspring. VAS ameliorates these autistic-like behaviors and increases the expression levels of RARβ, CD38, and OXT in the gestational VAD pups. In vitro, atRA increases the Ca 2+ excitability of neurons, which might further promote the release of OXT. Different CD38 levels are induced in the neurons by infection with different RARβ adenoviruses. Furthermore, atRA enhances the binding of RARβ to the proximal promoter of CD38, indicating a potential upregulation of CD38 transcriptional activity by RARβ., Conclusions: Gestational VAD might be a risk factor for autistic-like behaviors due to the RARβ signal suppression of CD38 expression in the hypothalamus of the offspring, which improves with VAS during the early-life period. The nutritional status during pregnancy and the early-life period is important in rats., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cellular retinoic acid-binding protein 2 (CRABP2) has been detected in several organs during embryonic development. Recent studies have demonstrated that CRABP2 plays important roles in the retinoic acid, β-catenin and Notch signaling pathways, as well as in the interaction between epithelial and mesenchymal cells, which are important for human dental pulp stem cells (hDPSCs) and tooth development. In the present study, the expression of CRABP2 during mouse molar development and the role of CRABP2 in hDPSC odontoblastic differentiation were evaluated. CRABP2 was gradually decreased during the development of the first maxillary molar, which exhibited the same trend as the expression of CRABP2 during the odontoblastic induction of hDPSCs. CRABP2 knockdown inhibited the proliferative ability of hDPSCs, while it enhanced odontoblastic differentiation via promoting mineralization nodule formation and upregulating the activity of alkaline phosphatase and the expression of mineralization-related genes. The present study uncovered a novel function of CRABP2 in hDPSCs. Our data suggest that CRABP2 may act as a regulator during the proliferation and differentiation of hDPSCs.

Retinoic acid receptor β (RARβ), a known tumor suppressor gene, is frequently silenced in numerous malignant types of tumor. Recent reports have demonstrated that loss of RARβ expression may be responsible, in part, for the drug resistance observed in clinical trials. However, little is known about the role of RARβ in regulating drug sensitivity in patients with cholangiocarcinoma (CCA) with a high risk of mortality and poor outcomes. In the present study, low RARβ expression was observed in the majority of CCA tissues investigated (28/33, 84.8%). In addition, the CCA cell line QBC939, which exhibits low RARβ expression, was found to be significantly resistant to chemotherapeutic agents compared with SK‑ChA‑1, MZ‑ChA‑1 and Hccc9810 CCA cell lines, which exhibit high RARβ expression. Furthermore, upregulation of RARβ significantly enhanced the sensitivity of QBC939 cells to common chemotherapeutic agents both in vitro and in vivo. Upregulation of RARβ was shown to increase the expression of proapoptotic genes bax, bak and bim, in addition to caspase‑3 activity, and decrease the expression of antiapoptotic genes bcl‑2, bcl‑xL and mcl‑1. As a result, CCA cells were more susceptible to caspase‑dependent apoptosis. Taken together, these data suggest that RARβ upregulation rendered CCA cells more sensitive to chemotherapeutic agents by increasing the susceptibility of cells to caspase-dependent apoptosis. These results support the hypothesis that RARβ may be an ideal chemosensitization target for the treatment of patients with drug-resistant CCA.

Gene promoter methylation may be used a potential biomarker for detecting solid tumor including cervical cancer. Here, we used methylation sensitive-high resolution melting (MS-HRM) analysis to detecting promoter methylation ratios of DAPK1, MGMT and RARB gene in patients with different cervical disease grade. The detection of gene promoter methylation was conducted in two hundred fifty patients' samples including normal cytology (n=48), cervical intraepithelial neoplasia grade 1 (CIN1, n=54), cervical intraepithelial neoplasia grade 2 (CIN2, n=47), cervical intraepithelial neoplasia grade 3 (CIN3, n=56) and cervical squamous cell carcinomas (SCS, n=45). We found there were a significant positive correlation between the promoter methylation status of DAPK1 and cervical disease grade (P=0.022). In addition, the methylated promoters of DAPK1 combined with MGMT, MGMT combined with RARB, DAPK1 combined with RARB were positive correlated with cervical disease grade (P < 0.05). All three genes promoters methylated were positive correlated with cervical disease grade (P < 0.001). Receiver operating characteristic (ROC) curves was conducted to evaluate whether the three genes methylation could be used to be a potential marker for diagnosing high grade cervical disease (HSIL and SCC). The cutoff values for the methylation rates of all these genes were 0-5%. Regrettably, only the methylation of MGMT combined with DAPK1 gave 43.4% sensitivity and 68.6% specificity. The current results indicated that MS-HRM-based testing for DNA methylations of MGMT plus DAPK1 genes holds some promise for high grade cervical disease screening.

Taking advantage of "Y" junction structure and restriction endonuclease assisted cyclic enzymatic amplification, a dual-probe electrochemical DNA (DE-DNA) biosensor was designed to detect double-stranded DNA (dsDNA) of acute promyelocytic leukemia (APL) related gene. Two groups of detection probes were designed, and each group was composed of a biotinylated capture probe and an assisted probe. They were separately complementary with two strands of target dsDNA in order to prevent the reannealing of the two separate strands from target dsDNA. First, thiol functionalized capture probes (C1 and C2) were severally assembled onto two different gold electrodes, followed by hybridizing with target dsDNA (S1a-S1b) and assistant probes to form two Y-junction-structure ternary complexes. Subsequently, restriction sites on the ternary complexes were digested by Rsa I, which can release S1a, S1b and biotins from the electrode surfaces. Meanwhile, the released S1a and S1b can further hybridize with the unhybridized corresponding detection probes and then initiate another new hybridization-cleavage-separation cycle. Finally, the current signals were produced by the enzyme-catalyzed reaction of streptavidin-horse reddish peroxidase (streptavidin-HRP). The distinct difference in current signals between different sequences allowed detection of target dsDNA down to a low detection limit of 47 fM and presented excellent specificity with discriminating only a single-base mismatched dsDNA sequence. Moreover, this biosensor was also used for assay of polymerase chain reaction (PCR) samples with satisfactory results. According to the results, the power of the DE-DNA biosensor as a promising tool for the detection of APL and other diseases., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. All-trans retinoic acid (ATRA) is the major physiologically active form of vitamin A, regulating expression of many genes. Disturbances of vitamin A metabolism are prevalent in some cancer cells. The main aim of this work was to investigate deeply the components of retinoid signaling in PDAC compared to in the normal pancreas and to prove the clinical importance of retinoid receptor expression. For the study, human tumor tissues obtained from PDAC patients and murine tumors from the orthotopic Panc02 model were used for the analysis of retinoids, using high performance liquid chromatography mass spectrometry and real-time RT-PCR gene expression analysis. Survival probabilities in univariate analysis were estimated using the Kaplan-Meier method and the Cox proportional hazards model was used for the multivariate analysis. In this work, we showed for the first time that the ATRA and all-trans retinol concentration is reduced in PDAC tissue compared to their normal counterparts. The expression of RARα and β as well as RXRα and β are down-regulated in PDAC tissue. This reduced expression of retinoid receptors correlates with the expression of some markers of differentiation and epithelial-to-mesenchymal transition as well as of cancer stem cell markers. Importantly, the expression of RARα and RXRβ is associated with better overall survival of PDAC patients. Thus, reduction of retinoids and their receptors is an important feature of PDAC and is associated with worse patient survival outcomes., (© 2014 Wiley Periodicals, Inc.)

Objective: The elevation of cellular retinoic acid-binding protein-I (CRABP-I) has been suggested as a candidate in the pathogenesis of paediatric moyamoya disease (MMD). However, few studies have addressed CRABP-I in adult onset MMD. The aim of this study was to examine the expression of CRABP-I in the cerebrospinal fluid (CSF) of adult onset MMD, and to evaluate its association with clinical presentation and postoperative haemodynamic change., Methods: This study examined the CSF from 103 patients: bilateral MMD, n=58 (56.3%); unilateral MMD, n=19 (18.4%); atherosclerotic cerebrovascular disease (ACVD), n=21 (20.4%); and control group, n=5 (4.9%). The intensity of CRABP-I was confirmed by western blotting and expressed as the median (25th-75th percentile). The differences in CRABP-I expression according to disease entity (unilateral MMD vs bilateral MMD vs ACVD), initial presenting symptoms (haemorrhage vs ischaemia) and postoperative haemodynamic change (vascular reserve in single photon emission CT and basal collateral vessels in digital subtraction angiography) were analysed., Results: CRABP-I intensities in bilateral MMD (1.45(0.86-2.52)) were significantly higher than in unilateral MMD (0.91(0.78-1.20)) (p=0.044) or ACVD (0.85(0.66-1.11)) (p=0.004). No significant differences were noted based on the initial presenting symptoms (p=0.687). CRABP-I was not associated with improvement in vascular reserve (p=0.327), but with decrease in basal collateral vessels (p=0.023) postoperatively., Conclusions: Higher CRABP-I in the CSF can be associated with typical bilateral MMD pathogenesis in adults. Additionally, postoperative basal collateral change may be related to the degree of CRABP-I expression., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

CRABP-II, a retinoic acid binding protein, shuffles retinoic acid from cytoplasm into nucleus and forms a complex with nuclear retinoic acid receptor to facilitate transcriptional activities of retinoic acid. In this study, we studied the expression patterns of CRABP-II in pancreatic ductal adenocarcinoma (PDAC) compared with those in normal pancreas, chronic pancreatitis, and precancerous lesions. We showed no detectable expressions of CRABP-II in normal pancreatic parenchyma, normal ductal epithelium, and chronic pancreatitis. In contrast, the expression of CRABP-II was readily detected in all PDACs including metastatic PDACs. CRABP-II staining was also observed and progressively increased from pancreatic intraepithelial neoplasia 1 to 3. In addition, when fine needle aspiration specimens were evaluated from patients with PDAC, CRABP-II was positive in 55.6% cases if cytology diagnosis was "atypia," and in 87.5% cases, if "malignancy." Our study suggests that CRABP-II is highly and specifically expressed in PDAC and is more commonly expressed in high-grade precursor cancerous lesions than in low-grade lesions. Therefore, overexpression of CRABP-II is a late event of pancreatic carcinogenesis, and it could be used as a diagnostic marker to distinguish PDAC from other benign pancreatic conditions in both resection and cytology specimens., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Transcriptional activity of the retinoic acid receptor (RAR) is regulated by diverse binding partners, including classical corepressors and coactivators, in response to its ligand retinoic acid (RA). Recently, we identified a novel corepressor of RAR called the retinoic acid resistance factor (RaRF) (manuscript submitted). Here, we report how adenovirus E1A stimulates RAR activity by associating with RaRF. Based on immunoprecipitation (IP) assays, E1A interacts with RaRF through the conserved region 2 (CR2), which is also responsible for pRb binding. The first coiled-coil domain of RaRF was sufficient for this interaction. An in vitro glutathione-S-transferase (GST) pull-down assay was used to confirm the direct interaction between E1A and RaRF. Further fluorescence microscopy indicated that E1A and RaRF were located in the nucleoplasm and nucleolus, respectively. However, RaRF overexpression promoted nucleolar translocation of E1A from the nucleoplasm. Both the RA-dependent interaction of RAR with RaRF and RAR translocation to the nucleolus were disrupted by E1A. RaRF-mediated RAR repression was impaired by wild-type E1A, but not by the RaRF binding-defective E1A mutant. Taken together, our data suggest that E1A is sequestered to the nucleolus by RaRF through a specific interaction, thereby leaving RAR in the nucleoplasm for transcriptional activation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The development of the most common multidrug resistance (MDR) phenotype is associated with a massive overexpression of P-glycoprotein (P-gp) in neoplastic cells. In the current study, we used three L1210 cell variants: S cells - parental drug-sensitive cells; R cells - drug-resistant cells with P-gp overexpression due to selection with vincristine; T cells - drug-resistant cells with P-gp overexpression due to stable transfection with the pHaMDRwt plasmid, which encodes human full-length P-gp. Several authors have described the induction of P-gp expression/activity in malignant cell lines after treatment with all-trans retinoic acid (AtRA; ligand of retinoic acid nuclear receptors, RARs). An isomer of AtRA also exists, 9-cis retinoic acid, which is a ligand of both RARs and nuclear retinoid X receptors (RXRs). In a previous work, we described that the combined treatment of R cells with verapamil and AtRA induces the downregulation of P-gp expression/activity. In the current study, we studied the expression of RARs and RXRs in S, R and T cells and the effects of treatment with AtRA, 9cRA and verapamil on P-gp expression, cellular localization and efflux activity in R and T cells. We found that the overexpression of P-gp in L1210 cells is associated with several changes in the specific transcription of both subgroups of nuclear receptors, RARs and RXRs. We also demonstrated that treatment with AtRA, 9cRA and verapamil induces alterations in P-gp expression in R and T cells. Particularly, combined treatment of R cells with verapamil and AtRA induced downregulation of P-gp content/activity. In contrast, similar treatment of T cells induced slight increase of P-gp content without any changes in efflux activity of this protein. These findings indicate that active crosstalk between the RAR and RXR regulatory pathways and P-gp-mediated MDR could take place.

Background: Diagnosis of ovarian cancer is often delayed because of subtle symptoms and a lack of a specific, sensitive test useful for the general population. The majority of cases are diagnosed at late stages, after the tumor has metastasized and implanted on many other abdominal organs and cavity surfaces. A paucity of prognostic markers makes it difficult to define which tumors will act aggressively and shorten survival. Hence, it is imperative to have new screening tests for diagnosis of ovarian cancer at earlier stages, prior to metastatic progression. Diagnosis at these early stages will dramatically increase the overall survival of women with ovarian cancer., Material and Methods: Based on previously published literature on proposed molecular cell markers in ovarian carcinoma, we sought to validate the overexpression of two genes (cellular retinoic acid Binding Protein, CRABP-1, and spondin 1) through immunohistochemistry., Results: We verified the overexpression of spondin 1 in ovarian cancer. Expression of cellular retinoic acid Binding Protein, CRABP-1 in whole ovarian cancer tissue sections was higher than in the TMA tissue cores., Conclusion: Our results thus demonstrate that spondin 1 is a useful marker for ovarian cancer; additionally, the high percentages of tumors that are positive for spondin 1 make it an ideal target for therapy. CRABP-1 was not expressed at high levels in any subtype of ovarian cancer, making it a poor marker.

Background: Recurrence of craniopharyngiomas (CPs) represents a frequent and unpredictable incident. Rapid tumor recurrence within a few months after surgery has been rarely reported. Nondefinitive predictive factors for rapid CP recurrence have been identified to date. We have systematically analyzed the tumor factors that presumably influence in CP recurrence., Methods: We present the histologic and immunohistochemical analysis of an infundibulo-tuberal CP that was operated twice within a 3-month interval due to rapid recurrence. We investigated the differential characteristics of the subgroup of CPs that recurred in large surgical series published in the literature, along with cases reporting rapid tumor recurrence after surgery., Results: Specimens of our patient showed an adamantinomatous CP with whorl-like arrays and thick peritumoral gliosis. Ki-67 labeling index in primary and recurrent samples was 20% and 15%, respectively. p53 labeling index was 18% and 15%, respectively. The thorough analysis of literature showed that presence of tumor remnants and missing radiotherapy treatment after subtotal removal are strong predictors of tumor recurrence. Third ventricle involvement, large tumor size, tight adherence to surrounding structures, and presence of whorl-like arrays might also foster recurrence. High Ki-67 levels, p53 expression, and an intense reactive gliosis might point to rapid tumor growth., Conclusions: Reliable tumor markers that predict CP recurrence are still lacking. The CP features presumably related to a higher risk of its recurrence are thought to be a larger tumor size, a tight adherence to the hypothalamus, the presence of whorl-like arrays, and high Ki-67 and p53 levels., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Retinoic acid is an important regulator of cellular proliferation and differentiation. The action of retinoic acid is mediated by retinoic acid receptors (RARs) and the retinoid X receptors. The objective was to elucidate the protein localization and expression of RARα, RARβ, and RARγ in bull and dog sperm. Bull and dog sperm were subjected to an immunostaining procedure to determine presence of RARα, RARβ, and RARγ. We concluded that all three receptors were present in different regions of bull and dog sperm at varying levels. Protein expression in bull and dog sperm lysates was investigated using protein dot-blot analyses. The protein levels of RARα and RARγ were higher than the protein level of RARβ in bull and dog sperm. Protein sequences of RARα, RARβ, and RARγ for bull and dog were 98%, 89%, and 98%, respectively, on similarity alignment. In conclusion, the presence of RARα, RARβ, and RARγ receptors supported their role in sperm structure and function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A new affinity chromatography method was developed by modifying a zonal elution method. The new method targets transient protein-protein interactions, such as those encountered during direct ligand transfer between the ligand transporter and its cognate receptor. A ligand-loaded transport protein is immobilized on the solid support, and a plug containing a putative receptor is flowed through the column. Elution profiles of proteins not interacting with the immobilized transporter can be approximated with a simple Gaussian curve, while the elution profiles of cognate receptors show significant delay and exhibit complex shape. Ligand transfer from the immobilized transporter molecules to the receptors is verified by both UV absorbance measurements and mass spectrometry. The sensitivity of the method is demonstrated using retinoic acid (RA) transfer from various isoforms of cellular RA binding proteins (CRABPs) and RA receptor γ (RARγ). Although these interactions have been hypothesized long ago to proceed via direct mechanism (i.e., via transient docking of the receptor and the transporter), the existing biophysical techniques failed to detect the presence of the transporter-receptor complexes. However, the modified zonal elution method provides unequivocal evidence of direct interaction between RARγ and one of the CRABP isoforms (CRABP II) during the ligand transfer to the receptor.

Breast cancer is the second leading cause of cancer death for women in the United States. Of the different subtypes, estrogen receptor-negative (ER(-)) tumors, which are ErbB2+ or triple-negative, carry a relatively poor prognosis. In this study, we used system-wide analysis of breast cancer proteomes to identify proteins that are associated with the progression of ER(-) tumors. Our two-step approach included an initial deep analysis of cultured cells that were obtained from tumors of defined breast cancer stages, followed by a validation set using human breast tumors. Using high-resolution mass spectrometry and quantification by Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), we identified 8,750 proteins and quantified 7,800 of them. A stage-specific signature was extracted and validated by mass spectrometry and immunohistochemistry on tissue microarrays. Overall, the proteomics signature reflected both a global loss of tissue architecture and a number of metabolic changes in the transformed cells. Proteomic analysis also identified high levels of IDH2 and CRABP2 and low levels of SEC14L2 to be prognostic markers for overall breast cancer survival. Together, our findings suggest that global proteomic analysis provides information about the protein changes specific to ER(-) breast tumor progression as well as important prognostic information., (©2012 AACR)

This project involved the collection of final effluent samples from 39 wastewater-treatment plants (WWTPs) in Victoria, Australia, in late summer (late February to early March 2007). The 39 WWTPs included 15 lagoon-based plants and 24 with activated sludge-based processes. Samples were collected and subjected to measurement of retinoic acid receptor (RAR) and aryl hydrocarbon receptor (AhR) activity of the dissolved phase using yeast-based recombinant receptor-reporter gene bioassays. More than 90% of the effluents examined in this study elicited RAR activity (<0.5-198 ng/l a-t-RA equivalents [EQ]). All of the effluents had AhR activity (16-279 ng/l βNF EQ). Notwithstanding the paucity of comparative data, on the whole, the levels of RAR and AhR activity observed in this pilot survey of Victorian WWTP effluents were greater than those recently reported internationally. One assumption commonly made is that WWTP discharges will be diluted significantly in the receiving environment, further decreasing the potential risk of the discharges. Making this assumption may not be appropriate for some of Victoria's more ephemeral waterways or where effluent is discharged to an enclosed water body, such as a lake or terminal wetland. However, even where WWTP discharges represent all of the environmental flow in the warmer months, the observed RAR and AhR activity (as all-trans-retinoic acid (RA) and 2,3,7,8-tetrachloro-dibenzo-p-dioxin [TCDD] EQ, respectively) was still significantly lower than the concentrations of RA, and 2,3,7,8-TCCD known to cause developmental malformations in fish larvae after short-term exposure to these chemicals. Of perhaps greater concern, WWTP effluent can contain significant suspended solids (essentially biosolids), which may be a considerable sink for some hormonally active, hydrophobic compounds, and which may in turn increase the long-term exposure risk for aquatic fauna. Further studies of the nuclear and AhR activity of WWTP effluent suspended soilds are required to address this hypothesis.

Purpose: CRABP1 is a modulator of retinoic acid function. The aim of the present study was to investigate CRABP1 expression and its clinical significance in ovarian carcinoma., Methods: Expression of CRABP1 protein was investigated by immunohistochemical analysis in 100 ovarian carcinomas of various histological sub-types, including serous and clear cell adenocarcinomas. Relationship of CRABP1 expression to clinical features, including prognosis, was analyzed., Results: Reduced expression of CRABP1 protein was detected especially frequently in the serous and clear cell adenocarcinomas sub-types, 50% (20 of 40) and 38% (10 of 26) of cases, respectively. We found that in both serous and clear cell adenocarcinomas overall survival was significantly poorer in the cases with reduced CRABP1 expression compared to similar cases where expression was maintained, irrespective of the disease stage (P = 0.0073 and 0.049, respectively). Disease-free survival of the serous and clear cell adenocarcinoma cases with reduced CRABP1 expression was significantly poorer, compared to the cases whose CRABP1 expression was maintained (P = 0.024). Multivariate analysis showed that reduced expression of CRABP1 was a significantly important prognostic factor (adjusted hazard ratio: 8.189 (95% CI, 2.186-30.672, P = 0.0019))., Conclusions: The present study is the first to demonstrate that the reduced expression of CRABP1 has a potential as a prognostic marker for serous adenocarcinoma which is the most frequent histological ovarian tumor type and also for clear cell carcinoma that often exhibits chemo-resistance. Further study is necessary to clarify how CRABP1 protein expression was altered and how CRABP1 affects ovarian carcinoma cells.

Purpose: This randomized phase II trial evaluated whether the combination of cisplatin and paclitaxel (PC) plus all-trans retinoic acid (ATRA) increases response rate (RR) and progression-free survival (PFS) in patients with advanced non-small-cell lung cancer (NSCLC) with an acceptable toxicity profile and its association with the expression of retinoic acid receptor beta 2 (RAR-beta2) as a response biomarker., Patients and Methods: Patients with stages IIIB with pleural effusion and IV NSCLC were included to receive PC, and randomly assigned to receive ATRA 20 mg/m(2)/d (RA/PC) or placebo (P/PC) 1 week before treatment until two cycles were completed. RAR-beta2 expression was analyzed in tumor and adjacent lung tissue., Results: One hundred seven patients were included, 55 in the P/PC group and 52 in the RA/PC group. RR for RA/PC was 55.8% (95% CI, 46.6% to 64.9%) and for P/PC, 25.4% (95% CI, 21.3 to 29.5%; P = .001). The RA/PC group had a longer median PFS (8.9 v 6.0 months; P = .008). Multivariate analysis of PFS showed significant differences for the RA/PC group (hazard ratio, 0.62; 95% CI, 0.4 to 0.95). No significant differences in toxicity grade 3/4 were found between groups, except for hypertriglyceridemia (10% v 0%) in RA/PC (P = .05). Immunohistochemistry and reverse-transcriptase polymerase chain reaction assays showed expression of RAR-beta2 in normal tissues of all tumor samples, but only 10% of samples in the tumor tissue., Conclusion: Adding ATRA to chemotherapy could increase RR and PFS in patients with advanced NSCLC with an acceptable toxicity profile. A phase III clinical trial is warranted to confirm these findings.

Two primary prevention trials unexpectedly showed adverse effects of supplemental beta-carotene on lung cancer incidence in cigarette smokers. To elucidate the molecular mechanisms that might underlie these effects, we studied the immunohistochemical expression of cytochrome P450 1A1, 1A2, and 2E1, retinoic acid receptor beta, activated protein-1 elements, cyclin D1, and Ki67 in lung tumors and, when available, adjacent normal tissues obtained from incident cases in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study. Archival lung tissue was available from 52 men randomized to receive 20 mg of beta-carotene per day and 30 men randomized to the placebo arm, all of whom were diagnosed with incident non-small-cell lung carcinoma during the course of the trial and subsequently underwent radical pulmonary resection. In normal-appearing bronchial epithelium, positive staining for cyclin D1 was observed in 23% of cases in the beta-carotene group and 0% of cases in the placebo group (based on only 3 of 13 versus 0 of 11 cases staining positively, however; P = 0.04), with no differences in expression noted in lung tumor tissue (P = 0.48). There were no statistically significant differences in Ki67 expression in normal or cancerous lung tissue between intervention groups, although a small increase in staining in tumors was noted among cases in the beta-carotene versus placebo group (88% versus 71% of cases stained positive, respectively; P = 0.13). Contrary to expectation, beta-carotene supplementation had no apparent effect on retinoic acid receptor-beta expression. These findings suggest that male smokers supplemented with beta-carotene may have had an increased risk of lung cancer due to aberrant cell growth, although our results are based on a relatively small number of cases and require confirmation in other completed trials of beta-carotene supplementation., (2010 AACR.)

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR., (2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.)

Background: Hepatic progenitor cells (HPCs) can be isolated from fetal liver and extrahepatic tissues. Retinoic acid (RA) signalling plays an important role in development, although the role of RA signalling in liver-specific progenitors is poorly understood., Aims: We sought to determine the role of RA in regulating hepatic differentiation., Methods: RNA was isolated from liver tissues of various developmental stages. Liver marker expression was assessed by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Reversibly immortalized HPCs derived from mouse embryonic day 14.5 (E14.5) liver (aka, HP14.5) were established. Albumin promoter-driven reporter (Alb-GLuc) was used to monitor hepatic differentiation. Glycogen synthesis was assayed as a marker for terminal hepatic differentiation., Results: Retinoic acid receptor (RAR)-alpha, retinoid X receptor (RXR)-alpha and RXR-gamma expressed in E12.5 to postnatal day 28 liver samples. Expression of RAR-beta and RXR-beta was low perinatally, whereas RAR-gamma was undetectable in prenatal tissues and increased postnatally. Retinal dehydrogenase 1 and 2 (Raldh1 and Raldh2) were expressed in all tissues, while Raldh3 was weakly expressed in prenatal samples but was readily detected postnatally. Nuclear receptor corepressors were highly expressed in all tissues, while expression of nuclear co-activators decreased in perinatal tissues and increased after birth. HP14.5 cells expressed high levels of early liver stem cell markers. Expression of RA signalling components and coregulators was readily detected in HP14.5. RA was shown to induce Alb-GLuc activity and late hepatocyte markers. RA was further shown to induce glycogen synthesis in HP14.5 cells, an important function of mature hepatocytes., Conclusions: Our results strongly suggest that RA signalling may play an important role in regulating hepatic differentiation.

Specific genetic alterations in multiple myeloma (MM) may cause more aggressive diseases. Paired gene array analysis on 51 samples showed that retinoic acid (RA) receptor alpha (RARalpha) expression significantly increased at relapse compared with diagnosis. RARalpha encodes 2 major isoforms: RARalpha1 and RARalpha2. In this study, we examined the function of RARalpha2 in MM. Reverse transcription-polymerase chain reaction (RT-PCR) revealed ubiquitous RARalpha1 expression in MM cells, but RARalpha2 was expressed in 26 (32%) of 80 newly diagnosed patients and 10 (28%) of 36 MM cell lines. Patients with RARalpha2 expression had a significantly shorter overall survival on identical treatments. The presence of RARalpha2 remained significant on multivariate analysis. Knockdown of RARalpha2 but not RARalpha1 induced significant MM cell death and growth inhibition, and overexpressing RARalpha2 activated STAT3 and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. Interestingly, all-trans retinoic acid (ATRA) treatment induced potent cell death and growth inhibition in RARalpha2(+) but not RARalpha2(-) MM cells; overexpressing RARalpha2 in RARalpha2-deficient MM cells restored sensitivity to ATRA. Furthermore, ATRA treatment significantly inhibited the growth of RARalpha2-overexpressing MM tumors in severe combined immunodeficiency (SCID) mouse model. These findings provide a rationale for RA-based therapy in aggressive RARalpha2(+) MM.

In early HIV-1 infection, Vdelta1 T lymphocytes are increased in peripheral blood and this is related to chemokine receptor expression, chemokine response, and recirculation. Herein we show that, at variance with healthy donors, in HIV-1-infected patients ex vivo-isolated Vdelta1 T cells display cytoplasmic interferon-gamma (IFN-gamma). Interestingly, these cells coexpress cytoplasmic interleukin-17 (IL-17), and bear the CD27 surface marker of the memory T-cell subset. Vdelta1 T cells, isolated from either patients or healthy donors, can proliferate and produce IFN-gamma and IL-17 in response to Candida albicans in vitro, whereas Vdelta2 T cells respond with proliferation and IFN-gamma/IL-17 production to mycobacterial or phosphate antigens. These IFN-gamma/IL-17 double-producer gammadelta T cells express the Th17 RORC and the Th1 TXB21 transcription factors and bear the CCR7 homing receptor and the CD161 molecule that are involved in gammadelta T-cell transendothelial migration. Moreover, Vdelta1 T cells responding to C albicans express the chemokine receptors CCR4 and CCR6. This specifically equipped circulating memory gammadelta T-cell population might play an important role in the control of HIV-1 spreading and in the defense against opportunistic infections, possibly contributing to compensate for the impairment of CD4(+) T cells.

Aim: T regulatory (Treg) cells have been detected in periodontitis lesions, and forkhead box P3 (Foxp3) expression has been negatively correlated to receptor activator of nuclear factor-kappa B ligand (RANKL). The aim of this study was to correlate T-helper type 1 (Th1), Th2, Th17 and Treg transcription factor expressions, in gingival tissues from patients undergoing active periodontal tissue destruction, with bone loss-associated cytokines., Materials and Methods: In 10 chronic periodontitis patients undergoing disease progression, the mRNA expressions of T-bet, GATA-3, Foxp3, RORC2, interleukin (IL)-1beta, IL-10, IL-17, RANKL, interferon (IFN)-gamma and transforming growth factor (TGF)-beta1 were quantified using real-time reverse transcription-polymerase chain reaction. The levels of these markers were compared between active and inactive periodontal lesions., Results: In active periodontal lesions, Foxp3, T-bet, RANKL, IL-17, IL-1beta and IFN-gamma were significantly over-expressed compared with inactive lesions. The expression of IFN-gamma was the highest within the active periodontal lesions, similar to that of TGF-beta1 within the inactive ones. There was a positive correlation between RANKL and IL-17. Additionally, RANKL and IL-17 were positively correlated with RORC2, but no correlation was detected with Foxp3., Conclusions: These results lead us to speculate that Foxp3(+) cells that do not have a regulatory function might have a role in the pathogenesis of active periodontal lesions by down-regulating TGF-beta1 and IL-10 synthesis that lead to the over-expression of Th17-associated cytokines RANKL and IL-17.

A total of 16 water samples from four rivers in Japan were examined for their agonistic activities against five human nuclear receptors (estrogen receptor [ER] alpha, thyroid hormone receptor alpha, retinoic acid receptor [RAR] alpha, retinoid X receptor alpha, and vitamin D receptor) by using a yeast two-hybrid assay. The results suggest that the river environment is contaminated with endocrine disrupting chemicals (EDCs) that can interact with a variety of nuclear receptors and that contamination with those that have RAR agonistic activity may be more serious than contamination with well-known EDCs that act as ER agonists.

Background and Aims: IL-17-producing CD4(+) T-helper cells (Th17) contribute to chronic autoimmune inflammation in the brain, and levels of Th17-derived cytokines increase in patients with colitis, suggesting a role in pathogenesis. We analyzed the roles of Th17 cells and the transcription factor retinoic acid receptor-related organ receptor (ROR)gamma, which regulates Th17 differentiation, in chronic intestinal inflammation., Methods: Using an adoptive transfer model of colitis, we compared the colitogenic potential of wild-type, interleukin-17A (IL-17A)-, IL-17F-, IL-22-, and RORgamma-deficient CD4(+)CD25(-) T cells in RAG1-null mice., Results: Adoptive transfer of IL-17A-, IL-17F-, or IL-22-deficient T lymphocytes into RAG1-null mice caused severe colitis that was indistinguishable from that caused by wild-type cells. In contrast, transfer of RORgamma-null T cells failed to increase mucosal IL-17 cytokine levels and did not induce colitis. Treatment with IL-17A was able to restore colitis after transfer of RORgamma-null T cells, indicating a crucial role for Th17 cells in pathogenesis. Treatment of RAG1 mice that received IL-17F-null (but not wild-type) T cells with a neutralizing anti-IL-17A antibody significantly suppressed disease, indicating redundant biological effects of IL-17A and IL-17F., Conclusions: We have identified a crucial role of RORgamma-expressing Th17 cells in chronic intestinal inflammation. RORgamma controls IL-17A and IL-17F production, and these cytokines have a redundant but highly pathogenic role in gut inflammation. Reagents that target RORgamma or a combination of anti-IL-17A and anti-IL-17F might be developed as therapeutics for chronic colitis.

The development of a testis requires the proper spatiotemporal expression of the SRY gene and other genes that act in a dosage-sensitive manner. Mutations in the SRY gene account for only 10-15% of patients with 46,XY gonadal disorder of sex development (DSD). To enable the diagnostics of deletions and duplications of genes known to be involved in different forms of DSD, we developed a synthetic probe set for multiplex ligation-dependent probe amplification (MLPA) analysis. Here, we report the results from the analysis of 22 patients with 46,XY gonadal DSD. The analysis with the DSD probe set has led to the identification of two copy number variations, an 800-kb NR0B1 (DAX1) locus duplication on Xp21 in a patient with isolated partial gonadal dysgenesis and a duplication of the SRD5A2 gene that represents a rare normal variant. The described MLPA kit represents an optimal complement to DNA sequence analysis in patients with DSD, enabling screening for deletions and duplications of several genes simultaneously. Furthermore, the second identification of an NR0B1 locus duplication in a patient with isolated gonadal dysgenesis, without dysmorphic features and/or mental retardation, highlights the importance of evaluating NR0B1 duplication in patients with gonadal dysgenesis.

The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) beta is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RAR beta could act as a tumor suppressor. However, the status of RAR beta in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RAR alpha, RAR beta, and RAR gamma using AM580 (a RAR-specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RAR beta and RAR gamma mRNA was significantly induced by AM580, compared with vehicle controls, whereas RAR alpha mRNA was significantly attenuated by AM580, compared with vehicle. RAR beta was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RAR beta mRNA expression in the Ishikawa cell line, and the expression level of RAR beta in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma.

Background: We examined the expressions of Ad4BP/SF-1 and DAX-1 in primary and metastatic lesions of advanced adrenocortical carcinoma with normal hormonal findings by immunohistochemistry and discussed their usefulness as immunohistochemical markers for diagnosis., Patients and Methods: Four patients (average age 54.3 +/- 3.9) with retroperitoneal and metastatic tumors were enrolled in this study. All patients had normal hormonal findings. Ad4BP/SF-1 and DAX-1 underwent immunohistochemical study using needle biopsy specimens from the retroperitoneal tumor or metastatic tumor of these patients to diagnose primary adrenocortical carcinoma., Results: Immunoreactivity for Ad4BP/SF-1 and DAX-1 indicated that it was localized exclusively in the nuclei. Ad4BP/SF-1 immunoreactivity was observed in the carcinoma cells of 2 patients, but not in the others, whereas DAX-1 immunoreactivity was observed in the carcinoma cells of all 4 patients., Conclusion: The expressions of Ad4BP/SF-1 and DAX-1 are considered essential to maintain the biological characteristics of adrenocortical cells even without abnormal hormonal findings or even after malignant transformation and metastasis. These markers are useful to distinguish other retroperitoneal tumors, especially in patients with bulky tumors, and to diagnose the metastatic site of origin as adrenocortical carcinoma in patients clinically presenting with widespread metastasis., (Copyright 2008 S. Karger AG, Basel.)

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac.

The retinoic acid receptor beta (RARbeta) is a retinoic acid (RA)-inducible tumor suppressor, which plays an important role in the arrest of neuroblastoma cell growth. Using human neuroblastoma SH-SY5Y cells, we have examined the regulation of RARbeta expression by histone deacetylase inhibitors (HDACi), considered to be promising agents in anticancer therapy. Our results show that HDACi cooperated with RA to increase RARbeta mRNA levels and to activate the RARbeta2 promoter in transient transfection assays. Chromatin immunoprecipitation assays showed that the basal RARbeta2 promoter that contains the RA response element was refractory to acetylation by both HDACi and RA. In addition, HDACi caused a transient increase in acetylation of a downstream RARbeta2 region, even though global histones remain hyperacetylated after a prolonged treatment with the inhibitors. RA potentiated this response and maintained acetylation for a longer period. Despite the cooperation of RA with HDACi to increase transcription of the RARbeta gene, these inhibitors caused a paradoxical reduction of the cellular levels of the RARbeta protein in cells treated with the retinoid. This reduction is secondary to a change in the protein half-life that is decreased by the HDACi due to increased ubiquitin-independent proteasomal degradation. These results show that HDACi regulate expression of the tumor suppressor gene RARbeta by both transcriptional and posttranscriptional mechanisms and might then modulate sensitivity to the retinoid in neuroblastoma cells.

Retinoic acid (RA) treatment of SH-SY5Y neuroblastoma cells results in activation of phosphatidylinositol-3-kinase (PI3K) signaling pathway, and this activation is required for RA-induced differentiation. Here we show that RA activates PI3K and ERK1/2 MAPK signaling pathways through a rapid, nongenomic mechanism that does not require new gene transcription or newly synthesized proteins. Activation of PI3K by RA appears to involve the classical nuclear receptor, retinoic acid receptor (RAR), on the basis of the pharmacological profile of the activation, loss, and gain of function experiments with mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells, and the physical association between liganded RAR and PI3K activity. The association of RAR with the two subunits of PI3K was differentially regulated by the ligand. Immunoprecipitation experiments performed in SH-SY5Y cells showed stable association between RARalpha and p85, the regulatory subunit of PI3K, independently of the presence of RA. In contrast, ligand administration increased the association of p110, the catalytic subunit of PI3K, to this complex. The intracellular localization of RAR proved to be relevant for PI3K activation. A chimerical RAR fusing c-Src myristylation domain to the N terminus of RARalpha (Myr-RARalpha) was targeted to plasma membrane. Transfection of Myr-RARalpha to mouse embryo fibroblast-RAR(alpha beta gamma)(L-/L-) null cells and COS-7 cells results in strong activation of the PI3K signaling pathway, although both in the absence as well in the presence of RA. Our results support a mechanism in which ligand binding to RAR would play a major role in the assembly and intracellular location of a signaling complex involving RAR and the subunits of PI3K.

Purpose: Retinoids inhibit proliferation and induce differentiation in melanoma cells. Retinoic acid receptors (RAR) and retinoid X receptors (RXR) mediate the various modulatory effects of retinoids in cells. We have studied the in situ expression of each RAR and RXR protein (alpha, beta, gamma) in a large series of melanocytic lesions and correlated the expression with clinicopathologic features and prognosis of the patients., Experimental Design: Tissue microarray blocks of 226 melanocytic lesions were semiquantitatively evaluated by immunohistochemistry for the cytoplasmic and nuclear expression of RAR and RXR protein (alpha, beta, gamma)., Results: A significant decrease of RARbeta protein (P < 0.0001), nuclear expression of RARgamma (P < 0.0001), and RXRalpha (P < 0.0001) was found in primary and metastatic melanomas as compared with nevi. Loss of nuclear immunoreactivity for RARgamma (P = 0.048) and RXRalpha (P = 0.001) was observed in the lesions showing vertical growth pattern. In addition, in patients with concomitant loss of cytoplasmic staining for RARalpha and RXRalpha, the probability of overall survival (log-rank test, P = 0.002) and disease-specific survival (log-rank test, P = 0.014) was significantly lower., Conclusions: Aberrant expression of retinoid receptors seems to be a frequent event in melanoma and suggests an impairment of the retinoid pathway in this cancer. Our data indicate the loss of retinoid receptor expression with melanoma progression and suggest a possible prognostic significance of the analysis of retinoid receptors in melanoma.

Polychlorinated biphenyls (PCBs) have been associated with a number of toxic effects in marine mammals such as endocrine disruption and immunotoxicity that, in turn, are widely thought to have contributed to population level impacts including reproductive failure and outbreaks of disease. In this study, the dietary hormone vitamin A and expression levels of one of its receptors, retinoic acid receptor alpha (RARalpha), were used as biomarkers of PCB-associated health effects in harbour seals. Harbour seal pups (n=24) were live-captured in coastal British Columbia, Canada, and Washington State, USA, and sampled for whole blood (to obtain peripheral blood mononuclear cells, PBMCs) and blood plasma, as well as biopsies of blubber and skin. Concentrations of circulatory vitamin A (retinol) in plasma and stored vitamin A in blubber were negatively associated with blubber PCB concentrations (R=-0.518, p=0.013 and R=-0.645, p=0.009, respectively). However, vitamin A concentrations in skin, an important target tissue, remained constant, which likely reflects a compensatory transfer from blubber to maintain physiological functions. In addition, we characterized the harbour seal RARalpha, and investigated its expression levels as a potential biomarker in seals. RARalpha expression in blubber, but not on PBMCs, was elevated in more contaminated animals (R=0.580, p=0.009). This may represent a direct contaminant-related effect, or, a compensation for the contaminant-related disruption of (circulatory and/or blubber) hormone levels. Since vitamin A is critical to developmental, reproductive and immunological health, our observations of a contaminant-related disruption of its physiology in free-ranging seals may portend population level consequences. Vitamin A concentrations and RARalpha expression levels can therefore represent relevant and sensitive biomarkers of PCB-associated toxic effects in toxicological studies of marine mammals.

The expression of retinoid acid receptors alpha (RARalpha) and beta (RARbeta) and estrogen receptor alpha (ERalpha) was assessed by immunohistochemistry and Western blotting in normal ovaries, serous cystadenoma (n = 20), serous borderline (n = 14), and serous ovarian cancer (n = 47) and was correlated in cancer cases with stage, grade, progress-free survival (PFS), and survival. RARalpha was increasingly expressed in benign cystadenomas, borderline, and low-stage and advanced-stage neoplasms (p < 0.001). In stage III, G3 serous carcinoma, increased RARalpha expression was an independent prognostic factor associated with lower chemoresponse to first-line chemotherapy (taxol and carboplatin) and shorter PFS (p < 0.002).RARbeta and ERalpha expression did not correlate with RARalpha tumor characteristics or PFS and survival., ((c) 2008 S. Karger AG, Basel)

Many complementary or competing signalling pathways bear an influence on the myometrium at any one time, and because the retinoic acid signalling pathway influences differentiation of a wide array of human tissues, this may be one of the determinants of myometrial differentiation during pregnancy. We have explored the novel hypothesis that the retinoids may act as important regulators in controlling the differentiated state of the human myometrium during pregnancy by characterizing the expression profiles for cellular retinoid-binding proteins CRBPI, CRABPI and CRABPII in non-pregnant, pregnant (non-labouring) and labouring human myometrium taken from the functionally distinct upper and lower uterine segments. In addition, we have investigated the effect of all-trans retinoic acid (ATRA) on the expression of several retinoic acid response genes including cyclooxygenase-2 (COX-2) and connexin-43 (Cx-43). Different spatial and temporal patterns of expression were observed for CRBPI, CRABPI and CRABPII within the upper and lower uterine segments through the three trimesters of pregnancy and in labour. Furthermore, the expression of COX-2, Cx-43, CRABPI, the transcription factor c-Jun and the retinoic acid receptor RARbeta altered in response to different concentrations of ATRA, suggesting that the differential expression of cellular retinoid-binding proteins may lead to different levels of retinoic acid being delivered to its nuclear targets, leading to the differential expression of specific target genes within the myometrium during pregnancy.

Objective: Mutations in DAX-1, an X-linked gene encoding an orphan nuclear receptor, have been associated with adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Here we describe two novel DAX-1 mutations, Y214X and I361T, associated with childhood-onset primary adrenal failure. We aimed at analysing their effects on protein localization, transcriptional activity and propose a structural-function relationship., Design: We have directly sequenced the DAX-1 gene from PCR-amplified DNA. The effect of the mutations on protein localization was assessed by immunocytochemistry in transfected cells. The impact of mutations on transcriptional activity was determined by transfection using a StAR promoter-luciferase reporter system., Results: The mutation Y214X produces a protein lacking the C-terminal half of DAX-1. The other one, I361T, affects a highly conserved amino acid within the putative ligand-binding domain. The mutant Y214X displayed a wild-type subcellular localization while I361T was partially retained in the cytoplasm. Both mutants retained subtantial transcriptional repressive activity, compared to mutants producing early onset adrenal failure. Interestingly, I361T displayed similar in vitro transcriptional repressive activity to the mutant I439S previously described in a case of late-onset AHC. This shows that molecular alterations of DAX-1 leading to similar in vitro activities may result in very different ages of onset of adrenal failure, which suggests that additional genetic and epigenetic factors are important in determining the clinical course of AHC., Conclusions: Our results help the understanding of structure-function relationships in the DAX-1 molecule, suggesting that the N-terminal domain is relatively autonomous and add credence to presumed molecular interactions within ligand binding domain of the protein.

The Ewing family of tumors harbors chromosomal translocations that join the N-terminal region of the EWS gene with the C-terminal region of several transcription factors of the ETS family, mainly FLI1, resulting in chimeric transcription factors that play a pivotal role in the pathogenesis of Ewing tumors. To identify downstream targets of the EWS/FLI1 fusion protein, we established 293 cells expressing constitutively either the chimeric EWS/FLI1 or wild type FLI1 proteins and used cDNA arrays to identify genes differentially regulated by EWS/FLI1. DAX1 (NR0B1), an unusual orphan nuclear receptor involved in gonadal development, sex determination and steroidogenesis, showed a consistent up-regulation by EWS/FLI1 oncoprotein, but not by wild type FLI1. Specific induction of DAX1 by EWS/FLI1 was confirmed in two independent cell systems with inducible expression of EWS/FLI1. We also analyzed the expression of DAX1 in Ewing tumors and derived cell lines, as well as in other nonrelated small round cell tumors. DAX1 was expressed in all Ewing tumor specimens analyzed, and in seven out of eight Ewing tumor cell lines, but not in any neuroblastoma or embryonal rhabdomyosarcoma. Furthermore, silencing of EWS/FLI1 by RNA interference in a Ewing tumor cell line markedly reduced the levels of DAX1 mRNA and protein, confirming that DAX1 up-regulation is dependent upon EWS/FLI1 expression. The high levels of DAX1 found in Ewing tumors and its potent transcriptional repressor activity suggest that the oncogenic effect of EWS/FLI1 may be mediated, at least in part, by the up-regulation of DAX1 expression.

Retinoic acid (RA) and its natural and synthetic derivatives (retinoids) are important dietary factors which regulate cellular differentiation and growth, so that they are thought to be particularly effective at preventing the development of several tumours. They play this role as ligands of the RAR and RXR nuclear retinoic acid receptors, including the RA receptor isoforms alpha, beta, and gamma. These ligand-activated nuclear receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. Among these target genes, the RARbeta gene is of great interest, being able to encode a potential tumour suppressor. It should be emphasized that most breast carcinomas and breast cancer cell lines show loss or down-regulation of RARbeta receptor expression, whereas RARalpha and gamma, as well as retinoid X receptors, appear to be variably expressed in both normal and tumour cells. It is also interesting to note that basal and RA-induced RARbeta mRNA levels tend to increase with senescence of normal cells. This information provides further support for the hypothesis that genetic events involved in cellular senescence may also play a significant role in tumour suppression in humans. The aim of this review is to clarify whether expression of RARbeta could be modulated by chemopreventive intervention and may therefore serve as an intermediate biomarker in chemoprevention trials for some cancers.

DAX-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1) is a recently characterized member of the orphan nuclear receptor family. DAX-1 functions as a global negative regulator of steroid hormone production. It inhibits adrenal 4 binding protein (Ad4BP)/steroidogenic factor-1 (SF-1) pathway-dependent P450arom expression in cultured human endometriotic stromal cells and acts as a corepressor for estrogen receptors (ER). In this study we first examined the localization of DAX-1 in 46 normal cycling endometria, 36 cases of endometrial hyperplasia and 103 cases of endometrial carcinoma by using immunohistochemistry to elucidate the possible involvement of DAX-1 and its correlation to the status of Ad4BP/SF-1, a universal transcription factor of steroidogenesis. We then evaluated DAX-1 mRNA expression, using quantitative reverse transcription-polymerase chain reaction for DAX-1 in 33 cases of endometrial carcinoma for further characterization. We subsequently correlated these findings with various clinicopathological parameters of the cases. Ad4BP/SF-1 immunoreactivity was not detected in any human endometria examined. A significant inverse correlation was detected between the status of DAX-1 immunoreactivity and histological grade (P = 0.0003) in endometrial carcinoma. The labeling index (LI) values of DAX-1 in normal endometrium during the secretory phase (P < 0.0001) and hyperplasia (P < 0.0001) were significantly higher than that of carcinoma. No significant correlations were detected between DAX-1 immunoreactivity and amounts of aromatase mRNA. There was a statistically significant positive correlation between DAX-1 and ERalpha (P = 0.006) and ERbeta LI (P < 0.001). These findings suggest that DAX-1 may inhibit the proliferation and progression of endometrial carcinoma through inhibition of estrogenic actions, possibly by interacting with ER present in carcinoma cells, rather than regulating in situ steroidogenesis.

We have investigated the role of Vitamin A (retinoid) proteins in hepatic retinoid processing under normal conditions and during chemical stress induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a chemical known to interfere with retinoid turnover and metabolism. Three separate studies were performed in wildtype control mice and transgenic mice that lack one or more isoforms of retinoic acid receptors (RAR), retinoid X receptors (RXR), or intracellular retinoid-binding proteins (CRABP I, CRABP II, CRBP I). Body and organ weight development was monitored from 2 weeks of age to adult, and hepatic levels of retinyl esters, retinol, and retinoic acid were investigated. In addition, hepatic concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid, a recently discovered retinoid metabolite that has proven sensitive to both TCDD exposure and Vitamin A status, were also determined. Mice absent in the three proteins CRBP I, CRABP I, and CRABP II (CI/CAI/CAII-/-) displayed significantly lower hepatic retinyl ester, retinol, and all-trans-retinoic acid levels compared to wildtype mice, whereas the liver concentrations of 9-cis-4-oxo-13,14-dihydro-retinoic acid was considerably higher. After treatment with TCDD, hepatic total retinoids were almost entirely depleted in the CI/CAI/CAII-/- mice, whereas wildtype mice and mice lacking CRABP I, and CRABP II (CAI/CAII-/-) retained approximately 60-70% of their Vitamin A content compared to controls at 28 days. RAR and RXR knockout mice responded similarly to wildtype mice with respect to TCDD-induced retinoid disruption, with the exception of RXRbeta-/- mice which showed no decrease in hepatic Vitamin A concentration, suggesting that the role of RXRbeta in TCDD-induced retinoid disruption should be further investigated. Overall, the abnormal retinoid profile in the triple knockout mice (CI/CAI/CAII-/-), but not double knockout (CAI/CAII-/-) mice, suggests that a loss of CRBP I may account for the difference in retinoid profile in CI/CAI/CAII-/- mice, and is likely to result in an increased susceptibility to hepatic retinoid depletion following dioxin exposure.

We examined the effect of all-trans retinoic acid (ATRA) on the androgen-induced growth of mouse seminal vesicles (SVs) in vivo and its mechanisms. Testosterone propionate (TP) alone or with ATRA was injected daily into adult castrated BALB/c mice. Injections of ATRA significantly inhibited the TP-induced growth of SVs in terms of wet weight and DNA synthesis by a pair of SVs evaluated by [3H]thymidine uptake. The bromodeoxyuridine labelling index showed that ATRA inhibited the proliferation of both epithelial and stromal cells. Immunoreactivity for retinoic acid receptor-alpha was found in the basal epithelial cells. Injections of ATRA affected neither 5alpha-reductase activity nor the expression of mRNAs for TGF-beta1, 2 and 3 and TGF-beta receptor 1 and 2 in the SVs. However, androgen receptor (AR) binding assays and Western blotting revealed a decrease in AR without a change in ligand-binding affinity. The present study showed that retinoid inhibited the androgen-induced growth of mouse SVs in vivo, and suggests that a decrease in AR is one of its mechanisms.

Context: Although glucocorticoid hormone, thyroid hormone, and retinoic acid play important roles in fetal development, the expression of their receptors in human lung is still unknown., Objective: The aim of this study was to investigate the ontogeny of glucocorticoid receptor (GR)alpha, thyroid hormone receptors (TRs), retinoic acid receptors (RARs), and retinoid X receptors (RXRs) mRNA expression in human lungs., Design: Lungs from human fetuses and neonates (13.5-41 wk gestation; n = 20) as well as adults (n = 5) were analyzed by real-time PCR to monitor the ontogeny of mRNA expression for each receptor. In addition, immunohistochemistry was performed to show the cellular distribution of the different receptors., Results: The expression of GRalpha, TRs, RARs, and RXRs was already detected in the earliest developmental stages analyzed. There was no significant difference in mRNA expression between developmental groups for any of the genes studied. However, for fetal and neonatal samples, there were positive correlations between gestational age and mRNA expression for RARalpha (r = 0.665; P = 0.001), RXRalpha (r = 0.444; P = 0.050), and RXRgamma (r = 0.464; P = 0.039). Immunohistochemical studies showed the presence of GRalpha, TRs, RARs, and RXRs in the nuclei of both epithelial and mesenchymal cells, albeit more pronounced in epithelium of larger airways., Conclusions: The detection of GRalpha, TRs, RARs, and RXRs expression in human lung as early as 13.5 wk gestation implies an early potential for therapeutic or toxic effects by exogenous analogs or by excess of endogenous ligands.