Specific Retinoid Receptors Research Articles

Direct inhibition of retinoic acid catabolism by fluoxetine.

Recent evidence from animal and human studies suggests neuroprotective effects of the SSRI fluoxetine, e.g., in the aftermath of stroke. The underlying molecular mechanisms remain to be fully defined. Because of its effects on the cytochrome P450 system (CYP450), we hypothesized that neuroprotection by fluoxetine is related to altered metabolism of retinoic acid (RA), whose CYP450-mediated degradation in brain tissue constitutes an important step in the regulation of its site-specific auto- and paracrine actions. Using traditional pharmacological in vitro assays, the effects of fluoxetine on RA degradation were probed in crude synaptosomes from rat brain and human-derived SH-SY5Y cells, and in cultures of neuron-like SH-SY5Y cells. Furthermore, retinoid-dependent effects of fluoxetine on neuronal survival following glutamate exposure were investigated in rat primary neurons cells using specific retinoid receptor antagonists. Experiments revealed dose-dependent inhibition of synaptosomal RA degradation by fluoxetine along with dose-dependent increases in RA levels in cell cultures. Furthermore, fluoxetine's neuroprotective effects against glutamate excitotoxicity in rat primary neurons were demonstrated to partially depend on RA signaling. Taken together, these findings demonstrate for the first time that the potent, pleiotropic antidepressant fluoxetine directly interacts with RA homeostasis in brain tissue, thereby exerting its neuroprotective effects.

LXRα Enhances Lipid Synthesis in SZ95 Sebocytes

Differentiation of sebocytes is strongly associated with enhanced lipid synthesis and accumulation in the cells. Liver X receptors (LXRs) are members of the nuclear receptor superfamily, which play a critical role in cholesterol homeostasis and lipid metabolism. We examined whether LXRalpha regulated lipid synthesis in the immortalized human sebaceous gland cell line SZ95. When the SZ95 sebocytes were treated with the ligand of LXR such as TO901317 or 22(R)-hydroxycholesterol, lipid droplets were accumulated in the majority of cells when examined by Oil Red O staining. The expression of the known LXR targets, such as fatty acid synthase and sterol regulatory-binding protein-1, was induced by TO901317. TO901317 treatment increased expression of LXRalpha but not that of LXRbeta. Transfection of antisense LXRalpha significantly decreased TO901317-induced target gene expression and lipid droplet accumulation, suggesting a major role of LXRalpha in differentiation of sebocytes. Further, TO901317 decreased the expression of cyclooxygenase-2 and inducible nitric oxide synthase that was induced by lipopolysaccharide treatment. Together, these results indicate that important roles of LXRalpha in differentiation and inflammatory signaling in sebaceous glands. Thus, we suggest that LXR ligands could provide a new class of therapeutic agents for sebaceous gland-associated disorders such as seborrhea and acne.

13‐cis‐retinoic acid alters the cellular morphology of slice‐cultured serotonergic neurons in the rat

Retinoids influence cellular processes such as differentiation, proliferation and apoptosis via retinoic acid receptor (RAR) and retinoid X receptor (RXR), and have therapeutic applications in several cancers and dermatologic diseases. Recent reports indicate that depression occasionally occurs in patients using the acne drug Accutane, the active component of which is 13-cis-retinoic acid (13-cis-RA). Although impairment of serotonin (5-HT)-expressing neurons, including morphologic changes, is thought to be associated with depressive symptoms, the effects of 13-cis-RA on 5-HT neurons have not been examined. The present study demonstrated that 13-cis-RA alters the morphology of 5-HT neurons in cultured rat midbrain slices. The 13-cis-RA-induced changes were partially blocked by RXR and RAR antagonists. Furthermore, cotreatment with RAR and RXR agonists altered the morphology of 5-HT neurons to a greater extent than the individual application of each agonist. The morphologic changes were completely blocked by RXR antagonist, whereas RAR antagonist partially blocked the effects. These results suggest that 13-cis-RA exerts its action on slice-cultured 5-HT neurons, at least in part, through specific retinoid receptors. Moreover, RXR has a greater influence on the morphology of 5-HT neurons than RAR. The receptor-mediated actions of 13-cis-RA presented here may provide a clue for further research on depression associated with the use of 13-cis-RA.

Development and quality of bovine morulae cultured in serum-free medium with specific retinoid receptor agonists

Retinoids regulate development and differentiation of the bovine blastocyst in vitro, although the underlying mechanisms remain to be clarified. A challenge in reproductive biotechnology is the identification of pathways that regulate early embryonic development and their influence on blastocyst differentiation, apoptosis and survival to cryopreservation as traits of embryo quality. The present paper analyses the effects of short-term exposure (24 h) to retinoids on in vitro-produced bovine morulae. Immature cumulus oocyte complexes were in vitro matured and fertilised. Presumptive zygotes were subsequently cultured in modified synthetic oviduct fluid up to Day 6, in which morulae were randomly allocated to the different experimental groups. The treatments consisted of 0.1 microM LG100268 (LG; a retinoid X receptor agonist), 0.7 microM all-trans retinoic acid (ATRA; a retinoic acid receptor agonist) or no additives. Day 8 blastocyst development was increased in the ATRA-treated group compared with the LG and untreated embryos. In Day 7 embryos, the number of total cells and cells allocated to the trophectoderm were higher in the ATRA-treated group compared with untreated embryos. Apoptosis in the inner cell mass increased after LG treatment, whereas ATRA had no effect. After vitrification and warming, survival and hatching rates of Day 7 blastocysts did not change with retinoid treatment. Within the LG-treated and untreated blastocyst groups, survival and hatching rates were higher for Day 7 than Day 8 embryos; however, Day 8 blastocysts treated with ATRA showed improved hatching rates. In conclusion, treatment of morulae with ATRA in serum-free medium improves embryo development and quality without increasing the incidence of apoptosis and necrosis.

A Crucial Role for Cellular Retinol-Binding Protein I in Retinoid Signaling

Retinol (the prototypic vitamin A) and its metabolites called retinoids play important physiologic roles in embryonal development, vision, maintenance of epithelial differentiation, immune functions, and reproduction (1). Many of these functions are mediated by retinol metabolites such as all-trans-retinoic acid (ATRA) (2). ATRA modulates gene expression by means of nuclear receptors that are members of the steroid hormone gene superfamily. These receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), appear in at least three subtypes, designated , , and . The receptors function as ligand-activated heterodimeric DNA-binding transcription enhancing factors and regulate the transcription of various genes, which play important roles during development and in adult tissues. Abberant expression and function of specific retinoid receptors, primarily RAR , have been associated with cancer development and progression (3,4). However, recent studies have highlighted additional mechanisms for abrogation of retinoid signaling in carcinogenesis. These involve abnormalities located upstream of retinoic acid and its nuclear receptors. These defects interfere with retinol storage and its metabolism to retinoic acid and result in a localized retinoid deficiency. Interestingly, we have suggested that decreased levels of RAR may be, at least in part, caused by local vitamin A deficiency (5). Dietary retinol is stored in the liver in specialized cells and delivered to various target tissues via a retinol-binding protein

Retinoid-induced mu opioid receptor expression by phytohemagglutinin-stimulated U937 cells

Opioid use may be associated with an increased risk of neurological disease in human immunodeficiency virus (HIV) infection through effects on immune cell function. Studies were performed to examine the effects of specific retinoid receptor activation on mu opioid receptor (MOR) production by phytohemagglutinin (PHA)-stimulated U937 cells, a mononuclear cell line. PHA stimulation increased activation of the MOR promoter as well as levels of MOR mRNA, total receptor protein in cell lysates, and surface and cytoplasmic receptor expression. Retinoid X receptor (RXR) agonist and retinoic acid receptor (RAR) antagonist further increased MOR expression by the PHA-stimulated cells. In contrast, MOR expression was suppressed by RAR agonist and by RXR antagonist. Finally, opioid receptor binding was also increased by RXR agonist and RXR antagonist; no increase in binding occurred in the presence of RAR agonists and RXR antagonist. All together, these studies suggest that MOR expression in U937 cells can be differentially regulated by specific retinoid receptor activation. Such effects may have important clinical relevance for opioid users with HIV infection, including individuals with neurological disease.

Detection of retinoid receptor expression patterns in canine normal lymph nodes and in nodal lymphomas

Introduction: Retinoids are derivatives of vitamin A, which are essential for cell growth and differentiation of a variety of cells. Retinoid activity occurs after binding to a class of nuclear receptors termed retinoic acid receptor (RAR) and retinoid X receptor (RXR). Receptor‐ligand interactions ultimately activate the transcription of target genes. Retinoids have been shown in vitro to participate in both lymphocyte activation and apoptosis. In human cancer patients, specific ligands to retinoid receptors have been approved for the treatment of T‐cell cutaneous lymphomas. The use of retinoids for the treatment of additional lymphoid malignancies, both of B and T‐cell types, has been speculated. In vitro experiments demonstrating lymphocyte apoptosis subsequent to treatment with both natural and synthetic retinoids further support the potential utility of retinoid therapy in spontaneous lymphoid malignancies.Objectives: To detect the pattern of retinoid receptor expression (RAR and RXR) and their isoforms (α, β, and γ) in normal lymph nodes and in nodal lymphomas in dogs.Methods: Immunohistochemistry was performed in tissue specimens derived from naturally occurring cases of nodal lymphomas and normal lymph nodes from dogs. Rabbit polyclonal antibodies (Santa Cruz Biotechnology), previously demonstrated to be cross‐reactive with canine tissue, were utilized to assess the expression patterns of RAR/RXR and their respective isoforms (α, β, and γ) in all pathologic and normal tissue samples evaluated.Results: Normal lymph nodes were consistently negative for all isoforms. In hyperplastic nodes, macrophages were positive for all isoforms. Lymphomas both of B and T‐cell type stained strongly positive for the isoforms RARα and RXRγ.Conclusions: A distinctive pattern of expression between lymphomas, lymphoid hyperplasia, and normal lymph nodes was detected in dogs. Different retinoid receptor expression profiles from normal lymph nodes and nodal lymphomas seem to be of diagnostic importance. Further investigation with larger number of lymphoma cases is warranted to thoroughly characterize retinoid receptor expression patterns in lymphoid malignancies with the possibility of using specific retinoid receptor ligands for the treatment of canine nodal lymphomas.

Synthetic retinoids and their nuclear receptors.

In addition to all-trans-retinoic acid and its 9 and 13-cis isomers, four synthetic retinoids are currently available to treat diseases of hyperproliferation, such as acne, psoriasis, and actinic keratosis, or cancers such as acute promelocytic leukemia, cutaneous T-cell lymphoma, and squamous or basal cell carcinoma. The retinoids extert their antiproliferative effects by interacting with their retinoic acid and retinoid X receptors that act as ligand-inducible transcription factors. These homologous receptors function either directly on retinoid response elements or indirectly by modifying the responses of other transcription factors. Their major domains for binding DNA and their ligands have been characterized by either nuclear magnetic resonance spectroscopy or X-ray crystallography. The identification and design of synthetic retinoids are overviewed, as are their selective interactions with specific retinoid receptor subtypes and their clinical effects against cancer. Emphasis is placed on the retinoid X receptors and their ligands.

Retinoid receptors and vitamin A deficiency: differential patterns of transcription during early avian development and the rapid induction of RARs by retinoic acid

The functional links of specific retinoid receptors to early developmental events in the avian embryo are not known. Before such studies are undertaken, knowledge is required of the spatiotemporal expression patterns of the receptor genes and their regulation by endogenous retinoic acid levels during the early stages of development. Here, we report the expression patterns of mRNAs for RARα, RARα2, RARβ2, RARγ, RARγ2, RXRα, and RARγ from neurulation to HH10 in the normal and vitamin A-deficient (VAD) quail embryo. The transcripts for all retinoid receptors are detectable at HH5, except for RXRγ, which is detected at the beginning of HH6. At the 4/5 somite stage of HH8, when retinoid signaling is initiated in the avian embryo, mRNAs of all receptors are present, with very strong and ubiquitous expression patterns for RARα, RARα2, RARγ, RARγ2, and RXRα, a persistent expression of RARγ in the neural tissues, a strong expression of RARβ2 in lateral plate mesoderm and somites, and an anterior expression of RXRγ. All retinoid receptors are expressed in the heart primordia. In the VAD quail embryo, the general pattern of retinoid receptor transcript localization is similar to that of the normal, except that the expression of RARα2 and RARβ2 is severely diminished. Administration of retinol or retinoic acid to VAD embryos at or before the 4/5 somite stage rescues the expression of RARα2 and RARβ2 within approximately 45 min and restores normal development. RARβ2 expression requires the expression of RARα2. After neurulation, the expression of all retinoid receptors in the VAD quail embryo becomes independent of vitamin A status and is similar to that of the normal. The mRNA levels and sites of expression of the key enzyme for retinoic acid biosynthesis, Raldh-2, are not affected by vitamin A status; the expression pattern is restricted and does not correspond to that of retinoid receptors at all sites. The general patterns and intensity of retinoid receptor gene expression during early quail development are comparable to those of the mammalian and thus validate the application of results from retinoid-regulated avian development studies to those of the mammalian.

Aberration in the Expression of the Retinoid Receptor, RXRα, in Prostate Cancer

There is ample evidence for a role for retinoids in the development and maturation of prostatic epithelium. In recent experiments with conditional disruption of a specific retinoid receptor, namely, RXRα in the prostatic epithelium of the mouse, we observed that a major component of retinoid action in the prostate is indeed mediated by RXRα. The results clearly indicated that the inactivation of RXRα in the prostate epithelium leads to the development of preneoplastic lesions (Huang et al. Cancer Res. 62: 4812-4819, 2002). To determine the relation of this finding to human prostate cancer, we examined the expression of RXRa protein in human prostate cancer cell lines by western blotting and prostate cancer specimens by immunohistochemistry. Relative to the "normal" prostate epithelial cells, there was approximately two- to nine-fold decrease in the full-length 54 kD RXRα protein in each of the seven different prostate cancer cell lines tested. Similarly, while RXRα immunostaining was uniformly strong in the nuclei of most of the benign prostatic epithelial cells of the thirteen adenocarcinoma specimens tested, a highly heterogeneous pattern of expression was detected in the malignant epithelium, with some areas with low or no staining, others with mostly cytoplasmic staining, and some with both nuclear and cytoplasmic immunoreactivity. To evaluate the effect of RXRα modulation on the biologic properties of prostate cancer cell lines, we used a lentivirus expression system to overexpress RXRα in CWR22R prostate cancer cells that basally expressed a marginal level of the receptor. The sorted RXRα-transduced cells were compared to the corresponding vector control cells for proliferative and apoptotic properties. A correlation of reduction of cell growth or increased susceptibility to apoptosis with increases in the level of RXRα nuclear receptor was demonstrated. These effects were further enhanced when the cell culture medium was supplemented with a retinoid receptor panagonist, 9-cis retinoic acid. Together, these data support the notion that, like in mouse prostate, loss or reduction of RXRα activity might be a critical factor in prostate tumorigenesis in humans.Key WordsRetinoid receptor RXRα, 9-cis retinoic acid, Prostate cancer cells, Lentivirus vectors, Apoptosis

Skin cancer chemoprevention: strategies to save our skin.

There are over 1 million cases of skin cancer diagnosed yearly in the United States. The majority of these are nonmelanoma (NMSCs) and are associated with chronic exposure to ultraviolet light (UV). Actinic keratosis (AK) has been identified as a precursor for SCC, but not for BCC. AKs are far more common than SCC, making them excellent targets for chemoprevention. Cancer chemoprevention can prevent or delay the occurrence of cancer in high-risk populations using dietary or chemical interventions. We have developed strategies that have rational mechanisms of action and demonstrate activity in preclinical models of skin cancer. Promising agents proceed to phase I-III trials in subjects at high risk of skin cancer. UV light induces molecular signaling pathways and results in specific genetic alterations (i.e., mutation of p53) that are likely critical to skin cancer development. UVB-induced changes serve as a basis for the development of novel agents. Targets include inhibition of polyamine or prostaglandin synthesis, specific retinoid receptors, and components of the Ras and MAP kinase signaling pathways. Agents under study include: epigallocatechin gallate (EGCG), a green tea catechin with antioxidant and sunscreen activity, as well as UVB signal transduction blocking activity; perillyl alcohol, a monoterpene derived from citrus peel that inhibits Ras farnesylation; difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase and polyamines; retinoids that target retinoid X receptors and AP-1 activity; and nonsteroidal anti-inflammatory agents that inhibit cylooxygenase and prostaglandin synthesis. We performed a series of Phase I-II trials in subjects with multiple AK. For example, a phase II randomized trial of topical DFMO reduced AK number, suppressed polyamines, and reduced p53 protein. Our goal is to develop agents for use in combination and/or incorporation into sunscreens to improve chemoprevention efficacy and reduce skin cancer incidence.

Retinol measurements and retinoid receptor gene expression in patients with multiple sclerosis.

Treatment with interferon (IFN)-beta1a has been associated with decreased disease activity in patients with multiple sclerosis (MS). In several biological systems, type 1 IFNs and retinoids have been demonstrated to have synergistic effects. In these studies, we measured blood and cerebrospinal fluid (CSF) retinol levels and naïve and memory T-helper cell subset percentages in samples from a group of patients with MS. We also examined retinol receptor expression in peripheral blood cells from MS patients with or without a history of prior treatment with IFN-beta1a. The mean plasma retinol level for untreated relapsing-remitting (RR) MS patients was lower than for patients with noninflammatory neurological disease. Among IFN-beta1a-treated RR patients, mean levels were slightly higher than for RR patients not on treatment Lower plasma retinol levels among the MS patents studied were associated with higher CSF retinol index measurements--a measure that was calculated to correct for nonspecific leakage of retinol from blood into CSF. Far the MS samples examined, there was a borderline statstically significant direct correlation between CSF retinol index measurements and CSF memory T-helper cell percentages. Examination of peripheral blood from untreated RR patents for retinoid receptor mRNA expression revealed the expression of the retinoic add receptor (RAR)-alpha, RAR-gamma, and retinoic X receptor (RXR)-alpha receptor subtypes. For RR patients on IFN-beta1a therapy, expression of the some RAR subtypes was noted as well as expression of RXR-beta and RXR-gamma. These studies suggest an association between plasma retinol levels and clincal disease activity in patents with MS and that treatment with IFN-beta1a may be associated with activation of specific retnoid receptor subtypes.

The Role of Specific Retinoid Receptors in Sebocyte Growth and Differentiation in Culture

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors, the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity, it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study, we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation, by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies, and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene, an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580, an RAR-alpha agonist) caused significant decreases in numbers of cells, colonies, and lipid-forming colonies, but with an exception at high doses of all-trans retinoic acid (10-6 M), with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%, mean +/- SEM, lipid-forming colonies, p < 0.01). Furthermore, the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid, adapalene, and CD2043 on both cell growth and differentiation. In contrast, the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p < 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors, especially the beta and/or gamma subtypes, mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors, whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid, the natural ligand of retinoid X receptors.

The Role of Specific Retinoid Receptors in Sebaceous Epithelial Cell (Sebocyte) Growth and Differentiation in Culture

The Role of Specific Retinoid Receptors in Sebaceous Epithelial Cell (Sebocyte) Growth and Differentiation in Culture

Specific retinoid receptors cooperate to signal growth suppression and maturation of human embryonal carcinoma cells.

This study addresses the contributions of specific retinoid receptors during all-trans-retinoic acid (RA)-mediated differentiation and growth suppression of human embryonal carcinoma cells. The pleiotropic effects of RA are mediated by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), members of the nuclear receptor family of transcription factors. After RA-treatment the multipotent human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) displays limited proliferative potential, reduced tumorigenicity, and morphologic and immunophenotypic neuronal maturation. RARgamma over-expression in NT2/D1 cells signals mesenchymal NT2/D1 terminal differentiation while RARalpha and RARbeta do not and RARgamma overcomes retinoid resistance in an NT2/D1 clone (NT2/D1-RI) having deregulated RARgamma expression. Since RARgamma transfectants do not display neuronal maturation, this study sought to identify cooperating retinoid receptors engaged in NT2/D1 differentiation. Through gain of function experiments, this report highlights RXRbeta as playing an important role along with RARgamma in signaling differentiation of NT2/D1 cells. Stable over-expression of RXRbeta, but not RXRalpha or RXRgamma, was found to signal NT2/D1 growth suppression and to induce a non-neuronal morphology and immunophenotype. Notably, co-transfection of RARgamma and RXRbeta resulted in marked growth suppression and for the first time, expression of typical neuronal markers of NT2/D1 differentiation. To clarify the role of RXRbeta and RARgamma in this differentiation program, a modified transient fibroblast growth factor-4 (FGF4) promoter-enhancer reporter assay that reflects effective RA-mediated differentiation of NT2/D1 cells was employed. Transfection of RARgamma or RXRbeta in NT2/D1 cells augments transcriptional repression of the FGF4 reporter and RARgamma and RXRbeta co-transfection markedly repressed reporter activity, indicating the combined role of these receptors in RA-induced NT2/D1 differentiation. Taken together, these findings reveal specific retinoid receptors must cooperate to signal terminal growth suppression and maturation of NT2/D1 cells. Since the transcriptional repression of FGF4 is coupled to the effective maturation of human embryonal carcinoma cells, the described co-transfection strategy should prove useful to identify genes with positive or negative effects on the differentiation program of these tumor cells.

Retinoic acid receptor beta expression and growth inhibition of gynecologic cancer cells by the synthetic retinoid N-(4-hydroxyphenyl) retinamide.

The synthetic retinoid N-(4-hydroxyphenyl) retinamide (4HPR) can inhibit the growth of tumor cells. Preliminary results from a clinical trial suggest that 4HPR may reduce ovarian cancer incidence. We examined the growth-inhibitory effects of 4HPR on gynecologic cancer cell lines in vitro and the role of retinoid receptors in modulating this effect. Twelve human gynecologic cancer cell lines (the ovarian cell lines--A224, AD10, UCI 101, UCI 107, SKOV3, 222, CP70, ML3B, and ML5; the cervical cell lines--HT3 and ME180; and the endometrial cell line--Hec 1A were tested for sensitivity to 4HPR (by assaying cell proliferation rates). Gel electrophoretic analysis of DNA fragmentation was used to measure programmed cell death (apoptosis). Specific retinoid receptor (retinoic acid receptor [RAR] and retinoid X receptor) messenger RNA (mRNA) levels were measured by northern blot hybridization. AD10 cells were stably transfected with human RARbeta complementary DNA, and the effect of 4HPR on cell proliferation was examined. 4HPR inhibited the growth of all 12 cell lines, but to varying degrees; IC50 values (i.e., concentrations that inhibit proliferation by 50%) ranged from 0.3 to 9 microM. Following 4HPR treatment, ovarian cancer cells that were sensitive to 4HPR (222, CP70, and UCI 101; IC50 <3 microM) contained higher levels of RARbeta transcripts than more resistant cells (AD10, ME180, Hec 1A, and A224; IC50 > or =3 microM) (2.8-fold; two-sided P = .006). Anchorage-independent growth of transfected AD10 cells expressing high levels of RARbeta was totally abolished, even in the absence of 4HPR; transfectants expressing low levels of RARbeta exhibited lower levels of anchorage-independent growth and grew more slowly in the presence of 4HPR than control untransfected AD10 cells. 4HPR inhibited the proliferation of ovarian cancer cells in vitro; RARbeta expression appeared to be associated with this effect.

Augmentation of tumor immunity in mice by intralesional injection of vitamin A.

We investigated the antitumor effect of vitamin A(VA) using the double grafted tumor technique to examine whether VA administered into a primary tumor (intralesionally or i.l.) accelerates antitumor immune reactions so that growth of the secondary tumor may be more effectively inhibited than by other systemic administration routes. In the double grafted tumor system, where BALB/c mice were inoculated with MethA fibrosarcoma cells into the right inguinal region (1 x 10[6] cells) on day 0 and later into the left (3 x 10[6] cells) on day 10, the injection of VA at a dose of 1000 IU/mouse i.l., s.c., i.p., and i.v. on days 3 through 7 inhibited the growth of the secondary tumor to the same extent, while VA at the i.l. dose of 100 IU/mouse into the primary tumor inhibited more effectively than by any other administration route. VA did not inhibit the secondary MethA growth in BALB/c (nu/nu) mice. The spleen cells taken from VA-treated tumor-bearing mice prevented the growth of MethA tumors in naive BALB/c mice when given as a mixture with the MethA inoculum (the Winn assay). The delayed-type hypersensitivity (DTH) response to methylated bovine serum albumin (MBSA) antigen was augmented when VA (1000 IU) was injected at the site of the antigen injection. These results suggest that the direct interaction of VA with the tumor cells may be necessary for the tumor immunity-potentiating effect of VA, and that T-lymphocyte-mediated tumor immunity is involved in the anti-tumor effect of VA. The antitumor mechanism of VA seems to involve retinoid receptors, because the benzoic acid derivative Am80, which has been reported to exert retinoidal activity by binding to specific retinoid receptors, also showed activity.

Specificity of retinoid receptor gene expression in mouse cervical epithelia.

Retinoids are powerful regulators of epithelial differentiation and are essential for its maintenance. Because retinoids are necessary for cervical epithelial differentiation, they have been used as chemopreventive agents of cervical dysplasia and neoplasia. We were interested in determining whether different cervical epithelial phenotypes express specific retinoid receptors. The cervical epithelium contains the two phenotypes, stratified squamous and simple columnar, which join at the squamocolumnar junction. In addition, the simple columnar epithelium undergoes squamous metaplasia in response to vitamin A deficiency. Therefore, the cervical epithelium is suitable to study the expression pattern of the retinoid receptors in the three phenotypes, simple columnar, stratified squamous, and squamous metaplastic, simultaneously. The distribution pattern of the major retinoic acid receptor (RAR) isoforms (alpha 1, alpha 2, beta 2, beta 3, gamma 1, and gamma 2) and retinoid-X receptors (RXR alpha, -beta, and -gamma) was studied by in situ hybridization. At the tissue level, RAR alpha (1 and 2) and RXR (alpha and beta) transcripts and, to a lesser extent, RAR gamma (1 and 2) transcripts were associated with the cervical stratified squamous subjunctional epithelium. The simple columnar epithelium, which is highly responsive to vitamin A status, expressed high levels of RAR alpha (1 and 2), RAR beta (2 and 3), and RXR (alpha and beta) transcripts. Only RAR beta (2 and 3) and RXR (alpha and beta) transcripts were down-modulated by the condition of vitamin A deficiency and expressed less in squamous metaplastic foci than the simple columnar epithelium. RXR gamma was undetectable in all three cervical epithelia. At the cellular level, basal and suprabasal expression was found for RARs, and preferential localization of RXRs was seen in basal cells. RXRs are auxiliary proteins for a variety of other nuclear receptors with which they form heterodimers, including RARs. The fact that RXRs are mainly localized in basal and columnar cells of the cervix suggests the need for the regulation and diversity generated by potential heterodimeric interactions in these rapidly proliferating cells in vivo. The unique pattern of expression and localization of the RARs and RXRs in different cervical epithelial tissues and cell types supports the hypothesis that they perform specific functions in cervical epithelial differentiation. This is in contrast to the major isoforms of each RAR, which have similar patterns of expression in the different cervical epithelial phenotypes and cell types, suggesting a redundancy in function.

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NTZ/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/Dl-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.

9-cis and all-trans retinoic acid induce a similar phenotype in human teratocarcinoma cells

Abstract Prior work has shown that all-trans retinoic acid (t-RA) treatment of the human teratocarcinoma (TC) cell line NTERA-2 clone D1 (abbreviated NT2/D1) induces a neuronal phenotype and other cell lineages. This study sought to explore the potential of 9-cis retinoic acid (9-cis RA) as a differentiation-inducing agent of this multipotent cell. Findings reported here show that 9-cis RA induced a phenotype similar to t-RA treatment of NT2/D1 cells. This similarity extended to their effects on the nuclear receptors retinoic acid receptor-β (RAR-β) and retinoid X receptor-α (RXR-α). Both retinoids prominently augmented RAR-β expression and transactivated a reporter plasmid containing putative RAR response elements (RAREs) with direct repeats separated by five nucleotides (DR5). Both retinoids had no appreciable effect on RXR-α expression and both minimally transactivated a reporter plasmid containing putative RXR response elements (RXREs) with direct repeats separated by one nucleotide (DR1). These studies suggest that 9-cis RA and t-RA activate common events during retinoid-mediated NT2/D1 differentiation. This hypothesis was supported by the finding that NT2/D1 cells rendered refractory to t-RA (NT2/D1-R1) were also resistant to 9-cis RA. To discover alterations that could confer retinoid-refractoriness, retinoid receptor expression was examined in NT2/D1-R1 cells. In contrast to NT2/D1, the NT2/D1-R1 cell was found to have reduced RXR-α expression at the level of total cellular RNA. These studies establish the effectiveness of 9-cis RA as a differentiation agent of human TC cells and demonstrate that retinoids with different nuclear receptor affinities can induce similar phenotypes in NT2/D1 cells. In addition, the findings in the retinoid resistant NT2/D1-R1 cell implicate a role for specific retinoid receptors in this human TC differentiation program.