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.     

Retinoic acids regulate apoptosis of T lymphocytes through an interplay between RAR and RXR receptors

Vitamin A deficiency has been known for a long time to be accompanied with immune deficiency and susceptibility to a wide range of infectious diseases. Increasing evidence suggests that retinoic acids derived from vitamin A are involved in the functional regulation of the immune system. Of the two groups of retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs) all-trans and 9-cis retinoic acids are high affinity ligands for RARs and 9-cis retinoic acid additionally binds to RXRs. In cells, at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid by unknown mechanisms. Apoptosis plays a major role in shaping the T cell repertoire and one way in which retinoids may affect immune functions is to influence the various apoptosis pathways. Indeed, it has been shown that retinoic acids can induce apoptosis, increase the rate of dexamethasone-induced death and inhibit activation-induced death of thymocytes and T lymphocytes. Therefore, retinoids together with glucocorticoids may be involved in regulating positive and negative selection of T lymphocytes. Here we demonstrate that retinoids can induce apoptosis of T cells through the stimulation of RARgamma. Specific stimulation of RARalpha, on the other hand, prevents both RARgamma-dependent and TCR-mediated cell death. In all these functions 9-cis retinoic acid proved to be more effective than all-trans retinoic acid suggesting the involvement of RXRs. Based on these results a possible mechanism through which costimulation of RARs and RXRs might affect spontaneous and activation-induced death of T lymphocytes is proposed.     

Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.     

Gene expression profiling identifies retinoids as potent inducers of macrophage lipid efflux

Vitamin A and its naturally occurring derivatives 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA) exert a variety of biological effects including immunomodulation, growth, differentiation, and apoptosis of normal and neoblastic cells. In order to directly study the effects of these retinoids on macrophage gene expression and lipid metabolism, primary human monocytes and in vitro differentiated macrophages were stimulated with beta-carotene, 9-cis RA, and ATRA and global gene expression profiles were analyzed by Affymetrix DNA-microarrays and differentially regulated genes were verified by quantitative TaqMan RT-PCR. Among others, we have identified a strong up-regulation of a cluster of genes involved in cholesterol metabolism including apolipoproteins (apoC-I, apoC-II, apoC-IV, apoE), the scavenger receptor CD36, steroid-27-hydroxylase (CYP27A1), liver X receptor alpha (LXRalpha), and ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Since the CYP27A1 gene displayed the strongest up-regulation on the mRNA level, we cloned various deletion constructs of the promoter region and analyzed the response to retinoids in macrophages. Thereby, a novel retinoic acid-responsive element could be located within 191 bp of the proximal CYP27A1 promoter. To further assess the functional consequences of retinoid receptor action, we carried out phospholipid and cholesterol efflux assays. We observed a strong induction of apoA-I-dependent lipid efflux in stimulated macrophages, implicating an important role for retinoids in cellular functions of macrophages.     

Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells.

Besides nuclear retinoid receptors and cellular retinoid binding proteins also retinoic acid (RA)-synthesizing enzymes (using all-trans-retinal as substrate) and RA-catabolizing enzymes (producing hydroxylated products) may explain the specific effects of retinoids. In the past we have established an active role for 4-hydroxy-RA and 4-oxo-RA, which originally were considered to be inactive retinoids, but in fact are highly active modulators of positional specification in Xenopus development. Here we present evidence for a specific role of hydroxylated RA metabolites in the onset of neuronal differentiation. 4-Hydroxy- and 18-hydroxy-RA are products of the hydroxylation of RA by a novel cytochrome P450 (CYP)-type of enzyme, CYP26, expression of which is rapidly induced by RA. P19 embryonal carcinoma (EC) cell lines stably expressing hCYP26 undergo extensive and rapid neuronal differentiation in monolayer at already low concentrations of RA, while normally P19 cells under these conditions differentiate only in endoderm-like cells. Our results indicate that the effects on growth inhibition and RARbeta transactivation of P19 EC cells are mediated directly by RA, while the onset of neuronal differentiation and the subsequent expression of neuronal markers is mediated by hCYP26 via the conversion of RA to its hydroxylated products.     

Synergistic effect of vitamin D derivatives and retinoids on C2C12 skeletal muscle cells

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.     

Alterations of cellular characteristics of a human ovarian teratocarcinoma cell line after in vitro treatment with retinoids

Differentiation of the human teratocarcinoma derived cell line. PA-1, with retinoids was examined at concentrations (10(-6)-10(-8) M) that did not exhibit an antiproliferative effect during log-phase growth. Treatment with naturally occurring retinoic acid or certain synthetic retinoids (13-cis retinoic acid, Ro10-9359, and Ro13-7410), while not significantly altering the log-phase growth rate, decreased the saturation cell density and mitotic indices after confluence. Retinoid treatment also induced changes in cell morphology, which appear to be related to reorganization of microtubules and microfilaments. Following retinoid treatment, the expression of cell glycoproteins (of 162 kDa, 152 kDa, 143 kDa. and 51 kDa) was altered. Treated cells also exhibited decreased expression of alkaline phosphatase, as well as an increased capacity for intercellular communication as evidenced by gap-junctional transfer of the phosphorylated toxic intermediate of 6-thioguanine to HPRT- cells. Treatment with retinoic acid dramatically reduced the quantity of shed plasma membrane material and altered its composition.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Friend erythroleukemia cell differentiation: induction by retinoids

Abstract. Growth in the presence of retinoids was found to induce erythroid differentiation in Friend murine erythroleukemia (MEL) cells in culture. The program of differentiated functions expressed by retinoid-treated cells was quite similar to that promoted by other inducers of MEL cell differentiation. For example, 70% or more of induced cells synthesized hemoglobin which accumulated to a level of 8 μg–10 μg per 10⁶ cells. The level of acetylcholinesterase activity increased two to five-fold in induced cells, and induction by retinoids, like induction by dimethylsulfoxide (DMSO), promoted the appearance of cell surface lumps or 'blebs'. All-trans retinaldehyde, which promoted maximum hemoglobin and acetylcholinesterase synthesis at a concentration of 5 × 10⁻⁷ M, was found to be a more potent inducer than all-trans retinoic acid or retinol, which both showed maximum induction at 1 × 10⁻⁵ M. Like differentiation promoted by DMSO, retinoid-induced differentiation was inhibited by 10⁻⁷ M dexamethasone.     

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

Functional studies of newly synthesized benzoic acid derivatives: identification of highly potent retinoid-like activity

Three newly synthesized benzoic acid derivatives (terephthalic acid anilides, chalcone carboxylic acid, and azobenzene carboxylic acid), with a certain structural similarity to retinoic acid, were examined for their retinoid-like bioactivity and their capacity to bind to cellular retinoid binding proteins. Two in vitro systems were used to evaluate their retinoid-like bioactivity: inhibition of adipose conversion of ST 13 murine preadipose cells and growth promotion of murine sarcoma virus (MSV)-transformed 3T3 cells in serum-free culture. All three compounds tested inhibited ST 13 adipose conversion at nanomolar concentrations in a manner similar to classical retinoids such as retinoic acid. The growth-stimulating activity of these compounds on MSV-transformed 3T3 cells was one to two orders of magnitude greater than that of retinoic acid. Simultaneous treatment with these compounds and retinoic acid produced only a barely detectable additive effect, suggesting a common mechanism of action, whereas unrelated mitogens, thrombin, and insulin worked synergistically in combination with retinoic acid. None of the compounds competed with retinol for binding to cellular retinol binding protein. However, two of the three competed with retinoic acid for binding to cellular retinoic acid binding protein. This study provides evidence that the newly synthesized compounds should be included among the retinoids and that their strong biological activity will undoubtedly contribute to the biological and medical application of retinoids.     

Conditioned media from solid tumor cell lines treated with retinoic acids both decreases and increases proliferation of capillary endothelial cells.

These studies investigated the effects of retinoic acids on endothelial cell proliferation. Three human neoplastic cell lines, U-373 MG glioblastoma, DU-145 prostate carcinoma, and TCCSUP bladder transitional cell carcinoma, were treated with all-trans, 9-cis, or 13-cis retinoic acids at 0.0001 to 10 microM. Hypoxia was used to ensure the expression of the angiogenic phenotype. Conditioned media (CM) were prepared by hypoxic culturing of the tumor cells with retinoic acids for 24 hours. Then CM were transferred to bovine capillary endothelial cells for 48 hours of normoxic culturing, counted and compared to controls. CM from U-373 MG and DU-145 cells, but not TCCSUP cells, treated with all-trans or 9-cis retinoic acids at several concentrations below 1 microM, caused significant (P<0.05) increases in endothelial cell proliferation of between 13 to 18%. Both nonconditioned and conditioned media, for retinoic acid concentrations above 1 microM, inhibited endothelial cell proliferation. All CM for 13-cis retinoic acid decreased endothelial cell proliferation. These results show that the cytotoxicity of retinoic acids and the growth promoting/inhibiting ability of the conditioned media is retinoic acid isoform, time, concentration, and cell type dependent. Most importantly, the conditioned media from tumor cells treated with low concentrations of all-trans or 9-cis retinoic acids significantly increased endothelial cell proliferation.     

Retinoids are produced by glia in the lateral ganglionic eminence and regulate striatal neuron differentiation

In order to identify molecular mechanisms involved in striatal development, we employed a subtraction cloning strategy to enrich for genes expressed in the lateral versus the medial ganglionic eminence. Using this approach, the homeobox gene Meis2 was found highly expressed in the lateral ganglionic eminence and developing striatum. Since Meis2 has recently been shown to be upregulated by retinoic acid in P19 EC cells (Oulad-Abdelghani, M., Chazaud, C., Bouillet, P., Sapin, V., Chambon, P. and Dollé, P. (1997) Dev. Dyn. 210, 173-183), we examined a potential role for retinoids in striatal development. Our results demonstrate that the lateral ganglionic eminence, unlike its medial counterpart or the adjacent cerebral cortex, is a localized source of retinoids. Interestingly, glia (likely radial glia) in the lateral ganglionic eminence appear to be a major source of retinoids. Thus, as lateral ganglionic eminence cells migrate along radial glial fibers into the developing striatum, retinoids from these glial cells could exert an effect on striatal neuron differentiation. Indeed, the treatment of lateral ganglionic eminence cells with retinoic acid or agonists for the retinoic acid receptors or retinoid X receptors, specifically enhances their striatal neuron characteristics. These findings, therefore, strongly support the notion that local retinoid signalling within the lateral ganglionic eminence regulates striatal neuron differentiation.