Low Concentrations Of Retinoic Acid Research Articles

Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

Directed Differentiation of V3 Interneurons from Mouse Embryonic Stem Cells

Excitatory commissural V3 interneurons (INs) of the ventral spinal cord have been shown to balance locomotor rhythm regularity and robustness in vivo. Unfortunately, due to the scarcity of these cells in the spinal cord, in vitro studies of dissociated V3 INs have yet to be reported. In this study, we developed an induction protocol for V3 INs from mouse embryonic stem cells. The effect of the concentration of a strong sonic hedgehog (Shh) agonist (smoothened agonist [SAG]) and retinoic acid (RA) on expression of progenitor p3 and postmitotic V3 IN transcription factor markers (ie, Nkx2.2 and Sim1) was examined. Cells were differentiated toward a more ventral fate by increasing the duration of SAG exposure from 4 days in a previously established motoneuron induction protocol to 6 days. At the end of the induction period, transcription factor expression was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, in situ hybridization, and flow cytometry. Lower concentrations of RA and a longer duration of SAG exposure led to increased levels of p3 and V3 marker expression. This novel induction protocol reveals the importance of Shh signaling duration in the dorsal-ventral patterning of the neural tube, and it provides a method to obtain V3 INs for future studies to allow better understanding their role in rewiring and regeneration after spinal cord injury.

Generation of V2a Interneurons from Mouse Embryonic Stem Cells

V2a interneurons of the ventral spinal cord and hindbrain play an important role in the central pattern generators (CPGs) involved in locomotion, skilled reaching, and respiration. However, sources of V2a interneurons for in vitro studies are limited. In this study, we developed a differentiation protocol for V2a interneurons from mouse embryonic stem cells (mESCs). Cells were induced in a 2(-)/4(+) induction protocol with varying concentrations of retinoic acid (RA) and the mild sonic hedgehog (Shh) agonist purmorphamine (Pur) in order to increase the expression of V2a interneuron transcription factors (eg, Chx10). Notch signaling, which influences the commitment of p2 progenitor cells to V2a or V2b interneurons, was inhibited in cell cultures to increase the percentage of V2a interneurons. At the end of the induction period, cell commitment was assessed using quantitative real-time polymerase chain reaction, immunocytochemistry, and flow cytometry to quantify expression of transcription factors specific to V2a interneurons and the adjacent ventral spinal cord regions. Low concentrations of RA and high concentrations of Pur led to greater expression of transcription factors specific for V2a interneurons. Notch inhibition favored V2a interneuron over V2b interneuron differentiation. The protocol established in this study can be used to further elucidate the pathways involved in V2a interneuron differentiation and help produce sources of V2a interneurons for developmental neurobiology, electrophysiology, and transplantation studies.

The CYP26 inhibitor R115866 potentiates the effects of all-transretinoic acid on cultured human epidermal keratinocytes

All-trans retinoic acid (RA) is known to regulate keratinocyte proliferation and differentiation, and retinoids are used as therapeutic agents in certain dermatological disorders, such as psoriasis and acne. Epidermal expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced by RA treatment and HB-EGF is responsible for RA-mediated epidermal hyperplasia in vivo. RA also induces HB-EGF expression in cultured keratinocytes and alters their differentiating phenotype. R115866 is a specific inhibitor of the cytochrome P450 isoform CYP26, which is involved in the metabolic inactivation pathway of RA. Thereby, R115866 is thought to be able to increase the intracellular levels of endogenous RA. To determine whether or not R115866 potentiates the effect of low concentrations of RA on keratinocytes. We analysed HB-EGF, involucrin and keratin 10 mRNA and protein levels in autocrine human keratinocyte cultures incubated for 18 h with RA or R115866 alone and with RA and R115866 combinations. RA induced HB-EGF and involucrin expression in a concentration-dependent manner, whereas it inhibited keratin 10 expression. R115866 alone had no effect on the expression of these genes. However, when R115866 was combined with low concentrations of RA, HB-EGF and involucrin expression was induced. These results strongly suggest that R115866 potentiates the effects of RA on epidermal keratinocytes when RA is present at low concentrations.

Epithelial differentiation is a determinant in the production of eotaxin-2 and -3 by bronchial epithelial cells in response to IL-4 and IL-13

The composition of the airway epithelium is dynamic and epithelial differentiation is regulated by endogenous mediators as well as inhaled substances. In atopic asthma the differentiation of the epithelium is altered. Various studies have addressed the ability of cultured airway epithelial cells to release the eosinophil-attractant chemokines eotaxin, eotaxin-2 and eotaxin-3 using epithelial cell lines or poorly differentiated primary cells. Since little is known about the role of the epithelial differentiation state in the response of epithelial cells to stimuli that increase production of mediators such as the eotaxins, we analyzed the effect of differentiation state on the production of the eotaxins. In particular, we investigated the effects of the Th2 cytokines IL-4 and IL-13 on eotaxin-2 and -3 production by primary human bronchial epithelial cells and examined whether their production is affected by epithelial cell differentiation using both submerged and air–liquid interface (ALI) cultures. The results show that both IL-4 and IL-13 increase eotaxin-2 and -3 mRNA expression and protein release in submerged- and ALI-cultures. Moreover, epithelial differentiation in ALI-cultures appeared an important determinant in the regulation of eotaxin-2 and -3. Mucociliary differentiation of the epithelial cells was induced by culture in the presence of a high concentration of retinoic acid (RA), whereas low concentrations of RA resulted in a flattened squamous epithelial phenotype. Mucociliary differentiated ALI-cultures expressed and released more eotaxin-3 upon stimulation with IL-4/IL-13, whereas eotaxin-2 production was predominantly found in squamous differentiated ALI-cultures. TNFα reduced IL-4-induced eotaxin-2 release in submerged cultures but not in ALI-cultures; no effects on eotaxin-3 synthesis were observed. The results indicate that epithelial differentiation is an important determinant in Th2 cytokine-induced eotaxin-2 and -3 release by airway epithelial cells. These findings may provide new insights into the role of airway epithelial differentiation and Th2 cytokines in the pathogenesis of inflammatory lung disorders such as asthma.

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment.

Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61alpha, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61alpha forms a protein complex with collagen and Hsp47, an ER-resident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61alpha is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61alpha may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37 degrees C and 43 degrees C (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61alpha. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61alpha. Sec61alpha, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61alpha levels, suggesting that Sec61alpha production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61alpha in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.

PPAR gamma activators induce differentiation of B12 oligodendrocyte‐like cells and rat spinal cord oligodendrocytes

The regulation of CNS lipid metabolism by nuclear receptors and their relation to cell differentiation remains undetermined. Since myelinating oligodendrocytes are the major lipid‐synthesizing cells in the CNS, we characterized the effect of PPAR activation in a CNS derived cell line that expresses oligodendrocyte markers and compared these effects with oligodendrocyte primary cultures (90–95% pure). The rat glioma derived B12 cell line express the three major PPAR isoforms (PPAR α, β and γ) and present a large number of peroxisomes, indicating an important lipid metabolism. Treatment with ciprofibrate, a general PPAR activator and clinical hypolipidemic, induces proliferation arrest, process extension and a moderate rise in the expression of acyl‐CoA oxidase, a specific marker of peroxisomal proliferation. Cell growth arrest by ciprofibrate is enhanced 100‐fold by low concentrations of retinoic acid (0.01 μm), suggesting the involvement of the PPAR‐retinoid acid receptor heterodimers. Since ciprofibrate possibly acts by modifying the concentration of endogenous PPAR ligands, we traced its effects to PPARγ by using isoform specific ligands: Troglitazone and 15‐deoxy‐prostaglandin J2, both of which induce growth arrest and process extension in B12 cells. These effects were corroborated on rat spinal cord derived oligodendrocytes primary cultures, where a significant rise in the number of mature oligodendrocytes is observed in response to PPARγ activators. These results show that PPARγ, a master gene in the differentiation of adipose tissue could be involved in the lipid metabolism of maturing oligodendrocytes.

Beta-carotene and lung cancer

Abstract Does beta-carotene increase, rather than decrease, human lung cancer rates? A large body of observational epidemiologic study has demonstrated that individuals who eat more fruits and vegetables rich in carotenoids and/or who have higher levels of serum beta-carotene have a lower risk of cancer, particularly lung cancer. This inverse relationship has been particularly strong in lung cancer patients with a history of heavy smoking. However, there is contradictory evidence from recent human intervention studies using beta-carotene supplements (20­30 mg per day). An increase in risk of lung cancer among smokers who took beta-carotene supplements was reported in the Alpha Tocopherol, Beta-carotene Cancer Prevention (ATBC) Trial and among smokers and asbestos-exposed workers in the Beta-Carotene and Retinol Efficiency Trial (CARET), but not among male physicians in the United States in the Physicians Health Study (only 11 % of whom were current smokers). Whether there is a true hazard associated with beta-carotene has been evaluated in control studies using the ferret. This animal mimics the human tissue metabolism of beta-carotene, and has been used for studies of tobacco smoking and inhalation toxicology. In the first study, ferrets were given a high-dose beta-carotene supplement equivalent to 30 mg per day in humans, and exposed cigarette smoke or both for six months. A strong proliferative response in lung tissue and squamous metaplasia were observed in all beta-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared to the control group, beta-carotene-supplemented animals (with or without smoke exposure) had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited reductions in RAR-beta gene expression (a tumor suppressor gene). Further, ferrets given a high-dose beta-carotene supplement and exposed to tobacco smoke had fourfold elevated expressions of c-jun and c-fos genes. In a second study, ferrets were given either physiological- or pharmacologic-dose beta-carotene supplementations, which were equivalent to 6 mg vs. 30 mg per day in humans, respectively. The animals were exposed to cigarette smoke for six months. The retinoic acid concentration and RAR beta-gene expression were reduced in the lung tissues, whereas the expression of AP1, cyclin D1, and proliferative cell nuclear antigen were greater in the high-dose, beta-carotene-supplemented animals with or without smoke, as well as the smoke-exposed, low-dose, beta-carotene-supplemented animalsbut not in the low-dose, beta-carotene-supplemented animals alone, as compared with the control group. Squamous metaplasia was only observed in the lung tissues of high-dose, beta-carotene exposed groups with or without smoke (but not the low-dose beta-carotene plus smoke group, the low-dose beta-carotene-supplemented group, or the control group). These data show that in contrast with the pharmacologic dose of beta-carotene, a physiologic dose of beta-carotene in smoke-exposed ferrets has no detrimental effectand, in fact, may afford weak protection against lung damage induced by cigarette smoke. Further studies from our laboratory have revealed an instability of the beta-carotene molecule in the lungs of cigarette smoke-exposed ferrets. Oxidized beta-carotene metabolites may play a role in lung carcinogenesis: by inducing carcinogen-bioactivating enzymes, facilitating the binding of metabolites of benz[a]pyrene to DNA, enhancing retinoic acid metabolism by P450 enzyme induction with subsequent down-regulation of RAR-beta, and acting as pro-oxidants, causing damage to DNA. Ferret studies under highly controlled experimental conditions using high- and low-dose beta-carotene in the presence of alpha tocopherol and ascorbic acid (thereby stabilizing the beta-carotene molecule) showed protective effects against smoke-induced lung squamous metaplasia in ferrets.

Melatonin-induced suppression of PC12 cell growth is mediated by its Gi coupled transmembrane receptors

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the melatonin response suggesting that melatonin may be acting through one of its Gi coupled cell surface receptors. This is confirmed by Western blots demonstrating the presence of MT1 receptors in PC12 cells. Coupling of the Gi protein to these receptors is demonstrated by failure of melatonin to suppress cell growth in PKA deficient A126-1B2-1 mutant PC12 cells. Similarly, melatonin failed to prevent cell proliferation when cells were incubated in the presence of the PKA inhibitor, Rp-cAMP. Retinoic acid and dexamethasone, agents known to effect PC12 cell growth and/or differentiation, displayed differential effects on the actions of melatonin. In the presence of melatonin and low concentrations of retinoic acid (100 nM), PC12 cell proliferation was stimulated compared to that seen with either agent alone, whereas no increase in cell proliferation was observed when higher concentrations of retinoic acid (100 μM) were used. The effects of dexamethasone on suppression of PC12 cell growth were additive with that of melatonin whereas, 1,25-dihydroxyvitamin D 3 (IC 50=10 nM), which by itself had no effect on PC12 cell growth, was found to inhibit the melatonin response. This study demonstrates that inhibition of PC12 cell growth, at physiological concentrations of melatonin, is mediated by cAMP-dependent cell surface receptors and this response is altered by other growth factors known to effect PC12 cell proliferation and differentiation.

Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the AscidianHerdmania curvata1

Ascidians, along with other urochordates, are the most evolutionary distant group from vertebrates to display definitive chordate-specific characters, such as a notochord, dorsal hollow nerve cord, pharynx and endostyle. Most solitary ascidians have a biphasic life history that has partitioned the development of these characters between a planktonic microscopic tadpole larva (notochord and dorsal nerve cord) and a larger sessile adult (pharynx and endostyle). Very little is known of the molecular axial patterning processes operating during ascidian postlarval development. Two axial patterning homeobox genes Otx and Cdx are expressed in a spatially restricted manner along the ascidian anteroposterior axis during embryogenesis and postlarval development (i.e., metamorphosis). Comparisons of these patterns with those of homologous cephalochordate and vertebrate genes suggest that the novel ascidian biphasic body plan was not accompanied by a deployment of these genes into new pathways but by a heterochronic shift in tissue-specific expression. Studies examining the role of all-trans retinoic acid (RA) in axial patterning in chordates also contribute to our understanding of the role of homeobox genes in the development of larval and adult ascidian body plans. Our studies demonstrate that RA does not regulate axial patterning in the developing ascidian larval neuroaxis in a manner homologous to that found in vertebrates. Although RA may regulate the expression of some ascidian homeobox genes, ectopic application of RA does not appear to alter the morphology of the larval CNS. However, treatment with similar or lower concentrations of RA, have a profound effect on postlarval development and the juvenile body plan. These changes are correlated to a dramatic reduction of Otx expression. Through these RA-induced effects we infer that while RA may regulate the expression of some homeobox genes during embryogenesis it has a far more dramatic impact on postlarval development where regulative processes predominate.

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 RARβ 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.

Retinoic Acid Inhibits CD40 + Interleukin-4–Mediated IgE Production In Vitro

To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)–mediated B-cell activation, the effect of 10−12 to 10−6 mol/L RA was studied in anti-CD40 (1 μg/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4–mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% ± 5% in PBMC and 55% ± 4.4% in B cells by all-trans RA, and 58% ± 6.7% and 51% ± 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10−14mol/L for B cells and >10−10 mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10−8mol/L for all-trans RA (94% ± 1.8%) and 96% ± 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10−10 mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-γ (IFN-γ) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors , β, and γ, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor , β, and γ. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the  receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4–stimulated B cells in vitro.© 1998 by The American Society of Hematology.

Retinoic Acid Inhibits CD40 + Interleukin-4–Mediated IgE Production In Vitro

To elucidate the role of retinoic acid (RA) in anti-CD40 + interleukin-4 (IL-4)–mediated B-cell activation, the effect of 10−12 to 10−6 mol/L RA was studied in anti-CD40 (1 μg/mL) + IL-4 (5 ng/mL)-mediated proliferation and Ig synthesis by human peripheral blood mononuclear cells (PBMC) and B cells in healthy donors. Anti-CD40 + IL-4–mediated proliferation of PBMC and B cells was inhibited by RA in a dose-dependent manner, with maximal inhibition of 62% ± 5% in PBMC and 55% ± 4.4% in B cells by all-trans RA, and 58% ± 6.7% and 51% ± 4.7%, respectively by 13-cis RA. IgE synthesis was even more markedly inhibited by RA starting at concentrations of >10−14mol/L for B cells and >10−10 mol/L for PBMC. Maximal inhibition of IgE production for B cells was at 10−8mol/L for all-trans RA (94% ± 1.8%) and 96% ± 3.2% for 13-cis RA. Low concentrations of RA inhibiting IgE synthesis (10−10 mol/L) affected neither B-cell proliferation nor the production of IgA, IgG, and IgM. Elucidation of the mechanism involved in this inhibition of IgE production shows that epsilon germline transcription is decreased by RA, whereas production of interferon-γ (IFN-γ) was not enhanced in the presence of RA. To differentiate whether the RA effect was mediated by RA receptors α, β, and γ, the expression of the retinoic acid receptors (RAR) was examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The data show that unstimulated human peripheral B cells express mRNA of the RA receptor α, β, and γ. Using retinoids with different receptor binding specificity (CD336, CD437, CD2019, CD367), dose-dependent inhibition of IgE synthesis was shown by all four derivates, but was most marked by an RA binding the α receptor with high specificity. Taken together, this study shows that RA inhibits IgE production of anti-CD40 + IL-4–stimulated B cells in vitro.© 1998 by The American Society of Hematology.

Exogenous retinoic acid during gastrulation induces cartilaginous and other craniofacial defects in Fundulus heteroclitus.

Embryonic levels of retinoic acid (RA) and the response of cells to RA are critical to the normal development of vertebrates. To understand the effects of RA signaling in Fundulus heteroclitus, we exposed embryos to a range of RA concentrations of 2 h during gastrulation. Embryos exposed to low concentrations of RA (10(-10)-10(-7) M) develop normally, whereas those exposed to higher concentrations (5 x 10(-7)-10(-4) M) develop characteristic dose-dependent defects. We describe, in detail, four stages of development that represent morphological effects of RA on (1) cell death and defects in the brain, heart, and eye, (2) relative size and differentiation, (3) duplications of pectoral fins, and (4) deletions in craniofacial cartilage elements. Analysis of cartilaginous skeletal elements demonstrates distinct patterns of deletions in the neurocranium and pharyngeal skeleton in response to increasing concentrations of RA. In F. heteroclitus, RA treatment during gastrulation results in five highly consistent phenotypes, which we have incorporated into an index of embryonic RA defects. This index should be valuable in the genetic analysis of RA pathways and in evaluating chemicals that interfere with embryonic RA signaling.

Retinoids modulate P2U purinergic receptor-mediated changes in transcervical paracellular permeability.

In human cervical cells, extracellular ATP induces an acute decrease in the resistance of the lateral intercellular space, the phase I response, followed by a delayed increase in tight junctional resistance, the phase II response. These responses depend on vitamin A because incubation of cells in retinoid-free medium (RFM) abolished both responses. Treatment with retinoic acid restored the phase I response in full, but the amplitude of the phase II response was restored only partly. Shorter incubations and lower concentrations of retinoic acid [half-maximal effective concentrations (K 1/2) = 0.1 microM] were required for restoring the phase I response than were required for reversing the phase II response (K 1/2 = 1 microM). The phase I response could be restored by ligands that bind to either retinoic acid receptors (RARs) or retinoid X receptors, but only RAR agonists had an effect on phase II response. RFM had no effect on decreases in resistance induced by ionomycin, but it attenuated phase II-like increases in resistance induced by KCl or by 1,2-dioctanoyl-sn-diglycerol (diC8). Actinomycin D blocked phase II response but not phase I response or the responses to ionomycin, KCl, or diC8. These results suggest that retinoids act on cervical cells via distinct retinoid receptor mechanisms and modulate phase I and phase II changes in resistance by regulating distinct signal mechanisms.

Effect of retinoic acid on melanoma cell-derived factor stimulation of fibroblast glycosaminoglycan synthesis.

The hyaluronan-rich matrix that surrounds many tumours and facilitates tumour cell growth and invasion is thought to be predominantly synthesized by normal stromal cells stimulated by tumour cell-derived factors. This study examines the possibility that the production of tumour cell-derived factors that stimulate fibroblast glycosaminoglycan (GAG) synthesis may be blocked by exposure to differentiation-inducing agents such as retinoic acid. We have demonstrated that Hs294T, C8161 and A375 human melanoma cell lines release factors into their medium that stimulate normal fibroblast GAG synthesis. Exposure of these melanoma cells to retinoic acid failed to mediate any significant reduction in growth over a 7-day period. Retinoic acid failed to block the tumour cell production of GAG-stimulating activities and even enhanced the activities produced by the C8161 cell line, particularly at low retinoic acid concentrations (48% stimulation at 10(-9) M retinoic acid; P < 0.02). Addition of retinoic acid directly to fibroblast cultures exposed to fibroblast-conditioned medium resulted in an inhibition of GAG synthesis with a 33% inhibition observed at 10(-5) M. Addition of retinoic acid to fibroblast cultures exposed to the tumour cell-conditioned medium failed to inhibit the stimulation of GAG synthesis. Other differentiation-inducing agents, such as hexamethylene-bis-acetamide and butyrate, also failed to block the production of tumour cell-derived GAG-stimulating activities. These results demonstrate that retinoic acid and other differentiation-inducing agents fail to inhibit melanoma cell production of fibroblast GAG synthesis-stimulating factors or their action upon fibroblasts.

An Analysis of Retinoic Acid-induced Gene Expression and Metabolism in AB1 Embryonic Stem Cells

Murine embryonic stem cells such as the AB1 cell line undergo differentiation in the presence of retinoic acid (RA) into an extraembryonic epithelial cell type. This results in the activation of genes such as Hoxa-1, Hoxb-1, laminin, collagen IV(alpha1), tissue plasminogen activator, RARbeta, and CRABPII. The CRABPI gene is regulated in an unusual fashion; CRABPI message and protein levels are induced at low concentrations of RA, but induction is diminished at higher concentrations. AB1 cells take up RA rapidly from the medium, and the addition of low, exogenous concentrations of RA to the culture medium results in very high intracellular RA concentrations. For example, AB1 stem cells cultured in 5 nM [3H]RA have an internal [3H]RA concentration of 1-2 microM within the first hour. AB1 cells also metabolize [3H]RA to more polar RA derivatives. The half-life of RA in AB1 cells not previously exposed to RA is about 2-2.5 h versus 40-45 min in cells cultured for 2-3 days in 1 microM exogenous RA. Thus, the enzyme(s) which metabolize RA are induced or activated by RA. Furthermore, the local concentration of RA required to elicit some biological responses may be higher than previously thought.

Liarozole potentiates the cancer chemopreventive activity of and the up-regulation of gap junctional communication and connexin43 expression by retinoic acid and beta-carotene in 10T1/2 cells.

Liarozole has been reported to inhibit P450 enzymes responsible for the catabolism of retinoic acid. This suggests that it may increase the effectiveness of cancer chemopreventive agents, such as retinoic acid, and pro-vitamin A carotenoids, such as beta-carotene, which may yield retinoids. To test this we have utilized the 10T1/2 cell assay system of neoplastic transformation. Simultaneous treatment with Liarozole (10(-5) M) potentiated by a factor of 1000 the ability of low concentrations of retinoic acid (10(-10) M) to inhibit carcinogen-induced neoplastic transformation, to up-regulate gap junctional communication and to increase connexin43 expression. When tested under conventional culture conditions, Liarozole itself partially suppressed neoplastic transformation and up-regulated gap junctional communication; this ability appears due to the presence of retinol in the serum component of cell culture medium. When assays for junctional communication and of connexin43 expression were performed under defined conditions, in the absence of serum, Liarozole was ineffective alone, yet still augmented the effects of retinoic acid. HPLC analysis of cell culture medium demonstrated that Liarozole (10(-5) M) completely protected retinoic acid (10(-6) M) from catabolism over a 48 h period. Potential effects of Liarozole on the activities of carotenoids were also examined. Inhibition of neoplastic transformation by the pro-vitamin A carotenoid beta-carotene, but not by the non-pro-vitamin A carotenoid canthaxanthin, was moderately potentiated by Liarozole. The augmentation of response to beta-carotene was more apparent when tested under defined conditions; here Liarozole strongly increased junctional communication and Cx43 expression. In contrast, Liarozole did not potentiate the activity of canthaxanthin under defined conditions, while increasing the activity of 4-keto retinoic acid, a likely metabolite. Liarozole also elevated connexin43 expression in early passage human fibroblasts. These data indicate that rapid catabolism of retinoic acid limits its in vitro activities, that a portion of the action of beta-carotene as a cancer preventive agent is due to its conversion to retinoic acid and that canthaxathin exerts its chemopreventive action largely independent of conversion to 4-keto retinoic acid.

Cell Type-Specific Regulation of Expression of Transcription Factor AP-2 in Neuroectodermal Cells

AP-2 is a cell type-specific DNA-binding transcription factor that regulates selected target genes in vertebrate organisms. Here we investigated cell type-specific expression and regulation of AP-2 in neuroectodermal cell lineages. During retinoic acid (RA)-mediated differentiation of P19 embryonal carcinoma cells into neuroectodermal cell types that include immunohistochemically defined neurons and astrocytes, we observed a strong induction of AP-2 transcripts and protein. In contrast, AP-2 mRNA was not induced in P19 cells which undergo mesoendodermal differentiation in response to 1% dimethylsulfoxide or low concentrations of RA, respectively. The potential of both neurons and astrocytes to express AP-2 was ascertained by using cerebellar neurons and astrocytes derived from newborn mice. Unlike these types of cells, microglial cells do not express AP-2. Dibutyryl cyclic AMP further enhanced levels of AP-2 transcripts in both P19 astrocytes and primary astrocytes which also respond to agents elevating intracellular cAMP (noradrenaline, isoproterenol, forskolin). The cAMP-dependent induction of AP-2 could be blocked by inhibitors of protein kinase A. In contrast to its action in P19 cells, RA had no effect on AP-2 mRNA levels in primary astrocytes. Our results indicate that AP-2 may play a role as a retinoic acid-sensitive regulator during differentiation of neurons and glia from an embryonic neural precursor. Furthermore, AP-2 may be involved in gene transcription in both mature neurons and astrocytes.

Retinoic acid is required for and potentiates differentiation of acute promyelocytic leukemia cells by nonretinoid agents

Patients with acute promyelocytic leukemia (APL) associated with the t(15;17) translocation and fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RAR-alpha) genes achieve complete remission but not cure with all-trans retinoic acid (RA), NB4, a cell line derived from a patient with t(15;17) APL that undergoes granulocytic differentiation when treated with pharmacologic doses of RA, was used as a model for differentiation therapy of APL. We found that NB4 cells are resistant to differentiation by nonretinoid inducers such as hexamethylene bisacetamide (HMBA), butyrates, vitamin D3, or hypoxanthine, all of which can induce differentiation in the commonly used HL60 leukemia cell line. Preexposure of NB4 cells to low concentrations of RA for a period as short as 30 minutes abolished resistance to nonretinoids and potentiated differentiation. Sequential RA and HMBA treatment yielded maximal differentiation by 3 days of drug exposure, whereas the effect of RA alone peaked after 6 days and yielded a smaller percentage of differentiated cells. RA also reversed NB4 cell resistance to butyrates and allowed for synergistic differentiation by these agents. Pretreatment with HMBA before exposure to RA failed to stimulate differentiation. Sequential RA/HMBA treatment also markedly increased the extent of differentiation of primary cultures of bone marrow and peripheral blood mononuclear cells from three APL patients. In one case RA/HMBA treatment overcame resistance to RA in vitro. Together, these results suggest that intermittent low doses of RA followed by either HMBA or butyrates may be a useful combination in the treatment of APL. This clinical strategy may help prevent or overcome RA resistance in APL.