Endogenous Retinoic Acid Levels Research Articles

Transcriptional regulation of genes involved in retinoic acid metabolism in Senegalese sole larvae.

The aim of this study was the characterization of transcriptional regulatory pathways mediated by retinoic acid (RA) in Senegalese sole larvae. For this purpose, pre-metamorphic larvae were treated with a low concentration of DEAB, an inhibitor of RALDH enzyme, until the end of metamorphosis. No differences in growth, eye migration or survival were observed. Nevertheless, gene expression analysis revealed a total of 20 transcripts differentially expressed during larval development and only six related with DEAB treatments directly involved in RA metabolism and actions (rdh10a, aldh1a2, crbp1, igf2r, rarg and cyp26a1) to adapt to a low-RA environment. In a second experiment, post-metamorphic larvae were exposed to the all-trans RA (atRA) observing an opposite regulation for those genes involved in RA synthesis and degradation (rdh10a, aldh1a2, crbp1 and cyp26a1) as well as other related with thyroid- (dio2) and IGF-axes (igfbp1, igf2r and igfbp5) to balance RA levels. In a third experiment, DEAB-pretreated post-metamorphic larvae were exposed to atRA and TTNPB (a specific RAR agonist). Both drugs down-regulated rdh10a and aldh1a2 and up-regulated cyp26a1 expression demonstrating their important role in RA homeostasis. Moreover, five retinoic receptors that mediate RA actions, the thyroid receptor thrb, and five IGF binding proteins changed differentially their expression. Overall, this study demonstrates that exogenous RA modulates the expression of some genes involved in the RA synthesis, degradation and cellular transport through RAR-mediated regulatory pathways establishing a negative feedback regulatory mechanism necessary to balance endogenous RA levels and gradients.

Inhibition of Axl Kinase Reverses the Mesenchymal Phenotype in Leukemic Cells through the Disruption of Retinoic Acid Signaling

Mesenchymal stem cells (MSCs) contribute to the regeneration of mesenchymal tissues, and are essential in providing support for the growth and differentiation of primitive hemopoietic cells within the bone marrow microenvironment. It is becoming increasingly clear that the tumor microenvironment plays a very important role in tumor progression and drug resistance, and the selection of cancer cells possessing the mesenchymal phenotype leads to drug resistance in many different tumor types. We have been exploring the role of the protein Axl in promoting the mesenchymal phenotype in both myeloid and lymphoid malignancies, and the role of Axl in promoting drug resistance in these malignancies. The signaling downstream of Axl that leads to the acquisition of the mesenchymal phenotype has not been well elucidated. Following results from a genetic screen using a zebrafish model, we have discovered a role for retinoic acid (RA) signaling which is regulated by Axl and controls the mesenchymal phenotype in leukemic cells. In addition, recent reports have shown an interaction between a retinoic acid regulated gene, RARRES1, and Axl, leading our group to seek to understand the role of retinoic acid signaling in the control of AXL. We hypothesized that treatment with our AXL inhibitor, TP-0903, would disrupt RA signaling and lead to a reversal of the mesenchymal phenotype in leukemia cells. Following TP-0903 treatment, we interrogated changes in mRNA expression using RT-PCR, protein expression using standard immunoblotting, and endogenous RA levels using a competitive ELISA. We also assessed the effect of TP-0903 on tumor growth in an in vivo model, assessing efficacy of TP-0903 in an MV4-11 xenograft mouse model. One of the genes that we detected being dramatically changed by treatment with TP-0903 was the RA metabolizing protein CYP26A1, suggesting that Axl inhibition indeed leads to changes in RA metabolism. We observed a strong induction of CYP26 mRNA expression following RA treatment in MV4-11 leukemia cells which was also observed in treatment with our AXL inhibitor, TP-0903, at levels as low as 100 nM. We also assessed TP-0903 activity in additional cell lines (HL60, A549, and H1650), and with an alternative AXL inhibitor, R428. Importantly, TP-0903 treatment correlated with increased CYP26 expression and reduced levels of endogenous RA. In vivo, TP-0903 strongly inhibited xenograft tumor volumes by up to 100% with multiple dose levels and treatment schedules. CYP26 expression in fixed tissues correlated well with mRNA levels observed in xenograft tumors following treatment. Taken together, our observations support our hypothesis that inhibition of AXL kinase by TP-0903 can disrupt RA metabolism by inducing CYP26 expression and this disruption of RA metabolism leads to reversal of the mesenchymal phenotype in leukemic cells. DisclosuresSoh:Tolero Pharmaceuticals: Employment. Bahr:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Peterson:Tolero Pharmaceuticals: Employment. Whatcott:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment.

Roles of retinoic acid signaling in normal and abnormal development of the palate and tongue

Palatogenesis involves various developmental events such as growth, elevation, elongation and fusion of opposing palatal shelves. Extrinsic factors such as mouth opening and subsequent tongue withdrawal are also needed for the horizontal elevation of palate shelves. Failure of any of these steps can lead to cleft palate, one of the most common birth defects in humans. It has been shown that retinoic acid (RA) plays important roles during palate development, but excess RA causes cleft palate in fetuses of both rodents and humans. Thus, the coordinated regulation of retinoid metabolism is essential for normal palatogenesis. The endogenous RA level is determined by the balance of RA-synthesizing (retinaldehyde dehydrogenases: RALDHs) and RA-degrading enzymes (CYP26s). Cyp26b1 is a key player in normal palatogenesis. In this review, we discuss recent progress in the study of the pathogenesis of RA-induced cleft palate, with special reference to the regulation of endogenous RA levels by RA-degrading enzymes.

Evolution of retinoic acid receptors and retinoic acid signaling.

Retinoic acid (RA) is a vitamin A-derived morphogen controlling important developmental processes in vertebrates, and more generally in chordates, including axial patterning and tissue formation and differentiation. In the embryo, endogenous RA levels are controlled by RA synthesizing and degrading enzymes and the RA signal is transduced by two retinoid receptors: the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). Both RAR and RXR are members of the nuclear receptor superfamily of ligand-activated transcription factors and mainly act as heterodimers to activate the transcription of target genes in the presence of their ligand, all-trans RA. This signaling pathway was long thought to be a chordate innovation, however, recent findings of gene homologs involved in RA signaling in the genomes of a wide variety of non-chordate animals, including ambulacrarians (sea urchins and acorn worms) and lophotrochozoans (annelids and mollusks), challenged this traditional view and suggested that the RA signaling pathway might have a more ancient evolutionary origin than previously thought. In this chapter, we discuss the evolutionary history of the RA signaling pathway, and more particularly of the RARs, which might have experienced independent gene losses and duplications in different animal lineages. In sum, the available data reveal novel insights into the origin of the RA signaling pathway as well as into the evolutionary history of the RARs.

A Critical Role for the Retinoic Acid Signaling Pathway in the Pathophysiology of Gastrointestinal Graft-Versus-Host Disease

Abstract 341Damage to the gastrointestinal tract during graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, the molecular mechanism(s) responsible for colonic GVHD induction remain obscure. All-trans retinoic acid (RA) is an active metabolite of vitamin A and has recently emerged as a critical regulator of gut immunity. We hypothesized that administration of RA might be a strategy to reduce inflammatory responses during GVHD, particularly within the colon microenvironment. In a B6→Balb/c MHC-mismatched murine allogeneic bone marrow transplantation (BMT) model, we found unexpectedly that exposure of recipient mice to exogenous RA resulted in significantly accelerated GVHD-associated mortality compared with DMSO-treated animals (p<0.0001). Histological examination of GVHD target organs 5 weeks after transplantation revealed a significant increase in pathologic damage in the colons of RA-treated mice compared with DMSO-treated animals (9.7 ± 0.8 versus 3.3 ± 0.9, p<0.001), whereas there was no significant difference in pathology scores in either the lung or liver. RA treatment resulted in significantly increased expression of the gut-homing molecules, CCR9 and α4β7, on donor T cells in mesenteric lymph nodes early after BMT. Notably, expression of other chemokine receptors that have been implicated in GVHD pathogenesis, such as CCR5, CXCR3, and CCR10, was not affected by RA treatment, demonstrating a selective role of RA in imprinting gut-homing donor T cells. Examination of GVHD target organs three weeks after transplantation revealed a significant increase (eight-fold) in the absolute numbers of both CD4+IFN-γ+ (TH1) and CD4+IL-17+ (TH17) cells in the colon of RA-treated mice versus DMSO-treated animals, whereas there were no statistical differences in TH1 or TH17 cells in the spleen, liver and lung. There was also a significant increase in the absolute number of regulatory T cells (Tregs) in the colon of RA-treated mice. However, the increase in Tregs was noted to be more modest (three-fold) than was observed for TH1 and TH17 cells, suggesting a pro-inflammatory role of RA. To further confirm a role for RA in imprinting gut-homing donor T cells during GVHD, we generated vitamin A-deficient mice and employed them as recipients. Depletion of RA in recipient mice by vitamin A deprivation resulted in a dramatic reduction of gut homing molecule expression on donor T cells after allogeneic BMT. Retinoic acid receptor-α (RAR-α) signaling appears to be critical in mediating the effects of RA on alloreactive T cells. In mixed lymphocyte culture, when B6 T cells were stimulated with Balb/c dendritic cells, the presence of an RAR-α agonist was able to significantly enhance the expression of gut-homing molecules on T cells. In contrast, the addition of a selective RAR-α antagonist abolished the effects of RA on T cells. We then measured endogenous RA levels in the colons of mice undergoing either syngeneic or allogeneic BMT by mass spectrometry. These studies revealed that RA was significantly increased in the colon tissue of allogeneic transplant recipients compared with syngeneic transplant recipients early after BMT (day 7). In addition, Real-Time q-PCR analysis revealed a mean 3.5-fold increase in RAR-α expression in purified donor splenic CD4+ T cells 4 days post transplantation when compared to purified naïve CD4+ T cells obtained from original donor animals. Finally, we examined the ability of RAR-α-deficient T cells to cause GVHD. We found that recipients of RAR-α-deficient marrow grafts had significantly decreased GVHD-associated mortality when compared with the recipients of wild type T cells (93% versus 36% at day 90, p=0.002). Histological examination of GVHD target organs on days 10–12 after transplantation revealed a significant reduction in pathologic damage in the colon of mice that received RAR-α-deficient T cells compared with control mice (7.3 ± 2.5 versus 0.8 ± 1.0, p<0.01). In summary, we have identified RA as a novel and critical molecule in the induction of gastrointestinal GVHD. These findings provide evidence that blockade of the RA signaling pathway may represent a novel strategy to mitigate the severity of colonic GVHD. Disclosures:No relevant conflicts of interest to declare.

Presence of Ribeiroia ondatrae in the developing anuran limb disrupts retinoic acid levels

The widespread reports of malformed frogs have sparked interest worldwide to try and determine the causes of such malformations. Ribeiroia ondatrae is a digenetic trematode, which has been implicated as one such cause, as this parasite encysts within the developing tadpole hind limb bud and inguinal region causing dramatic limb malformations. Currently, the mechanisms involved in parasite-induced limb deformities remain unclear. We sought to investigate whether the level of retinoic acid (RA), a morphogenetic factor known to play a critical role in limb bud formation, is altered by the presence of R. ondatrae within the infected tadpole. Alteration of RA levels within the limb bud caused by the presence of the parasite may be achieved in three ways. First, metacercariae are actively secreting RA; second, cercariae, upon entering the limb/inguinal region, may release a large amount of RA; finally, the metacercariae may induce either an increase in the synthesis or a decrease in the degradation of the host's endogenous retinoic acid levels. Here, we show through high performance liquid chromatography and mass spectrometry that limb bud tissue of Lithobates sylvaticus, which has been parasitised, contains 70% more RA compared to the unparasitised control. Furthermore, parasites that have encysted within the limb buds appear to contain substantially less RA (56%) than the free swimming cercariae (defined as the infectious stage of the parasite). Taken together, these data illustrate for the first time that encystment of R. ondatrae leads to an increase in RA levels in the tadpole limb bud and may offer insight into the mechanisms involved in parasite-induced limb deformities.

Quantitative assessment of endogenous retinoids in breast cancer models

Breast cancer pathogenesis involves disregulation of signaling by retinoic acid (RA), the active metabolite of vitamin A and an essential regulator of cell proliferation, differentiation, and apoptosis. It is not clear if changes to endogenous RA concentrations (via RA biosynthesis defects) or changes to proteins that translate the RA signal drive physiological outcomes. We are interested in understanding if endogenous RA levels are altered in tumors and, if so, when does disregulation of RA concentrations occur during carcinogenesis. From evaluations of gene expression of various components of the retinoid pathway, disregulation of RA signaling is an early event in cancer making the retinoid pathway is an attractive metabolic target for intervention and chemoprevention. Altered expression of genes involved in the trafficking of retinoids is indirectly linked to RA synthesis defects with the implication that RA synthesis defects exacerbate tumor progression. However, there has been no direct quantification of endogenous RA to characterize if RA is altered and, if so, the extent of the alteration. Here we describe quantitative mass spectrometric approaches for quantifying endogenous RA and other retinoids in tissue and present data from models used to study mammary carcinoma.

Study on Cytochrome P-450 Dependent Retinoic Acid Metabolism and its Inhibitors As Potential Agents for Cancer Therapy

The relative lack of clinical success with conventional anticancer agents may be due in part to the traditional concept of cancer being a biological state rather than a dynamic process. Redefining cancer as a dynamic disease commencing with carcinogenesis introduces the possibility of chemoprevention. Retinoids offer the promise of a therapeutic option based on differentiation of premalignant as well as malignant cells. Research to date has concentrated on the use of exogenous retinoids in cancer. Although this research continues with new retinoid derivatives, an alternative approach to overcoming the drawbacks associated with exogenous retinoids has been to increase the levels of endogenous retinoic acid (RA) by inhibiting the cytochrome P450- mediated catabolism of RA using a novel class of agents known as retinoic acid metabolism blocking agents (RAMBAs which increase the level of endogenous retinoic acid (RA) within the tumor cells by blocking their metabolism. This approach presents several theoretic advantages.In the present study a wide range of established P-450 inhibitors has been screened to examine their inhibitory activity on all-trans-Retinoic acid (ATRA) metabolism. Forty-one known P450 inhibitors were tested for their inhibitory activity against RA metabolism. Most of them are nitrogen-containing compounds. The results showed that among these compounds only six compounds (N-benzyl-2-phenylethanamine, itraconazole, chlorpromazine, 5-chloro-1,3-benzoxazol-2-amine, proadifen and furazolidone) showed inhibition of RA metabolism which was > 50%. Ketoconazole and liarozole were also screened as standard potent inhibitors in the same system and gave 87.5% and 89% inhibition, respectively. The results indicate that mostly azoles with substituents in positions other than the 1-position on the ring are very weak inhibitors of RA metabolism. The most effective inhibitors (ketoconazole, itraconazole, bifonazole and clotrimazole) are 1-substituted and possess relatively large aromatic groups in the molecule. 1-Substituted imidazoles bind to cytochrome P-450 with a very high affinity but substitution in the other position of the imidazole decreases the binding affinity.

The Histopathalogical Effects of Retinoic Acid on the Tissues

The aim of this study is to sum up the important information that has emerged from the last 10 years of experimental investigations over the effects of retinoic acid (RA) on embryonic structure and adult tissues. Administration of exogenous RA can affect the connective tissues including enhancement of myeloid compartment and suppression of erythroid cells and conversion of hematopoietic stem cells to erythroid progenitors. Also, it is able to induce osteogenic differentiation of stem cells derived from adipose tissues and etc. Examining the neural tissue highlighted that disruption of RA signaling in the adult leads to degeneration of motor neurons and development of some diseases. In vitro administration of All-Trans Retinoic Acid (ATRA) increased dendritic growth and synaptophysin puncta intensity and increased expressions of neuronal nuclei, neuron specific enolase, synaptophysin. RA also promotes expression of a marker of mature astrocytes. On muscular tissue, it can inhibit proliferation of smooth muscle cells (SMC) while promoting differentiation of SMC in vitro instead. The ATRA stimulates skeletal myogenesis while inhibiting cardiomyogenesis and hypertrophy and proliferation of cultured neonatal cardiomyocytes and cardiofibroblasts. In addition, differences in levels of embryonic RA may contribute to variability in great artery anomalies. In epithelial tissue, the squamous epithelium exposed to ATRA showed the columnar differentiation independent to proliferation. Also RA seems able to rescue the regeneration process of injured gut and revealing a better wound healing of the intestine undergone intra-operative radiotherapy. It can interrupt the process of progressive fibrosis, enhancements of the langerhans islets, exocrine pancreas, modulate the health of the mammary glands and repairs the lung cell. Thus, differences in levels of endogenous RA in embryonic and adult tissues may contribute to anomalies and pathogenesis of disease, furthermore RA has paradoxical effects on the parts forming the connective and muscles tissue in equal conditions.

Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism

Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multi-organ birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. We used a microarray approach to identify genes that function as negative feedback regulators of retinoic acid signaling. We screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists and validated them by RNA in situ hybridization. Focusing on genes known to be involved in RA metabolism, we determined that dhrs3a, which encodes a member of the short-chain dehydrogenase/reductase protein family, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the reduction of the RA precursor all-trans retinaldehyde to vitamin A; however, a developmental function has not been demonstrated. Using morpholino knockdown and mRNA over-expression, we demonstrate that Dhrs3a is required to limit RA levels in the embryo, primarily within the central nervous system. Dhrs3a is thus an RA-induced feedback inhibitor of RA biosynthesis. We conclude that retinaldehyde availability is an important level at which RA biosynthesis is regulated in vertebrate embryos.

Both all-trans retinoic acid and cytochrome P450 (CYP26) inhibitors affect the expression of vitamin A metabolizing enzymes and retinoid biomarkers in organotypic epidermis

The biosynthesis of retinoic acid (RA) from retinol is controlled by several enzymes, e.g. dehydrogenases (RalDH2, RoDH-4) and retinol-esterifying enzyme (LRAT), whereas its degradation mainly involves CYP26 enzymes. In keratinocytes, RA activates the nuclear retinoid-receptors inducing the transcription of many genes. Here, we examined the effects of RA and the CYP26 inhibitors, liarozole and talarozole, on retinoid metabolism and RA-regulated genes in organotypic epidermis. RA induced the expression of CYP26 enzymes already after 8 h, whereas LRAT exhibited a later response and peaked at 48 h, indicating a feedback induction of retinol esterification. In line with a reduced biosynthesis of RA from retinol after exogenous RA, the expression of RDH16 reduced 80% in response to exogenous RA. The mRNA expression of RA-regulated genes (KRT2, KRT4, CRABPII and HBEGF) was altered within 24 h after RA exposure. In contrast, the CYP26 inhibitors caused only minor effects, except for a clear-cut induction of CYP26A1 only when combined with minute amounts of exogenous RA. Cellular accumulation of exogenous [3H]RA was higher after talarozole than after liarozole, probably indicating a greater CYP26-inhibitory potency of the former drug. The present study shows that CYP26A1 expression is extremely sensitive to both exogenous RA and increased endogenous RA levels, i.e. due to CYP26 inhibition, and thus an excellent biomarker for retinoid signalling in organotypic epidermis.

Regulation of expression of the retinoic acid metabolizing enzyme CYP26A1 in uteri of ovariectomized mice after treatment with ovarian steroid hormones

The retinoic acid (RA) synthesizing enzymes, retinaldehyde dehydrogenases (RALDH), are expressed in specific spatial and temporal patterns in uterine tissues during estrous cycle and early pregnancy in mice. Expression of RALDH1 and 2 has been shown to be induced by estrogen treatment within the uterus. In this study, we determined the influence of progesterone and 17-ss-estradiol on the uterine expression of the RA-metabolizing enzyme CYP26A1 after specific time intervals (1, 4, 24, and 48 hr after treatment of ovariectomized mice). In a following experiment, we investigated the influence of gestagen (promegestone 0.3 mg/kg body weight), estrogen (estradiol 3 microg/kg), their combination, as well as the antagonizing anti-progesterone hormone (RU 486 10 mg/kg) on the uterine expression of CYP26A1. Expression of CYP26A1 was localized using in situ hybridization and quantified using RT-PCR. CYP26A1 mRNA expression was strongly--although transiently--induced in uterine endometrial epithelial and glandular cells after administration of gestagen or the combination of gestagen + estrogen, but not by estrogen alone. These observations were confirmed by semi-quantitative RT-PCR experiments on whole uteri. Thus, we show that the expression of CYP26A1 in endometrial epithelial cells is regulated by progesterone and not significantly influenced by co-administration of estrogen. These data indicate an additional level of hormonal control of endogenous RA levels in the mouse uterus, where its synthesis would rely on estrogen-dependent expression of RALDH enzymes, whereas its active metabolism would be triggered by progesterone-induced CYP26A1 expression.

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.

Postnatal effects of retinoic acid on cerebellar development.

Retinoic acid(RA) is a potent teratogen to which the early CNS is known to be highly sensitive. However, very little is know about the postnatal effects of RA. The cerebellum is a candidate for postnatal RA toxicity, as it develops late and exhibits temporal patterns of RA synthesis that are synchronized with developmental stages. Northern blotting shows that the cerebellum expresses receptors for RA, predominantly of the RXR group activated by 9-cis RA. To determine whether development can be disrupted by RA excess, newborn rats were injected with RA at a time when endogenous RA levels are normally low. Histological examinations at postnatal day 14 revealed loss of a subpopulation of proliferating granule cells, and adult cerebella showed clusters of ectopic granule cells located in the molecular layer. Thus, although the granule cells may normally be regulated by endogenous RA, they are sensitive to abnormally high RA concentrations.

The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer.

Although significant advances have been made in the treatment of some malignancies, the prognosis of patients with metastatic tumors remains poor. Differentiating agents redirect cells toward their normal phenotype and therefore may reverse or suppress evolving malignant lesions or prevent cancer invasion. In addition, they offer a potential alternative to the classic cytostatic drugs. The purpose of this review was to examine the current and potential future roles of differentiating agents in the treatment of cancer. Initial studies with differentiating agents focused on retinoid therapy. Although retinoids have shown some clinical success, their widespread use has been limited by resistance and, in the chemopreventive setting, toxicity. This has led to the synthesis of a number of new retinoids that currently are undergoing clinical investigation. A further approach to overcoming the drawbacks associated with exogenous retinoids has been to increase the levels of endogenous retinoic acid (RA) by inhibiting the cytochrome P450-mediated catabolism of RA using a novel class of agents known as retinoic acid metabolism blocking agents (RAMBAs). Liarozole, the first RAMBA to undergo clinical investigation, preferentially increases intratumor levels of endogenous RA resulting in antitumor activity. Although studies using exogenous retinoids in this setting have not yet fulfilled their initial promise, studies with a growing set of synthetic retinoids are ongoing. Furthermore, modulation of endogenous retinoids may offer a significant new potential treatment for cancer.

A sensitive bioassay for enzymes that synthesize retinoic acid

Retinoic acid (RA) is a potent regulator of gene transcription and it plays a pivotal role in neural development. As the compound is active at nanomolar concentrations, standard RA detection methods based on high-pressure liquid chromatography (HPLC) are poorly suited for neurodevelopmental questions and single RA measurements require pooling of tissues from very large numbers of embryos. An alternative approach is to determine the potential for RA synthesis by assaying for the enzymes that catalyze the last step of RA synthesis, the irreversible oxidation of retinaldehyde to RA. In a quantitative comparison of retinaldehyde dehydrogenase levels with endogenous RA levels, we found a good concordance between the two parameters. For the detection of retinaldehyde dehydrogenases we have developed an assay which involves analyses of protein fractions, separated by isoelectric focusing (IEF), with a RA responsive cell line; operationally, this technique is a zymography bioassay. This method is exquisitely sensitive: tissue volumes as small as a sugar grain can be assayed, and it can be used on all species. Separation by IEF allows in a single run the identification and relative quantitation of several enzyme isoforms.

Retinoic acid and development of the retina

Abstract Retinoic acid (RA) is known as the activating trigger for a large number of processes in developing and mature vertebrates, and it plays a pivotal role in eye development. We present here a brief review of the RA system in general, and we summarize the evidence for a determining role of RA in the embryonic eye. The earliest and most significant ocular feature influenced by RA is the dorso-ventral axis. A lasting differential expression of different RA generating enzymes along the retinal dorso-ventral axis then creates very high endogenous RA levels, as well as a ventro-dorsal RA gradient, features that are likely to direct morphogenesis along this axis in the embryonic eye. RA is also likely to play a significant role in the function of the mature eye, as some of the chromophore released from photo-bleached rhodopsin is converted to RA, a mechanism for light to directly influence gene expression. The pivotal role of RA in eye morphogenesis may represent a developmental correlate of an evolutionary origin of RA-mediated transcriptional regulation from retinoid usage in vision.

Sex differences in endogenous retinoid release in the post-embryonic spinal cord of the western mosquitofish, Gambusia affinis affinis.

In this study we have shown sex differences in endogenous retinoic acid synthesis and retinaldehyde dehydrogenase activity in the post-embryonic spinal cords of immature female and male G. a. affinis. The F9 reporter cell assay and the zymography bioassay showed that the endogenous retinoic acid levels correlated with the levels of the endogenous enzyme(s) responsible for retinoic acid synthesis. These data also showed that both the endogenous retinoic acid levels and the enzyme(s) were higher in spinal cord segments 8-16 of immature males than in immature females. We have also shown that exogenous treatment of 17 alpha-methyltestosterone results in the masculinization of the immature female's anal fin and its appendicular support elements as well as the endogenous synthesis of retinoic acid and retinaldehyde dehydrogenase activity. The F9 reporter cell assay showed that the endogenous retinoic acid levels were relatively unchanged in spinal cord segments 8-16 of immature males treated with 17 alpha-methyltestosterone. However, the F9 reporter cell assay showed that the endogenous retinoic acid levels in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone were markedly higher than levels observed in the immature males. The data also showed that the activity of the enzyme(s) responsible for the synthesis of endogenous retinoic acid was higher in spinal cord segments 8-16 of immature females treated with 17 alpha-methyltestosterone than in immature males treated with 17 alpha-methyltestosterone. Currently under investigation is the question of what role the endogenous enzyme(s) responsible for the synthesis of retinoic acid plays either alone or in concert with androgen in organizing hormone-dependent sexually dimorphic areas in the teleost body plan.

Localization of CRABP‐I and CRABP‐II mRNA in the early mouse embryo by whole‐mount in situ hybridization: Implications for teratogenesis and neural development

Retinoic acid (RA) has been implicated in vertebrate neural pattern formation. In this paper we analysed the expression patterns of the cellular retinoic acid binding proteins (CRABP-I and II) during early morphogenesis in normal and RA-treated mouse embryos by whole-mount in situ hybridization. This technique allowed a detailed analysis of the spatial and temporal changes in mRNA expression pattern. Both CRABPs were expressed in a rhombomere specific pattern; putative neural crest cells in the branchial arches expressed the CRABPs at levels corresponding to the rhombomere from which they were derived. CRABP-II, but not CRABP-I, was expressed in the neural epithelium caudal to the hindbrain. CRABP-I is strongly expressed in a fine net-like pattern which extends from the caudal diencephalon to the rostral hindbrain and remains predominantly dorsal to the lateral midline of the neural tube. This network corresponds to the pattern formed by the putative first axons of the embryonic mouse brain which are produced by the developing neurons of the mesencephalic nucleus of the trigeminal nerve. Although the expression of CRABP-I was unaffected by a teratogenic dose of RA, CRABP-II expression was increased slightly with no alteration in the normal spatial or temporal boundaries. These results support the suggestion that the CRABPs may play an important role in modulating endogenous RA levels, particularly in the developing nervous system and its neural crest derivatives. Furthermore, the limited ability of CRABP mRNA levels to respond to exogenous retinoids may be a factor in retinoid teratogenicity.

Cellular retinoic acid binding protein: Detection and quantitation in regenerating axolotl limbs

The concentrations of apo (unoccupied), holo (occupied), and total cellular retinoic acid binding protein (CRABP) were measured at various stages of axolotl limb regeneration. The ratio of apo-CRABP to holo-CRABP declined with advancing regenerate stage until the CRABP was all in the holo form. The increase in holo-CRABP is correlated with a stage-dependent shift in the effect of exogenous retinoic acid on regenerate pattern, from pattern duplication to inhibition of regeneration. The data suggest, though they do not prove, that these different morphological effects could be due to a shift from a CRABP-dependent to a CRABP-independent mechanism of exogenous retinoic acid (RA) action that is related to stage-specific variations in endogenous RA levels.