Cytosolic Retinoic Acid Binding Protein Research Articles

The Intracellular Vitamin A-Binding Proteins: An Overview of Their Functions*

The intracellular retinoid-binding proteins bind retinol, retinaldehyde, and retinoic acid for purposes of protection against decomposition, solubilize them in aqueous medium, render them nontoxic, and transport them within cells to their site of action. These binding proteins also function by presenting the retinoids to the appropriate enzymes for metabolism. The cytosolic retinol and retinoic acid-binding proteins--CRBP, CRABP, CRBP (II)--function in transport and metabolism of retinoids within parenchymal, intestinal, reproductive, and fetal cells and across blood-organ barriers. A different group of retinoid-binding proteins, more related to serum retinol-binding protein, functions in epididymis and uterus. Retinaldehyde-binding protein aids in the oxidation-reduction reaction of 11-cis-retinol-11-cis-retinaldehyde in the retina, where the interphotoreceptor retinol-binding protein transports retinol between pigment epithelium and photoreceptors. Finally, a group of retinoic acid-binding proteins termed "receptors" functions in the nucleus by attaching to promoter regions of a number of specific genes to stimulate their transcription and thus affect growth, development, and differentiation.

Topical tretinoin or adapalene in acne vulgaris: an overview

Retinoids target several pathoetiologic events of acne vulgaris. The undisputed efficacy of tretinoin, and yet its underutilization, due to apprehension of retinoid dermatitis, triggered a search for newer, well‐tolerated retinoids. The discovery of nuclear retinoic acid receptors has provided clues to a rational design of synthetic, receptor‐selective retinoic acid agonists. Adapalene is an addition to the arsenal of topical retinoids. It possesses the biological properties of tretinoin, but has a distinct physiochemical profile, including high lipophilicity and increased chemical and photostability. It exhibits selective affinity for nuclear retinoic acid receptors and does not bind to cytosolic retinoic acid binding proteins. It exemplifies the formulation of a novel retinoid with specific pharmacologic profile and clinical objectives. Accordingly, numerous clinical trials have compared adapalene and tretinoin in the management of acne vulgaris and concluded that tretinoin 0.05% gel exhibits a greater anti‐acne efficacy than adapalene 0.1% gel, but has higher skin irritation potential. This article reviews the pharmacology of adapalene, including its retinoid receptor binding profile, antiproliferative effects, cell differentiation modulation, comedolytic and anti‐inflammatory activity, and specifically focuses on the comparison of the efficacy and irritation profile of adapalene and tretinoin.

Isolation, Characterization, and cDNA Sequence of a Carotenoid Binding Protein from the Silk Gland of Bombyx mori Larvae

A carotenoid binding protein (CBP) has been isolated from the silk glands of Bombyx mori larvae. The protein has an apparent molecular mass of 33 kDa and binds carotenoids in a 1:1 molar ratio. Lutein accounts for 90% of the bound carotenoids, whereas alpha-carotene and beta-carotene are minor components. Immunological analysis demonstrated the presence of CBP only in the yellow-colored tissues of the silk gland, midgut, testis, and ovary. Several phenotypes of B. mori mutants linked to carotenoid transport have been utilized to characterize CBP. The Y (yellow hemolymph) gene controls uptake of carotenoids from the midgut lumen into the midgut epithelium, and larvae with the +(Y) gene lack this property. Immunoblotting analysis confirmed the presence of CBP in mutants with the dominant Y gene only. Immunohistochemistry verified the localization of CBP in the villi of the midgut epithelium, indicating that CBP might be involved in absorption of carotenoids. A cDNA clone for CBP encoding a protein of 297 amino acids has been isolated from the B. mori silk gland cDNA library. The deduced amino acid sequence revealed that CBP is a novel member of the steroidogenic acute regulatory (StAR) protein family with its unique structural feature of a StAR-related lipid transfer domain, known to aid in lipid transfer and recognition. Lutein-binding capacity of the recombinant CBP (rCBP) determined by incubating rCBP with lutein followed by immunoprecipitation using anti-CBP IgG conjugated to protein A-Sepharose, demonstrated the formation of a lutein-rCBP complex. Sequence analyses coupled with binding specificity suggest that CBP is a new member of the StAR protein family that binds carotenoids rather than cholesterol.

Stimulation of vitamin A 1 acid signaling by the HIV protease inhibitor indinavir

HIV protease inhibitors (PIs) are effective drugs for the treatment of AIDS. However, PI therapy is sometimes associated with side-effects including increased plasma lipids and altered body fat distribution, although fat redistribution may occur in some patients not treated with PIs. Overdosage with vitamin A 1 acid (all- trans-retinoic acid, ATRA) or its metabolites may cause similar changes in lipid metabolism. Moreover, the PI indinavir and retinoids have been associated with nail, skin, and hair defects, suggesting that indinavir and retinoids may exert their effects through similar molecular mechanisms. This hypothesis was tested by examining the effects of PIs on retinoid signaling in vitro. Mesenchymal stem cells (C3H10T1/2) were cultured in the presence of various PIs (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) and synthetic retinoids, and the metabolic response was assessed by measuring the activity of a retinoid-regulated protein, alkaline phosphatase (ALP). Of the PIs tested, only indinavir stimulated ATRA-dependent ALP activity and altered stem cell morphology; the effects of indinavir occurred in the presence of ATRA, but not in its absence. Moreover, indinavir increased the effects of ATRA on lipid accumulation during fat cell differentiation. AGN 193109 (4-[[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), a retinoic acid receptor (RAR) antagonist, inhibited the synergistic effects of indinavir and ATRA, indicating that indinavir increases RAR signaling. However, indinavir did not potentiate ALP activity in the presence of the RAR agonist CH55 (3,5-di- tert-butylchalcone 4′-carboxylic acid). Unlike ATRA, CH55 does not bind to cytosolic retinoic acid binding protein (CRABP), suggesting that CRABP may regulate the effects of indinavir on RAR signaling. These observations support the proposal that altered retinoid signaling promotes some of the adverse reactions associated with indinavir therapy, such as altered lipid metabolism.

Expression and purification of CRABPs from E. coli.

Nuclear-retinoid receptors provide a mechanism of retinoid action, but most likely where expressed, the cytosolic cellular retinoic acid-binding proteins (CRABPs) also affect the ability of retinoids to initiate biological signals (, ). holo-CRABP I sequesters retinoic acid (RA) with a K d value that may be <1 nM, and serves as a high-affinity (K m ~2 nM), efficient substrate of RA metabolism, thereby controlling not only the steady-state concentration of RA, but also its availability (). The impact of CRABP on retinoid metabolism may extend beyond RA. CRABP binds RA metabolites, including 4-OH-RA, 4-oxo-RA, and several metabolites whose structures have not been identified, and could thereby influence then disposition in vivo (). For example the elimination t1/2 of RA in the presence and absence of CRABP I was 35 and 40 min, respectively, in incubations with rat testis microsomes Unbound 4-OH-RA and 4-oxo-RA had elimination t1/2 values of 40 and 6 min, respectively. In contrast, CRABP-bound 4-OH-RA and CRABP-bound 4-oxo-RA were essentially metabolically inert. This shows that CRABP does not affect all retinoids similarly and that it could have a profound influence on the steadystate concentrations of several retinoids in vivo.

Induction of the oxidative catabolism of retinoid acid in MCF-7 cells.

Cytochrome P450-dependent oxidation is a pathway for all-trans-retinoic acid (all-trans-RA) catabolism. Induction of this catabolic pathway was studied in MCF-7 breast cancer cells. MCF-7 cells showed low constitutive all-trans-RA catabolism. Concentration-dependent induction was obtained by preincubation of the cells with all-trans-RA (10(-9) to 10(-6) M). Onset of induction was fast, being detectable within 60 min, with maximal induction (45-fold) obtained after 16 h. Enzymatic characterization of induced all-trans-RA catabolism showed an estimated Km value (Michaelis-Menten constant) of 0.33 microM and a Vmax value (maximal velocity of an enzyme-catalysed reaction) of 54.5 fmol polar all-trans-RA metabolites 10(6) cells(-1) h(-1). These kinetic parameters represent the overall formation of polar metabolites from all-trans-RA. Induction of all-trans-RA catabolism was also obtained with other retinoids, CH55 >> 13-cis-RA = all-trans-RA > 9-cis-RA > 4-keto-all-trans-RA > 4-keto-13-cis-RA > retinol. The potency of the retinoids to induce all-trans-RA catabolism was correlated to their retinoic acid receptor affinity (Crettaz et al, 1990; Repa et al, 1990; Sani et al, 1990). Induction of all-trans-RA catabolism was inhibited by actinomycin D. Furthermore, all-trans-RA did not increase cytosolic retinoic acid-binding protein (CRABP) mRNA levels. These data suggest that induction of all-trans-RA catabolism in MCF-7 cells is a retinoic acid receptor-mediated gene transcriptional event. Induced all-trans-RA catabolism was inhibited by various retinoids with decreasing potency in the order: all-trans-RA > 4-keto-all-trans-RA > 13-cis-RA > 9-cis-RA > 4-keto-13-cis-RA > retinol > CH55. The antitumoral compound liarozole-fumarate inhibited all-trans-RA catabolism with a potency similar to that of all-trans-RA.

Vitamin A deficiency and the expression of retinoic acid receptors during early cardiogenesis in quail embryo.

The vitamin A deficiency-associated lethal syndrome observed in avian embryos may be linked to dysfunction of vitamin A-dependent genes. We tested this hypothesis in a quail embryo model by examining the expression of retinoic acid receptors (RARs) and cytosolic retinoic acid binding protein (CRABP) in normal and vitamin A-deficient embryos during early development. RARα and RARγ mRNA were expressed at the same level in normal and vitamin A-deficient embryos during all stages studied. Expression of CRABP I was low in normal and vitamin A-deficient quail embryos during early development, but increased rapidly at later stages. Two transcripts of RARβ, 3.2 and 3.5 kb, were detected in quail embryos during developmental stages 6-12. In normal emryos the level of the 3.2-kb isoform increased as embryonic development advanced. The expression of the 3.5-kb transcript was significantly decreased in vitamin A-deficient embryos, while the 3.2-kb transcript was undetectable by northern analysis. In situ hybridization of stage 7-8 normal quail embryos using a chicken RARβ2 riboprobe revealed that RARβ2 expression was predominantly associated with the cell populations in heart-forming regions, somites, neural folds, notochord and the presumptive thyroid. In stark contrast, in the vitamin A-deficient quail embryo RARβ2 was not expressed in any of the above cell populations. We conclude that the expressions of RARβ and CRABP I are developmentally regulated. Additionally, the expression of RARβ2 during early embryogenesis is regulated by vitamin A status. We propose that RARβ2 plays an important role in the mechanism of action of retinoids in early avian development. The lack of expression of RARβ2 may be linked to abnormalities of the cardiovascular system.

Acquired Resistance to All-Trans Retinoic Acid Therapy in Acute Promyelocytic Leukemia

The current treatment of acute promyelocytic leukemia (APL, also called AML3 subtype) is focused on differentiating agents such as the vitamin A derivative all-trans retinoic acid (ATRA). This agent is a novel and very promising therapy for this disease that is characterized cytogenetically by a translocation t(15;17)(q21;q22) involving the α-retinoic acid on chromosome 17 and the PML gene on chromosome 15. Clinical trials have demonstrated that ATRA followed by or combined with conventional chemotherapy may be more beneficial than chemotherapy for inducing complete remission. Unfortunately, as a single agent ATRA does not appear to be able to maintain patients in remission (median 6 months) and when relapse occurs resistance to a second induction of ATRA therapy is observed in almost all cases. Recently, our laboratory investigated whether specific features of the AML3 cells at relapse could explain the in vivo resistance observed. We have demonstrated that AML3 patient cells (from 4 patients) at relapse show high levels of CRABP, a cytosolic retinoic acid binding protein, and this protein was not detected prior to ATRA therapy. Relapse-AML3 cells (n=12) showed reduced differentiation induction when compared with ‘virgin’-AML3 cells. Results from this study suggest that CRABP could modulate ATRA cellular concentrations reaching the nucleus. This induced, ATRA hypercatabolytic state should be monitored during consolidation therapy and at relapse, to detect ATRA resistance in AML3.

Single Versus Multiple Dose Administration of All-trans-retinoic Acid during Organogenesis: Differential Metabolism and Transplacental Kinetics in Rat and Rabbit

Standard teratogenicity testing is usually performed by administration of a test compound daily throughout an extended period of organogenesis (e.g., between Days 6 and 15 in rat and 6 and 18 in rabbit). On the other hand, single dose experiments during a specific period were often demonstrated to be more effective in unveiling a particular teratogenic effect. We have assessed here if toxicokinetics is an important factor for the interpretation of the differences between two administration regimens of all-trans-retinoic acid (all-trans-RA) in two species. The transplacental pharmacokinetics of a low teratogenic dose of all-trans-RA administered orally were compared in a single versus multiple dose regimen in both the Wistar rat and the Swiss hare rabbit. In both species, the single dose animals were treated on Gestational Day 12, while the multiple dose animals received daily doses from Gestational Days 7 through 12. Pharmacokinetic profiles were determined for maternal plasma and embryo after dosing on Gestational Day 12 (for both the single and multiple dose regimens) and analyzed by reverse-phase HPLC. The dose used for both species was 6 mg/kg body wt/day which has recently been reported to be a marginal to low teratogenic dose when administered daily throughout organogenesis. In both rat and rabbit, the AUC of all-trans-retinoic acid in maternal plasma was much reduced (factor of 9 in the rat, factor of 2 in the rabbit) after multiple application as compared to the single administration, presumably due to enzyme induction. A similar, but not as pronounced effect was also observed in the embryo of both species. This diminished effect in the embryo indicates a relative increase of placental transfer at the lower maternal plasma concentration observed after multiple dosing, which may possibly be due to an increased availability of binding sites such as cytosolic retinoic acid binding protein and nuclear receptors in the embryo. In the rat, also the metabolite levels were reduced, while in the rabbit, the metabolites of the 13-cis-configuration were concomitantly increased. Our results suggest that multiple administration of a drug such as retinoic acid, which induces its own elimination pathways, results in substantially lowered drug levels in maternal plasma and embryo.(ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous Status of Retinoids and Their Cytosolic Binding Proteins in Limb Buds of Chick vs Mouse Embryos

The morphogenetic role of all-trans-retinoic acid (RA) during limb development, especially its activity in directing anterior/posterior pattern formation remains controversial. We have measured retinoids and cytosolic binding proteins in anterior and posterior pieces of limb buds from comparably aged chick and mouse embryos expecting that localization patterns of morphological significance will be evident across species. Many species differences were found including: (1) nondetectable 3,4-didehydroretinoic acid (ddRA) in mouse limb buds, the predominant morphogenetically active retinoid in chick limbs; the precursor of ddRA, 3,4-didehydroretinol (ddROH), was also not present in mouse limb buds but found in high concentration in chick limb buds; (2) a higher concentration of morphogenetically active retinoid (RA + ddRA) in chick compared to mouse limb buds; (3) a high level of retinyl esters (1.5 μM) in chick limb bud, but undetectable concentration in mouse limb buds; and (4) a higher level of cytosolic retinoic acid binding proteins (CRABPs) in chick limb buds, especially CRABP II, which is 10× greater than in mouse limb buds. An interesting finding seen in mouse and chick limb buds was a disparity between the concentration of ligand and cytosolic binding protein. Retinol (ROH) and ddROH were present in much higher concentrations than cytosolic retinol binding protein (CRBP). Conversely, RA and ddRA were in far lower concentration than CRABPs. Any morphological significance of these disparities is unknown. A putative gradient of RA, high posteriorly, was found in chick limb buds as in earlier studies (Thaller and Eichele, 1987), but the magnitude of this gradient was less than previously reported. We also found ddRA in equal concentration in the anterior and posterior pieces of chick limb bud studied here, further weakening the idea of an RA gradient as the direct force in anterior/posterior pattern formation. The "free" concentration of retinoic acid, calculated from ligand and binding protein concentration and affinity, was slightly higher in posterior chick limb tissue (2.0X) and in mouse limb buds at a concentration similar to the Kd of murine nuclear retinoic acid receptors

Effects of vitamin A deficiency and repletion on rat glucagon secretion.

To determine whether vitamin A is involved in pancreatic alpha cell function, we tested for (a) effects of vitamin A deficiency on glucagon release from perifused islets and perfused pancreases, and (b) the presence of cytosolic retinol-binding proteins (CRBP) and retinoic acid-binding proteins (CRABP), in the glucagon-secreting alpha cell line, ln-R1-G9. Arginine 19 mM plus glucose 2.8 mM-stimulated glucagon secretion was markedly impaired in islets and pancreases of vitamin A-deficient rats or rats that had at some time been cycled through vitamin A deficiency (ever A-def) despite repletion with retinoids for 2-4 weeks. Insulin secretion was impaired likewise. Repletion starting early in the development of vitamin A deficiency and for a longer period of time (18 or 60 days) did not restore glucagon secretion, but did normalize insulin secretion. CRBP and CRABP were present in ln-R1-G9 cells. We conclude that (a) vitamin A deficiency is associated with a defect in glucagon secretion; (b) The defect in secretion occurs early in the course of vitamin A deficiency; (c) The defect persists despite repletion; and (d) The requirement of vitamin A for secretion and the presence of CRBP and CRABP in glucagon-secreting cells support a physiologic role for vitamin A at the alpha cell level.

Retinoic Acid Receptor, Cytosolic Retinol-Binding and Retinoic Acid-Binding Protein mRNA Transcripts and Proteins in Rat Insulin-Secreting Cells

To define the mechanism of vitamin A action at the beta-cell level, we tested for the presence of messenger RNA for retinoic acid receptors alpha, beta, and gamma; cytosolic retinol-binding protein; and cytosolic retinoic acid-binding protein in RINm5F cells, an insulin-secreting cell line, and determined whether cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein are present in isolated purified normal rat beta-cells. Northern blot analyses showed two transcripts of retinoic acid receptor alpha messenger RNA (3.8 and 2.4 kb), one transcript of retinoic acid receptor messenger RNA (3.8 kb), and one transcript of cytosolic retinol-binding protein (0.9 kb) in RINm5F cells. Ribonuclease protection assays also showed the presence of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in RINm5F cells. Quantitatively, cytosolic retinol-binding protein levels were 0.10 +/- 0.02 pg/micrograms total RNA. Using specific radioimmunoassays, normal isolated purified rat beta-cells contained CRBP (19.2 +/- 2.38) and cytosolic retinoic acid-binding protein (16 +/- 0.53 ng/10(6) cells). The presence of message for retinoic acid receptors alpha and gamma, cytosolic retinol-binding protein, cytosolic retinoic acid-binding protein, and the gene products of cytosolic retinol-binding protein and cytosolic retinoic acid-binding protein in insulin-secreting cells support a mechanism of vitamin A action and role for cytosolic and nuclear receptors at the beta-cell level similar to that suggested in nonendocrine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal ethanol ingestion effects on fetal rat brain vitamin A as a model for fetal alcohol syndrome.

Fetal embryo, head, and brain tissue from different gestational ages were analyzed for retinol content, nuclear retinoic acid receptor and cytosolic retinoic acid binding protein levels after maternal ethanol ingestion and compared with fetal levels in control diet pregnancies. Retinol levels in fetal embryo and brain of ethanol-ingesting pregnancies were 2- to 3-fold higher than fetal embryo and brain retinol of control pregnancies. Nuclear retinoic acid receptor was lower in 10-day embryo of ethanol pregnancies and apparently unaffected in fetal head and brain by maternal ethanol consumption at other days of gestation. In fetal head there was a significant overall ethanol effect on cytosolic retinoic acid binding protein, with increased levels in fetal tissue from ethanol-consuming pregnancies. These observations of altered embryo, fetal head, and fetal brain retinol and receptor protein levels support the hypothesis of a possible role of vitamin A in fetal alcohol syndrome.

Control of Epidermal Differentiation by a Retinoid Analogue Unable to Bind to Cytosolic Retinoic Acid-Binding Proteins (CRABP)

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.

Multi-Stage Program of Differentiation in Human Epidermal Keratinocytes: Regulation by Retinoids

The proliferation and differentiation of epidermal keratinocytes in vivo and in vitro is influenced by a variety of different factors, including several peptide growth factors, protein kinase C activators, retinoids, and various cytokines. Retinoids can affect the proliferation of human epidermal keratinocytes either positively or negatively and influence the multi-step program of differentiation in epidermal keratinocytes at very specific stages. Epidermal keratinocytes express nuclear retinoic acid receptors, RARalpha, and RARgamma. It is likely that at least some of the alterations in gene expression induced by retinoids are mediated through these RAR. The cytosolic retinoic acid-binding protein (CRABP), which is differentially expressed during squamous differentiation of human epidermal keratinocytes, may control the effective concentration of retinoic acid in the cell and therefore regulate indirectly gene expression.

Differentiation of F9 Embryonal Carcinoma Cells by Synthetic Retinoids: Amplitude of Plasminogen Activator Production Does Not Depend on Retinoid Potency or Affinity for F9 Nuclear Retinoic Acid Receptors

Retinoic acid and analogues (retinoids) are able to induce the differentiation of F9 murine embryonal carcinoma stem cells into endoderm-like cells. The secretion of plasminogen activator (PA) which accompanies this differentiation is a good index of the biological response of F9 cells to retinoids. We have previously reported that the potency of a series of natural and synthetic retinoids, evaluated by the concentration which provokes half-maximal induction of PA, correlates well with the affinity of these compounds for the endogenous F9 nuclear retinoic acid receptors, but not for the cytosolic retinoic acid binding protein, CRABP. In this paper we show that various retinoids differ, not only in terms of potency, i.e. the dilution at which they are active, but also in terms of the amount of PA that they induce. This parameter, called amplitude, is used to quantify the extent of PA induction by a given retinoid relative to retinoic acid. The amplitude parameters of synthetic retinoids are found to vary over a wide range and are independent of both potency and binding affinity for F9 retinoic acid receptors. It is proposed that the amplitude of the biological response to a given retinoid is the resultant of three factors: (i) the total or partial agonist character of the retinoid; (ii) the binding spectrum of the retinoid for the various types of retinoic acid receptors; (iii) the chemical and metabolic stability of the retinoid in the test system.

Phorbol ester decreases cytosolic retinoic acid binding protein (CRABP) capacity in normal but not psoriatic epidermis.

AbstractThe retinoic acid binding protein (CRABP) characterized in human epidermal cytosol exhibits a three‐fold increased binding capacity in lesional psoriatic epidermis as compared to normal or uninvolved skin. Treatment of epidermal homogenate from normal subjects by phorbol ester + ATP decreases the specific binding capacity of CRABP without affecting its dissociation constant (Kd=10 nM). The same effect was not observed in involved and uninvolved psoriatic epidermis. These results could be related to the previously reported decreased protein kinase C activity in psoriatic skin. They also suggest that posttranslational events could be responsible for pathological and pharmacological variations in CRABP binding capacity.

Effect of all- trans-retinoic acid on nuclear RNA polymerase activity in chemically-induced rat mammary tumors

In the present study the effect of all-trans-retinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (1) The tumor mince was incubated with 1 μM RA for 30 min at 30°C; the RNA polymerase activity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 25°C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the addition of RA-CRABP into the reaction mixture did not alter the enzyme activity. These results suggest that alteration of RNA polymerase activity may be an essential step in the retinoid action in mammary tissues.

Cytosolic retinoic acid binding protein in the human pancreas.

Cytosolic retinoic acid receptor in carcinoma, chronic pancreatitis, and normal pancreatic tissue were examined using sucrose density gradient centrifugation, isoelectric focussing on agarose gel and saturation analysis. Thirteen patients were studied. Cytosolic retinoic acid binding protein (cRABP) was detected in all the samples with chronic pancreatitis and pancreatic carcinoma, but not in the normal tissue. Using sucrose gradient centrifugation, the highest concentrations of cRABP were found in pancreatic carcinoma tissues, ranging from 5.5-23.9 pmol/mg protein. These concentrations were markedly different than in chronic pancreatitis tissue (0.7-2.7 pmol/mg protein). Saturation analysis of cRABP showed a mean dissociation constant of 21.5 nM and maximum binding sites of 5.2 pmol/mg protein. Cytosolic retinoic acid binding protein was separated at an isoelectric point of 4.5 on agarose gel. The presence of cRABP suggest that retinoic acid may have a role to play in the function of the pancreas.

An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[ b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity ( K d of about 2 n m), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.