Anhydroretinol, a retinoid active in preventing mammary cancer induced in rats by N-methyl-N-nitrosourea.

As determined by in vitro tests, anhydroretinol, a metabolic product of retinol, was bound specifically by serum retinol-binding protein and by cellular retinol-binding protein but not by cellular retinoic acid-binding protein or the nuclear receptors, RARs and RXRs. For rats dosed with the mammary carcinogen, N-methyl-N-nitrosourea (45 mg/kg body weight) and given diets containing either the retinoid vehicle, anhydroretinol (67, 134, 268, or 536 mg/kg of diet), or retinyl acetate (328 mg/kg of diet), there were, over a 90-day observation period, no significant differences in body weights. The compound did not accumulate in liver tissue or cause an increase in hepatic levels of retinyl palmitate (potential problems observed with other retinoids). The numbers of mammary cancers were as follows: no retinoid, 4.5/rat; retinyl acetate, 2.1/rat; and increasing doses of anhydroretinol, 2.9, 3.3, 3.0, and 1.7/rat, respectively. Thus, anhydroretinol, at non-toxic levels, was effective as a preventive agent in this experimental model of breast cancer.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Retinyl substituted-benzyl ethers. Inhibition of mammary carcinogenesis by retinyl 3,4,5-trimethoxybenzyl ether (RTMBE).

Recently, we reported that retinyl 2-propynyl ether (RPE) inhibits MNU-induced mammary cancer in rats and is less toxic than RME and retinyl acetate. The preparation and biological investigations of retinyl ethers have now been extended to retinyl substituted-benzyl ethers, some of which bind to cellular retinol-binding protein. In long-term (160-180 days) experiments, retinyl 3,4,5-trimethoxybenzyl ether (RTMBE) has been shown to be active against MNU-induced mammary cancer in Sprague-Dawley rats. In effectiveness, RTMBE is comparable, at least, to retinyl acetate; but, unlike retinyl acetate, RTMBE is comparatively non-toxic to rats and mice, is not converted enzymatically to retinol, and does not cause significant increases in retinyl palmitate concentrations in the liver. RTMBE reaches high concentrations in mammary tissue. Two of the four RTMBE congeners that were evaluated in 90 day studies were moderately effective in inhibiting mammary carcinogenesis.

Analysis of homo- and heterodimerization of retinoid receptors in solution.

To characterize the dimerization of retinoid receptors in solution, RAR alpha homodimers and RAR alpha-RXR alpha heterodimers, formed in the absence or the presence of a naturally occurring RA response element (betaRARE) under different ionic conditions, were analyzed by size-exclusion fast protein liquid chromatography and sucrose density gradient sedimentation. In the presence of [3H]RA both RAR alpha and RXR alpha existed primarily as monomers of 50 kDa in solutions containing 80 mM KCl. However, when betaRARE was included in these incubations, a 40-fold increase in the occurrence of both the RAR alpha homodimers and the RAR alpha-RXR alpha heterodimers (125 kDa) was observed. The presence of RAR alpha and RXR alpha in the betaRARE-associated homo- and heterodimers was confirmed by the positive interaction of the receptors with the specific antibodies. Both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers bound betaRARE even in the absence of the ligand RA with the heterodimer showing a 2- to 4-fold greater affinity than the homodimer for the DNA binding element. When the receptors were incubated in solutions of increasing ionic concentration (50-300 mM KCl), a decrease in the amount of both RAR alpha homodimers and RAR alpha-RXR alpha heterodimers was accompanied by a corresponding increase in the monomeric fraction even in the presence of betaRARE, suggesting that the high salt concentrations inhibit the surface to surface interactions between the monomers. These observations suggest that in vivo, as in solution, the formation of a stable retinoid receptor dimer complex is dependent upon both receptor-receptor and receptor-RARE interactions.

Didehydroretinoic acid: retinoid receptor-mediated transcriptional activation and binding properties.

All-trans-3,4-Didehydroretinoic acid (vitamin A2 acid; DDRA) is one of the retinoids present in human skin, the most responsive tissue to retinoid treatment. To understand the mechanism of action of DDRA in the control of differentiation and tumorigenesis, we studied its interaction with cellular retinoic acid-binding proteins (CRABPs) and nuclear all-trans-retinoic acid (RA) receptors (RARs), and 9-cis-retinoic acid receptors (RXRs). The IC50 plots of DDRA for inhibition of [3H]RA binding to CRABP I and II and to RAR alpha, beta and gamma illustrate that this retinoid binds with the same affinity as RA to these proteins. DDRA, however, showed higher affinity than RA for RXR alpha. Evaluation of the transcriptional activation potential of DDRA in CV-1 cells showed that this retinoid induced RAR alpha-mediated transcription to the same magnitude as RA in the 10(-9) to 10(-6) M concentration range. However, in comparison to RA, DDRA produced a 2- to 3-fold higher activation of the transcription mediated by RXR alpha homodimers, as well as RAR beta-RXR alpha heterodimers. These results suggest that the biological activity of retinoids in the skin may be attained through the joint potential of both RA and DDRA.

Retinyl ethers as cancer chemopreventive agents. Suppression of mammary cancer.

It had been demonstrated previously that retinyl methyl ether (RME) can suppress carcinogen-induced mammary cancers in vivo. It had also been shown that RME is demethylated enzymatically to retinol and produces the toxic effects of retinol; however, a rationale was developed for further investigations of retinyl ethers and was the basis for the synthesis and biological evaluations of new retinyl ethers for the chemoprevention of mammary cancer, reported herein. Two of the new retinyl ethers, retinyl 3-methyl-2-butenyl ether (RMBE) and retinyl 2-propynyl ether (RPE), were evaluated for the suppression of mammary cancers in vivo. RMBE, RPE, RME, the 2,3,6-trimethyl-4-methoxyphenyl analogue of RME, and retinyl acetate (a positive control) were incorporated individually into the feed of rats that had been injected with N-methyl-N-nitrosourea to induce mammary cancers. Ninety-day tests of these compounds for suppression of mammary cancer showed that RPE has significant cancer chemopreventive activity, comparable to that of retinyl acetate in simultaneous tests. RMBE demonstrated borderline activity. Both RPE and RMBE were less toxic than retinyl acetate or RME and, in contrast to the other retinoids, did not cause accumulation of large amounts of retinyl palmitate in the liver. Further investigations of RPE showed that it accumulated in mammary tissue after a single oral dose was administered to female rats, reached maximum concentrations within 24 h, and was still present at 75-80% of maximum concentrations after 72 h. In ethanol at 25 degrees C, RPE slowly underwent intramolecular cyclization; small amounts of the cyclized product also appeared in mammary tissue of rats dosed with RPE. During the mammary cancer bioassay, however, RPE was essentially stable in the feed. Some of the new retinyl ethers, as well as RME, bind to cellular retinol-binding protein.

Characterization of cellular retinoic acid-binding protein I from chick embryo and its ligand binding properties.

In order to characterize cellular retinoic acid-binding protein (CRABP) from chick embryos, we resolved the [3H]RA-binding activity of embryo extracts by Mono Q anion-exchange chromatography. Fractions containing [3H]RA-binding proteins eluted in two peaks at 7 and 10 min with the latter showing 10-fold higher activity than the former. When the [3H]RA-binding activity in the major peak was resolved on a Superose-12 size-exclusion column, a protein of about 15,000 Da, similar in size to CRABP I or II, was eluted. The identity of this RA-binding component as CRABP I was confirmed by its immunopositive reaction with a CRABP I-specific monoclonal antibody. The chick embryo CRABP I, upon electrophoresis on native gel, however, showed slower migration than the mouse CRABP I, although both exhibited similar isoelectric pH (pI) of about 4.5. Equilibrium binding studies performed under saturating levels of RA indicated that the retinoid bound to the chick CRABP I with a Kd of 27 nM, a value similar to that reported for the native form of this protein from other species. Moreover, as indicated by their IC50 values, the relative binding affinities of various RA analogs for chick CRABP I are consistent with those obtained with human and mouse CRABP I. These results demonstrate that the major RA-binding protein expressed in chick embryo, while having a different charge as judged by electrophoretic mobility, is similar to mouse CRABP I in its size, pI, antigenic specificity, and ligand binding properties.

Conformationally defined 6-s-trans-retinoic acid analogs. 3. Structure-activity relationships for nuclear receptor binding, transcriptional activity, and cancer chemopreventive activity.

We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.

Analysis of the effects of CRABP I expression on the RA-induced transcription mediated by retinoid receptors.

The involvement of cellular retinoic acid-binding protein I (CRABP I) in the RA signaling was investigated by examining its effects on the interaction of retinoid receptors (RARs and RXRs) with RA-response elements (RAREs) as well as on the RA-induced transcription mediated by retinoid receptors. Analysis of the expression of mouse CRABP I from a cDNA expression plasmid in COS-1 cells revealed that this protein was about 5-fold more abundant in cytosol than in nuclei. The identity and the localization of CRABP I in the cytoplasm as well as the nuclei were also confirmed by the immunoperoxidase staining of the transfected COS-1 cells with CRABP I-specific antibody. When the nuclear extract containing a 10-fold molar excess of CRABP I was incubated with RAR alpha extract in the presence of [3H]RA and resolved on an FPLC size-exclusion column, a 20% decrease in the bound radioactivity in the RAR alpha fraction was accompanied by a proportional increase in the CRABP I fraction. In contrast, the addition of CRABP I did not significantly affect the interaction of RAR alpha or RAR alpha-RXR alpha heterodimers with RAREs. Moreover, the coexpression of CRABP I in CV-1 cells did not markedly inhibit or enhance the transcription activated by RARs and RAR alpha-RXR alpha heterodimers under RA concentrations ranging from 10(-10) to 10(-6) M. These results demonstrate that CRABP I, while it might be important for RA homeostasis, is not directly involved in the retinoid receptor-mediated RA-signaling pathway.

Retinyl methyl ether: binding to transport proteins and effect on transcriptional regulation.

Retinyl methyl ether (RME) which prevents cancers of the rat mammary gland, binds to cellular retinol-binding protein and serum retinol-binding protein but not to cellular retinoic acid-binding protein or to the nuclear retinoid receptors, RARs/RXRs. Since the biochemical effects of retinoids likely involve activation or suppression of RAR/RXR-mediated gene transcription, we evaluated such activity of RME by performing cotransfection assays involving CV-1 cells, expression vectors containing RAR and/or RXR cDNA, and an appropriate reporter vector. In the concentration range of 10(-9)-10(-6), RME did not activate transcription by either of the heterodimers (RARalpha, beta or gamma/RXR alpha) or the homodimer (RARalpha/RARalpha). The retinoid, however, exhibited concentration-dependent inhibitory effects on the basal level of transcriptional activity (no other retinoid added) of both the RAR beta- and RARgamma/RXRalpha heterodimers and of the retinoic acid-induced transcriptional activation of the RARgamma/RXRalpha receptors. Thus, RME acted as a retinoic acid antagonist, a role possibly involved in its cancer preventive activity.

Biochemical characteristics and differentiating activity of 4-oxo analogs of retinoic acid.

3-Methyl-4-oxoretinoic acid and 3-cinnamyl-4-oxoretinoic acid bind to a cellular retinoic acid-binding protein (CRABP-II) and to a retinoic acid-receptor protein (RARa). These analogs of 4-oxoretinoic acid, as well as the parent compound, have less binding affinity than retinoic acid. Cotransfection assays in CV-1 cells with plasmids containing cDNAs for RAR alpha, RAR beta and RAR gamma (homodimers) and RAR alpha-RXR alpha and RAR beta-RXR alpha (heterodimers), indicate that 3-cinnamyl-4-oxoretinoic acid induces relatively less transcriptional activity than 4-oxoretinoic acid and its 3-methyl analog, both of which are less effective than retinoic acid. In differentiating mouse F9 embryocarcarcinoma cells, the order of effectiveness is retinoic acid > 4-oxoretinoic acid = 3-methyl-4-oxoretinoic acid > 3-cinnamyl-4-oxoretinoic acid. This order of potency is similar to that for inhibition of induction of ornithine decarboxylase (ODC) activity and for prevention of papillomas on the skin of mice. Binding to CRABP-II and activation of RARs appear to be important factors for expression of differentiating activity, inhibition of induction of ODC activity and prevention of papillomas on the skin of mice.

N-(4-hydroxyphenyl)retinamide: interactions with retinoid-binding proteins/receptors.

The cellular transport, metabolism and biological activity of retinoids are mediated by their specific binding proteins and nuclear receptors. For an understanding of the mode of action of retinoids with potential cancer chemopreventive or other biological activity, it is important to study their interactions with these binding proteins and receptors. In our attempts to understand the action of N-(4-hydroxyphenyl)retinamide (4HPR) and other retinamides in the prevention of cancer, we observed that 4HPR binds to a serum protein with a molecular size of approximately 20,000. The retinoid, however, did not show any binding affinity for cellular retinol-binding protein (CRABP) or for cellular retinoic acid-binding protein (CRABP). However, it showed binding affinity for the nuclear receptors of retinoic acid (RARs) equivalent to 15% of that of retinoic acid. The physicochemical properties of the 4HPR binding protein in the serum were identical to those of serum retinol binding protein (RBP). Antibodies against RBP quantitatively immunoprecipitated the protein-4HPR complex, confirming that the retinoid specifically binds to RBP. Although retinol and 4HPR cross-competed for RBP binding, N-phenylretinamide, in which the 4-hydroxyl group is absent, and N-(4-methoxyphenyl)retinamide, a major cellular metabolite of 4HPR, in which the hydroxyl group is blocked, did not show affinity for the binding protein. The results indicate that the hydroxyl group of 4HPR is essential for binding of this type of retinoid to RBP. Thus, our studies suggest that serum transport of 4HPR may be facilitated by RBP. To bind more efficiently to CRBP, CRABP, or RARs/RXRs, the retinoid may require further metabolic change.

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

A conformationally defined 6-s-trans-retinoic acid isomer: synthesis, chemopreventive activity, and toxicity.

A conformationally defined retinoic acid analog (1) which contains a dimethylene bridge to maintain the 6-s-trans orientation for two terminal double bonds in the polyene chain was synthesized. A Reformatsky reaction was utilized to extend the polyene chain of the starting enone, which provided exclusively the 9Z-configuration for the intermediate aldehyde. A Horners-Emmons condensation with this aldehyde then produced retinoic acid analogs with both 9Z- and 9Z,13Z-configurations. An I2-catalyzed isomerization of the intermediate 9Z-aldehyde yielded the all-E-aldehyde, which was olefinated as above to yield the (all-E)- and (13Z)-retinoic acid analogs of 1. Each configurational isomer of 1 was evaluated for its ability to inhibit the binding of retinoic acid to CRABP (chick skin) and to inhibit the chemical induction of ornithine decarboxylase in mouse skin. In each assay (all-E)-1 was the most active isomer, and this activity was comparable to or better than that for (all-E)-retinoic acid. (all-E)-1 and (13Z)-1 were both shown to be equally effective as (13Z)-retinoic acid in suppressing the proliferation of human sebaceous cells in vitro. (all-E)-1 was further evaluated for its ability to prevent the induction of mouse skin papillomas and to induce signs of vitamin A toxicity in mice. The cancer chemopreventive activity of (all-E)-1 was comparable to that of (all-E)-retinoic acid, and the toxicity was comparable to or slightly better than that of the natural vitamin.

Retinoyl beta-glucuronide: lack of binding to receptor proteins of retinoic acid as related to biological activity.

Retinoid beta-glucuronides have emerged as biologically active, water-soluble, natural retinoids with relatively few toxic and teratogenic effects. The mechanism of action of these glucuronides in the control of epithelial differentiation, growth, and tumorigenesis is unknown. Since retinoyl beta-glucuronide (RAG) contains a free carboxyl group, we studied the interactions of RAG with cellular retinoic acid-binding protein (CRABP) and nuclear receptors of retinoic acid (RARs), the possible mediators of the biological action of retinoic acid (RA). RAG did not exhibit any significant affinity to bind either CRABP or RARs. During 24- and 48-hr incubations of RAG in chick cytosol, detectable amounts of RA were generated which interacted with the RA receptors. In chick skin, the biological activity of RAG may be due to this slowly released RA. Other possible modes of action of RAG are suggested.

A new retinoid-binding protein with a low molecular weight.

Cellular retinoid-binding proteins and nuclear receptors may mediate the intracellular transport and the action of retinoids in the control of differentiation and tumorigenesis. We report a new retinoid-binding protein (Ret BP) with a molecular size of 4,000 that binds retinol, retinoic acid, and some of their derivatives. Purification of Ret BP from chick skin cytosol involved DEAE-Sephadex, Sephadex G-100, and Mono Q column chromatography. The Ret BP-retinoid complex eluted at 195 mM NaCl during Mono Q column chromatography using a 0-300 nM NaCl gradient. Superose-12 column chromatography indicated a molecular size of 4,000 for Ret BP. The binding protein showed a pI of 6.8 on electrofocusing in ampholines of pH 3-10. Ret BP may act as an affinant for retinoids in the cell, and may serve to dispense the ligands to their respective functionally active sites.

Detection of cellular retinoic acid-binding protein in chick embryonic tissues by monoclonal antibodies.

We describe the production of monoclonal antibodies (MAbs) directed against cellular retinoic acid-binding protein (CRABP) and their application for the quantitation and localization of CRABP during the development and growth of chick embryo. Three MAbs, classified as D-10 and H-6 (IgG1-isotype), and G-4 (IgM-isotype), exhibited the highest degree of immunoreactivity for chick embryo CRABP. The antibodies showed partial reactivity to CRABP from rat testis. None of the MAbs showed cross-reactivity with cellular retinol-binding protein or fatty acid-binding protein which are structurally similar to CRABP. The antigen-specificity was confirmed by immunoblot analysis as well as by fast protein liquid chromatographic analysis. The radioimmunoassay developed for MAb (D-10) provided a detectability range of 0.5-5.0 ng of CRABP in the standard displacement curves. An abundance of CRABP was found in embryonic skin, brain, testis and eye. Several other tissues (heart, lung, liver), previously reported to have undetectable levels of CRABP, showed significant amounts of the binding protein. The levels of CRABP peaked in early (4-6-day-old) and late (11-14-day-old) stages of embryo development. Immunolocalization of CRABP in chick embryo skin demonstrates a specific intense staining for the antigen in the dense areas of mesenchyme cells (mesodermal layers); little or no staining was apparent in the differentiated cells of epidermis and peridermis as well as in the loose connective tissues. The MAbs are useful not only in the purification of CRABP as an affinity adsorbent, but also in the elucidation of the possible role of CRABP in the transfer of the ligand to its nuclear receptors and in the morphogenetic gradient formation of RA in chick embryo tissues.

Isolation, partial purification and characterization of nuclear retinoic acid receptors from chick skin.

Nuclear receptors (RARs) for retinoic acid (RA) are considered to be the ultimate mediators of the action of RA in the control of cell differentiation and inhibition of tumorigenesis. We have isolated and partially purified and characterized RAR from a RA-responsive tissue, chick embryo skin. The purification steps included Affi-Gel blue chromatography, ultrafiltration, size exclusion chromatography, and preparative isoelectric focusing. The electrofocusing of RAR-[3H]RA complex in ampholines (pH 3-10) revealed that the receptors have an isoelectric pH of 7.5. Whereas pronase-digested the RAR-[3H]RA complex completely, DNase showed 20-35% and RNase showed negligible digestive action on the complex. The ligand binding to RAR was completely inhibited by a mercury compound. RAR-alpha- and RAR-beta-specific antibodies, on Western blot analysis, immunoreacted with a protein having a molecular weight of 50,000, presumably RAR. Binding affinity studies revealed that biologically active analogs of RA with a free COOH group (e.g., 13-cis-RA, RO-13-7410, Ch 55, and Am 80) showed, like RA, high binding affinity for RAR, whereas biologically ineffective analogs of RA (e.g., furyl and pyridyl) were poor binders. Other groups of retinoids, in which the COOH group was either lacking or blocked, did not bind to RAR whether or not they were biologically active.

Synthetic and naturally occurring retinoids inhibit third- to fourth-stage larval development by Onchocerca lienalis in vitro.

A series of synthetic retinoids was screened for the ability to inhibit the third-to fourth-stage larval molt by Onchocerca lienalis in vitro. Of the 14 retinoids tested, eight gave significant inhibition of the molt at a concentration of 30.6 microM or less. Probit analysis of dose-response data collected for these active compounds indicated values for ED50 in the range of 3.7-17.1 microM. In general, the most active of these N-substituted retinamides were those with small alkyl or monohydroxy alkyl substituents. The most active of these was all-trans-N-(2-hydroxyethyl)retinamide with an ED50 of 3.7 microM. Both the all-trans and 13-cis isomers of the alkyl substituted derivatives were active, the all-trans-N-hydroxyethyl derivative being approximately 5 times as active as the corresponding 13-cis isomer. The N-2,3 dihydroxypropyl derivative, two derivatives with aromatic side chains and three N-(retinoyl)amino acids were inactive by the criteria set in the initial screening. There was no strict correlation between growth regulating activity against O. lienalis and binding affinity for a retinol binding protein from Onchocerca gibsoni.