The stereospecific suicide inhibition of human melanoma thioredoxin reductase by 13-cis-retinoic acid

13-cis retinoic acid has been shown to be a stereospecific suicide inhibitor of thioredoxin reductase purified from human melanoma tissue. All trans retinoic acid does not inhibit this enzyme. The covalent addition of 13-cis retinoic acid to the thiolate active site of thioredoxin reductase produces a thioether enzyme-inhibitor complex. This has been established by a kinetic analysis and by active site labeling with 3H-13 cis retinoic acid. A mechanism involving Michael addition of the thiolate group in the active site of thioredoxin reductase to the 13-cis double bond of enzyme-bound inhibitor has been proposed. This reaction may be important in the human epidermis because thioredoxin reductase has been shown to be a major antioxidant catalyst in human keratinocytes, melanocytes, melanoma cells, and in human skin as well as in melanoma tissues.     

Quantification of retinoic acid by gas-liquid chromatography-mass spectrometry: total versus all-trans-retinoic acid in human plasma

An assay based on negative ion chemical ionization mass spectrometry has been developed to quantify retinoic acid in plasma or serum. The lower limit of detection is 75 pg (240 fmol); normal values of retinoic acid can be determined on as little as 40 microliters of human plasma. The plasma concentrations of total retinoic acid in 12 healthy male volunteers taking no medication or vitamin supplementation ranged from 2.8 to 6.6 ng/ml; the mean was 4.9 ng/ml. The assay can be manipulated to measure all-trans-retinoic acid alone; about 75% of retinoic acid in human plasma or rat serum is all-trans-retinoic acid. Both retinol and retinoic acid can be quantified on the same 0.1-ml sample; the concentration of retinoic acid in human plasma or rat serum is at least 150-fold less than that of retinol.     

Separation of retinoid receptors from cultured retinoblastoma cells

Receptor proteins for [³H] retinol and [³H] retinoic acid in cultured human retinoblastoma cells have been separated rapidly and reproducibly by two different methods. By isoelectric focusing, the isoelectric point of the retinol receptor is at pH 4.0; the retinoic acid receptor has a higher isoelectric point of 4.3. Polyacrylamide slab gel electrophoresis revealed a slower migration rate for the [³H] retinoic acid receptor compared to the [³H] retinol receptor. The separate nature of the two proteins has thus been established in this unique human cell line.     

Saturation analysis of cellular retinoid binding proteins: application to retinoic acid resistant human neuroblastoma cells and to human tumors

A method for saturation analysis of cellular retinoic acid and retinol binding proteins, CRABP and CRBP, respectively, in cultured cells and human tumor samples, and its application to a retinoic acid resistant subline of the human neuroblastoma LA-N-5 cell line is described. Assessment of retinoid binding was accomplished by incubation of cytosols with increasing concentrations of [3H]retinoid (28-43 Ci/mmol; 1 Ci = 37 GBq) for 24 h. Bound retinoid was separated from free retinoid by adsorption with dextran-coated charcoal. Nonspecific binding was quantitated in parallel incubations which had been treated with p-chloromercuribenzene sulfonate (PCMBS), resulting in selective elimination of sulfhydryl-dependent ligand binding to both CRABP and CRBP. Quantitation was accomplished by Scatchard analysis of specific (PCMBS sensitive) binding. Employing this technique, specific retinoid binding was attributed to the presence of 2S macromolecules which displayed the known properties of CRABP and CRBP, namely ligand specificity, saturability, high ligand affinity, and PCMBS sensitivity. The apparent dissociation constants (Kd) for retinoic acid binding in cytosols prepared from murine 3T6 fibroblasts, rat testes, and a human ovarian tumor were 7, 11, and 35 nM, respectively. These preparations also bound retinol with high affinity, exhibiting Kds of 12, 26, and 48 nM, respectively. A retinoic acid resistant subline of LA-N-5 cells designated LA-N-5-R9 was established by long-term culture in the presence of 10(-6) M retinoic acid. This subline is resistant to the effects of retinoic acid in that it requires a 10-fold higher concentration of retinoic acid for 50% inhibition of growth than the parent line and displays no retinoic acid induced morphologic differentiation. Saturation analysis of CRABP in the parent and resistant subline reveal no significant alteration in either CRABP content or affinity. These results indicate that resistance to retinoic acid induced differentiation in LA-N-5-R9 occurs distal to CRABP binding or that CRABP does not mediate this response to retinoic acid.     

Haemoglobin-catalysed retinoic acid 5,6-epoxidation.

Examination of the subcellular distribution of retinoic acid 5,6-epoxidase activity in rat liver and human liver homogenates showed that there is a prominent peak of activity in a high-density fraction. A corresponding peak was also detected in rat blood and human blood. Retinoic acid 5,6-epoxidation was catalysed by human blood cells but not by human plasma, and purified human haemoglobin also catalysed the epoxidation of retinoic acid to 5,6-epoxyretinoic acid. These results suggest that retinoic acid 5,6-epoxidase activity in human liver and rat liver homogenates is partially due to the presence of residual blood cells, and particularly haemoglobin, in the homogenates. In the retinoic acid 5,6-epoxidation catalysed by human haemoglobin, molecular O2 was required and its reaction was stimulated by Triton X-100. Boiling of haemoglobin solution resulted in an 94% decrease in the activity. NADPH (1 mM) and NADH (1 mM) completely [2-mercaptoethanol (5 mM) almost completely] inhibited the 5,6-epoxidation catalysed by haemoglobin, but catalase, superoxide dismutase and mannitol showed no inhibitory effect. CN- ion (100 mM) inhibited the reaction, but N3- ion (100 mM) did not.     

Synergistic action of tiazofurin and retinoic acid on differentiation and colony formation of HL-60 leukemia cells

Tiazofurin and retinoic acid synergistically induced differentiation and inhibited colony formation in HL-60 human promyelocytic leukemia cells in cell culture. The synergism was the result of different mechanisms of action, since the effect of tiazofurin, unlike that of retinoic acid, was prevented by addition of guanosine. Since it has been shown that tiazofurin down-regulated the expression of c-Ki-ras oncogene, and retinoic acid that of the myc oncogene, the joint impact of these drugs is of clinical interest particularly in end-stage leukemia where the therapeutic usefulness of tiazofurin has recently been demonstrated.     

Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo

A clear separation of retinol, retinal, and retinoic acid has been achieved by liquid-gel partition chromatography on Sephadex LH-20 with solvent mixtures of chloroform, Skellysolve B, and methanol. A mixture of retinyl esters, retinol, retinal, and retinoic acid has been resolved on hydroxyalkoxypropyl Sephadex using Skellysolve B and acetone. There is no decomposition of any of the vitamin A compounds during chromatography, and recovery is complete. The combination of mildness and potential for resolution makes liquid-gel partition chromatography a superior tool for the separation of vitamin A compounds. This method has been applied to the study of vitamin A metabolism at physiological levels in the vitamin A-deficient rat. Retinyl palmitate, an ester of retinoic acid, retinal, retinol, retinoic acid, and a polar metabolite have been demonstrated in various tissues of the rat 12 hr after a dose of 2 micro g of [11-(14)C]retinyl acetate.     

Effects of retinoic acid on the concentrations of radioactive metabolites of retinol in tissues of rats maintained on a retinol-deficient diet

The effects of feeding retinoic acid for 2 and 6 days on the metabolism of labeled retinol in tissues of rats maintained on a vitamin A deficient diet was studied. The metabolites of retinol were analyzed by high performance liquid chromatography. Feeding retinoic acid for 2 days significantly reduced the blood retinol and retinyl ester levels without affecting the vitamin A content of the liver. In intestine and testis the content of labeled retinoic acid was decreased significantly by dietary retinoic acid. Addition of retinoic acid to the diet for 6 days resulted, in addition to decreased blood retinol and retinyl ester values, in an increase in the retinyl ester values in the liver. The accumulation of retinyl ester in the retinoic acid fed rat liver was accompanied by an absence of labeled retinoic acid. Kidney tissue was found to contain the highest levels of labeled retinoic acid, retinol, and retinyl esters; dietary retinoic acid did not alter the concentrations of these retinoids in the kidney during the experimental period. Since kidney retained more vitamin A when the liver vitamin A was low and also dietary retinoic acid did not affect the concentrations of radioactive retinoic acid in the kidney, it is suggested that the kidney may play a major role in the production of retinoic acid from retinol in the body.     

Expression of a drug resistance gene in human neuroblastoma cell lines: modulation by retinoic acid-induced differentiation.

Expression of a multidrug resistance gene (mdr1) and its protein product, P-glycoprotein (Pgp), has been correlated with the onset of multidrug resistance in vitro in human cell lines selected for resistance to chemotherapeutic agents derived from natural products. Expression of this gene has also been observed in normal tissues and human tumors, including neuroblastoma. We therefore examined total RNA prepared from human neuroblastoma cell lines before and after differentiation with retinoic acid or sodium butyrate. An increase in the level of mdr1 mRNA was observed after retinoic acid treatment of four neuroblastoma cell lines, including the SK-N-SH cell line. Western blot (immunoblot) analysis demonstrated concomitant increases in Pgp. However, studies of 3H-vinblastine uptake failed to show a concomitant Pgp-mediated decrease in cytotoxic drug accumulation. To provide evidence that Pgp was localized on the cell surface, an immunotoxin conjugate directed against Pgp was added to cells before and after treatment with retinoic acid. Incorporation of [3H]leucine was decreased by the immunotoxin in the retinoic acid-treated cells compared with the undifferentiated cells. These results demonstrate that whereas expression of the mdr1 gene can be modulated by differentiating agents, increased levels of expression are not necessarily associated with increased cytotoxic drug accumulation.     

Determination of 13-cis-retinoic acid and its major metabolite, 4-oxo-13-cis-retinoic acid, in human blood by reversed-phase high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) method for the quantitation of 13-cis-retinoic acid (13-cis-RA) and its major metabolite, 4-oxo-13-cis-RA, in human blood has been developed. The method includes extraction of 1 ml of blood with diethyl ether at pH 6 and the analysis of the extract by reversed-phase HPLC with solvent programming and detection at 365 nm. The quantitation ranges for 13-cis-RA and 4-oxo-13-cis-RA are 10–2000 and 50–2000 ng/ml of blood, respectively. The method also provides estimates of the concentrations of all-trans-RA and 4-oxo-all-trans-RA. The mean intra- and inter-assay variabilities for all four compounds were 6% or less. The method separates 13-cis-RA and 4-oxo-13-cis-RA from 9-cis-RA, all-trans-RA, 4-oxo-all-trans-RA, and some other possible metabolites, such as hydroxy and epoxy retinoic acids. The method has been successfully applied to the analyses of over 1200 blood samples from four 13-cis-RA clinical studies.     

Disabled-2 mediates c-Fos suppression and the cell growth regulatory activity of retinoic acid in embryonic carcinoma cells

F9 embryonic stem cell-like teratocarcinoma cells are widely used to study early embryonic development and cell differentiation. The cells can be induced by retinoic acid to undergo endodermal differentiation. The retinoic acid-induced differentiation accompanies cell growth suppression, and thus, F9 cells are also often used as a model for analysis of retinoic acid biological activity. We have recently shown that MAPK activation and c-Fos expression are uncoupled in F9 cells upon retinoic acid-induced endodermal differentiation. The expression of the candidate tumor suppressor Disabled-2 is induced and correlates with cell growth suppression in F9 cells. We were not able to establish stable Disabled-2 expression by cDNA transfection in F9 cells without induction of spontaneous cell differentiation. Transient transfection of Dab2 by adenoviral vector nevertheless suppresses Elk-1 phosphorylation, c-Fos expression, and cell growth. In PA-1, another teratocarcinoma cell line of human origin that has no or very low levels of Disabled-2, retinoic acid fails to induce Disabled-2, correlating with a lack of growth suppression, although PA-1 is responsive to retinoic acid in morphological change. Transfection and expression of Disabled-2 in PA-1 cells mimic the effects of retinoic acid on growth suppression; the Disabled-2-expressing cells reach a much lower saturation density, and serum-stimulated c-Fos expression is greatly suppressed and disassociated from MAPK activation. Thus, Dab2 is one of the principal genes induced by retinoic acid involved in cell growth suppression, and expression of Dab2 alone is sufficient for uncoupling of MAPK activation and c-Fos expression. Resistance to retinoic acid regulation in PA-1 cells likely results from defects in retinoic acid up-regulation of Dab2 expression.     

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27^Kip1 and p16^INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.     

Separation of the natural retinoids by high-pressure liquid chromatography

A reverse phase high-pressure liquid chromatography system for rapid separation of various retinoids (vitamin A and its analogs) with little or no degradation is described. This method permits detection of as little as 22 pmol of retinoic acid. The procedure has been applied to the study of retinoic acid metabolism in vitamin A-deficient hamsters.     

Retinol oxidation to retinoic acid in human thyroid glandular cells

Abstract

Retinoic acid is regarded as the retinol metabolite that controls proliferation and differentiation of epithelial cells. In the present study, we investigated the potential role of xanthine dehydrogenase (XDH) in retinoic acid biosynthesis in human thyroid glandular cells (HTGC). In particular, we observed that cellular retinoids binding proteins (CRBPs) are also implicated in the biosynthetic pathway leading to retinoic acid formation in primary cultures of HTGC, as we have already reported for human mammary epithelial cells (HMEC). After partial protein purification, the enzyme responsible for retinoic acid biosynthesis was identified and quantified as XDH by immunoassay, by its ability to oxidize xanthine to uric acid and its sensitivity to the inhibitory effect of oxypurinol. The evidence of XDH-driven formation of retinoic acid in HTGC cultures further corroborates the potential role of XDH in retinoic acid biosynthesis in the epithelia.     

Identification of genes up-regulated by retinoic-acid-induced differentiation of the human neuronal precursor cell line NTERA-2 cl.D1

The human teratocarcinoma cell line NTERA-2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up-regulated by this paradigm of neuronal differentiation. Microfibril-associated glycoprotein 2 was found to be drastically up-regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags.     

Retinoic acid-binding protein in rat tissue. Partial purification and comparison to rat tissue retinol-binding protein.

When the 100,000 X g supernatant fractions of several rat organs are incubated with all-trans-[3H]retinoic acid, a binding component for retinoic acid with a sedimentation coefficient of 2 S can be detected by sucrose gradient centrifugation. This tissue binding protein for retinoic acid is distinct from the tissue binding protein for retinol which has been previously described. The tissue retinoic acid-binding protein has been partially purified from rat testis and this partially purified protein would appear to have a molecular weight of 14,500 as determined by gel filtration and high binding specificity for all-trans-retinoic acid. Binding of [3H]retinoic acid is not diminished by a 200-fold molar excess of retinal, retinol, or oleic acid but is reduced by a 200-fold excess of unlabeled retinoic acid. Tissue retinoic acid-binding protein can be detected in extracts of brain, eye, ovary, testis, and uterus but is apparently absent in heart muscle, small intestine, kidney, liver, lung, gastrocnemious muscle, serum, and spleen. This distribution is different than that observed for the tissue retinol-binding protein. Tissue retinol-binding protein was also purified extensively from rat testis. The partially purified protein has an apparent molecular weight of 14,000 and high binding specificity for all-trans-[3H]retinol as only unlabeled all-trans-retinol but not retinal, retinoic acid, retinyl acetate, retinyl palmitate, or oleic acid could diminish binding of the 3H ligand under the conditions employed. The partially purified protein has a fluorescence excitation spectrum with lambda max at 350 nm. In contrast, the retinol-binding protein isolated from rat serum and described by others has a fluorescence excitation spectrum with lambda max at 334 nm and an apparent molecular weight of 19,000. When partially purified tissue retinol-binding protein is extracted with heptane, the heptane extract has a fluorescence excitation spectrum similar to that of all-trans-retinol.     

Cloning of human myeloid-associated differentiation marker (MYADM) gene whose expression was up-regulated in NB4 cells induced by all-trans retinoic acid

A full-length cDNA of 3192 bp isolated from human bone marrow cDNA library was predicted an ORF encoding 298 amino acids. The deduced protein, containing seven putative transmembrane segments and sharing 75.8% amino acid identity with mouse Myadm protein, was named as human MYADM. The results of Northern blot analysis showed that MYADM was ubiquitously expressed in 15 of 16 adult tissues tested, except thymus. To determine whether the novel human gene was involved in hematopoietic differentiation process as mouse Myadm did, we examined the mRNA expressive abundance of this gene between normal bone marrow cells and peripheral blood leukocytes, and detected the expression change in NB4 cells induced by all–trans retinoic acid at different induce time by the semi-quantitative RT-PCR. The results showed that the expression of the novel gene was not only significantly higher in peripheral blood leukocytes than in bone marrow cells, but also significantly up-regulated when the NB4 cells(derived from a patient with acute promyelocytic leukemia) were induced by all-trans retinoic acid (ATRA) for 48hr. It is suggested that human MYADM was also associated with the differentiation of hematopoietic cells or acute promyelocytic leukemia cells. In addition, MYADM was mapped to human chromosome 19q13.33-q13.4 by Radiation Hybrid mapping, and it consists of 3 exons and 2 introns and spans a 7.1-Kb genomic region.     

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.