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PubMed1995

A human retinal pigment epithelial cell line that retains epithelial characteristics after prolonged culture.

PURPOSE: A spontaneously arising, apparently transformed, cell line has been cloned from a primary culture of human retinal pigment epithelial (RPE) cells and has been subcultured more than 200 times. The similarities of these cells to human RPE cells in vivo have been determined. METHODS: The structure of the transformed cells has been determined by light and electron microscopy and by immunocytochemistry using antibodies that detect cytoskeletal and other proteins. The ability of the cell line to bind and phagocytose photoreceptor material has also been assessed by fluorescence and electron microscopy. The metabolism of all-trans-retinol has been investigated by incubation of the cells with 3H-all-trans-retinol and analysis of the metabolic products by high-performance liquid chromatography. RESULTS: The transformed cells possess an epithelial cobblestone morphology with intercellular junctional complexes containing N-cadherin. The cytoskeleton of these cells comprises cytokeratins that are characteristic of epithelial cells, together with actin, spectrin, and vimentin. The keratins expressed are those typical of RPE cells. The cells also express cellular retinaldehyde binding protein and retinol dehydrogenase activity but do not express retinoid isomerase or lecithin retinol acyl transferase activities. These cells also exhibit phagocytic activity. CONCLUSIONS: This cell line retains many of the metabolic and morphologic characteristics of RPE cells in vivo although there are some differences, particularly the loss of some enzymatic activities and cytoskeletal polarization. These cells should be useful in further studies of RPE cell metabolism and other functions.

Cell Line, Transformed↗
PubMed1994

Biological activity of all-trans retinol requires metabolic conversion to all-trans retinoic acid and is mediated through activation of nuclear retinoid receptors in human keratinocytes.

The biological activity of all-trans retinol, in human keratinocytes, was investigated through metabolic and functional analyses that assessed the capacity for retinol uptake and metabolism and the mechanism of retinol-induced activation of gene transcription. Human keratinocytes converted all-trans retinol predominantly to retinyl esters, which accounted for 60 and 90% of cell-associated radiolabel after a 90-min pulse and a 48-h chase, respectively. Human keratinocytes also metabolized all-trans retinol to low levels of all-trans retinoic acid (11.47-131.3 ng/mg of protein) in a dose-dependent manner, between 0.3 and 10 microM added retinol. Small amounts of 13-cis retinoic acid (5.47-8.62 ng/mg of protein) were detected, but 9-cis retinoic acid was detected only when keratinocytes were incubated with radiolabeled retinol. There was no accumulation of the oxidized catabolic metabolites 4-hydroxy- or 4-oxoretinoic acid; however, 5,6-epoxy retinoic acid was detected at pharmacological levels (10 and 30 microM) of added retinol. Biological activity of retinol was assessed through analysis of two known retinoic acid-mediated responses: 1) reduction of type I epidermal transglutaminase and 2) activation of a retinoic acid receptor-dependent reporter gene, beta RARE3-tk-CAT. Both all-trans retinol and all-trans retinoic acid reduced type I epidermal transglutaminase in a dose-dependent manner; however, the ED50 for all-trans retinol (10 nM) was 10 times greater than for all-trans retinoic acid (1 nM). All-trans retinol also stimulated beta RARE3-tk-CAT reporter gene activity in a dose-dependent manner. Half-maximal induction was observed at 30 nM retinol, which was again 10-fold greater than observed with all-trans retinoic acid. Cotransfection of human keratinocytes with expression vectors for dominant negative mutant retinoic acid and retinoid X receptors reduced retinol-induced beta RARE3-tk-CAT reporter gene activation by 80%. Inhibition of conversion of all-trans retinol or all-trans retinaldehyde to all-trans retinoic acid by citral reduced beta RARE3-tk-CAT activity 98 and 86%, respectively. These data demonstrate that retinol-induced responses in human keratinocytes are mediated by its tightly regulated conversion to retinoic acid, which functions as a ligand to activate nuclear retinoic acid receptors.

Cell Nucleus↗
PubMed1994

[Retinotherapy of skin diseases].

The discovery of retinoid receptors has contributed greatly to our understanding of the mechanism of action of vitamin A. The organism produces at least two ligands from ingested vitamin A which act as hormones modulating the activity of numerous genes via their nuclear receptor. These ligands are produced locally by target cells from retinol and retinaldehyde. These advances do not respond to the clinicians' interrogation as to why 13cis retinoic acid blocks sebaceous secretion and cures severe acne while other known retinoids are uneffective. Current research would suggest that the expression of nuclear receptors is not altered in skin diseases but that upstream anomalies in the intracrine system (enzymes and binding proteins) could be involved. Clinically, teratogenic risks are a major obstacle to the oral administration of retinoids and the future in skin diseases lies most likely in topical applications.

Administration, Oral↗
PubMed1995

The retinal pigment epithelial-specific 11-cis retinol dehydrogenase belongs to the family of short chain alcohol dehydrogenases.

We have isolated and partially characterized a 32-kDa membrane-associated protein (p32), which forms a complex with p63, an abundant membrane protein in bovine retinal pigment epithelium. The sequence of a cDNA clone for p32 revealed an open reading frame encoding 318 amino acid residues. Several hydrophobic regions could be identified, suggesting that p32 is an integral membrane protein. A search of data bases identified p32 as a member of the superfamily of short chain alcohol dehydrogenases. Transcripts for p32 were specifically expressed in retinal pigment epithelium. Overexpression of p32 in Cos cells produced a membrane-bound stereospecific 11-cis retinol dehydrogenase, active in the presence of NAD+ as cofactor but not in the presence of NADP. We propose that p32 is the stereospecific 11-cis retinol dehydrogenase, which catalyzes the final step in the biosynthesis of 11-cis retinaldehyde, the universal chromophore of visual pigments.

Alcohol Oxidoreductases↗
PubMedDate not supplied

Identification of 9,13-dicis-retinoic acid as a major plasma metabolite of 9-cis-retinoic acid and limited transfer of 9-cis-retinoic acid and 9,13-dicis-retinoic acid to the mouse and rat embryos.

9-Cis-retinoic acid (9-cis-RA) has been proposed to be the endogenous ligand of retinoid X receptors. We examined the plasma pharmacokinetics of 9-cis-RA and its metabolites in nonpregnant female NMRI mice after oral dosing with 50 mg 9-cis-RA/kg body weight. Furthermore, we studied the metabolism of 9-cis-RA and its transfer to the embryo following oral administration of the precursor 9-cis-retinaldehyde (9-cis-RAL; 100 mg/kg body weight) to pregnant mice and rats on gestational days 11 and 13, respectively. Following 9-cis-RA administration, plasma levels of 9-cis-RA reached their maximum within 40-60 min and then declined in a monoexponential manner with an apparent half-life of 64 +/- 32 min. A great variety of polar metabolites of 9-cis-RA was found; among them, the beta-glucuronides of 9-cis-RA (9-cis-RAG) and of 9-cis-4-oxo-RA (9-cis-4-oxo-RAG) could be identified. A further prominent polar metabolite of 9-cis-RA in mouse plasma was shown to be an additional RA isomer (distinct from 13-cis-RA and all-trans-RA) whose concentrations weeesimilarly high as those of 9-cis-RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Characterization of human retinoic acid receptor alpha 1 expressed in recombinant baculovirus-infected Sf9 insect cells.

Full-length human retinoic acid receptor alpha 1 (hRAR alpha 1) was expressed in Sf9 insect cells using the baculovirus expression vector system (BEVS). Western blot analysis using a specific anti-RAR peptide antiserum detected two major protein bands with apparent mol wts of approximately 54 and approximately 51 kDa in extracts from insect cells infected with recombinant hRAR alpha 1 Autographica californica (AcNPV) baculovirus. Analysis of recombinant extracts from Sf9 cells labeled in vivo with [32P]orthophosphate suggested that the recombinant protein was phosphorylated. A component in the recombinant nuclear extracts specifically bound [3H]all-trans-retinoic acid (RA) and sedimented in sucrose density gradient centrifugation as a single, symmetric peak with a sedimentation coefficient of approximately 3.6S, corresponding to a protein of approx 50 kDa. Scatchard analyses determined that [3H]RA was bound in recombinant extracts by a single class of binding sites with an apparent dissociation constant of approximately 0.3 nM and nuclear and cytoplasmic extracts contained approximately 1200 and approximately 200 pmoles, respectively, of unoccupied receptor per mg protein. In competitive ligand binding assays, relative binding affinities of 9-cis- and 13-cis-RA for hRAR alpha 1 in nuclear extracts were about threefold and sixfold lower than all-trans-RA, whereas all-trans-retinol, -retinaldehyde, and -retinyl acetate demonstrated relatively weak binding. In gel mobility shift assays, the electrophoretic migration of a [32P]-labeled oligonucleotide containing the retinoic acid response element of the RAR beta gene was retarded in the presence of recombinant nuclear and cytoplasmic extracts. The apparent complex formation between recombinant hRAR alpha 1 and beta RARE was greatly enhanced by the addition of nuclear extract from wild-type AcNPV-infected Sf9 cells, possibly because of heterodimer formation between recombinant hRAR alpha 1 and a metazoan RXR homolog. Thus, recombinant hRAR alpha 1 expressed at high levels in Sf9 insect cells exhibited biochemical properties of the native protein, including nuclear translocation, specific high affinity ligand and RARE binding, and possible heterodimer formation.

Amino Acid Sequence↗
PubMed1994

Characterization of two structurally related Xenopus laevis protein tyrosine phosphatases with homology to lipid-binding proteins.

We have chosen Xenopus laevis as a model system to study how protein tyrosine phosphatases (PTPases) function in growth and development. As an initial step, we have previously isolated in a polymerase chain reaction (PCR)-based protocol cDNA fragments which correspond to sequences within the catalytic domains of PTPases (Yang, Q., and Tonks, N. K. (1993) Adv. Protein Phosphatases 7, 359-372). Two of these PCR products, designated X1 and X10, have now been used to screen a X. laevis ovary cDNA library to obtain complete coding sequences for two distinct PTPases. The X1 cDNA encodes a protein (PTPX1) of 693 amino acids (approximately 79 kDa); the X10 cDNA encodes a protein of 597 amino acids (approximately 69 kDa). Both PTPX1 and PTPX10 lack potential membrane spanning sequences and therefore can be classified as non-transmembrane/cytoplasmic PTPases. While the overall structure of these PTPases are similar, sharing segments of 95% amino acid identity, they differ in that PTPX1 contains a unique 97-amino acid insert between the N-terminal segment and C-terminal catalytic domain. The absence of complete identity between PTPX1 and PTPX10 suggests that these two sequences are the products of separate genes and not the result of alternative splicing. This conclusion is confirmed by PCR analysis of Xenopus genomic DNA. Both PTPases share sequence identities in their N-terminal segments with two lipid-binding proteins, cellular retinaldehyde-binding protein and SEC14p, a phospholipid transferase. In addition, the unique insert sequence of PTPX1 shares identity with PSSA, a protein involved in phosphatidylserine biosynthesis. Sequence comparison suggests that PTPX10 is the Xenopus homolog of the human PTPase Meg-02, while PTPX1 is a structurally related yet distinct PTPase. Intrinsic PTPase activity of PTPX1 and PTPX10 was demonstrated in lysates of Sf9 cells infected with recombinant baculoviruses encoding either enzyme. PTPX1 can be recovered in both soluble and membrane fractions from Xenopus oocytes with the membrane form exhibiting approximately 4-fold higher activity than the soluble form.

Amino Acid Sequence↗
PubMed1993

An opsin homologue in the retina and pigment epithelium.

PURPOSE: The aim of this project was to investigate the retinal pigment epithelium (RPE) at the molecular level by identification of novel RPE-specific cDNAs that may encode proteins of signal transduction pathways or other proteins that are expressed preferentially in the RPE. METHODS: A bovine RPE cDNA library was constructed in bacteriophage lambda g10 using RPE-enriched poly(A)+ RNA. The library was screened by differential hybridization to bovine RPE and kidney cDNA probes. RESULTS: A member of the hepatahelical receptor family was identified in bovine RPE by molecular cloning. Its deduced amino acid sequence predicts a protein that has 291 amino acid residues and resembles most closely the family of visual pigments. A lysine residue, analogous to the retinaldehyde attachment site in rhodopsin, is conserved in the seventh hydrophobic segment of the novel sequence. Messenger RNA encoding the putative G protein-coupled receptor was detected by in situ hybridization in the RPE, inner nuclear layer, and specific cells of the ganglion cell layer. Immunohistochemical staining of bovine retina showed that the receptor protein is localized in Müller cells, as well as in the RPE. CONCLUSIONS: A novel heptahelical receptor defines a distant evolutionary branch of the visual pigment tree. The selective localization of this putative receptor, its abundance in RPE and retina, and its homology to the visual pigments suggest that the function of this receptor is important in a visual process involving the RPE and Müller cells.

Amino Acid Sequence↗
PubMed1993

Photoreceptors regulating circadian behavior: a mouse model.

Our recent studies have examined circadian photoreception in mice with hereditary retinal disorders (rd/rd and rds/rds). Despite the loss of visual function in these mice, circadian responses to light remain unaffected. Using c-fos expression within the suprachiasmatic nuclei (SCN) as a marker of neural activation of the circadian entrainment pathway, we have found identical levels of Fos in the SCN of rd/rd and +/+ mice in response to retinal illumination. On the basis of action spectrum studies, and measurements of photopigment retinoids using high-pressure liquid chromatography, we believe that the photopigment mediating circadian responses to light is based upon an opsin, and that 11-cis-retinaldehyde is the photopigment chromophore. Preliminary measurements of mouse rod opsin, blue cone, and green-red cone opsin messenger RNA in retinally degenerate mice suggest that none of these opsins is exclusively used to mediate circadian responses to light. Collectively, our data suggest that circadian photoreception can be maintained by a very small number of rod or cone cells without outer segments, or, alternatively, is performed by an unrecognized class of photoreceptive cell within the mammalian retina.

Animals↗
PubMed1994

Retinoic acid inhibition of IL-1-induced IL-6 production by human lung fibroblasts.

IL-6 is a multi-functional cytokine that plays an important role in normal biologic homeostasis and disease pathogenesis. Retinoids are vitamin A analogs that regulate the function of a wide variety of inflammatory and structural cells. To further understand the biology of retinoids and IL-6 we determined whether all-trans-retinoic acid (RA) and other retinoids regulate lung fibroblast IL-6 production. RA did not stimulate fibroblast IL-6 production. Instead, it inhibited the production of IL-6 by IL-1-stimulated cells. This effect was dose-dependent with an IC50 of 10(-7) M RA and significant inhibition being noted with doses of RA as low as 10(-8) M. These inhibitory effects could not be explained by cytotoxicity or a shift in the kinetics of IL-6 production. They also did not appear to involve alterations in the early events in IL-1-induced IL-6 production, because RA inhibited IL-6 production even when added 6 h after IL-1 and RA did not inhibit IL-1 binding to cell surface IL-1 receptors. RA inhibition of IL-6 protein production was associated with a comparable decrease in IL-6 mRNA accumulation and gene transcription. 13-cis-retinoic acid, retinol, retinaldehyde, all-trans etretin, Ro 13-6298, and 9-cis retinoic acid also inhibited IL-1-induced IL-6 production. However, 4-hydroxyphenyl retinamide and etretinate did not share this property. The inhibitory effects of these analogues may be mediated by nuclear retinoic acid receptors as mRNA encoding RAR-alpha, RAR-gamma, and RXR-alpha were present, and RAR-beta was induced by RA in human lung fibroblasts. These studies demonstrate that RA and other retinoid analogs inhibit IL-1-induced IL-6 production and that this effect is analog-specific and, at least partially, transcriptionally mediated.

Cell Line↗
PubMed1996

Vitamin A down-regulation of IFN-gamma synthesis in cloned mouse Th1 lymphocytes depends on the CD28 costimulatory pathway.

Some infections deplete serum retinol, low retinol reduces immunity, and reduced immunity establishes susceptibility to further infection in a cyclical relationship that is poorly understood. We showed that when retinol was low, there was excessive Th1 cell IFN-gamma synthesis and inadequate Th2 cell IL-4 and IL-5 synthesis. The retinol metabolite retinoic acid inhibited the IFN-gamma stimulatory activity of APCs, enhanced Th2 cell differentiation, and inhibited Th1 cell IFN-gamma synthesis. Here we focus on the mechanism for retinoic acid inhibition of IFN-gamma synthesis in myelin basic protein-specific MM4 Th1 cells. Physiologic amounts of all-trans-retinoic acid directly and specifically down-regulated the MM4 Th1 cell IFN-gamma secretion rate in vitro without affecting cell growth, viability, or overall protein synthesis. All-trans-, 9-cis-, and 13-cis-retinoic acid, and the synthetic retinoid Ch55, inhibited IFN-gamma synthesis effectively, whereas retinaldehyde, retinol, and retinyl acetate did not. This pattern suggests retinoic acid receptor involvement in the inhibition mechanism. Retinoic acid did not inhibit when Th1 cells were activated only through the TCR/CD3 complex, with or without IL-2 costimulation. Retinoic acid inhibited IFN-gamma synthesis when the CD28 costimulatory pathway was activated in addition to the TCR/CD3 pathway, suggesting it blocks some step in the CD28 pathway. Retinoid probably acted to decrease IFN-gamma transcript accumulation by decreasing transcription because it did not decrease transcript stability. We suggest that unrestrained IFN-gamma synthesis is one key immunobiologic mechanism that accounts for poor antibody-mediated immunity in hypovitaminosis A, since IFN-gamma in relatively small amounts can limit Th2 cell growth and interfere with the B cell stimulatory functions of Th2 cell cytokines.

Animals↗
PubMed1997

Tractional force generation by porcine Müller cells. Development and differential stimulation by growth factors.

PURPOSE: To assess the ability of retinal Müller cells to generate tractional forces during dedifferentiation in culture and to assess their responsiveness to contraction-stimulating growth factors. METHODS: Müller cells were isolated from papain-DNase-digested porcine retina. The identity of the isolated cells was confirmed by immunodetection of carbonic anhydrase II (CA-II), cellular retinaldehyde-binding protein (CRALBP), glial fibrillary acidic protein (GFAP), vimentin, and alpha smooth muscle actin (alpha SMA). Tractional force generation was assessed as a function of Müller cell contraction of collagenous extracellular matrices in vitro. The effects of potential promoters were assessed by addition directly to culture medium. The contributions of specific promoting to the contraction-promoting activity in serum were assessed by adding neutralizing antibodies and measuring loss of stimulatory activity. RESULTS: Freshly isolated Müller cells did not generate substantial matrix contraction. However, this activity increased 150-fold within 12 days in culture and continued to increase during the next 21 days. Development of the capacity for extracellular matrix contraction coincided with the acquisition of immunodetectable alpha SMA and loss of GFAP. Matrix contraction by Müller cells was stimulated in a dose-dependent fashion by human serum, platelet-derived growth factor (PDGF), and insulin-like growth factor-I (IGF-I). Müller cells were not stimulated by transforming growth factor beta 1 (TGF beta 1), transforming growth factor beta 2 (TGF beta 2), or endothelin-1 (E1). Neutralizing antibodies against PDGF and IGF-I reduced the activity in human serum by 37% and 58%, respectively, and 87% when added together. CONCLUSIONS: Porcine Müller cells in culture acquire the ability to contract extracellular matrices and thus generate tractional forces. Acquisition of this activity coincides with alpha SMA expression and loss of GFAP. Further, this activity is dependent on the presence of exogenous promoters, including PDGF or IGF-I.

Animals↗
PubMed1997

Analysis of esterification of retinoids in the retinal pigmented epithelium of the Mitf-vit (vitiligo) mutant mouse.

PURPOSE: Mice homozygous for the vitiligo mutation of the microphthalmia (Mitf) gene have a retinal degeneration characterized by slow loss of photoreceptor cells and compromised retinal pigment epithelial (RPE) structure and function. The levels of retinyl esters, which are essential for generation of 11-cis-retinaldehyde for the formation of rhodopsin, were reported previously to be elevated by 6 weeks postnatally in the RPE of vitiligo mutant mice. The purpose of the present study was to determine whether this elevation was due to increased activity of lecithin:retinol acyl transferase (LRAT) the enzyme that converts all-trans-retinol to retinyl esters. METHODS: Retinoids extracted from the RPE and neural retina of mutant and normal mice ages 2, 4, 6 and 8 weeks were analyzed by reversed-phase HPLC. The esterification capacity of the RPE to convert 3H-retinol to 3H-retinyl ester was determined by HPLC in mutant and normal mice at 3 and 9 weeks. RESULTS: Retinyl ester levels were elevated significantly in the mutant RPE as early as postnatal week 2 and were four-fold greater by 8 weeks. The esterification assay indicated no significant differences between mutants and controls at 3 weeks. At 9 weeks, the esterification activity of the mutant RPE was significantly reduced compared to controls rather than elevated. CONCLUSIONS: The data suggest that the accumulation of retinyl esters is not due to increased LRAT activity. Alternative explanations for the retinyl ester accumulation are discussed.

Acyltransferases↗
PubMed1997

Relative contribution of human erythrocyte aldehyde dehydrogenase to the systemic detoxification of the oxazaphosphorines.

Detoxification of cyclophosphamide is effected, in part, by hepatic class 1 aldehyde dehydrogenase (ALDH-1)-catalyzed oxidation of aldophosphamide, a pivotal aldehyde intermediate, to the nontoxic metabolite, carboxyphosphamide. This enzyme is found in erythrocytes as well. Detoxification of aldophosphamide may also be effected by enzymes, viz. certain aldo-keto reductases, that catalyze the reduction of aldophosphamide to alcophosphamide. Such enzymes are also found in erythrocytes. Not known at the onset of this investigation was whether the contribution of erythrocyte ALDH-1 and/or aldo-keto reductases to the overall systemic detoxification of circulating aldophosphamide is significant. Thus, NAD-linked oxidation and NADPH-linked reduction of aldophosphamide catalyzed by relevant erythrocyte enzymes were quantified. ALDH-1-catalyzed oxidation of aldophosphamide (160 microM) to carboxyphosphamide occurred at a mean (+/- SD) rate of 5.0 +/- 1.4 atmol/min/rbc (red blood cell). Aldo-keto reductase-catalyzed reduction of aldophosphamide (160 microM) to alcophosphamide occurred at a much slower rate, viz. 0.3 +/- 0.2 atmol/min/rbc. Thus, at a pharmacologically relevant concentration of aldophosphamide, viz. 1 microM, estimated aggregate erythrocyte ALDH-1-catalyzed aldophosphamide oxidation, viz. 2.0 micromol/min, was only about 3% of estimated aggregate hepatic enzyme-catalyzed aldophosphamide oxidation, viz. 72 micromol/min; however, this rate is greater than the estimated flow-limited rate of aldophosphamide delivery to the liver by the blood, viz. 1.5 micromol/min. These observations/considerations suggest an important in vivo role for erythrocyte ALDH-1 in systemic aldophosphamide detoxification. Erythrocyte ALDH-1-effected oxidation of other aldehydes to their corresponding acids, e.g. retinaldehyde to retinoic acid, may also be of pharmacological and/or physiological significance since a wide variety of aldehydes are known to be substrates for ALDH-1.

Adult↗
PubMed1998

Metabolism of all-trans-retinol in normal human cell strains and squamous cell carcinoma (SCC) lines from the oral cavity and skin: reduced esterification of retinol in SCC lines.

Retinoids, metabolites and synthetic derivatives of vitamin A (retinol), have been shown to inhibit carcinogenesis in various epithelial tissues in animal model systems and to have clinical efficacy as chemotherapeutic agents against certain types of cancer, including squamous cell carcinomas (SCCs). We examined the metabolism of [3H]retinol in normal human cell strains and SCC lines from the oral cavity and skin, and we report here that the cultured normal human epithelial cell strains esterified [3H]retinol to a much greater extent than the SCC lines. Furthermore, microsomal extracts of normal cell strains (e.g., OKF4) exhibited about 7-fold more palmityl-CoA-dependent, phenylmethylsulfonyl fluoride-resistant retinol esterification activity than extracts from SCC lines (e.g., SCC25). The fact that the esteriflcation of retinol was phenylmethylsulfonyl fluoride resistant suggests that the enzyme acyl-CoA:retinol acyltransferase is involved. Culture of both the normal and SCC lines in the presence of 1 microM all-trans-retinoic acid (RA) for 48 h enhanced the formation of [3H]retinyl esters from [3H]retinol. All of the cell lines examined can also metabolize [3H]retinol to [3H]RA, [3H]14-hydroxy-4,14-retroretinol, [3H]retinaldehyde, and [3H]3,4-didehydroretinol, but this metabolism occurs to varying extents in different cell lines. Culture of the cells in the presence of RA for 48 h did not affect the subsequent metabolism of [3H]retinol to [3H]RA and [3H]14-hydroxy-4,14-retroretinol, but it did reduce the metabolism of [3H]retinol to [3H]3,4-didehydroretinol. When cultured for 6-10 h in the presence of nanomolar concentrations of exogenous [3H]retinol, both the normal and SCC lines had much higher intracellular [3H]retinol concentrations, in the micromolar range. No correlation was seen between CRABP II or CRBP I mRNA levels and the levels of either intracellular [3H]retinol or [3H]retinol metabolism in these lines. The reduced ability to esterify retinol in these tumor cells may result in inappropriate cell growth and the loss of normal differentiation responses because of the lack of a sufficient amount of internal retinol stored as retinyl esters.

Carcinoma, Squamous Cell↗
PubMed1998

Promoter analysis of RPE65, the gene encoding a 61-kDa retinal pigment epithelium-specific protein.

PURPOSE: To identify the functional promoter region and cis-acting elements that regulate the expression of RPE65, the retinal pigment epithelium (RPE)-specific gene responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy. METHODS: A human genomic DNA clone containing the 5'-flanking region of RPE65 was isolated and, 4.0 kb proximal to the transcription start site, was sequenced and analyzed for the presence of transcription factor-binding sites. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the upstream sequence in the human RPE cell lines ARPE19 and D407, as well as in the SK-Mel-28 and HeLa cell lines. Specific DNA protein-binding sites present in the 340 bp upstream of the transcription start site were identified by DNase I footprint analysis. RESULTS: Sequence analysis places the polymorphic marker, D1S2803, within the RPE65 upstream region and identifies a number of sequences homologous to the gene encoding the cellular retinaldehyde-binding protein. Functional analysis indicates that basal promoter activity is conferred by the sequence from -83 to +39 and is approximately equivalent in all cell lines tested, with no other control elements detected in 3.6 kb of the upstream sequence. At least eight protected regions are identified in DNase I footprint assays, including sequences corresponding to the predicted TATA box, AP-4, and nuclear factor-1 DNA protein-binding sites. CONCLUSIONS: These findings localize the basal promoter activity of RPE65, identify potential cis-acting elements that act as positive regulators of gene expression, and suggest that additional regulatory elements are likely to be involved in restricting gene expression to the retinal pigment epithelium. Identification of promoter elements and genetic markers in the upstream sequence will enable the screening of patients with retinal degeneration for possible mutations that affect RPE65 expression.

Base Sequence↗
PubMed1983

Use of a human tumor cloning system to screen retinoids for antineoplastic activity.

Retinoid analogs are being actively evaluated as antitumor agents in man. A human tumor cloning system was used to assess the antineoplastic activity of all-trans-retinoic acid, all-trans-retinol and 13-cis-retinoic acid. The effect of retinoids on the formation of tumor colony forming units (T-CFU) was surveyed for 67 different human tumors. These analogs showed minor activity in a variety of neoplasms. The decrease in T-CFU varied between histologic tumor types and between tumor of the same type. No significant difference between retinoid analogs was noted.

Antineoplastic Agents↗