Metabolism to a response pathway selective retinoid ligand during axial pattern formation

We report identification of 9-cis-4-oxo-retinoic acid (9-cis-4-oxo-RA) as an in vivo retinoid metabolite in Xenopus embryos. 9-Cis-4-oxo-RA bound receptors (RARs) α, β, and γ as well as retinoid X receptors (RXRs) α, β, and γ in vitro. However, this retinoid displayed differential RXR activation depending on the response pathway used. Although it failed to activate RXRs in RXR homodimers, it activated RXRs and RARs synergistically in RAR-RXR heterodimers. 9-Cis-4-oxo-RA thus acted as a dimer-specific agonist. Considering that RAR-RXR heterodimers are major functional units involved in transducing retinoid signals during embryogenesis and that 9-cis-4-oxo-RA displayed high potency for modulating axial pattern formation in Xenopus, metabolism to 9-cis-4-oxo-RA may provide a mechanism to target retinoid action to this and other RAR-RXR heterodimer-mediated processes.     

Retinol dehydrogenase 10 is indispensible for spermatogenesis in juvenile males

Retinoic acid (RA), an active vitamin A derivative, is essential for mammalian spermatogenesis. Genetic studies have revealed that oxidation of vitamin A to retinal by retinol dehydrogenase 10 (RDH10) is critical for embryonic RA biosynthesis. However, physiological roles of RDH10 in postnatal RA synthesis remain unclear, given that Rdh10 loss-of-function mutations lead to early embryonic lethality. We conducted in vivo genetic studies of Rdh10 in postnatal mouse testes and found that an RDH10 deficiency in Sertoli cells, but not in germ cells, results in a mild germ cell depletion phenotype. A deficiency of RDH10 in both Sertoli and germ cells in juvenile mice results in a blockage of spermatogonial differentiation, similar to that seen in vitamin A-deficient animals. This defect in spermatogenesis arises from a complete deficiency in juvenile testicular RA synthesis and can be rescued by retinoid administration. Thus, in juvenile mice, the primary, but not exclusive, source of RA in the testes is Sertoli cells. In contrast, adult Rdh10-deficient mice exhibit phenotypically normal spermatogenesis, indicating that during development a change occurs in either the cellular source of RA or the retinaldehyde dehydrogenase involved in RA synthesis.     

From the Cover: Crystal structure of native RPE65, the retinoid isomerase of the visual cycle

Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal. A key enzyme in this pathway is the microsomal membrane protein RPE65. This enzyme catalyzes the conversion of all-trans-retinyl esters to 11-cis-retinol in the retinal pigment epithelium (RPE). Mutations in RPE65 are known to be responsible for a subset of cases of the most common form of childhood blindness, Leber congenital amaurosis (LCA). Although retinoid isomerase activity has been attributed to RPE65, its catalytic mechanism remains a matter of debate. Also, the manner in which RPE65 binds to membranes and extracts retinoid substrates is unclear. To gain insight into these questions, we determined the crystal structure of native bovine RPE65 at 2.14-Å resolution. The structural, biophysical, and biochemical data presented here provide the framework needed for an in-depth understanding of the mechanism of catalytic isomerization and membrane association, in addition to the role mutations that cause LCA have in disrupting protein function.     

17beta-Hydroxysteroid dehydrogenase type 9 and other short-chain dehydrogenases/reductases that catalyze retinoid, 17beta- and 3alpha-hydroxysteroid metabolism

Subgroups of related short-chain dehydrogenase/reductase (SDR) family members serve as retinoid/androgen/estrogen metabolizing enzymes. These include retinol dehydrogenases (RoDHs) 1-3, cis-retinol/androgen dehydrogenase 1 and 2 (CRAD), retSDRs1-4, 9/11-cis-retinol dehydrogenase, and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) types 6 and 9. Interaction with cellular retinol-binding protein (CRBP), the major physiological form of retinol, led to the identification and cDNA cloning of RoDH1. Probes for RoDH1 contributed to cDNA cloning many of the others. Some of these SDRs show specificity with all-trans-retinol (RoDH, retSDR, 17beta-HSD6 and 9) and others with 9 and/or 11-cis-retinol (CRAD, 9/11-cis-retinol dehydrogenase). Many have 3alpha-HSD activities with 3alpha-androstandiol as the most efficiently used substrate, followed by androsterone. In addition to 3alpha-HSD activity, CRAD2 shows relatively weak 17beta-HSD activity with testosterone. Rat 17beta-HSD6 and mouse 17beta-HSD9, which are not interspecies homologs, have efficient 17beta-HSD activities. 17beta-HSD6 has approximately 50% greater 17beta-HSD activity with estradiol than with 3alpha-androstandiol. With 3alpha-androstandiol, 17beta-HSD9 operates equally efficiently as a 17beta-HSD or a 3alpha-HSD. The multi-substrate nature of these SDRs allows for retinoid/steroid interactions. The ability of some these SDRs to access retinol bound with CRBP provides specificity in retinoid metabolism and allows retinoic acid biosynthesis and retinol esterification to continue, as CRBP protects retinol from the general cellular milieu.     

Estrogen directly induces expression of retinoic acid biosynthetic enzymes, compartmentalized between the epithelium and underlying stromal cells in rat uterus

Estrogen (E2) has been shown to induce the biosynthesis of retinoic acid (RA) in rat uterus. Here we examined whether E2 could directly induce the enzymes involved in this process by using the ovariectomized rat. A retinol dehydrogenase that we have previously described, eRolDH, and the retinal dehydrogenase, RalDH II, were found to have markedly increased uterine mRNA levels within 4 h of E2 administration, independent of the prior administration of puromycin. eRolDH and RalDH II and their mRNAs were also increased in uteri of rats during estrus. This indicated that RA biosynthesis in rat uterus is directly controlled by E2 and provides a direct link between the action of a steroid hormone and retinoid action. We also examined the cell-specific localization of RalDH II by immunohistochemistry. The enzyme was observed in the stromal compartment, particularly in cells close to the uterine lumenal epithelium. eRolDH was observed only in the lining epithelial cells. Taken together with the previous observations of cellular retinol-binding protein and cellular retinoic acid-binding protein, type two also being expressed in the lumenal epithelium, we propose that RA production is compartmentalized, with retinol oxidation occurring in the lumenal epithelium and subsequent oxidation of retinal to RA occurring in the underlying stromal cells.     

Complementary deoxyribonucleic acid cloning and enzymatic characterization of a novel 17beta/3alpha-hydroxysteroid/retinoid short chain dehydrogenase/reductase.

17Beta-hydroxysteroid dehydrogenases (17betaHSDs) convert androgens and estrogens between their active and inactive forms, whereas retinol dehydrogenases catalyze the conversion between retinol and retinal. Retinol dehydrogenases function in the visual cycle, in the generation of the hormone retinoic acid, and some also act on androgens. Here we report cloning and expression of a complementary DNA that encodes a new mouse liver microsomal member of the short chain dehydrogenase/reductase (SDR) superfamily and its enzymatic characterization, i.e. 17betaHSD9. Although 17betaHSD9 shares 88% amino acid identity with rat 17betaHSD6, its closest homolog, the two differ in substrate specificity. In contrast to other 17betaHSD, 17betaHSD9 has nearly equivalent activities as a 17betaHSD (with estradiol approximately = adiol) and as a 3alphaHSD (with adiol approximately = androsterone). It also recognizes retinol as substrate and represents in part the NAD+-dependent liver microsomal dehydrogenase that uses unbound retinol, but not retinol complexed with cellular retinol-binding protein. Thus, this enzyme has catalytic properties that overlap with two subgroups of SDR, 17betaHSD and retinol dehydrogenases. Inactivation of estrogen and a variety of androgens seems to be its most probable function. Because of its apparent inability to access retinol bound with cellular retinol-binding protein, a function in the pathway of retinoic acid biosynthesis seems less obvious. These data provide additional insight into the enzymology of estrogen, androgen, and retinoid metabolism and illustrate how closely related members of the SDR superfamily can have strikingly different substrate specificities.     

Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment

Retinoic acid (RA), an active vitamin A metabolite, is a key signaling molecule in vertebrate embryos. Morphogenetic RA gradients are thought to be set up by tissue-specific actions of retinaldehyde dehydrogenases (RALDHs) and catabolizing enzymes. According to the species, two enzymatic pathways (β-carotene cleavage and retinol oxidation) generate retinaldehyde, the substrate of RALDHs. Placental species depend on maternal retinol transferred to the embryo. The retinol-to-retinaldehyde conversion was thought to be achieved by several redundant enzymes; however, a random mutagenesis screen identified retinol dehydrogenase 10 [Rdh10(Trex) allele; Sandell LL, et al. (2007) Genes Dev 21:1113-1124] as responsible for a homozygous lethal phenotype with features of RA deficiency. We report here the production and characterization of unique murine Rdh10 loss-of-function alleles generated by gene targeting. We show that although Rdh10(-/-) mutants die at an earlier stage than Rdh10(Trex) mutants, their molecular patterning defects do not reflect a complete state of RA deficiency. Furthermore, we were able to correct most developmental abnormalities by administering retinaldehyde to pregnant mothers, thereby obtaining viable Rdh10(-/-) mutants. This demonstrates the rescue of an embryonic lethal phenotype by simple maternal administration of the missing retinoid compound. These results underscore the importance of maternal retinoids in preventing congenital birth defects, and lead to a revised model of the importance of RDH10 and RALDHs in controlling embryonic RA distribution.     

Pathways of vitamin A delivery to the embryo: insights from a new tunable model of embryonic vitamin A deficiency

Circulating retinoids (vitamin A and its derivatives) are found predominantly as retinol bound to retinol-binding protein (RBP), which transports retinol from liver stores to target tissues, or as retinyl ester incorporated in lipoproteins of dietary origin. The transport of retinoids from maternal to fetal circulation is poorly understood, especially under conditions of inadequate dietary vitamin A intake. Here we present RBP-/- mice as a tunable model of embryonic vitamin A deficiency. This model has enabled us to analyze metabolic links between maternal nutrition and retinoid delivery to the fetus. Our data show that retinol-RBP is the primary contributor to fetal development, whereas retinyl ester are largely responsible for accumulation of fetal retinoid stores. Furthermore, these studies indicate the importance of embryonic RBP in distributing vitamin A to certain developing tissues under restrictive diets. We also show differences among developing tissues in their dependency on the embryonic retinol-RBP pathway. Finally, we demonstrate that accumulation of embryonic vitamin A stores does not depend on the expression of RBP in the fetal liver.     

The C9 methyl group of retinal interacts with glycine-121 in rhodopsin

The visual pigment rhodopsin is a prototypical G protein-coupled receptor. These receptors have seven transmembrane helices and are activated by specific receptor–ligand interactions. Rhodopsin is unusual in that its retinal prosthetic group serves as an antagonist in the dark in the 11-cis conformation but is rapidly converted to an agonist on photochemical cis to trans isomerization. Receptor–ligand interactions in rhodopsin were studied in the light and dark by regenerating site-directed opsin mutants with synthetic retinal analogues. A progressive decrease in light-dependent transducin activity was observed when a mutant opsin with a replacement of Gly¹²¹ was regenerated with 11-cis-retinal analogues bearing progressively larger R groups (methyl, ethyl, propyl) at the C9 position of the polyene chain. A progressive decrease in light activity was also observed as a function of increasing size of the residue at position 121 for both the 11-cis-9-ethyl- and the 11-cis-9-propylretinal pigments. In contrast, a striking increase of receptor activity in the dark—i.e., without chromophore isomerization—was observed when the molecular volume at either position 121 of opsin or C9 of retinal was increased. The ability of bulky replacements at either position to hinder ligand incorporation and to activate rhodopsin in the dark suggests a direct interaction between these two sites. A molecular model of the retinal-binding site of rhodopsin is proposed that illustrates the specific interaction between Gly¹²¹ and the C9 methyl group of 11-cis-retinal. Steric interactions in this region of rhodopsin are consistent with the proposal that movement of transmembrane helices 3 and 6 is concomitant with receptor activation.     

Highly efficient retinal metabolism in cones

After bleaching of visual pigment in vertebrate photoreceptors, all-trans retinal is reduced to all-trans retinol by retinol dehydrogenases (RDHs). We investigated this reaction in purified carp rods and cones, and we found that the reducing activity toward all-trans retinal in the outer segment (OS) of cones is >30 times higher than that of rods. The high activity of RDHs was attributed to high content of RDH8 in cones. In the inner segment (IS) in both rods and cones, RDH8L2 and RDH13 were found to be the major enzymes among RDH family proteins. We further found a previously undescribed and effective pathway to convert 11-cis retinol to 11-cis retinal in cones: this oxidative conversion did not require NADP⁺ and instead was coupled with reduction of all-trans retinal to all-trans retinol. The activity was >50 times effective than the oxidizing activity of RDHs that require NADP⁺. These highly effective reactions of removal of all-trans retinal by RDH8 and production of 11-cis retinal by the coupling reaction are probably the underlying mechanisms that ensure effective visual pigment regeneration in cones that function under much brighter light conditions than rods.     

WY-14643 and 9-<Emphasis Type="Italic">cis</Emphasis>-retinoic acid induce IRS-2/PI 3-kinase signalling pathway and increase glucose transport in human skeletal muscle cells: differential effect in myotubes from healthy subjects and Type 2 diabetic patients

Aims/hypothesis To determine the effects of peroxisome proliferator-activated receptor (PPAR) and retinoid X receptor (RXR) agonists on insulin action, we investigated the effects of Wy-14643 and 9-cis-retinoic acid (9-cis-RA) on insulin signalling and glucose uptake in human myotubes.Methods Primary cultures of differentiated human skeletal muscle cells, established from healthy subjects and Type 2 diabetic patients, were used to study the effects of Wy-14643 and 9-cis-RA on the expression and activity of proteins involved in the insulin signalling cascade. Glucose transport was assessed by measuring the rate of [³H]2-deoxyglucose uptake.Results Wy-14643 and 9-cis-RA increased IRS-2 and p85 phosphatidylinositol 3-kinase (PI 3-kinase) mRNA and protein expression in myotubes from non-diabetic and Type 2 diabetic subjects. This resulted in increased insulin stimulation of protein kinase B phosphorylation and increased glucose uptake in cells from control subjects. Myotubes from diabetic patients displayed marked alterations in the stimulation by insulin of the IRS-1/PI 3-kinase pathway. These alterations were associated with blunted stimulation of glucose transport. Treatment with Wy-14643 and 9-cis-RA did not restore these defects but increased the basal rate of glucose uptake.Conclusions/interpretation These results demonstrate that PPAR and RXR agonists can directly affect insulin signalling in human muscle cells. They also indicate that an increase in the IRS-2/PI 3-kinase pathway does not overcome the impaired stimulation of the IRS-1-dependent pathway and does not restore insulin-stimulated glucose uptake in myotubes from Type 2 diabetic patients.Abbreviations PI 3-kinase phosphatidylinositol 3-kinase - PKB protein kinase B - PKC protein kinase C - PPAR peroxisome proliferator-activated receptor - 9-cis-RA 9-cis-retinoic acid - RXR retinoid X receptor     

Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase Adh3

Influence of vitamin A (retinol) on growth depends on its sequential oxidation to retinal and then to retinoic acid (RA), producing a ligand for RA receptors essential in development of specific tissues. Genetic studies have revealed that aldehyde dehydrogenases function as tissue-specific catalysts for oxidation of retinal to RA. However, enzymes catalyzing the first step of RA synthesis, oxidation of retinol to retinal, remain unclear because none of the present candidate enzymes have expression patterns that fully overlap with those of aldehyde dehydrogenases during development. Here, we provide genetic evidence that alcohol dehydrogenase (ADH) performs this function by demonstrating a role for Adh3, a ubiquitously expressed form. Adh3 null mutant mice exhibit reduced RA generation in vivo, growth deficiency that can be rescued by retinol supplementation, and completely penetrant postnatal lethality during vitamin A deficiency. ADH3 was also shown to have in vitro retinol oxidation activity. Unlike the second step, the first step of RA synthesis is not tissue-restricted because it is catalyzed by ADH3, a ubiquitous enzyme having an ancient origin.     

NinaB combines carotenoid oxygenase and retinoid isomerase activity in a single polypeptide

In animals, successful production of the visual chromophore (11-cis-retinal or derivatives thereof such as 11-cis-3-hydroxy-retinal) is essential for photoreceptor cell function and survival. These carotenoid-derived compounds must combine with a protein moiety (the opsin) to establish functional visual pigments. Evidence from cell culture systems has implicated that the retinal pigment epithelium protein of 65 kDa (RPE65) is the long-sought all-trans to 11-cis retinoid isomerase. RPE65 is structurally related to nonheme iron oxygenases that catalyze the conversion of carotenoids into retinoids. In vertebrate genomes, two carotenoid oxygenases and RPE65 are encoded, whereas in insect genomes only a single representative of this protein family, named NinaB (denoting neither inactivation nor afterpotential mutant B), is encoded. We here cloned and functionally characterized the ninaB gene from the great wax moth Galleria mellonella. We show that the recombinant purified enzyme combines isomerase and oxygenase (isomerooxygenase) activity in a single polypeptide. From kinetics and isomeric composition of cleavage products of asymmetrical carotenoid substrates, we propose a model for the spatial arrangement between substrate and enzyme. In Drosophila, we show that carotenoid-isomerooxygenase activity of NinaB is more generally found in insects, and we provide physiological evidence that carotenoids such as 11-cis-retinal can promote visual pigment biogenesis in the dark. Our study demonstrates that trans/cis isomerase activity can be intrinsic to this class of proteins and establishes these enzymes as key components for both invertebrate and vertebrate vision.     

Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue

Most opsins selectively bind 11-cis retinal as a chromophore to form a photosensitive pigment, which underlies various physiological functions, such as vision and circadian photoentrainment. Recently, opsin 3 (Opn3), originally called encephalopsin or panopsin, and its homologs were identified in various tissues including brain, eye, and liver in both vertebrates and invertebrates, including human. Because Opn3s are mainly expressed in tissues that are not considered to contain sufficient amounts of 11-cis retinal to form pigments, the photopigment formation ability of Opn3 has been of interest. Here, we report the successful expression of Opn3 homologs, pufferfish teleost multiple tissue opsin (PufTMT) and mosquito Opn3 (MosOpn3) and show that these proteins formed functional photopigments with 11-cis and 9-cis retinals. The PufTMT- and MosOpn3-based pigments have absorption maxima in the blue-to-green region and exhibit a bistable nature. These Opn3 homolog-based pigments activate G_i-type and G_o-type G proteins light dependently, indicating that they potentially serve as light-sensitive G_i/G_o-coupled receptors. We also demonstrated that mammalian cultured cells transfected with the MosOpn3 or PufTMT became light sensitive without the addition of 11-cis retinal and the photosensitivity retained after the continuous light exposure, showing a reusable pigment formation with retinal endogenously contained in culture medium. Interestingly, we found that the MosOpn3 also acts as a light sensor when constituted with 13-cis retinal, a ubiquitously present retinal isomer. Our findings suggest that homologs of vertebrate Opn3 might function as photoreceptors in various tissues; furthermore, these Opn3s, particularly the mosquito homolog, could provide a promising optogenetic tool for regulating cAMP-related G protein-coupled receptor signalings.     

9-cis retinoic acid induces complete remission but does not reverse clinically acquired retinoid resistance in acute promyelocytic leukemia

9-cis retinoic acid (RA) is a high-affinity ligand for both retinoic acid receptors (RARs) and retinoid "X" receptors (RXRs). Although all- trans RA does not bind to RXRs, RAR/RXR heterodimers or RXR/RXR homodimers bind to specific DNA response elements and modulate proliferation and differentiation of normal and malignant cells. Because the development of clinical resistance to all-trans RA has been associated with a progressive decrease in plasma drug concentrations, we evaluated the ability of 9-cis RA to induce in vitro cytodifferentiation in subclones of a retinoid-sensitive and resistant APL cell line (NB4) and in short-term cultures of fresh leukemic cells aspirated from patients. We also evaluated the clinical activity and pharmacokinetics of 9-cis RA (LGD 1057) in patients with APL who were previously treated with all-trans RA. In vitro tests of both retinoid- sensitive NB4 cells, as well as samples of fresh cells from 11 patients with APL, showed relatively equivalent degrees of sensitivity to both 9- cis RA and all-trans RA at concentrations ranging from 10(-6) to 10(-8) mol/L; however, no substantial cytodifferentiation was observed using either drug alone or in combination (10(-6) mol/L of each) in retinoid- resistant NB4 cells. Seven patients with APL who had previously relapsed from a remission induced by all-trans RA were treated with 9- cis RA at daily oral doses ranging from 30 to 230 mg/m2. Pharmacokinetic studies showed that the mean terminal plasma half-life of 9-cis RA (1.3 hours) changed very little after several weeks of dosing, although the mean change per dose level in area under the plasma concentration x time curves and peak plasma concentrations showed a decrease by 49% and 45%, respectively. Peak plasma concentrations equaled or exceeded concentrations that were effective against retinoid-sensitive cells in vitro. Despite these favorable pharmacokinetic results, only one of the seven patients achieved complete remission, corroborating in vitro studies of blasts from three of the nonresponders that showed a relatively equivalent degree of resistance to both retinoids. Our results suggest that while 9-cis RA may not induce its own catabolism to the same degree as all-trans RA, this feature does not appear to overcome clinically acquired resistance to all-trans RA in APL. Nonetheless, the drug can induce complete remissions in patients with APL and may be useful for extended therapy in other diseases. Future studies should address the use of lower doses in patients who have not previously received retinoid therapy.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Hypothetical Mechanism for Fetal Alcohol Syndrome Involving Ethanol Inhibition of Retinoic Acid Synthesis at the Alcohol Dehydrogenase Step

Ethanol acts as a teratogen causing brain, craniofacial, and limb abnormalities in those suffering from fetal alcohol syndrome. Normal embryonic development of the vertebrate nervous system and limbs has recently been shown to be governed by retinoic acid, the active form of vitamin A. Retinol dehydrogenase is an enzyme needed to convert vitamin A (retinol) to retinoic acid, a molecule that specifies embryonic pattern formation by controlling gene expression. Ethanol acts as a competitive inhibitor of the retinol dehydrogenase activity attributed to mammalian alcohol dehydrogenase (ADH), an enzyme that uses both retinol and ethanol as substrates. An hypothesis is presented in which many of the abnormalities observed in fetal alcohol syndrome may be caused by high levels of ethanol acting as a competitive inhibitor of ADH-catalyzed retinol oxidation in the embryo or fetus. This would presumably result in a reduction of retinoic acid synthesis in embryonic tissues such as the nervous system and limbs that require critical levels of this molecule to specify spatial patterns.     

Novel retinoic acid, 9-cis retinoic acid, in combination with all-trans retinoic acid is an effective inducer of differentiation of retinoic acid-resistant HL-60 cells

Recent studies have shown that a high proportion of patients with acute promyelocytic leukemia (APL) achieve complete remission after treatment with all-trans retinoic acid (RA). Nevertheless, despite an initial good response, most patients that received continuous treatment with all-trans RA relapse and develop RA-resistant disease. The 9-cis RA is a high-affinity ligand for retinoid X receptors (RXRs) and also binds efficiently to retinoic acid receptors (RARs); all-trans RA is a ligand for RARs. Both alone are able to induce differentiation of wild-type HL- 60 cells. We found that neither all-trans RA nor 9-cis RA (< 2 x 10(-6) mol/L) induced differentiation of RA-resistant HL-60 cells into either mature granulocytes or monocytes. However, morphologic differentiation of the RA-resistant HL-60 cells was induced by 10(-6) mol/L all-trans RA combined with various concentrations (10(-12) to 10(-6) mol/L) of 9- cis RA. Electron microscopic examination also confirmed that the combination of both retinoids induced RA-resistant HL-60 cells to differentiate to mature granulocytes. Functional analysis of differentiation (NBT reduction activity) confirmed the necessity of both analogs to induce differentiation. Also, expression of myeloid- specific differentiation antigens (CD11b and CD14) as well as migration inhibitory factor-related protein (MRP)-8/14 mRNAs were upregulated only in the presence of both retinoids in a dose-dependent manner. In these conditions 3H-thymidine incorporation was inhibited and numbers of viable cells were decreased, suggesting that all-trans RA with 9-cis RA may inhibit cell growth and induce differentiation of RA-resistant HL-60 cells into mature granulocytes. These studies suggest that 9-cis RA in combination with all-trans RA is an effective inducer of RA- resistant HL-60 cells and may have implications for both the biology of retinoids and clinical treatment of RA-resistant acute myelogenous leukemia, including APL patients.