[CANCER RESEARCH 62, 1196–1204, February 15, 2002] retSDR1, a Short-Chain Retinol Dehydrogenase/Reductase, Is Retinoic Acid- inducible and Frequently Deleted in Human Neuroblastoma Cell Lines1 Fabio Cerignoli, Xiaojia Guo, Beatrice Cardinali, Christian Rinaldi, Jessica Casaletto, Luigi Frati, Isabella Screpanti, Lorraine J. Gudas, Alberto Gulino, Carol J. Thiele,3 and Giuseppe Giannini2,3 Department of Experimental Medicine and Pathology, University La Sapienza, 00161 Rome, Italy3 [L. F., I. S., A. G., G. G.]; Department of Experimental Medicine, University of L’Aquila, 67100 L’Aquila, Italy [F. C.]; Neuromed Institute, 86077 Pozzilli, Italy [C. R., L. F.]; Pasteur Institute Cenci-Bolognetti Foundation, 00161 Rome, Italy [I. S.]; Institute of Cell Biology, Consiglio Nazionale delle Ricerche (CNR), 00016 Monterotondo, Italy [B. C.]; Department of Pharmacology, Weill Medical College of Cornell University, New York, New York, 10021 [X. G., L. J. G.]; and Cellular and Molecular Biology Section, National Cancer Institute, NIH, Bethesda, Maryland3 20892 [J. C., C. J. T.] ABSTRACT enzymes and binding proteins (reviewed in Refs. 2, 3). With the clear exception of the visual cycle, RA appears to be the active metabolite Vitamin A is required for a number of developmental processes and for of retinol for most functions. Free RA does not accumulate in the the homeostatic maintenance of several adult differentiated tissues and cells, and its activity is regulated by at least two different binding organs. In human neuroblastoma (NB) cells as well as some other tumor types, pharmacological doses of retinoids are able to control growth and proteins, CRABPI and -II and by an active metabolism that rapidly induce differentiation in vitro and in vivo. In a search for new genes that converts it into more soluble forms (4–8). RA significantly contrib- are regulated by retinoids and that contribute to the biological effects utes to its own synthesis and degradation through the transcriptional retinoids have on NB cells, we have isolated five differentially expressed regulation of cellular retinol binding proteins, CRABPs, and many transcripts. Here we report on the characterization of one of them enzymes involved in several steps of retinol and RA synthesis and (DD83.1) in NB cell lines. DD83.1 is identical to the human retSDR1, a catabolism (6, 9–16). short chain dehydrogenase/reductase that is thought to regenerate retinol Biologically active vitA metabolites regulate transcription of target from retinal in the visual cycle. Its expression is strongly, but differently, genes through the activation of nuclear receptors of two classes, RARs regulated by retinoids in NB cell lines, and it is widely expressed in human and RXRs, each of which is coded by three different genes (␣, ␤, and tissues, which suggests that it is involved in a more general retinol meta- ␥). A transcriptionally active unit consists of a RXR/RAR heterodimer bolic pathway. Both the retinoic acid-dependent and the exogenous ex- pression of retSDR1 in SK-N-AS cells induce the accumulation of retinyl with required ligand binding on RAR, and occasionally on RXR for a esters, which indicates that it is involved in generating storage forms of more potent genomic response (17–20). A variety of studies have retinol in tissues exposed to physiological retinol concentrations. We also indicated that specific receptor subtypes can serve particular func- show that the human retSDR1 gene, which maps on chromosome 1p36.1, tions. Although there is some redundancy, defective phenotypes, is contained in the DNA fragment deleted in many NB cell lines bearing generated either in cell culture or in animal settings, cannot always be MYCN amplification but is conserved in a cell line with a small 1p deletion fully compensated by the overexpression of other retinoid receptors and normal MYCN. Our observations suggest that retSDR1 is a novel (21, 22). The involvement of RAR␣ in the pathogenesis of promy- regulator of vitamin A metabolism and that its frequent deletion in NB elocytic leukemia has also indicated that spontaneous mutations in the cells bearing MYCN amplification could compromise the sensitivity of retinoid receptors can be associated with cancer development (23). those cells to retinol, thereby contributing to cancer development and progression. More recently, the absence or the reduced expression of specific retinoid receptor isoforms, particularly RAR␤, was found to be rele- vant for the development and progression of several types of cancer INTRODUCTION (see Refs. 22, 24 and references therein). Natural and synthetic reti- noids are being used for the chemoprevention and treatment of many vitA4 or retinol is essential for proper vertebrate embryonic devel- human neoplastic diseases (25). The recent finding that retinoids used opment and growth as well as for the maintenance of several adult in the settings of minimal residual disease could efficiently increase differentiated functions, including vision, fertility, and correct tro- the event-free survival of patients with advanced stage NB (26) phism of the epithelia (1). Dietary-assumed retinol is delivered to the indicates a need for a greater understanding of the sensitivity of these liver, in which more than 90% of the total content of vitA is stored in tumor cells to retinoids. the form of retinyl esters, mostly retinyl palmitate. Retinol is appar- NB is a neural crest-derived tumor. The correct homeostatic control ently the major form of transported vitA and the homeostatic control of vitA metabolism is crucial for proper development and mainte- of circulating and storage retinol is strictly regulated by a plethora of nance of the integrity of neural crest cell structures. Cranial and trunk neural crest derivatives are almost invariably damaged by the terato- Received 2/12/01; accepted 12/28/01. The costs of publication of this article were defrayed in part by the payment of page genic effects of exogenously added retinoids during development (1). charges. This article must therefore be hereby marked advertisement in accordance with Incorrect development of these regions occurs in most double-RA 18 U.S.C. Section 1734 solely to indicate this fact. receptor knockout mice (21, 27), which suggests that either excessive 1 Supported in part by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), the National Research Council (CNR), Biotechnology and Oncology Project, the or reduced retinoid signaling are deleterious for proper development Ministry of University, Research and Technology (MURST), the Associazione per la lotta of these regions. More subtle effects of retinoids are also important for al Neuroblastoma (ANB), the MURST-CNR “Biomolecole per la Salute Umana” Pro- gram, the Pasteur Institute Cenci-Bolognetti Foundation, the Italian Ministry of Health, the correct development of the neural crest-derived peripheral nervous and NIH Grant ROI CA77509 (to L. J. G.). system, and the adrenergic cells and enteric neurons are highly re- 2 To whom requests for reprints should be addressed, at Department of Experimental sponsive to retinoids (28, 29). NB cells are among the most sensitive Medicine and Pathology, University La Sapienza, Policlinico Umberto I, Viale Regina Elena, 324, 00161 Rome, Italy. Phone: 39-06-4958637; Fax: 39-06-4461974; E-mail: and frequently used neural crest cells for testing retinoid effects in [email protected]. culture. In fact, many NB cell lines respond to RA with a reduction of 3 These laboratories contributed equally to this work. their proliferation rate and a morphological and biochemical differ- 4 The abbreviations used are: vitA, vitamin A; RA, retinoic acid, ATRA, all-trans-RA; 9CRA, 9-cis-RA; RAR, RA receptor; RXR, retinoid X receptor; CRABP, cellular retinoic acid entiation toward more neuronal phenotypes (30–32). This process is binding protein; NB, neuroblastoma; SRO, shortest region(s) of overlap; IF, immunofluores- partially attributable to the repression of mycN (in MYCN amplified cence; tPA, tissue plasminogen activator; HPLC, high-performance liquid chromatography; hretSDR1, human retSDR1; CHX, cycloheximide; ORF, open reading frame; MoAb, mono- cell lines; Ref. 33), and to the increase in Trk receptors (34, 35) and clonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. in the cyclin-dependent kinase inhibitor p27 (36). However, some NB 1196 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2002 American Association for Cancer Research. retSDR1 AND RETINOL METABOLISM IN NB CELLS cells, which can still transduce RA-mediated signaling, are resistant to sucrose, 10 mM Tris-HCl (pH 7.4) and Protease Inhibitor Cocktail (Sigma RA-dependent growth inhibition and differentiation, possibly because Chemical Co.). Nuclei and mitocondria were then removed by a 10-min of a different pattern of expression of HMGI proteins (37, 38). centrifugation at 8,000 ϫ g. The supernatant was further centrifuged at Furthermore, it appears that even in sensitive cells the pathways 105,000 ϫ g for 1.5 h to obtain the microsomal and the cytosolic fraction. For leading to differentiation and growth inhibition can be partially sep- each fraction, an equal amount of proteins normalized on cell number was separated on 13% SDS-PAGE and was analyzed by Western blot using the arated (20, 39). 9E10 anti-myc MoAb and an anti-␣-tubulin MoAb (Oncogene Research Prod- To identify retinoid-responsive genes, whose regulation occurs in ucts). For IF, cells were fixed in 4%