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Tumor Biology |
Department of Experimental Medicine and Pathology, University La Sapienza, 00161 Rome, Italy,3 [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, Maryland,3 20892 [J. C., C. J. T.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biologically active vitA metabolites regulate transcription of target genes through the activation of nuclear receptors of two classes, RARs and RXRs, each of which is coded by three different genes (
, ß, and 
). A transcriptionally active unit consists of a RXR/RAR heterodimer with required ligand binding on RAR, and occasionally on RXR for a more potent genomic response (17, 18, 19, 20)
. A variety of studies have indicated that specific receptor subtypes can serve particular functions. Although there is some redundancy, defective phenotypes, generated either in cell culture or in animal settings, cannot always be fully compensated by the overexpression of other retinoid receptors (21
, 22)
. The involvement of RAR in the pathogenesis of promyelocytic leukemia has also indicated that spontaneous mutations in the retinoid receptors can be associated with cancer development (23)
. More recently, the absence or the reduced expression of specific retinoid receptor isoforms, particularly RARß, was found to be relevant for the development and progression of several types of cancer (see Refs. 22
, 24
and references therein). Natural and synthetic retinoids are being used for the chemoprevention and treatment of many human neoplastic diseases (25)
. The recent finding that retinoids used in the settings of minimal residual disease could efficiently increase the event-free survival of patients with advanced stage NB (26)
indicates a need for a greater understanding of the sensitivity of these tumor cells to retinoids.
NB is a neural crest-derived tumor. The correct homeostatic control of vitA metabolism is crucial for proper development and maintenance of the integrity of neural crest cell structures. Cranial and trunk neural crest derivatives are almost invariably damaged by the teratogenic effects of exogenously added retinoids during development (1) . Incorrect development of these regions occurs in most double-RA receptor knockout mice (21 , 27) , which suggests that either excessive or reduced retinoid signaling are deleterious for proper development of these regions. More subtle effects of retinoids are also important for the correct development of the neural crest-derived peripheral nervous system, and the adrenergic cells and enteric neurons are highly responsive to retinoids (28 , 29) . NB cells are among the most sensitive and frequently used neural crest cells for testing retinoid effects in culture. In fact, many NB cell lines respond to RA with a reduction of their proliferation rate and a morphological and biochemical differentiation toward more neuronal phenotypes (30, 31, 32) . This process is partially attributable to the repression of mycN (in MYCN amplified cell lines; Ref. 33 ), and to the increase in Trk receptors (34 , 35) and in the cyclin-dependent kinase inhibitor p27 (36) . However, some NB cells, which can still transduce RA-mediated signaling, are resistant to RA-dependent growth inhibition and differentiation, possibly because of a different pattern of expression of HMGI proteins (37 , 38) . Furthermore, it appears that even in sensitive cells the pathways leading to differentiation and growth inhibition can be partially separated (20 , 39) .
To identify retinoid-responsive genes, whose regulation occurs in association with growth inhibition or differentiation induced by retinoids, we applied the RNA fingerprinting methodology to KCNR cells treated with different combinations of receptor-selective retinoids. This analysis resulted in five differentially displayed PCR amplicons. In this article, we report on one of them that is identical to the hretSDR1, a short chain dehydrogenase/reductase, that can catalize the reduction of retinal in the visual cycle (40) . The expression of this gene is strongly, but differently, induced by retinoids in all of the NB cells tested and is widely expressed among human tissues, which suggests its involvement in a more general retinol metabolic pathway. Consistently, retSDR1 overexpression in SK-N-AS cells strongly induced the formation retinyl esters. We also demonstrated that the human retSDR1 gene, which maps on chromosome 1p36.1 (40) , is contained in the DNA fragment deleted in many NB cell lines bearing MYCN amplification, but is conserved in a cell line with a small deletion and single copy MYCN. These observations suggest that retSDR1 might be involved in a general retinol metabolic pathway and that its partial absence in 1p36-deleted NBs might reduce their sensitivity to retinol in vivo.
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MATERIALS AND METHODS |
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Nucleic Acid Isolation, Northern Blot, and Southern Blot Analysis.
After the appropriate treatment, cells were washed twice with cold PBS, and total RNA was extracted using the RNeasy system (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Northern analysis of total RNA (20 µg) and Southern analysis of genomic DNA (20 µg) were performed as described previously (20)
. Multiple tissue Northern blots (Clontech, Palo Alto, CA) contained 2 µg of polyadenylated RNA from various adult and fetal tissues. The DD83.1 fragment or the PCR-cloned (see below) complete coding sequence of hretSDR1, were labeled with [-32P]dCTP using a random primer labeling kit (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom) according to the manufacturer’s directions and used as cDNA probes for Northern hybridization. Blots were hybridized [42°C in 50% (v/v) formamide, 1 M NaCl, 10% dextran sulfate, 1% SDS, 100 µg/ml salmon sperm DNA] and washed (0.2x SSC, 0.1% SDS at 60°C–65°C) at high stringency.
RNA Fingerprinting.
For the RNA fingerprinting we followed the protocol published by Malgaretti et al. (41)
and readapted as described previously (42, 43, 44)
. Primers used were as follows: 83, 5'-CGTGGGCAACCT-3'; 122, 5'-CATGGCTGCCAG-3'; 133, 5'-CAGTCCTGGCCA-3'. Cloned DNA inserts were sequenced by using an ABI PRISM 377 DNA Sequencer and the DNA Sequencing kit-Big Dye terminator (PE-Applied Biosystems, Warrington, Great Britain). The differential regulation of the isolated genes by the retinoids was confirmed by semiquantitative reverse transcription (RT)-PCR analysis and/or by Northern blots.
hretSDR1 ORF Cloning, Transfection, Cell Fractionation, and IF.
A PCR-amplified fragment containing hretSDR1 ORF was cloned in frame with a Myc-tag (retSDR1-Myc) in the pcDNA3.1/Myc-His B (Invitrogen, San Diego, CA). retSDR1-Myc was transfected in SK-N-SH neuroblastoma cells, by liposomal transfer using the TransFast reagent (Promega Corporation, Madison WI), according to the manufacturer’s instructions. The following day, cells were processed for cell fractionation or IF experiments. For subcellular fractionation, cells were lysed in 1 ml of iso-osmotic buffer containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4) and Protease Inhibitor Cocktail (Sigma Chemical Co.). Nuclei and mitocondria were then removed by a 10-min centrifugation at 8,000 x g. The supernatant was further centrifuged at 105,000 x g for 1.5 h to obtain the microsomal and the cytosolic fraction. For 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 9E10 anti-myc MoAb and an anti--tubulin MoAb (Oncogene Research Products). For IF, cells were fixed in 4% formaldehyde, permeabilized with 0.25% Triton X-100, incubated with either the biotinylated 9E10 antibody or the anti-retSDR1 mouse MoAb followed by an incubation with an antimouse biotinylated antibody (DAKO Corporation, Carpinteria, CA). Both were revealed with streptavidin-FITC (DAKO Corporation).
retSDR1 overexpressing stable clones were obtained by transfecting SK-N-AS cells with either the retSDR1-Myc or the control constructs followed by G418 selection. After 10–20 days, individual clones (including CTR#2 and SDR#62) were picked and stably grown under selection. In addition, to minimize the possibility of misinterpretation caused by clonal variability we have also used transfected SK-N-AS cells, which, after the proper selection time were not individually cloned, but established as heterogeneous mixed nonclonal continuous cell lines (CTRpool and SDRpool).
Retinoid Metabolism.
Retinol metabolism was measured using [3H]retinol by reverse-phase HPLC according to previously described methods (45
, 46)
. The metabolism of nonradiolabeled retinol and retinaldehyde was measured in CTR#3 and SDR#62 clones by reverse phase HPLC followed by photodiode array detection (45
, 46)
. All of the studies were performed at least twice with multiple time points. Appropriate standards were run to assist in the identification of the endogenous retinoids. Both the cells and the medium were analyzed for retinol content at various times.
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RESULTS |
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further supported these observations and enabled the identification of genes whose expression is modulated by retinoids in KCNR cells. In fact, through this methodology, we have examined 
240 genes expressed in KCNR cells and identified 5 of them whose expression is regulated by retinoids. Oligo(83), oligo(122), and oligo(133) generated amplicons differentially expressed in our assay (Fig. 1A)
. Band DD83.1, which was clearly absent in the reaction performed on control cells and on cells treated with the RXR-selective SR11246 and the RAR-selective SR11254, appeared as an additional displayed band in the reaction obtained by cells treated with other retinoids and/or retinoid combinations (Fig. 1A)
. Basal levels of band DD122.2 were detected in controls, and it was induced to comparable levels by ATRA, 9CRA, SR11383+SR11246, Am580+SR11246, and SR11254+SR11246, and to a lesser extent by the single retinoids. Band DD133.1, which was absent in the control, SR11246-, and SR11254+SR11246-treated cells, was highly represented in 9CRA-treated cells, but was also induced by SR11383+SR11246 and Am580+SR11246 (Fig. 1A)
. The intensity of surrounding bands was used to assess the relative loading of the lanes and allowed us to consider the strong signal observed in SR11254-treated cells as an artifact caused by overloading of the lane. Band 122.4 was also positively regulated by ATRA, 9CRA, and all effective retinoid combinations (Fig. 1A)
. Interestingly DD122.3, which was not influenced by ATRA, 9CRA, or single retinoid application, was reduced only by the combination of SR11383 and SR11246 (Fig. 1A)
, which is the retinoid combination that most effectively controls growth and induces differentiation in KCNR cells (20)
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. Through this method we could clearly distinguish the differentially displayed cloned inserts (that hybridized only to retinoid-induced RNA fingerprinting reactions; Fig. 1B
, Lanes 6, 7, and 8) from cloning artifacts (that hybridized to both control- and retinoid-induced RNA fingerprinting reaction; Fig. 1B
, Lanes 1, 2, 3, 4, 5, 9, and 10). The differential expression of DD83.1, DD122.2, and DD122.3 was further confirmed by a semiquantitative reverse transcription-PCR (Fig. 1C)
. Sequencing of the cloned amplicons and their comparison to nonredundant sequence databanks revealed that DD122.2 was a partial clone for the human SH3-binding protein 2, which maps to chromosome 4p16.3 close to the Huntington’s disease region, whereas DD122.3 was a partial sequence for the human mitochondrial dicarboxylate carrier protein. DD83.1, which demonstrated the typical signature of a dehydrogenase, was identical to the recently reported hretSDR1 mRNA. The other two sequences did not have recognizable homologues in nonredundant databases.
DD83.1/retSDR1 Expression in NB Cells.
DD83.1 is a partial clone spanning from base 289 to 842 of the deposited hretSDR1 sequence, a region that includes a large part of its ORF. This fragment and a full coding sequence fragment were used to confirm its inducibility by retinoids in KCNR cells by Northern blot. As shown in Fig. 1D
, this analysis confirmed that retSDR1 is undetectable in untreated KCNR cells and is induced by ATRA, 9CRA, and all of the retinoid combinations effective in the RNA fingerprinting after 4 days of treatment. Analysis of the retSDR1 mRNA expression in a panel of NB cell lines revealed the existence of several transcripts, the expression of which was differentially regulated by ATRA (Fig. 2)
. In particular, after 3 h, we detected a very strong RA-induced increase in three different transcripts (1.4-, 1.8-, and 3.5-kb) in SK-N-SH cells that persisted even after 3 days of ATRA treatment. A similar situation occurred in SK-N-AS cells, which lacked the 1.8-kb transcript. In contrast, ATRA induced the 1.8-kb transcript in KCNR cells (Fig. 2A)
and in LAN-5 (not shown) at the latest time point and, compared with SK-N-SH and SK-N-AS, transcript accumulation reached consistently lower levels. Finally, in SK-N-BE cells, we could detect only a slight increase of the 1.4-kb transcript after 3 days of ATRA treatment (Fig. 2A)
. A more detailed time course analysis verified that retSDR1 induction by ATRA required a longer time in KCNR cells, in which it first appeared after 12 h and reached a plateau between 72 and 96 h; and in both KCNR and SY5Y cells, its expression persisted throughout 8 days of ATRA treatment (not shown). Interestingly, protein kinase A activation, vitamin D3, dexamethasone, and TGFß could not induce retSDR1 mRNA in several NB cell lines (not shown), which suggested that the retSDR1 increase is a specific effect of RA.
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. In addition, it did not affect ATRA induction of retSDR1 in the first 3 h of treatment, which suggested a direct regulation by ATRA. However, although it was still completely ineffective by itself, CHX enhanced ATRA-induced accumulation of the 1.4-kb transcript as early as after 6 h, thus anticipating the plateau levels otherwise obtained after 24 h of ATRA stimulation (Fig. 2B)
. Conversely, CHX inhibited the ATRA-induced accumulation of the 3.5-kb specie at 6, 12, and 24 h (Fig. 2B)
. Furthermore, in SK-N-SH cells, CHX did not affect the ability of ATRA to induce any retSDR1 transcript at either 6 or 12 h, whereas doxorubicin, an RNA polymerase inhibitor, abolished the ATRA-induced expression of both transcripts (not shown), which suggested that at least the 1.4-kb transcript is directly regulated by RA in both SK-N-AS and SK-N-SH cells.
retSDR1 Gene Maps within the Region Frequently Deleted in Human NBs.
The retSDR1 gene was mapped on human chromosome 1p36.1 (40)
, an area frequently lost in aggressive NB tumors, which suggests that retSDR1 might cosegregate with the still undiscovered NB tumor suppressor gene(s). In particular, at least two different SRO have been identified in the 1p region. The largest SRO, which would include the 1p36.1, is most frequently deleted in MYCN-amplified tumors (47, 48, 49)
. To test the hypothesis that retSDR1 is deleted in NB tumors, we have analyzed the status of the retSDR1 gene in several NB cell lines, by Southern blot (Fig. 3)
. We have found that three MYCN-amplified cell lines (LAN-5, KCNR, and SK-N-BE cells) that have a monoallelic deletion in the 1p region (47)
also have a haploid content of the retSDR1 gene compared with undeleted cells (SK-N-SH and SY5Y) or compared with a cell line that is known to have a small interstitial deletion (SK-N-AS) not extending to the 1p36.1 band (Fig. 3)
. Therefore, these results confirm that retSDR1 cosegregates with a NB tumor suppressor/deletion region. They also indicate that the delayed and reduced expression that we observed in KCNR, SK-N-BE (Fig. 2)
, and LAN-5 (not shown) is associated with a retSDR1 haploid DNA content (Fig. 3)
.
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. Although the retSDR1 cDNA was initially isolated from retina (40)
, its expression appears to be less restricted than originally reported. We detected high levels of expression in fetal kidney, liver, and lung and in adult heart, placenta, lung, liver, kidney, pancreas, thyroid, testis, stomach, trachea, and spinal cord. We also found lower levels in skeletal muscle, intestine, and lymph node. retSDR1 was barely detectable in adrenals, brain, thymus, and hematopoietic tissues. Two different retSDR1 cDNA probes recognized at least four different transcripts of 3.5-, 2.5-, 1.8-, 1.4-kb, and most tissues expressed more than one specie of retSDR1 mRNA (Fig. 4)
. In heart and fetal kidney, all forms were present, whereas in adult stomach, thyroid, and spinal cord, only the 2.5-, 1.8-, and 1.4-kb transcripts were detected. Interestingly, testis expressed only one form, which was smaller than 1.4 kb.
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. These data are consistent with the in situ pattern of localization of the exogenous retSDR1/Myc-tagged protein detected by IF experiments, which mainly localized to a perinuclear membranous compartment and was assembled in patched structures (Fig. 5B, a)
. To further address the issue of retSDR1 localization and to confirm that its expression is induced by ATRA in NB cells, we performed IF studies with a MoAb developed against the human protein and able to recognize overexpressed hretSDR1 (Ref. 40
; Fig. 5B, b
). The endogenous retSDR1 could be detected by a three-step IF both in control- and in ATRA-treated SK-N-SH cells (Fig. 5B, c and d)
. The endogenous protein showed an intracellular distribution largely overlapping that of the exogenous one, with localization in patched structures with a strict perinuclear distribution in untreated cells. Under ATRA stimulation, a remarkable enlargement of the immunoreactive regions were observed that were extended through the entire cytoplasm, confirming that ATRA induces a higher content of protein per cell.
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To test whether expression of retSDR1 could change the biological responses of NB cells to retinoids, we isolated individual clones or heterogeneous mixed nonclonal continuous cell lines (defined pools) of SK-N-AS cells stably expressing retSDR1. Both retSDR1- and mock-transfected cells could activate the expression of several genes (including the endogenous retSDR1, tPA, RARß, and HMGI(Y)) in response to RA treatment (Fig. 6A)
, thus suggesting that retSDR1 does not significantly modify responsiveness to exogenous RA. Therefore, we tested the effect of retSDR1 overexpression on retinol activities. Exogenous retinol could efficiently induce the expression of specific genes between 0.1- and 1-µM concentrations in parental SK-N-AS cells (not shown). A mock-transfected pool of SK-N-AS (CTRpool) and one of the selected control clones (CTR2) readily showed an increased expression of the endogenous 3.5-kb retSDR1 transcript, tPA, and RARß transcripts when treated with either 0.1 or 1 µM retinol (Fig. 6, B and C)
. In contrast, the retSDR1-transfected pool of SK-N-AS (SDRpool) showed a lower induction of endogenous retSDR1, tPA, and RARß compared with controls at 1 µM retinol. In the stably transfected and highly expressing line SDR#62, RARß mRNA was detected at low levels both in basal and in the retinol-induced conditions, and very little induction of the endogenous retSDR1 mRNA was seen even in response to 1 µM retinol. Moreover, all of the retSDR1-transfected cells were almost insensitive to 0.1 µM retinol (Fig. 6C)
, which suggests that overexpression of retSDR1 might reduce the amount of retinol available for direct or indirect transcriptional activation, possibly as a consequence of retinol metabolism toward retinyl esters, as we previously observed in ATRA-pretreated SK-N-AS cells.
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) and SDR#62 cells (Fig. 7, D, E, and F)
were treated with control solvent, (Fig. 7, A and D)
1 µM retynaldehyde (Fig. 7, B and E)
, or 1 µM retinol (Fig. 7, C and F)
for 24 h, harvested and analyzed for retinoid metabolism by reverse-phase HPLC and a photodiode array detector. Under basal conditions (control solvent; Fig. 7, A and D
), we detected no retinoids peak in the array profiles of either the medium (not shown) or the extracts of the CTR#3 and SDR#62 lines. Incubation with 1 µM retynaldehyde for 24 h resulted in no detectable increase in retinoic acid (Fig. 7, B and E
, at 21 min) and in a similar increase in retinol in either CTR#3 or SDR#62 (Fig. 7, B and E
, at 30.5 min), whereas we detected a 5-fold increase in retinyl esters only in SDR#62 (Fig. 7E
, at 50–58 min). After incubation with 1 µM retinol for 24 h, we could not detect any RA production in the medium (not shown) or extracted from the cells from either CTR#3 or SDR#62 lines; in contrast, we observed a 20-fold increase in the amount of retinyl esters only in clones SDR#62 (Fig. 7F
, at 50–58 min) and SDR#44 (not shown), but not in the CTR#3 line (Fig. 7C)
and CTRpool (not shown). Overall, our data indicate that either endogenous or exogenously expressed retSDR1 favors the metabolism of retinol and retinal to storage forms of vitA.
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DISCUSSION |
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The strong induction of retSDR1 by RA is common to all NB cell lines that we have tested and also to a variety of other nonneuroblastic human cell lines.6 This induction, therefore, can be interpreted as the attempt of cells to accumulate local retinol storage in the case of retinol availability. RetSDR1 induction by vitA derivatives appears highly specific because it was not evoked by either protein kinase A activation or treatment with vitamin D3, dexamethasone, or TGFß.6 Our findings complement several observations indicating that many cells possess a RA-inducible retinol metabolic enzyme system that can generate either transcriptionally active retinol metabolites or storage forms of vitA (46 , 50, 51, 52) . Consistent with this, RA transcriptionally or posttranscriptionally regulates the expression of a number of proteins relevant to its own metabolism and activity, including cellular retinol-binding proteins, CRABPs, LRAT, RALDHs, RARs, and now retSDR1 (6 , 9, 10, 11, 12, 13, 14, 15, 16) . RA also induces its own metabolism in embryonal carcinoma and in some neoplastic cells in culture (5 , 6 , 46 , 53 , 54) . The biochemical reactions through which retSDR1 promotes retinyl ester accumulation are not well characterized and will be the subject of future studies.
Haploinsufficiency at retSDR1 Locus Might Contribute to Cancer.
Quantitative or qualitative alterations in proteins involved in vitA metabolism and signal transduction are involved in cancer development. Spontaneous mutations in the retinoid receptors can be associated with cancer or even involved in its molecular pathogenesis, as in the case of RAR and promyelocytic leukemia (23)
. An absence or reduced expression of RARß was observed in several types of cancer (see Refs. 22
, 24
and references therein), and its presence seems to be required for growth inhibition and differentiation (22)
. vitA and some of its derivatives are being actively and successfully used in cancer chemoprevention and therapy (25)
.
The retSDR1 gene, which encodes an enzyme widely expressed among human tissues and is involved in retinol metabolism, is located on chromosome 1p36.1 (40) , a region very frequently rearranged in human cancer. Heterogeneous deletions and translocations of chromosome 1p were described in different neoplasias, but they appear with maximum frequency in NB, melanoma, and MEN2A-associated neoplasia (55) . Extensive deletions of 1p are associated with advanced stage and/or a more aggressive neoplasia and poor prognosis, suggesting the presence of gene(s) the deletion of which may contribute to tumor progression (56, 57, 58) . In NBs, the extent of 1p deletions is extremely heterogeneous, and at least two potential NB suppressor loci are present in two different areas (59, 60, 61) . Large chromosomal deletions from 1p35–pter are observed in most MYCN-amplified tumors, associated with more aggressive behavior and advanced stage, whereas smaller interstitial deletions involving 1p36.1–1p36.3 are mainly detected in single copy-MYCN tumors (47, 48, 49 , 57 , 62) .
Rather than the biallelic deletion of one gene, haploinsufficiency at several loci leading to a dosage defect involving multiple gene products is the most common outcome of the microsatellite instability observed in gastrointestinal cancer (63) . Although several candidates have been cloned out of the 1p SRO, there is no conclusive evidence that any of them is the bona fide NB suppressor gene. It is possible that the sum of mono- or biallelic deletions at many loci within the large 1p deletion might provide a growth or survival advantage to preneoplastic cells and, therefore, contribute to tumor development and/or progression. For example, the deletion of the MEMO1 gene, which maps at 1p35–36.1, is associated with and might be involved in controlling MYCN amplification and/or expression (64) , thus providing the 1p-deleted tumors with another growth advantage. It is tempting to speculate that the reduced content of several gene products involved in growth control and survival (including p73, RIZ, TNFR2, ID3, DAN, E2F2) caused by large 1p chromosomal deletions might add further growth or survival advantages to neoplastic cells. The monoallelic deletion of retSDR1 that we observed in several NB cell lines is associated with the reduced and delayed accumulation of the corresponding mRNAs in response to RA. Thus, it could provide a selective growth and survival advantage by altering the metabolic pathways that control the local production of biologically active vitA metabolites. In support of this hypothesis, we have shown that retSDR1 is an enzyme involved in the generation of storage forms of retinol that may be important cell growth and differentiation regulators. The possibility that retSDR1 might be involved in a growth/tumor suppressive pathway, perhaps through the further inactivation of the single allele remaining in many 1p-deleted human cancers, should also be considered.
In conclusion, we have provided evidence that retSDR1 is a novel regulator of vitA metabolism involved in the production of a local storage form of retinol, retinyl esters in NB cells. RetSDR1 is induced by RA in a wide array of cell lines derived from different human tissues, and it is frequently deleted in MYCN-amplified NB cell lines. It is possible that its deletion in NB cells and in a number of other human tumors might compromise their capability to form local retinyl esters for retinol storage. In the absence of local stores, and particularly under the low concentrations of circulating retinol that can be found in NB and other cancer patients (65) , the production of vitA active metabolites would be blunted. This might impair an important growth-inhibitory pathway and thus contribute to cancer development and progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 Ministry of University, Research and Technology (MURST), the Associazione per la lotta al Neuroblastoma (ANB), the MURST-CNR "Biomolecole per la Salute Umana" Program, the Pasteur Institute Cenci-Bolognetti Foundation, the Italian Ministry of Health, and NIH Grant ROI CA77509 (to L. J. G.). 
2 To whom requests for reprints should be addressed, at Department of Experimental 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: giuseppe.giannini{at}uniroma1.it
3 These laboratories contributed equally to this work.
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 binding protein; NB, neuroblastoma; SRO, shortest region(s) of overlap; IF, immunofluorescence; tPA, tissue plasminogen activator; HPLC, high-performance liquid chromatography; hretSDR1, human retSDR1; CHX, cycloheximide; ORF, open reading frame; MoAb, monoclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
5 F. Cerignoli and G. Giannini, unpublished observations.
6 G. Giannini and C. J. Thiele, unpublished observations.
Received 2/12/01. Accepted 12/28/01.
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, CTRpool; 
, CTR#2; 
, SDRpool; 
, SDR#62.