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Function of a truncated glucocorticoid receptor form at a negative glucocorticoid response element in the proopiomelanocortin gene M K Turney and W J Kovacs1 Department of Veterans Affairs Medical Center, Nashville, Tennessee, USA 1 Division of Diabetes, Endocrinology and Metabolism, Vanderbilt University School of Medicine, Nashville, Tennessee, USA (Requests for offprints should be addressed to W J Kovacs, Division of Diabetes, Endocrinology, and Metabolism, Room 715 MRB 2, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; Email:
[email protected])
ABSTRACT
ACTH-producing tumors of nonpituitary origin characteristically exhibit insensitivity to the negative feedback effects of glucocorticoids. In the DMS-79 cell line derived from an ACTHproducing small cell lung cancer we have previously identified an aberrantly spliced glucocorticoid receptor (GR) that lacks a ligand-binding domain. We examined the interactions of this truncated form of GR with the proximal human proopiomelanocortin (POMC) promoter. In electrophoretic mobility shift assays GR bound to the negative glucocorticoid response element (nGRE) at position –78 to –50 in the human POMC promoter. Nur77, an orphan nuclear receptor that exerts positive regulatory effects on the POMC gene is also known to bind to this DNA element. The functional properties of GR and GR binding to this DNA element were
INTRODUCTION
It has been recognized for over 35 years that tumors of nonpituitary origin are capable of producing biologically active adrenocorticotropin (ACTH), the product of the proopiomelanocortin (POMC) gene and the chief regulator of adrenal steroidogenesis (Meador et al. 1962). Such ACTH-producing nonpituitary tumors arise from diverse cell types in lung, thymus, pancreas, prostate and other tissues (Liddle et al. 1965). While these tumors acquire the principal feature of the pituitary corticotroph phenotype, ACTH secretion, they differ substantially from normal corticotrophs in their response to the negative regulatory feedback of adrenal corti-
examined in transient transfection experiments in murine AtT-20 corticotroph tumor cells. Reporter gene expression under the control of proximal POMC promoter elements was stimulated by addition of forskolin to the culture medium or by transfection with expression constructs for human Nak1, the human homologue of Nur77. Treatment of transfected cells with dexamethasone resulted in suppression of forskolin- or Nak1-stimulated POMC-reporter gene expression in the presence of co-transfected GR but not with GR. The experiments indicate that in the human POMC promoter GR is capable of binding to the nGRE but cannot effect trans-repression of POMCreporter gene expression.
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costeroids on ACTH production. While ACTHproducing pituitary corticotroph tumors largely exhibit only relative resistance to the negative feedback effects of glucocorticoids, ‘ectopic’ ACTHproducing tumors are, in general, absolutely resistant to glucocorticoid-mediated inhibition of POMC gene expression (Liddle 1960, Meador et al. 1962, Flack et al. 1992). To investigate the role of the glucocorticoid receptor (GR) in this apparent glucocorticoid resistance in ectopic ACTH syndrome we have previously studied the ACTH-producing DMS-79 cell line, developed from a small cell lung cancer. In these cells we have determined that the GR mRNA is expressed in an aberrantly spliced form that
Journal of Molecular Endocrinology (2001) 26, 43–49 Online version via http://www.endocrinology.org 0952–5041/01/026–043 2001 Society for Endocrinology Printed in Great Britain
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retains the intron between exons 7 and 8. This splice variant introduces a premature stop codon, so that the resultant GR retains a DNA-binding domain but lacks a ligand-binding domain (Gaitan et al. 1995, Parks et al. 1998). This GR form is incapable of ligand-induced activation of a positively regulated glucocortioid response element (GRE)-reporter construct and does not act as a dominant negative to inhibit normal receptor function at such a reporter construct (Gaitan et al. 1995). In the present work we have studied the function of this truncated GR (GR) form at a negative GRE (nGRE) in the proximal promoter region of the POMC gene. This nGRE was identified around position63 in the rat promoter by studies of GR binding and functional assessments using reporter constructs (Charron & Drouin 1986, Drouin et al. 1989, 1993) and appears to be a site at which GR and the orphan nuclear receptor Nur77 exert opposite effects on downstream gene expression (Murphy & Conneely 1997, Philips et al. 1997a,b). We report that GR binds to the nGRE but fails to exert negative regulatory effects on a downstream reporter gene, and fails to antagonize the stimulatory effect of a human homologue of Nur77. MATERIALS AND METHODS
Cell culture AtT-20 DV16 F-2 cells (American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml) and 10% bovine calf serum (BCS). Stocks were maintained in 75 cm2 flasks at 37 C in an atmosphere of 5% CO2. Plasmids The 2593 bp human GRT cDNA (pRShGR; the gift of Dr R M Evans, Salk Institute, La Jolla, CA, USA) was subcloned into PCMV4 vector. The GR from DMS-79 cells (Gaitan et al. 1995) was also subcloned in PCMV4. This insert contains 2155 bp identical to GR wild-type (WT) and 491 bp of a sequence derived from intron G of the human GR gene (Parks et al. 1998). The cDNA for the human Nur77 homologue designated Nak1 (Locus HUMNAK1; GenBank Accession number D49728) (Nakai et al. 1990) was generated by RT-PCR amplification of DMS-79 cell RNA. The amplified DNA fragment, corresponding to bases 110–1906 of the Genbank Sequence and containing the full 1794 bp open reading frame was subcloned into Journal of Molecular Endocrinology (2001) 26, 43–49
pCMVScript (Stratagene, La Jolla, CA, USA). Integrity of the sequence was confirmed by sequencing of the plasmid insert. Luciferase reporter gene constructs were prepared using the PGL3 basic vector (Promega, Madison, WI, USA). Human POMC promoter fragments (+1 to 680 and +1 to 389) were prepared by PCR amplification of human genomic DNA and cloned into the PGL3 vector. Integrity of all reporter constructs was confirmed by DNA sequencing. In vitro transcription and translation In vitro transcription and translation of GR and Nak1 were carried out using the TNT kit (Promega). In some experiments [35S]methionine was incorporated into the synthesized protein. Electrophoretic mobility shift assays (EMSA) EMSAs were carried using a duplex oligonucleotide corresponding to a fragment of the human POMC promoter from 78 to 50 bp upstream of the transcriptional start site: 5 -GTGCTGCCGGGA AGGTCAAAGTCCCGCGC-3 . The duplex oligonucleotide was end labeled with [-32P]dATP and polynucleotide kinase. The radiolabeled fragment was separated from free nucleotides by centrifugation on a Sephadex G25 spun column (5 -3 , Inc., Boulder, CO, USA). The radiolabeled probe (0·5 ng) was incubated with 0·75 µl in vitro translated protein (GR, GR or Nak1) along with 0·1 µg poly d(AT) for 20 min on ice. To assess nonspecific binding some tubes also contained 5–50 ng non-radiolabeled duplex oligonucleotide in the preincubation. The binding reaction was then carried out on ice for 10 min in 10 mM Hepes pH 7·9, 10% glycerol, 50 mM KCl, 0·1 mM EDTA. Samples were electrophoresed on 5% nondenaturing polyacrylamide gels at 200 V at a temperature of 4 C. Gels were dried and exposed overnight to Kodak XAR film. Transfections and luciferase reporter assays AtT-20 cells were plated at 500 000 cells per 60 mm well and transfected with 5 µg pGL3 -POMC promoter construct and 1 µg pCMV4 hGR, pCMV4 hGR, or pCMVscript Nak1 using Superfect transfection reagent (Qiagen, Chatsworth, CA, USA) at a ratio of 2 µl:1 µg DNA according to the manufacturer’s protocol. Complexes were allowed to form for 10 min. Cells and complexes were then incubated for 3 h at 37 C in 5% CO2. Cells were then washed in PBS and incubated overnight with www.endocrinology.org
GR binds to nGRE ·
1. In vitro transcription and translation of GR and GR. Complementary cDNAs encoding GR WT and the truncated form of GR from DMS-79 cells (GR) were subcloned in pGEM3z and the linearized plasmids were used as templates for in vitro transcription and translation as described in Materials and Methods. To assess the integrity of the in vitro produced protein products [35S]methionine was incorporated into the reaction mixtures. The resultant radiolabeled proteins were separated on 7·5% SDS-PAGE and detected by fluorography. Lane 1 (GR) shows the intact GR WT product; lane 2 (GR) shows the truncated DMS-79 type GR. Molecular weight marker migration positions are shown at the left.
5 ml DMEM with 10% charcoal-stripped BCS. Added to some wells were: forskolin at a final concentration of 25 µM, dexamethasone at a final concentration of 50 nM, or ethanol. The next morning the cells were washed and harvested for luciferase assay using reagents and protocols from a commercial kit (Tropix Luciferase Assay kit; Tropix, Bedford, MA, USA). Bioluminescence was detected using a Monolight 3010 luminometer (BD Pharmingen, San Diego, CA, USA). RESULTS
DNA-binding activity of GR to the POMC promoter nGRE is preserved The truncated GR from DMS-79 cells (GR) retains the capacity to bind to specific positive and negative GREs. This GR form has previously been found to lack the ability to induce transcription of a reporter gene downstream from a positive GRE (Gaitan et al. 1995). GR WT and GR were prepared by in vitro transcription and translation (Fig. 1) and used in EMSAs with a duplex www.endocrinology.org
and
2. GR retains the capacity to bind to a negative GRE/Nur response element in the proximal human POMC promoter. EMSAs were carried out using in vitro transcribed and translated human Nur77 homologue Nak1 or GRs and 32P-radiolabeled nGRE/NurRE as a probe. Addition of the human Nak1 results in formation of a [32P]DNA–protein complex (lane 2) that is diminished by addition of excess nonradioactive duplex oligonucleotide (lane 3). With the same DNA response element GR WT (lane 4) and GR (lane 6) also form complexes that are diminished by excess nonradioactive competitor probe (lanes 5 and 7).
oligonucleotide corresponding to the classic GRE in the mouse mammary tumor virus (MMTV) promoter and to the sequence in the human POMC promoter from 50 to 78 bases upstream of the transcriptional start site. This corresponds approximately to the positions 53 to 81 in the rat POMC sequence and is identical to the rat sequence in the nucleotides 5 -GGAAGGTCA-3 that have been demonstrated to serve as the nGRE as well as a positive response element for the Nur77 family of orphan nuclear receptors. Both GR and GR bound to the positive GRE from the MMTV promoter (not shown). Nak1 (the human homologue of Nur77) and both GR WT and GR were all found to bind to the DNA fragment corresponding to the nGRE and all were displaceable by an excess of nonradioactive duplex oligonucleotide (Fig. 2). Journal of Molecular Endocrinology (2001) 26, 43–49
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3. GR fails to mediate suppression of forskolin-driven POMC gene expression. AtT-20 murine corticotroph tumor cells were transfected with a POMC-luciferase reporter construct including the proximal 680 bases of the human POMC promoter. The cells were co-transfected with either GR (left two bars) or GR WT (right two bars). POMC reporter gene expression was driven by the addition of forskolin to the medium; cells were treated either with plain medium (open bars) or with medium containing dexamethasone (solid bars). After 24 h the cells were lysed and extracts prepared for assay of luciferase activity. Data shown are means... for six independent experiments.
GR fails to suppress a reporter gene under control of the proximal POMC promoter We tested whether GR’s ability to interact with the nGRE was sufficient to mediate suppression of a POMC-luciferase reporter construct transfected into AtT-20 murine corticotroph tumor cells. Cells were transfected with either GR WT or GR and treated with forskolin to drive POMC-reporter gene expression. GR-transfected cells did not show significant suppression of reporter gene expression after dexamethasone treatment, while GR WT mediated an average suppression of 78·21·5% (Fig. 3). Corticotropin-releasing hormone- or forskolininduced rises in intracellular cAMP levels have been shown to increase expression of the orphan nuclear receptor Nur77 in AtT-20 cells with consequent induction of POMC gene expression (Murphy & Conneely 1997). It has also been demonstrated that Nur77-family transcription factors can transactivate POMC gene expression, and that GR and Nur77 may exert antagonistic effects at their shared response element (Murphy & Conneely 1997, Philips et al. 1997a,b). We examined the ability of GR to function as an antagonist of Nur77-driven POMC gene expression. AtT-20 cells were transJournal of Molecular Endocrinology (2001) 26, 43–49
4. GR fails to mediate suppression of Nak1-driven POMC gene expression. AtT-20 murine corticotroph tumor cells were transfected with a POMC-luciferase reporter construct including the proximal 680 bases of the human POMC promoter. The cells were co-transfected with a Nak1 expression vector and either GR (left two bars) or GR WT (right two bars). Cells were treated either with plain medium (open bars) or with medium containing dexamethasone (solid bars). After 24 h the cells were lysed and extracts prepared for assay of luciferase activity. Data shown are means... for four independent experiments.
fected with Nak1 expression vector and either GR WT or GR as well as POMC-luciferase reporter gene constructs. In the presence of GR WT, the addition of dexamethasone resulted in a suppression of POMC reporter gene activity by an average of 45·76·5% (Fig. 4, right; P=0·018) while POMC reporter expression actually rose by an average of 4117·6% in GR-transfected cells, although this difference between untreated and dexamethasonetreated cells did not reach statistical significance (Fig. 4, left; P=0·13). GR has been postulated to interfere with Nur77 signaling at response elements in the POMC promoter, and such regulatory sequences have been identified at nucleotides 414 to –387 and 70 to –58 in the rat promoter (Philips et al. 1997a,b). We explored the relative importance of these two elements in the human promoter to attempt to identify where GR failed to exert normal ligandmediated suppressive effects on POMC gene expression. First, a POMC reporter construct with the proximal 389 bp of promoter sequence upstream of the start site was used. This construct lacks the upstream NUR response element identified in the rat promoter. Dexamethasone-dependent, GRmediated suppression of forskolin-driven reporter gene expression was observed with GR but not with GR (Fig. 5A). When the data from these experiments were expressed as percent suppression www.endocrinology.org
GR binds to nGRE · and
(389 bp) construct (69·54·0 vs 82·82·6% for the 680 construct; P=0·0319; Fig. 5B), suggesting that the more proximal nGRE exerted quantitatively greater effects in GR-mediated POMC gene suppression. DISCUSSION
5. Elements within the proximal 389 bp of the human POMC promoter suffice for suppression of forskolin-driven POMC gene expression. (A) AtT-20 murine corticotroph tumor cells were transfected with a POMC-luciferase reporter construct including the proximal 389 bases of the human POMC promoter. The cells were co-transfected with either GR (left two bars) or GR WT (right two bars). POMC reporter gene expression was driven by the addition of forskolin to the medium; cells were treated either with plain medium (open bars) or with medium containing dexamethasone (solid bars). After 24 h the cells were lysed and extracts prepared for assay of luciferase activity. Data shown are means... for four independent experiments. (B) The ability of dexamethasone to suppress forskolindriven reporter expression from either the 389 bp or the 680 bp POMC promoter constructs was compared as the percent change from the forskolin-stimulated baseline after the addition of dexamethasone. Cells were transfected with GR WT and the respective POMCluciferase reporter constructs and treated as in (A). Data shown are means... for four independent experiments.
from baseline by dexamethasone, we could detect a small but statistically significant diminution in suppressive effects mediated through the shorter www.endocrinology.org
These experiments reported here demonstrate that the truncated GR form expressed in an ACTHproducing small cell lung cancer line is capable of binding to cis-acting elements in the proximal promoter of the POMC gene, but is incapable of suppressing forskolin- or Nur77-induced transcriptional activation of a downstream reporter gene. This GR splice variant has been identified in glucocorticoid-resistant myeloma cell lines (Moalli et al. 1993, Krett et al. 1995) as well as in the DMS-79 small cell lung cancer (Gaitan et al. 1995, Parks et al. 1998). The GR form arises from a failure to splice out the intron between exons 7 and 8 of the human GR. The resultant protein retains a DNA-binding domain but lacks a ligand-binding domain. At a positively regulated promoter element GR acted neither as a constitutive activator nor as a dominant negative inhibitor of normal receptor function (Gaitan et al. 1995). Its function at the promoter elements of genes negatively regulated by glucocorticoids has not been previously examined. The molecular pathology underlying the syndrome of ectopic ACTH secretion by tumors arising in nonpituitary tissues is largely unexplored. Clinical observation has revealed several salient features of the disorder. First, these nonpituitary tissues acquire the principal feature of the differentiated pituitary corticotroph phenotype. Whether this is the result of alterations in the POMC gene itself (Monig et al. 1993), or to abnormal expression of trans-acting factors that drive POMC gene activity (Picon et al. 1995, 1999a,b) is an area of active inquiry. Secondly, nonpituitary ACTH-secreting tumors generally exhibit profound resistance to the negative feedback effects of glucocorticoids (Liddle 1960, Meador et al. 1962, Liddle et al. 1963). Defects in glucocorticoid action appear to be responsible for this insensitivity (Ray et al. 1994, 1996, Gaitan et al. 1995). The present report is the first functional examination of a GR isoform from an ACTH-producing tumor using the POMC promoter. Whether similar defects occur in other nonpituitary tumors is not yet known. Finally, the levels of ACTH produced by nonpituitary tumors are in many instances much higher than those produced by corticotroph neoplasms. The relative contributions of transcription factors active Journal of Molecular Endocrinology (2001) 26, 43–49
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at the POMC promoter in combination with normal or abnormal GR forms to this markedly increased POMC gene expression are not known. Our experiments showed that, in cells transfected with Nak1 to drive POMC reporter gene expression, the addition of GR elicited a further increase in reporter gene expression. These data, however, do not achieve statistical significance, implying that a single cellular defect in GR signaling cannot account for the established clinical observations of greatly increased POMC gene expression as well as the failure of glucocorticoids to suppress ACTH production. It is entirely conceivable that overexpression of a positively acting transcription factor, such as Nur77 (Murphy & Conneely 1997, Philips et al. 1997a,b) or E2F (Picon et al. 1999b) might be capable of overriding any potential glucocorticoid negative feedback signal, irrespective of the integrity of the GR signaling system. The functional properties of GRs with deleted ligand-binding domains have been previously studied (Godowski et al. 1987, Hollenberg et al. 1989). Deletion of significant portions of the ligand-binding domain of GR by mutagenesis in vitro produces receptor forms that are constitutive activators of target gene transcription. This constitutive activity is at a low level compared with that achieved by the ligand-activated WT receptor. Despite using similar assay systems to assess the functional properties of GR, we did not detect such constitutive activity. Our present finding that, although the GR is capable of binding to the human POMC gene nGRE, it is incapable of suppressing forskolin- or Nak1-driven POMC reporter expression suggests that ligand-induced changes in the receptor (such as association with cofactors) are required for the suppressive effect on POMC gene expression. Extensive studies of the rat POMC gene promoter have defined two possible sites for GR’s suppressive action: the upstream Nur77 response element (Philips et al. 1997a) and the more proximal negative GRE/Nur77 response element (Murphy & Conneely 1997, Philips et al. 1997a). Using the human POMC promoter in transient transfection assays we found that most of the suppressive effects of GR (and the failure of GR) could be accounted for by actions exerted at the more proximal element. The relative role of each of these cis-acting elements in control of POMC gene expression will require further study. The availability of cell lines derived from POMC-expressing tumors of nonpituitary origin has advanced our understanding of the endocrine pathophysiology induced by these neoplasms. Further studies in clinical specimens will need Journal of Molecular Endocrinology (2001) 26, 43–49
to be carried out correlated with precise clinical phenotyping to definitively establish whether these observations of molecular defects in tumor cell lines accurately reflect the in vivo pathophysiology.
ACKNOWLEDGEMENTS
Special thanks to Dr David N Orth and Dr Lewis S Blevins, Jr for serving as consultants to this project. Mr Wendell Nicholson provided many helpful discussions. The work was supported by a Career Development Award (Clinical Investigator) and a Merit Review Grant from the United States Department of Veterans Affairs (to W J K). The Vanderbilt University Diabetes Research and Training Center provided many valuable core services and reagents.
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