[CANCER RESEARCH 51, 1196-1201, February 15, 19911

Differential Glucocorticoid Regulation of Glucagon Gene Expression in Cell LinesDerived from Rat and Hamster Islet Cell Tumors1

Chun Wang, Robert V. Campos, Kathy M. Stobie, Patricia L. Brubaker, and Daniel J. Drucker2

Departments of Medicine (C. W., R. C., D. J. D.J, Clinical Biochemistry [D. J. D.J, Genetics [D. J. D.J, and Physiology [K. M. S., P. L. B.J, University of Toronto,Toronto, Ontario, Canada

ABSTRACT

Glucocorticoid regulation of peptide hormone gene expression wasstudied in two cell lines derived from rodent islet cell tumors. In ratKIN 1056 \ cells, dexamethasone reduced the levels of glucagon niKVYtranscripts while markedly inducing the expression of the angiotensino-

gen gene. In contrast, dexamethasone had no effect on the regulation ofglucagon gene expression in hamster InRl-G9 cells. Wild type InRl-G9

cells did not support the induction of the murine mammary tumor viruspromoter by glucocorticoids, suggesting that these cells lacked the necessary cellular factor(s) for glucocorticoid responsiveness. Introductionof the glucocorticoid receptor into wild type InRl-G9 cells restored

glucocorticoid induction of the murine mammary tumor virus promoter,but not glucocorticoid regulation of glucagon gene expression. Dexamethasone treatment of Sprague-Dawley rats had no effect on the levels of

pancreatic glucagon mRNA transcripts. The results of these studiesdemonstrate that glucocorticoid regulation of glucagon gene expressionis restricted to the immortalized RIN1056A cell line, providing additionalevidence for cell-specific diversity in the regulation of peptide hormone

gene expression in neuroendocrine tumors.

INTRODUCTION

Endocrine tumors of the pancreas provide useful models forthe study of hormone biosynthesis and cellular differentiation.Pancreatic endocrine cell lines with variable hormonal pheno-types have been derived from rodent pancreatic tumors, permitting the detailed analysis of the molecular factors whichregulate hormone biosynthesis and cellular differentiation. Different strategies have been used to generate cell lines derivedfrom rodent pancreatic endocrine tumors. Insulinomas havearisen in rats treated with chemical carcinogens (1) as well asthose exposed to ionizing radiation (2). Islet cell tumors havealso developed in rodents inoculated with BK virus (3). Thedevelopment of transgenic mouse lines harboring pancreaticendocrine tumors has recently provided another source for thederivation of hormone-producing islet cell lines (4-6).

The pattern of phenotype-specific gene expression exhibitedby the differentiated adult endocrine pancreas has been demonstrated to be aberrant in neoplastic islet cells. Whereas thenormal endocrine islet contains specialized A, B, and D cellscommitted to the expression of the glucagon, insulin, andsomatostatin genes, immortalized islet cell lines have beenshown to be multipotential in their capacity for hormone genetranscription (7, 8). Islet cell lines have also been shown toexhibit features suggestive of ectopie hormone gene expression.

Received 10/12/90; accepted 12/5/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by grants from the Medical Research Councilof Canada and the National Cancer Institute. D. J. D. is a Career Scientist of theOntario Ministry of Health, and P. L. B. is a Research Scholar of the CanadianDiabetes Association. C. W. and K. M. S. are fellows of the Canadian DiabetesAssociation, and R. C. is an ICI Pharma-MRC fellow.

2To whom requests for reprints should be addressed, at Toronto GeneralHospital, 200 Elizabeth Street CCRW3-838, Toronto, Ontario, Canada M5G2C4.

For example, RIN cells have been demonstrated to express thecholecystokinin, gastrin, and angiotensinogen genes (7,8), peptide hormone genes not normally expressed in the adult pancreatic islet.

Our laboratory has been studying the control of peptidehormone gene expression in tumors of the endocrine pancreas(9-12). These studies have been directed at understanding theperturbations in hormone biosynthesis and secretion whicharise as a result of the dedifferentiation of the pancreatic endocrine cell. Although glucocorticoid hormones have beenshown to have important effects on the control of peptidebiosynthesis and cell growth in different tumor cells, littleinformation is available concerning the effects of glucocorticoidon normal and neoplastic islet cells. Glucocorticoid treatmentof rat insulinoma cells was found to stimulate DNA replicationin insulin-producing cells in culture (13) and dexamethasonetreatment of insulin-producing RINmSF cells has been shownto decrease glucagon-like peptide 1 binding, due to a reductionin the number of glucagon-like peptide 1 receptors (14). Wenow report the results of our studies on the effects of glucocorticoids on glucagon gene expression, demonstrating islet cell-specific differences in the steroid regulation of glucagon geneexpression.

MATERIALS AND METHODS

Cell Culture and TransfectionRIN 1056A cells (15) and InR 1-G9 cells (9) were grown in Dulbecco's

modified Eagle's medium supplemented with 10% calf serum under a

humidifiai atmosphere of 5% CO2. For steroid incubations, cells werewashed twice in phosphate-buffered saline and preincubated for 24 hin culture medium containing serum stripped with dextran-coated charcoal, prior to addition of glucocorticoids. All experiments were carriedout in steroid-depleted medium for an additional 6-48 h as described.

For transient transfection assays, InRl-G9 cells were transfected bythe DEAE-dextran technique as described previously (16). Cell extractswere prepared and assayed for chloramphenicol acetyltransferase activity as described previously (16), using 1 pCi of [14C]chloramphenicol ina 60-min assay. All CAT3 assays were linear for up to 2 h. The plasmidsMMTV-CAT and the human glucocorticoid receptor under the controlof the RSV promoter were obtained from V. Giguere, University ofToronto. Stable cell lines were prepared by selection in the antibioticG418 (BRL-Gibco Canada, Toronto, Ontario, Canada) following calcium phosphate transfection of the human glucocorticoid receptor andRSV-neo. The transfections and establishment of stable G418-resistantclones (by dilution cloning) was carried out as described previously(16). Clonal InRl-G9 cell lines expressing the glucocorticoid receptor(designated InRl-G9-GR) were grown in a low concentration of G418,200 pg/ml, to maintain selection for G418 resistance.

RNA Extraction and Analysis

For the preparation of total cellular RNA, cells were lysed in guani-dine isothiocyanate, and RNA was extracted using the phenol/acid

3The abbreviations used are: CAT, chloramphenicol acetyltransferase; RSV,Rous sarcoma virus; cDNA, complementary DNA; GLI, glucagon-like immuno-reactivity; IRG, immunoreactive glucagon; MMTV, murine mammary tumorvirus; PEPCK, phosphoenolpyruvate carboxykinase.

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GLUCOCORTICOID REGULATION OF GLUCAGON GENE EXPRESSION

precipitation procedure (17). For Northern blot analysis, 10 Mgof totalcellular RNA were size fractionated on a 1.2% agarose-formaldehydegel, and the gel was stained with ethidium bromide prior to transfer toensure the integrity and quantity of the RNA. The RNA was fixed to anylon membrane with UV light, following which prehybridization andhybridization were carried out in 1 x Denhardt's-4 x standard saline-citrate (1 x SSC = 0.15 M NaCl, 0.3 M NaCitrate)-200 Mg/ml salmonsperm DNA-40% deionized formamide-5% dextran sulfate-0.014 MTris, pH 7.4. Hybridization was performed in the same solution with 1x IO6 cpm/ml of "P-labeled cDNA probe for 24 h at 42°C.Final

washing conditions were 0.1 x standard saline-citrate-0.1 % sodiumdodecyl sulfate at 50°C.Autoradiography was performed using KodakX-Omat film at -70°C. Autoradiograms were quantitated by scanning

with a laser densitometer.

Peptide Extraction and Radioimmunoassays

Peptides were extracted from whole intestine, a section of pancreatictail, and cell lines by homogenization at 4°Cin l N HC1 containing 5%

(v/v) formic acid, 1% (v/v) trifluoroacetic acid, and 1% (v/w) NaCl,followed by reversed-phase adsorption to Cig-silica (C,8-SepPak;Waters Associates, Milford, MA). For assessment of peptide biosynthesis, RIN1056A cell or InRl-G9-GR cells were plated at a density ofIO6cells/plate, in triplicate, and incubated with vehicle or dexametha-

sone for periods of up to 48 h. Cell viability was consistently greaterthan 95% after 48 h as assessed by trypan blue exclusion. Total GLIsynthesis was obtained by summing the mean values for secreted andintracellular GLI, as described previously (18).

Assays. Tissue extracts were assayed for IRG and GLI using antiseraO4A (Dr. R. H. Unger, Dallas, TX) and K4023 (Novo Alle, Bagsvaerd,Denmark), respectively, as described previously (19, 20). AntiserumO4A recognizes the free COOH-terminal end of glucagon and detectsprimarily glucagon whereas antiserum K4023 recognizes the midse-quence of glucagon and cross-reacts equally well with glucagon andNH2- or COOH-terminally extended forms of glucagon (I.e., the intestinal peptides glicentin and oxyntomodulin). Radioimmunoassays forimmunoreactive insulin were carried out as previously described (21).Protein assays were carried out using the method of Lowry et al. (22).

Animal Experiments. Groups of male 200-g Sprague-Dawley rats (3-4/group) were given injections of either vehicle alone (1.0 ml of phosphate-buffered saline) or 100 pg of dexamethasone every 12 h, and ratswere sacrificed at 6, 12, 24, and 48 h. Plasma, pancreas, and intestinewas collected for measurement of GLI and IRG, and small sections ofpancreas and intestine were homogenized in guanidinium isolinocyanate for isolation of total cellular RNA.

RESULTS

To study the effect of glucocorticoids on the expression ofthe glucagon gene, we utilized the glucagon-producingRIN1056A cell line. These cells were derived from a radiation-induced rat insulinoma and have been shown to stably expressthe insulin and glucagon genes following multiple passages invitro (7, 15). Cells were incubated with different doses of thesynthetic glucocorticoid dexamethasone for up to 48 h, following which RNA was prepared for Northern blot analysis. Theresults of a 48-h incubation are shown in Fig. 1. The levels ofglucagon mRNA transcripts were markedly reduced (25% of

Fig. 1.. I, glucocorticoid inhibition of glucagon gene expression in RIN10S6Acells. Subconfluent I0-cm plates of RIN1056A cells were incubated for 48 h withdexamethasone in doses ranging from 10"" to 10~* M. Control cells (C) were

incubated with vehicle alone (10 ¡Aethanol/10 ml medium). Total cellular RNAwas analyzed by Northern blotting (10 fig/lane) and the blot was sequentiallyhybridized with cDNA probes for rat glucagon (G), rat insulin (/), chicken actin(Ac), and rat angiotensinogen (An). For quantitation. autoradiograms werescanned with a laser densitometer. The experiment was carried out in triplicateon two different occasions, and the results of a representative set of experimentsare shown here. B, densitometric evaluation of the relative changes in peptidehormone mRNA transcripts from the experiments shown in Fig. \A. For glucagon, insulin, and angiotensinogen, the mRNA transcripts at 10~" M dexametha

sone were assigned a relative value of 1, and the changes in mRNA transcriptlevels are plotted as a function of the |dexamethasone] x 10~*. 10~7,and 10"' M.

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GLUCOCORTICOID REGULATION OF GLUCAGON GENE EXPRESSION

control by densitometry) by incubation of cells with dexameth-asone at a concentration of 10~7 M. To study the effects of the

glucocorticoid on the expression of other peptide hormonegenes in RIN1056A cells the blot was rehybridized with a cDNAprobe for rat insulin. Dexamethasone treatment also resultedin a decrease (50% of control) in the amount of insulin mRNAtranscripts. Analysis of the time course of dexamethasone inhibition of glucagon gene expression demonstrated that a reduction of glucagon mRNA transcripts was first detectablebetween 24 and 36 h after cells were incubated with steroid(data not shown). To determine the specificity of the inhibitionof glucagon gene expression and to control for differences inthe amount of RNA loaded in each lane, the blot was rehybridized with a cDNA probe for 0-actin. In contrast to the decreasein glucagon and insulin gene expression observed followingdexamethasone incubation, no significant change in the levelsof actin mRNA transcripts was detected (Fig. 1). The relativechanges in peptide hormone mRNA transcript levels as determined following laser densitometry are shown in Fig. IB.

RIN1056A cells have been shown previously to ectopicallyexpress the gene encoding angiotensinogen, a peptide hormonegene known to be transcriptionally induced by glucocorticoids(23). To demonstrate that the reduction in glucagon and insulinmRNA transcripts observed with dexamethasone treatment wasnot simply due to a nonspecific glucocorticoid inhibition ofpeptide hormone gene expression in these cells, the identicalNorthern blot was rehybridized with a cDNA for rat angiotensinogen. A marked induction of angiotensinogen gene expression was noted following incubation of RIN1056A cells withdexamethasone, consistent with previous observations (23). Toassess the effects of dexamethasone treatment on total cellularGLI synthesis, RIN1056A cells were incubated with and without 10~7dexamethasone for 48 h, and media plus cell GLI was

determined. Dexamethasone treatment reduced the synthesis(media plus cell content) of GLI (30,538 versus 19,733 pg/106

cells, control versus dexamethasone).The results described above demonstrated that the expression

of the rat glucagon gene in RIN1056A cells was negativelyregulated by the synthetic glucocorticoid dexamethasone. Toextend these observations to a second model of glucagon geneexpression, we carried out similar experiments using the hamster InRl-G9 cell line. These cells, derived from a BK virus-transformed hamster islet cell tumor, have been shown toexpress the hamster glucagon gene at high levels (9). Incubationof InRl-G9 cells with dexamethasone at a concentration of10~6M had no effect on the levels of glucagon or insulin mRNA

transcripts, during 12- or 48-h incubations (Fig. 2), althoughthe relative levels of both peptide hormone mRNAs declinedafter 48-h incubations in the medium containing strippedserum. The slight induction of actin mRNA transcripts detectedafter 12 h was not observed consistently. One explanation forthe absent glucocorticoid inhibition of glucagon gene expression was the possibility that the InRl-G9 cells do not expressfunctionally competent glucocorticoid receptors. To determineif InRl-G9 cells contained the essential cellular elements necessary for glucocorticoid responsiveness a glucocorticoid-re-sponsive reporter gene (MMTV-CAT) was transiently introduced into these cells, and the transfected cells were tested forglucocorticoid responsiveness. InRl-G9 cells were transfectedwith the plasmid MMTV-CAT and incubated both with andwithout dexamethasone, following which chloramphenicol ace-tyltransferase activity was determined. The results of theseexperiments (Fig. 3) demonstrated no activation of MMTV-

12h 48h- +

-

Fig. 2. Effects of dexamethasone on glucagon (top panel) and insulin (middlepanel) gene expression in InRl-G9 cells. Wild type InRl-G9 cells were incubatedwith (+) and without (-) IO"6 M dexamethasone for 12 or 48 h. Total cellular

RNA (10 fjg/lane) was analyzed by Northern blotting with cDNA probes forglucagon (G), insulin (/), and actin (A). Each experiment was done at least twiceon separate occasions.

CAT by dexamethasone in wild type InRl-G9 cells. Controltransfections with RSV CAT resulted in high levels of CATactivity (data not shown), demonstrating that the lack of glucocorticoid responsiveness in the transient assay was not simplyattributable to difficulties with cell transfection. These resultsraised the possibility that the lack of glucocorticoid regulationof glucagon gene expression in the InRl-G9 cell line may be

attributable in part to the absence of functional glucocorticoidreceptors in these cells. To determine if glucocorticoid responsiveness could be restored following introduction of the glucocorticoid receptor into InRl-G9 cells, cotransfection experi

ments were carried out. The human glucocorticoid receptorunder the control of the RSV viral promoter was cotransfectedwith MMTV-CAT into InRl-G9 cells, and transfected cellswere incubated in the presence and absence of dexamethasone.The results of this experiment demonstrated that introductionof the cloned glucocorticoid receptor into InRl-G9 cells conferred dexamethasone induction of MMTV-CAT in transienttransfection studies (data not shown). To prepare stable InRl-G9 cell lines which expressed the glucocorticoid receptor, wildtype InRl-G9 cells were transfected with the human glucocorticoid receptor and the plasmid RSV-neo. Transfected cellswere exposed to the antibiotic selection agent G418, and surviving cells (designated InRl-G9-GR cells) were cloned and

individually expanded in culture. Clonal cell lines were screenedfor glucocorticoid responsiveness using activation of MMTV-

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GLUCOCORTICOID REGULATION OF GLUCAGON GENE EXPRESSION

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Fig. 3. Glucocorticoid responsiveness in InRl-G9 cells. Wild type InRl-G9cells (wt) and a clonal cell line expressing the glucocorticoid receptor, InRl-G9-GR (GR), were transfected with 1 ¿igof MMTV-CAT, and 24 h later, cells wereincubated with (+) and without (-) IO"6 M dexamethasone for an additional 24

h. Cell extracts were prepared and analyzed for chloramphenicol acetyltransferaseactivity (10 ng protein/lane) as described previously (16). MMTV-CAT transfec-tion of the InRl-G9-GR cells treated with dexamethasone gave a value of 37%acetylation per 10 /jg cell extract in a 60-min assay. All transfections were carriedout in triplicate, and the data shown are representative of two different experiments.

CAT expression as a reporter for glucocorticoid receptorexpression. The results of one such experiment are shown inFig. 3, demonstrating activation of the MMTV-CAT reportergene following transfection of the InRl-G9-GR clone express

ing the human glucocorticoid receptor. Following the establishment of several different stable InRl-G9-GR clones expressingthe human glucocorticoid receptor, the regulation of glucagongene expression in several of these lines was examined. Noeffect of dexamethasone on the expression of the glucagon orinsulin genes could be demonstrated using a number of differentInRl-G9-GR clones. The results of a representative experimentare depicted in Fig. 4. Incubation of InRl-G9-GR cells withdexamethasone at doses of up to 10~6 M for up to 48 h had no

effect on the level of glucagon or insulin mRNA transcripts.No significant change in GLI synthesis was detected in InRl-G9-GR cells incubated with dexamethasone for up to 48 h.

The results of the data obtained from studies of immortalizedrodent islet cell lines suggested that the glucocorticoid regulation of glucagon gene expression may be cell specific. Todetermine the effects of steroid treatment on the regulation ofglucagon gene expression in normal rats, male Sprague-Dawleyrats were given injections of dexamethasone (100 ^g every 12h) and the levels of glucagon mRNA transcripts as well as thelevels of plasma and tissue GLI peptides were determined at 6,12, 24, and 48 h. No change in the levels of glucagon mRNAtranscripts in pancreas or intestine could be detected in control

ftFig. 4. Regulation of glucagon gene expression in the InRI-G9-GR cell line.

A clonal line of InRl-G9-GR cells expressing the human glucocorticoid receptor,was incubated with and without 10"' M dexamethasone (DEX) for 48 h, following

which total cellular RNA was prepared and analyzed by Northern blotting. Theblot was hybridized with cDNA probes for glucagon (G) and insulin (/).

(vehicle injected) compared to dexamethasone-treated animalsat any of the above time points. The data for the 24-h timepoint for pancreatic glucagon mRNA transcripts is shown inFig. 5/4. The steroid-treated animals had an increase in plasmaglucose levels (control, 158 ±7, versus dexamethasone treated,195 ±4, P < 0.001) in association with an increase in plasmainsulin levels, presumably reflecting steroid-induced insulin resistance. Small but statistically significant decreases in the levelsof pancreatic GLI, IRG, and insulin were observed in thesteroid-treated animals (Fig. SB). No changes in the levels ofintestinal GLI were detected in steroid-treated animals (datanot shown).

DISCUSSION

Studies of normal islets and immortalized islet cell lines havedemonstrated a number of abnormalities in hormone biosynthesis and secretion characteristic of the immortalized pheno-type. For example, hormone production and secretion rateshave been shown to be substantially diminished in transformedcell lines when compared to normal islets (18, 24). Glucoseregulation of insulin secretion and insulin gene expression hasalso been shown to be perturbed in most immortalized islet celllines (4, 24). Studies of glucagon gene expression using normaland tumor-derived cells have also shown considerable differ

ences with respect to the control of hormone secretion andglucagon gene expression. For example activation of the aden-

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C C D D

GLU

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H

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B

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IRI GLI IRG IRI

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Fig. 5. A, pancreatic glucagon mRNA transcripts from control and dexameth-asone-treated rats. Rats were given injections of vehicle alone (C) or 100 ^gdexamethasone (£>)at time 0 and 12 h, following which rats were sacrificed at 24h, and total cellular RNA was prepared for Northern blot analysis. GLU, glucagon.II. plasma and tissue levels of GLI. IRG. and immunoreactive insulin (IRI} incontrol and dcxamethasone-treated rats. Groups of vehicle-injected (2-4/group)and dexamethasone-injected male (3-4/group) 200-g Sprague-Dawley rats weregiven injections of 100 fig of dexamethasone every 12 h, and rats were sacrificedat 6, 12, 24. and 48 h. Plasma and tissue levels of GLI. IRG. and IRI demonstratedsimilar trends at all time points studied, and data from all time points wastherefore pooled for statistical analysis, [control, n = 10 (D); dexamethasone-treated, n = 15 (U)]. Statistical significance was assessed using the unpairedStudent's / test. ' P < 0.05, *«/><0.01 .'»«/><0.001. Bars. SE.

ylate cyclase pathway leads to increased expression of theglucagon gene as well as an increase in glucagon secretion fromnormal rat islets in short term culture (25). In contrast, noinduction of glucagon gene expression or glucagon secretionwas detected when similar experiments were carried out usinghamster InRl-G9 or RINI056A glucagon-producing islet celllines (9, 26). Phorbol esters increased glucagon gene transcription in rat RIN1056A cells but had no effect on glucagon geneexpression in normal rat islets or the hamster InRl-G9 cell line(9, 25, 26). Moreover sodium butyrate induced the differentiation of the RIN1056A cell line and increased the transcriptionof the glucagon gene in RIN cells but had no effect on InRl-G9 cells or normal islets (9, 27).

The results of the experiments presented here demonstratefor the first time that glucocorticoids negatively regulate theexpression of the glucagon gene in the rat RIN1056A cell line.This effect is not rapid, and a decrease in the amount ofglucagon mRNA transcripts following glucocorticoid treatmentwas detectable only after more than 24 h of steroid treatment.

The mechanisms whereby glucocorticoids exert these effects onthe expression of the glucagon gene remain unknown. Severallines of evidence suggest that the actions of glucocorticoids maybe indirect, perhaps by modulating the synthesis of a transcription factor required for glucagon gene transcription or a factorinvolved in the regulation of glucagon mRNA stability. Wehave not been able to demonstrate glucocorticoid-inducedrepression of glucagon-CAT activity using transfectedRIN1056A and InRl-G9-GR cells, with up to 2.4 kilobases ofrat glucagon gene 5'-flanking and promoter sequences directing

CAT transcription, and no glucocorticoid-regulatory elementshave yet been identified in the 5'-flanking region of the human

or rat glucagon genes (28, 29). The glucocorticoid inhibition ofinsulin gene expression has recently been studied in HIT insu-linoma cells and was also shown to be independent of effectson insulin gene transcription. The results of these experimentssuggested that glucocorticoids exerted their effects on insulingene expression by destabilizing insulin mRNA transcripts (30).

Additional evidence for the lack of a direct effect of glucocorticoids on glucagon gene expression derives from our studiesof the hamster InRl-G9 cell line. The stable introduction ofthe human glucocorticoid receptor into InRl-G9 cells resultedin the production of a series of clonal InRl-G9-GR cell lineswith competent glucocorticoid responsiveness, as assessed bythe activation of a transfected MMTV-CAT reporter gene. Incontrast to the glucocorticoid activation of MMTV-CAT, noeffect of dexamethasone on the regulation of the endogenoushamster glucagon gene could be demonstrated. These resultssuggested that one or more factors present in the RIN1056Acells, distinct from the glucocorticoid receptor, may be necessary for glucocorticoid inhibition of glucagon gene expression.

The results of studies of the glucocorticoid induction ofhepatic gene expression in primary cultures of rat hepatocytessupport the hypothesis that some genes require additional cell-specific factors for glucocorticoid regulation of gene expression.For example the delayed kinetics of the glucocorticoid induction of the PEPCK gene, as well as the effects of cycloheximideon glucocorticoid induction, provides support for the existenceof additional necessary protein factors which mediate glucocorticoid induction of PEPCK in primary hepatocyte cultures (31).In contrast, dexamethasone rapidly stimulates PEPCK genetranscription in a rat hepatoma cell line (32), and functionalglucocorticoid-regulatory elements have been mapped to thepromoter region of the PEPCK gene using gene transfer studieswith hepatoma cells (33). An alternative explanation for ourresults resides in the possibility that the rat but not the hamsterglucagon gene contains regulatory elements which mediate theglucocorticoid inhibition of glucagon gene expression.

To extend our observations to an in vivo model of glucagongene expression, we studied the effects of dexamethasone treatment on glucagon gene expression in rat pancreas and intestine.No effects of dexamethasone on the levels of glucagon mRNAtranscripts in vivo were observed, although small decreases inthe levels of pancreatic IRG and immunoreactive insulin weredetected. These results provide further support for the lack ofa direct glucocorticoid effect on glucagon gene expression. Theuncoupling of the control of glucagon biosynthesis, secretion,and changes in glucagon mRNA transcripts has been describedpreviously (34), but the mechanisms underlying these changeshave not yet been defined. It remains possible that the normalcirculating concentrations of glucocorticoids tonically inhibitglucagon gene expression and that no further repression isobtained with larger doses of steroids such as those used in our

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GLUCOCORTICOID REGULATION OF GLUCAGON GENE EXPRESSION

experiments. This hypothesis may be tested directly in futureexperiments with adrenalectomized rats.

The demonstration that dedifferentiated RIN1056A cells express functional glucocorticoid receptors stands in contrast tostudies of normal rat islets which have shown that only pancreatic 0-cells contain glucocorticoid receptor immunoreactivity(35). Since RIN1056A cells display many characteristics ofimmature dedifferentiated multipotential islet cells, future experiments examining the expression of the glucocorticoid receptor in the developing fetal islets may implicate a role forglucocorticoids in the development and maturation of the endocrine pancreas.

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12. Drucker, D. J., Asa, S. L., Silverberg, J., and Brubaker. P. L. Molecular andcellular analysis of a neoplastic pancreatic A cell tumor. Cancer (Phila.), 126:1762-1770, 1990.

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14. Richter, G., Coke, R., Coke, B., and Arnold, R. Dexamethasone pretreatmentof rat insulinoma cells decreases binding of glucagon-like peptide-l(7-36)amide. J. Endocrino!., 126:445-450, 1990.

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16. Drucker, D. J., Philippe, J., Jepeal, L., and Habener, J. F. Glucagon gene5'-flanking sequences promote islet cell-specific glucagon gene transcription.J. Biol. Chem., 262: 15659-15665, 1987.

17. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation byacid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Chem.,162: 156-159, 1987.

18. Drucker, D. J., and Brubaker, P. Proglucagon gene expression is regulatedby a cyclic AMP-dependent pathway in rat intestine. Proc. Nati. Acad. Sci.USA, 86: 3953-3957, 1989.

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Research. on September 28, 2020. © 1991 American Association for Cancercancerres.aacrjournals.org Downloaded from

1991;51:1196-1201. Cancer Res   Chun Wang, Robert V. Campos, Kathy M. Stobie, et al.   Tumors

CellExpression in Cell Lines Derived from Rat and Hamster Islet Differential Glucocorticoid Regulation of Glucagon Gene

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