Hormonal Control of Adrenocortical Cell Proliferation

DESENSITIZATION TO ACTHANDINTERACTION

BETWEENACTHANDFIBROBLAST GROWTHFACTOR

IN BOVINE ADRENOCORTICALCELL CULTURES

PETERJ. HORNSBYand GoPRDONN. GILL

From the Department of Medicine, Division of Endocrinology, University of California,San Diego, School of Medicine, La Jolla, California 92093

A B S T R AC T A primary bovine adrenocortical cellculture system responsive to physiological concentra-tions of ACTH has been established. When addedto cultures, ACTH inhibited DNAsynthesis and celldivision over the same concentration range requiredfor stimulation of fluorogenic steroid production (0.01-10 nM). With chronic exposure to ACTH, cells be-came desensitized to the growth inhibitory effectsof ACTH. Though cell growth was initially completelyinhibited by ACTH, cells ultimately began to growin its continued presence. The lag time to initiationof cell growth, the rate of growth, and the final densityachieved depended on the ACTH concentration.Desensitization to ACTH1_39 was not induced bymonobutyryl cyclic AMPnor by ACTH11-24. Specificityof desensitization was apparent because cells whichhad become desensitized to ACTH1_39 remainedfully responsive to monobutyryl cyclic AMP, pros-taglandin E1, and cholera toxin. Though the effectsof ACTHon cell growth were readily reversible uponhormone removal, the desensitized response to readdi-tion of ACTHpersisted for at least 8 h.

Fibroblast growth factor (FGF) stimulated both thegrowth rate and saturation density achieved. FGF didnot alter the growth inhibitory effects of ACTHnor thereduced growth rate observed in desensitized cellsmaintained in ACTH. However, FGF greatly in-creased the saturation density achieved by culturesmaintained with ACTH.

Dr. Homsby was a Postdoctoral Research Fellow of theScience Research Council, London. Dr. Gill is the recipientof U. S. Public Health Service Research Career DevelopmentAward AM70215 from the National Institute of Arthritis,Metabolism, and Digestive Diseases.

Received for publication 20 September 1976 and in re-vised form 28 January 1977.

Through the process of desensitization, adreno-cortical cells are able to grow in the presence ofhigh concentrations of ACTH and to respond to theeffects of a growth factor by increasing the celldensity achieved. This pattern of response may be ageneral one for growth control under the combinedeffects of antimitotic and mitotic factors.

INTRODUCTION

Experiments with cultured adrenocortical cells de-rived from either normal tissue (1, 2) or an adrenaltumor (Y-1 cells) (3) have established that the directaction of ACTH' on the adrenocortical cell is anti-mitotic. It has therefore been suggested that the stimu-latory action of ACTH on adrenal cell division invivo may be indirect (4, 5), possibly being mediatedby a growth factor. One of the known growth factors,fibroblast growth factor (FGF) has been demonstratedto be mitogenic for both normal (4) and tumor (6)adrenocortical cells in culture. If such a growth fac-tor acts in vivo, its effect would be opposed by thedirect anti-growth action of ACTH. This activity ofACTH has been shown, in studies in cultured Y-1cells, to be most pronounced in the inhibition of DNAsynthesis, with RNA and protein synthesis beinglittle affected (5). Because RNAand protein synthesisare stimulated by serum growth factors and thesteroidogenic pathway by ACTH, the result is ahypertrophied hyperfunctional adrenocortical cell(5, 7). This correlates well with the action of ACTH

1Abbreviations used in this paper: ACTH or ACTH,39,full porcine pituitary ACTH molecule; ACTH11_24, syn-thetic corticotropin-(11-24)-tetradeca-peptide; cAMP,cyclic AMP; FGF, fibroblast growth factor; LH, luteinizinghormone.

The Journal of Clinical Investigation Volume 60 August 1977 -342-352342

in vivo (8, 9). Nevertheless, adrenal cell division doesoccur in vivo in the presence of elevated levels ofACTH (9-12), particularly under such conditions asadrenal regeneration (13). Wehave therefore investi-gated in detail the effects of ACTHon the prolifera-tion of normal adult bovine adrenocortical cells inmonolayer culture, studying in particular the resultsof prolonged ACTH treatment and the interaction ofACTH with the one known mitogenic hormone forthe adrenal cell, FGF.

METHODSPreparation of cultures. The method of preparing a sus-

pension of adrenocortical cells from bovine adrenal glandshas been previously described (4). 20 ml of cell suspensionwas obtained per gland. Additional fetal calf serum wasadded to make the total serum concentration 20%, anddimethyl sulfoxide (Mallinckrodt Inc., St. Louis, Mo.) addedto give a 5% solution. The cell suspension was then dividedinto 2-ml aliquots which were placed in vials and frozen inliquid nitrogen. Cells remained viable for at least 6 moand multiple experiments could be performed with thesame cell preparation. Vials were subsequently recoveredfrom liquid nitrogen as required, unfrozen and diluted in anappropriate volume of Coon and Weiss' modification (14)of Ham's F-12 medium (Grand Island Biological Co., GrandIsland, N. Y.) with 10% fetal calf serum (Irvine ScientificSales Co., Fountain Valley, Calif.). The same batch of fetalcalf serum was used throughout the present experiments.Medium contained 100 ,ug/ml penicillin G (Sigma ChemicalCo., St. Louis, Mo.) and 50 Ag/ml gentamicin (SigmaChemical Co.). The diluted cell suspension was placed intissue culture plates (Falcon Plastics, Div. of BioQuest,Oxnard, Calif.) and the cells allowed to attach to theplastic for 6 h. The medium was then renewed.

Measurement of cell number. Medium was removedfrom cultures and replaced with 1 ml Tris-buffered salinecontaining 0.05% trypsin and 5 mMEDTA. After 10 min atroom temperature the cells were removed from the plate byflushing with a Pasteur pipette, diluted with an appropriatevolume of phosphate-buffered saline, and counted immedi-ately with a Coulter Counter, model ZF (Coulter ElectronicsInc., Hialeah, Fla.).

Measurement of [3H]thymidine incorporation and auto-radiography. [3H]Thymidine (60 Ci/mmol, 10 ,uCi/plate)(Schwarz/Mann Div., Becton, Dickinson & Co., Orangeburg,N. Y.) was added to the cultures without medium change.4 h later the radioactive medium was removed, the cultureswashed with phosphate-buffered saline, and 1 ml of a 1%aqueous solution of Triton X-100 (Sigma Chemical Co.)added. The solution was left to solubilize the cells for 5 minand then transferred to 10 ml of absolute ethanol. Thismaterial was filtered under vacuum through a 2.4-cmdiameter glass fiber filter (Whatman GF/A, W. & R. BalstonLtd., England), and the filter washed twice with 10 ml ab-solute ethanol. The filter was then counted in toluene-Liquifluor (New England Nuclear, Boston, Mass.) (3.8 liters:160 ml).

The procedure for autoradiography was that described byHolley and Kieman (15).

Cell cycle analysis by flow microfluorometry. Cellswere prepared for flow microfluorometer analysis by mithra-mycin staining (16) and analyzed in a Los Alamos designmicrofluorometer with an argon laser at 488 nm. Analysisof the distribution of cells with varying DNA contents

was performed by the graphic method of Holley andKieman (17).

Measurement of fluorogenic steroids. Fluorogenic ster-oids (principally cortisol and corticosterone) were meas-ured by a modification of the sulfuric acid-induced fluores-cence method (18). The culture medium was extracted with3 vol of dichloromethane and the extract mixed with 2 ml of a65% solution of sulfuric acid in 95% ethanol. After 1 h thefluorescent emission at 520 nm of the sulfuric acid-ethanolmixture resulting from 470 nm excitation was measured andcompared to that of a cortisol standard. Because the pri-mary steroid product of bovine adrenocortical cells is corti-sol (19), results are expressed as micrograms of cortisolstandard.

MaterialsPurified porcine ACTH1-39 was obtained from Armour

Pharmaceutical Co., Phoenix, Ariz. An appropriate volumeof 0.1 M acetic acid was added to a 25-U vial on the dayof use. ACTH solutions were not stored. Before additionto the cultures, the ACTHsolution was mixed with culturemedium to the extent of 1% by volume, and then addedto the culture immediately. The same volume of 0.1 Macetic acid was added to control cultures.

ACTH11-24 (the gift of Dr. Rittel of Ciba-Geigy, Basal,Switzerland) was dissolved in 0.1 Macetic acid before use.

Purified FGF was the gift of Dr. Denis Gospodarowicz,The Salk Institute, La Jolla, Calif. FGF was dissolved inphosphate-buffered saline, pH 7.4, with 0.5% bovine serumalbumin (Sigma Chemical Co.) and the solution was mixedwith culture medium to the extent of 5%by volume immedi-ately before adding to cultures.

Monobutyryl cyclic AMP(Sigma Chemical Co.) was dis-solved in phosphate-buffered saline before use. Pros-taglandin El (the gift of Dr. J. Pike of Upjohn Co., Kala-mazoo, Mich.) was dissolved in 95% ethanol. The solutionwas mixed to the extent of 1% by volume with culturemedium prior to addition to cultures. Control cultures alsoreceived 1%ethanol. Cholera toxin was from Schwarz/MannDiv., Becton, Dickinson & Co. A I mgvial was reconstitutedwith 1 ml distilled water and further diluted with phos-phate-buffered saline.

Mithramycin was obtained from Pfizer Inc., New York.Aminoglutethimide (Elipten phosphate) was a gift from ciba-Geigy Corp., Summit, N. J., and was dissolved in dinethylsulfoxide for addition to culture medium at 1%by volume.

RESULTS

Acute responsiveness to ACTH. In the bovineadrenocortical cell system, responsiveness to physio-logical concentrations of ACTHwas demonstrated bydose-response curves for ACTHwhich indicated half-maximal fluorogenic steroid production at approxi-mately 0.5 nM ACTH (Fig. 1). Similar dose-responsecurves were obtained at the initiation of cell growth(day 1), during log growth (day 4), and as cells enteredthe stage of density-dependent inhibition of cell growth(day 6) (see Fig. 4). Previous studies indicated thatthese cells contained abundant lipid as demonstratedby oil-red 0 staining and gave a strongly positivehistochemical stain for A53-,B-hydroxysteroid dehydro-genase (4).

ACTHand Fibroblast Growth Factor Control of Adrenal Cell Proliferation 343

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FIGURE 1 Effect of ACTHon fluorogenic steroid productionand on [3H ]thymidine incorporation into DNA. Bovineadrenocortical cells were plated and grown as describedunder Methods. After 1, 3, 4, or 6 days, fresh medium con-taining the indicated concentrations of ACTH was added.16 h after ACTHaddition, [3H]thymidine was added for 4 h.The medium was removed for analysis of steroid productionover this 20-h period and cells were assayed for [3H]-thymidine incorporation into DNA. Fluorogenic steroidproduction, ([l); [3H]thymidine incorporation into DNA,(U).

When the adrenal cells which had grown to con-

fluence were subcultured, the steroidogenic capacityper cell was found to be equal to that of freshlyprepared cultures whether the subcultured cells hadpreviously been maintained with ACTH or in itsabsence (Fig. 2). In this experiment cells which hadgrown to confluence in the absence or presence of10 nM ACTH were subcultured and grown withoutACTH to a density of 105 cells/plate. Cholera toxin,which is a potent stimulator of steroidogenesis incultured adrenocortical cells (20), was added toassess maximal steroidogenic capacity independentlyof any alterations in ACTH sensitivity due to pre-treatment. A progressive increase in steroidogeniccapacity was seen in both freshly plated and sub-cultured cells; the same maximum rate of steroid pro-

duction per cell was reached by day 4. The increasingsteroidogenic capacity with prolonged cholera toxintreatment resembles that seen with prolonged ACTHtreatment in other adrenal cell culture systems (7,21-23); this increased steroidogenic capacity is due toprogressive stimulation of steroidogenic pathwayenzymatic activity. Cloned cell lines derived fromthese primary normal bovine adrenocortical cell cul-tures retain the same steroidogenic capacity over

many genlerations; continuously passaged cultures also

344 P. J. Hornsby and G. N. Gill

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FIGURE 2 Steroidogenic capacity of bovine adrenocorticalcells after subculture. The steroidogenic capacity of cellsgrown for prolonged periods in the presence or absence ofACTH was compared to that of freshly plated cells. Threegroups of cells were compared: Freshly plated cultures of105 cells/plate, (A); cells grown to confluence in the absenceof ACTH which were subcultured and grown to 10-5cells/plate, (c); and cells grown to confluence, in the pres-ence of 10 nM ACTH which were subcultured and grownto 105 cells/plate in the absence of ACTH, (c). The experi-ment was initiated by addition of fresh medium with I nMcholera toxin. Every 24 h, medium was removed for deter-mination of fluorogenic steroid production and freshmedium containing I nM cholera toxin added. In the pres-ence of cholera toxin, maximal steroidogenesis is observed(20) and cell growth does not occur (see Fig. 6).

retain the same steroidogenic capacity for over 50

2

generations.2 In both instances the cell populationgives a uniformly positive stain for A53-fl-hydroxy-steroid dehydrogenase.

ACTH inhibits DNA synthesis as previously re-ported for Y-1 and normnal rat adrenocortical cells(1-3). When ACTH was added for 16-h periods tocultures at any stage of their growth, >98% of [3 H]-thymidine incorporation into DNA was inhibited(Fig. 1). The ACTH dose response for inhibition of[3 H]thymidine incorporation was similar to that ob-served for stimulation of steroidogenesis and similarACTH sensitivity was observed at all stages of cellgrowth. The complete inhibition of [3 H]thymidine in-corporation into DNA by ACTH indicates that thepopulation is almost purely adrenocortical in origin.In contrast, ACTHup to 1 ,uM had no effect on [3 H]-thymidine incorporation into primary cultures ofbovine lung cells from similar animals. Autoradiog-

2 Hornsby, P. J., and G. N. Gill. Manuscript in preparation.

raphy showed that >98% of freshly plated bovineadrenal cells became labeled with [3H]thymidine andthat >98% of cells are labeled during log growthduring one generation time indicating no substantialsubpopulation of nondividing cells (see Fig. 5). Byautoradiography, ACTHwas shown previously to in-hibit DNAsynthesis in >98% of the cells (4).

When ACTH-treated cells were examined by flowmicrofluorometry, a predominant G1 arrest was ob-served. As shown in Fig. 3, randomly growing cellsare distributed throughout the cell cycle. WhenACTHwas added to parallel cultures for 24 h, cells with aG1 complement of DNApredominated. Analysis of theresults showed that in control cultures 34% of cellsare in G1, 31% in S and 35% in G2 + M. In ACTH-treated cultures, 79% of cells were in G1, 5% in S, and16% in G2 + M. This G1 growth arrest with ACTHaddition is identical to that observed in Y-1 functionalmouse adrenal cortical tumor cells (5, 7).

Treatment of cultures with aminoglutethimide, aninhibitor of the conversion of cholesterol to preg-nenolone (24), reduced the ACTHand monobutyrylcyclic AMP (cAMP)-stimulated level of steroid pro-duction to the nonstimulated level seen in the absenceof aminoglutethimide (Table I). Under these condi-tions, ACTHand monobutyryl cAMPcontinued to re-duce [3H]thymidine incorporation into DNA to thesame extent as in the non-aminoglutethimide-treatedcultures. ACTH does not appear to inhibit adrenalcell proliferation through its stimulation of steroido-genesis but by raising the intracellular concentra-tion of cAMP (3). The cultured cells behaved there-fore as a single population of adrenocortical cellswith respect to cell division, responsiveness to ACTH,and retention of steroidogenic capacity.

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TABLE IEffect of Aminoglutethimide on ACTHand Monobutyryl

cAMPStimulation of Steroidogenesis andInhibition of DNASynthesis

[3HIThymidineSteroid production incorporation

Amino- Amino-Treatment None glutethimide None glutethimide

jLg/24 h %control

Control 1.4 0.2 100 105ACTH(10 nM) 5.2 1.3 3.8 3.5Monobutyryl

cAMP(1 mM) 8.6 1.5 1.7 2.6

Cultures were treated for 2 days with 300 jug/ml amino-glutethimide. The medium was then replaced withoutadditions or with the addition of 10 nMACTHor 1 mMmono-butyryl cAMP. 18 h later, [3H]thymidine was added, and afteran additional 4 h the medium was removed for assay ofsteroids and the cells were harvested for measurement ofincorporation of radioactivity. Control cultures showed anincorporation of 2.7 x 104 cpm/105 cells.

Cell growth in the presence of chronically elevatedACTH levels. When primary bovine adrenocorticalcells were maintained in the presence of elevatedACTH concentrations, a characteristic pattern ofgrowth resulted (Fig. 4). In the absence of ACTHthere was a lag of 2 days during which no increase in

300 500

FIGURE 3 Flow microfluorometric analysis of the effect ofACTHon randomly growing primary bovine adrenal corticalcells. Cells were plated at 2 x 104 cells per plate and grownin 10% fetal calf serum for 3 days before the addition of10 nM ACTH. 24 h later, ACTH-treated and control cellswere harvested and treated as described under Methods.The number of cells assayed by flow microfluorometry was50,000 for the control cultues and 56,000 for the ACTH-treated cultures.

TIME (days)

FIGURE 4 Growth of adrenocortical cells in the continuouspresence of varying concentrations of ACTH. 6 h after cellswere plated, fresh F-12 medium containing 10% fetal calfserum was added, without ACTH (-) or with the additionof the following concentrations of ACTH: 1 pM, (0); 10pM, (A); 100 pM, (Li); 1 nM (0); 10 nM, (A). The medium,with the same concentrations of ACTH, was renewed at2-day intervals as indicated by the arrows.

ACTHand Fibroblast Growth Factor Control of Adrenal Cell Proliferation

looot

345

cell number was detected. Cells then entered a periodof exponential growth followed by a leveling of thegrowth curve to a plateau.

WhenACTHwas continuously present, all three ofthese growth stages were affected. The lag period wasprolonged, the prolongation being dependent uponthe ACTH concentration. The early slight decreasein cell number at the higher ACTH levels resultedfrom retraction (25), detachment, and loss of a smallnumber of cells. After this lag period, cells at alldoses of ACTHtested entered a period of exponentialgrowth despite the continuous addition of ACTH ateach change of the culture medium. The rate of growthdepended upon the ACTH concentration. In theabsence of ACTH, the average doubling time of thecell population equaled 24 h; in the presence of 10nM ACTH the doubling time equaled 48 h. The ex-ponential growth phase was followed by cessation ofgrowth which occurred at a reduced cell density whichwas dependent upon the ACTHconcentration present.At 10 nM ACTH plateauing of the increase in cellnumber occurred at 20% of the density achieved inits absence. The plateauing of increase in cell num-ber was not due to accelerated cell death but tomarkedly reduced DNA synthesis and cell division.When DNA synthesis in ACTH-treated cells whose

cell number had plateaued was compared to that ofcells during log growth, [3H]thymidine incorporationinto DNA was reduced to <5% of that of culturesgrowing in the presence of ACTH. Density-dependentinhibition of cell growth therefore occurs both withand without ACTH.

The pattern of a lag followed by exponential growthindicated that the cells must undergo considerabledesensitization to the original inhibitory effect ofACTH (Fig. 1). The cells maintain a constant lowerlevel of responsiveness to ACTH as shown by thereduced but constant growth rate which was dependentupon the ACTHconcentration.

The cell population appeared uniformly affected.Autoradiography of cells labeled with [3H]thymidinefor one generation while growing in the presence ofACTHrevealed no substantial subpopulation of non-dividing cells indicating that ACTH is not pref-erentially inhibiting some cells (Fig. 5). It appearsthat the whole population divides at the reduced ratein the presence of ACTH. The fact that these areadrenal cells was confirmed by the demonstration thatcells taken from a culture grown to confluence in thepresence of ACTHhave the same steroidogenic capac-ity as cells from a non-ACTH-treated culture or freshlyplated cells (Fig. 2).

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48-h period 2 days after plating out. (B) Cells in the log growth phase (day 4). (C) Cells inthe log growth phase in the continuous presence of 10 nM ACTH(day 6).

346 P. J. Hornsby and G. N. Gill

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Effect of ACTH11-24 and monobutyryl cAMP oncell growth. Whena high concentration of ACTHl-39(10 nM) was added to cultures during exponentialgrowth, an approximate doubling of cell number oc-curred, followed by cessation of growth within 24 h(Fig. 6). This response correlates with inhibition of[3Hl]thymidine incorporation into DNA(Fig. 1), andwith the G, point of growth arrest (Fig. 3).

In contrast, when the adrenocortical cells weremaintained in the presence of a high concentration(10 ,uM) of an ACTH fragment (ACTH11-24), no sig-nificant effect on cell growth occurred (Fig. 6). ThisACTH11_24 fragment acts as a competitive inhibitorof ACTHI_39 by binding to ACTH receptor sites butlacks the amino terminal portion required for biologicalactivity (26, 27).

Monobutyryl cAMP stimulates steroidogenesis andinhibits [3H]thymidine incorporation into DNAin thebovine adrenocortical cell system. When cells weremaintained with a concentration of monobutyrylcAMP (1 mM), equivalent to 10 nM ACTHin inhibit-ing [3H]thymidine incorporation, cell growth did notoccur (Fig. 6). This persistent growth inhibition con-trasts with the resistance to the growth inhibitoryeffects of ACTHl_39 which developed over this timeperiod.

Effect of desensitization to ACTHon responsivenessto monobutyryl cAMP, prostaglandin E1, and choleratoxin. The incorporation of [3H]thymidine into DNA

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TIME (DAYS)

FIGURE 6 Effect of ACTH11_24 and monobutyryl cAMP oncell growth. Medium with serum and the indicated additionswas replenished every 2 days as indicated by the arrowsat the top of the diagram. Normal medium without addi-tions, (-); plus monobutyryl cAMP (1 mM), (0); plusACTH,124 (10 ,uM), (A); plus ACTH,_39 (10 nM), (0); normalmedium without additions until day 4 when ACTH (10nM) was added as indicated by arrow, (V).

in growing bovine adrenocortical cells is inhibitedby monobutyryl cAMP, prostaglandin El, and choleratoxin (Fig. 7). ACTH, prostaglandin El, and choleratoxin have all been reported to elevate cAMP levelsin adrenocortical cells (28-30). When cells whichhave become desensitized to the growth inhibitoryeffects of 10 nM ACTH were tested for sensitivityto monobutyryl cAMP, prostaglandin El, and choleratoxin, sensitivity equivalent to control cultures wasnoted (Fig. 7). Though initial [3H]thymidine incorpora-tion over a 4-h period was lower, consistent with theprolonged average cell cycle time of ACTH-treatedcultures, no change in the dose-response curvesoccurred indicating that desensitization to ACTHdidnot cause desensitization to these other substances.

Pretreatment with monobutyryl cAMP or wvithACTHI124 did not cause the development of insensi-tivity to ACTHl-39. When inhibitory concentrationsof monobutyryl cAMP (1 mM) were removed, cellgrowth resumed. Addition of ACTH1_39 inhibited [3H]-thymidine incorporation over the same concentrationrange as in control cultures (Fig. 8). Exposure to 10,uM ACTHI1-24 did not affect the subsequent respon-siveness to ACTH1_39. This concentration of ACTH11-24was shown to be effective in inhibiting by 50%steroidogenesis stimulated by 10 nM ACTH1-39 whenadded simultaneously to cultures (data not shown).The apparent receptor affinity for ACTH1_39 relativeto ACTH11-24 of 1,000:1 is greater than that reportedfor binding to receptors in a membrane preparation(27) and may reflect different half-lives of ACTHI_39and ACTH11-24 which have been shown to be de-graded by different mechanism (31). This competitiveantagonist of ACTH action did not induce the de-sensitization caused by ACTH1_39.

Pretreatment with 100 ng/ml FGFwhich stimulated

0 0.1 00.01 0.1 10 10000.1 10 100 1000MONOBUTYRYLCYCLIC PROSTAGLANDNEl (IM) CHOLERATOXIN (pM)

AMP (mM)

FIGURE 7 Effect of monobutyryl cAMP, prostaglandin E1,and cholera toxin on [3H]thymidine incorporation intoDNA in control cells and in cells desensitized to ACTH.Control cells were grown for 4 days in the absence ofACTHand desensitized cells were grown for 6 days in thepresence of 10 nM ACTH. Medium was removed and freshmedium containing the indicated concentrations of mono-butyryl cAMP, prostaglandin E1, or cholera toxin added. 16 hlater the cells were pulsed for 4 h with [3H]thymidine andincorporation into DNA determined as described underMethods. Control cells, (-); desensitized cells, (0).

ACTHand Fibroblast Growth Factor Control of Adrenal Cell Proliferation 347

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FIGURE 8 Effect of treatment with ACTH,124, mono-

butyryl cAMP, and FGF on subsequent responsiveness toACTH,_39. Cells were grown for 4 days with or withoutthe indicated additions. Medium was removed and cellswashed twice by incubation with fresh medium for 30min to remove hormones. Fresh medium containing the indi-cated concentrations of ACTH,_39 was then added. 16 hlater cells were pulsed for 4 h with [3H]thymidine and in-corporation into DNA determined as described underMethods. Control, (U); pretreatment with ACTH1,_24 (10,uM), (A); pretreatment with FGF (100 ng/ml), (0); pretreat-ment with monobutyryl cAMP (1 mM), (0).

[3H]thymidine incorporation and cell growth (4) didnot affect the 50% effective dose of ACTH requiredfor inhibition of DNAsynthesis (Fig. 8).

Reversibility of growth inhibition due to ACTH.When ACTH is removed from cultures which havebecome desensitized, i.e., have entered the ex-

ponential phase of cell growth in the presence ofACTH, the rate of growth increases and a higher satura-tion plateau is achieved (Fig. 9).

Though the rate of cell growth and saturation densityincrease when ACTHis removed, cells remain resistantto subsequently added ACTH. When cultures whichhad become desensitized to 10 nM ACTHand were

growing at a reduced rate in its presence were washedand incubated for an additional period with 0.01-

100 nM ACTH, there was much less inhibition of[3H]thymidine incorporation at any dose tested com-

pared to nontreated cultures (Fig. 10). The flatteneddose-response curve persisted when cells were incu-bated for 8 h in ACTH-free medium before subsequentaddition of ACTH. The increased rate of cell growthupon ACTH removal suggests that the insensitivityto ACTH is not due to ACTH from the original ex-

posure remaining bound to receptors. Bound ACTHisreleased from receptors with a half-life of 3 min at370 C (27). Initial exposure to 10 nM ACTH for 20

348 P. J. Hornsby and G. N. Gill

min, a time sufficient to saturate ACTH receptors,followed by normal washing had no effect on the subse-quent response to ACTH; i.e., cells remained sensi-tive to ACTH giving an identical response to thecontrol curve shown in Fig. 10. Pretreatment for 6days with a lower concentration of ACTH (0.1 nM)resulted in a dose-response curve which on subse-quent incubation with 0.01-100 nM ACTH showedpartial desensitization. This dose-response curve wasintermediate between that seen with 10 nM ACTHand no pretreatment. As seen in Fig. 10, desensitiza-tion results in a decrease in maximum responsivenessto subsequent ACTH.

Interaction of ACTH and FGF on proliferation.As previously demonstrated (4), FGFwas effective inpromoting the growth of cultured adrenocortical cellsespecially from low densities and its use resulted in theachievement of higher saturation densities (Fig. 11).

When saturating concentrations of FGF (100 ng/ml)and ACTH (10 nM) were added together to culturesat each medium change, growth up to about 6 daysafter plating was not different from cultures treatedwith 10 nM ACTHalone (Fig. 11). After initial inhibi-tion by ACTH, desensitization occurred and cellsinitiated growth at a reduced rate. However, afterapproximately 6 days, there was a marked differencebetween FGF treated and nontreated cultures. Inthe absence of FGF, ACTH-treated cells plateaued

2x105 + +

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TIME (DAYS)

FIGURE 9 Reversibility of ACTH effects on cell growth.Cells were grown in the absence or presence of 10 nMACTH; the medium was renewed at the times indicatedby the arrows at the top of the diagram. At day 5, ACTHwas removed from one set of cultures and cells were thenmaintained in ACTH-free medium. Control cells withoutACTH, (0); with ACTH(10 nM), (0); ACTHremoval at timeindicated by arrow, (A).

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0 0.01 0.1 10 100CONCENTRATIONOF ACTH (nM)

FIGURE 10 Effect of desensitization to ACTH on subse-quent responsiveness to ACTH. Control cells were grownfor 4 days without ACTH and desensitized cells weregrown for 6 days in the presence of 0.1 or 10 nM ACTH.Hormone was removed by incubation with two washes ofnormal medium each for 10 min or by two washes of normalmedium each for 4 h. Fresh medium containing the indi-cated concentrations of ACTHwas then added and 16 h latercells were pulsed for 4 h with [3H]thymidine. Control cells,no pretreatment with ACTH, (U); pretreatment with ACTH(10 nM), 20-min wash, (0); pretreatment with ACTH (10mM), 8-h wash, (A); pretreatment with ACTH (0.1 nM),20-min wash, (A).

at a low density. In the presence of FGF, ACTH-treated cells continued to grow at the reduced rateand achieved a cell density at least 10-fold higherthan in its absence. FGF thus appears to have noeffect on the initial sensitivity of the cells to ACTHbecause it does not change the dose-response curveof ACTH inhibition of DNA synthesis (Fig. 8), nordoes it change the lag time to initiation of growth inthe presence of ACTH (Fig. 11). Similarly, FGFfailed to alter the reduced growth rate seen in thepresence of continuously elevated ACTH levels. Themajor effect of FGF was to allow continued growthso that significantly higher saturation densities wereachieved.

WhenACTHwas removed from cultures maintainedin the presence of FGF, reversibility of ACTHinhibi-tion was again noted (Fig. 11). The growth rate in-creased to the rapid rate observed in the presenceof FGF alone; this rate exceeded that of cells main-tained in the presence of FGF and ACTH.

The present results indicate that in cultured normalbovine adrenocortical cells, the continuous presenceof high levels of ACTHdid not prevent considerabledivision of adrenal cells from occurring, despite amarked initial inhibition of DNA synthesis. Cellsgrowing in the presence of ACTHretain their adrenal-specific properties and also remain responsive to FGFwhich causes the cultures to achieve far higher celldensities than is possible in the absence of FGF. Ifculture conditions reflect in vivo conditions, theadrenal gland could well achieve a long-term increasein cell number, even if ACTHhas a direct antimitoticaction in vivo and stimulates growth of the adrenalby an indirect mechanism as we have postulated (4, 5,7). Most of the increase in DNAcontent in vivo is alate effect of increasing the circulating level of ACTH(9). It may be that in vivo as in culture there is an initialperiod when ACTH is strongly inhibitory to DNAsynthesis, resulting in cellular hypertrophy (5, 8), butthe cells become desensitized to ACTHand cell divi-sion then occurs. The results reported here demon-strate that hormone desensitization may have pro-found effects on growth control, an aspect of desensiti-

2x101, +

106 ~ _o

0

z~~

2x 1031 I l l l0 2 4 6 8 10 12

TIME ( DAYS)

FIGURE 1 1 Effect of FGFand ACTHon adrenocortical cellgrowth. Medium containing the indicated concentrationsof ACTHand FGF was replenished every 2 days as indi-cated by the arrows at the top of the diagram. FGF (100ng/ml), (O); ACTH (10 nM), (A);FGF (100 ng/ml) plusACTH (10 nM), (A); FGF (100 ng/ml) plus ACTH (10 nM)until day 5 (arrow) when ACTHwas removed and growthcontinued in the presence of FGF alone, (v).

ACTHand Fibroblast Growth Factor Control of Adrenal Cell Proliferation 349

zation which has not been considered previously, butwhich maywell be an important mechanism for regulat-ing growth in vivo.

ACTH-induced desensitization to ACTH has notpreviously been investigated in normal adrenal tissue,but has been shown to occur in the Y-1 adrenal tumorcell line with respect to cAMP production (32). In avariety of other tissues it has now been demonstratedthat one effect of treatment of a target tissue with ahormone to which it is responsive is a subsequentdecrease either in its cAMPproducing potency or itsability to stimulate other biological responses (33-37).Desensitization does not generally extend to hor-mones other than the one with which the tissue is pre-treated, or to factors such as cholera toxin (32). Similarfindings were demonstrated for adrenocortical cells.The desensitized adrenal cells remained responsiveto the growth inhibitory effects of monobutyryl cAMP,prostaglandin E1, and cholera toxin.

In some cases of desensitization in whole cells anactual loss of receptors for the hormone occurs (33,34, 36), but desensitization also can occur in vitro inmembrane particles from ovarian follicles when stimu-lated with luteinizing hormone (LH) (37). It has beenproposed that the initial readily reversible desensitiza-tion is converted in whole cells to a more permanentstate (36). For LH desensitization in follicle mem-branes, cAMPproduction by LH does not seem to benecessary in that exogenously added cAMP did notinduce desensitization. The present experimentaltreatment of adrenal cells with monobutyryl cAMPalso resulted in a steadily maintained inhibition ofgrowth with no development of desensitization toeither monobutyryl cAMPor ACTH. This prompted usto test the effects of an ACTH fragment, ACTH11-24,which by binding to ACTH receptors displacesACTH1I39 or ACTH1l24 and acts as a competitive in-hibitor (26, 27). Because ACTH11-24 lacks the activesite of the ACTHmolecule (present in positions 1-10)(38), it has Ino agonist activity. Pretreatment with thisfragment at a high concentration had no desensitizingaction. Mere receptor occupancy does not appear suf-ficient for desensitization; elevated cAMP levels arealso insufficient for development of desensitization.Similar specificity for development of desensitizationto catecholamines and LH has been reported (37, 39).It would be of interest to study the effects of otherACTH fragments or analogs on desensitization toinvestigate whether separate molecular structuralrequirements exist for this process.

The present experiments indicate that the de-sensitized state can be maintained at a constant levelover a long period, as shown by the slowed but con-stant growth rate of cells in the presence of ACTH.The slower rate of growth produced by ACTHis readilyreversible; when cells were changed from ACTH-

350 P. J. Hornsby and G. N. Gill

containing medium to a lower concentration or toACTH-free medium, an increased rate of [3H]thymi-dine incorporation was demonstrable 24 h later andan increased cell number at 48 h. The rate of growthincreased to that seen with serum or, if FGF wasadded, to that seen with serum plus FGF. However,the desensitization persisted when the cells wereincubated in ACTH-free medium for 8 h before thestandard assay for ACTH inhibition of [3H]thymidineincorporation. The reversibility of the desensitizationphenomenon thus appears slow.

The persistence of effects of FGF in the presenceof ACTH is of considerable interest. The present ex-periments show that FGF can reverse the low satura-tion density characteristic of ACTH-treated cultures.Because the saturation density achieved with ACTHdid not result from accelerated cell death, the effect ofFGF on increasing the final cell density did notresult from an increase in cell survival. FGF did notaffect the slow rate of growth of ACTH-treated cul-tures nor did pretreatment with FGF affect ACTH-induced inhibition of growth. Consequently, it may behypothesized that FGF does not directly reversethe mechanism by which ACTHopposes growth. Pre-viously, FGF was shown to be an effective migra-tion stimulating factor, even in the presence of a highconcentration of ACTH (4). The action of FGF onmigration, saturation density, and growth of cellsfrom very low densities (4) seems to be separatefrom actual control of growth rate. Whether thesephenomena are all expressions of one common initialaction of FGF remains to be studied.

It is also unknown at this time whether FGFor an FGF-like molecule is of importance in vivo instimulating adrenal growth. Some specificity for FGFexists because epidermal growth factor does notaffect adrenocortical cell growth (4). An FGF-likemolecule has been isolated from the pituitary andshown to be effective in stimulating the growth ofovarian cells in culture (40). The possibility that such afactor may act on adrenocortical cells in vivo is sup-ported by a number of experiments demonstratinggrowth in response to factors other than ACTH. Growthhormone has been implicated as an adrenal growthstimulator (41) and an adrenal weight-maintaining fac-tor distinct from ACTHwas demonstrated in patientswith Cushing's syndrome (42). In addition, neuralstimulation of the adrenal results in growth by anACTH-independent mechanism (43, 44).

Though ACTH inhibits DNA synthesis acutely, itdoes not block serum-induced cellular hypertrophy (5);under these conditions, ACTH induces the steroido-genic capacity of the cell (7). This results in a hyper-trophied adrenocortical cell with increased steroido-genic potential. With chronically elevated levels ofACTH, desensitization may occur and DNAsynthesis

and cell division follow. The extent of cellular hyper-plasia achieved will depend upon the concentrationsof ACTHand of the relevant in vivo growth factors.Additional studies are required to determine whetherin vivo growth observed in the presence of high con-centrations of ACTHresults from elevation of growthfactor(s) entrained to ACTHor to alterations in cellularresponsiveness to constant concentrations of growthfactors. The interactions of FGFand ACTHon adreno-cortical cells may serve as a model of interactionsbetween growth and differentiation factors in a num-ber of cell types.

ACKNOWLEDGMENTS

The authors express appreciation to Dr. Denis Gospodarowiczof The Salk Institute for Biological Studies for helpfuldiscussions during the course of these studies and for thegenerous gift of purified FGF.

This investigation was supported by U. S. Public HealthService research grant AM13149 from the National Instituteof Arthritis, Metabolism, and Digestive Diseases.

REFERENCES

1. Ramachandran, J., and A. T. Suyama. 1975. Inhibition ofreplication of normal adrenocortical cells in culture byadrenocorticotropin. Proc. Natl. Acad. Sci. U. S. A. 72:113- 117.

2. O'Hare, M. J., and A. M. Neville. 1973. Effects ofadrenocorticotrophin on steroidogenesis and prolifera-tion by adult adrenal cells in monolayer culture. Bio-chem. Soc. Trans. 1: 1088-1091.

3. Masui, H., and L. D. Garren. 1971. Inhibition of replica-tion in functional mouse adrenal tumor cells by adreno-corticotropic hormone mediated by adenosine 3':5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U. S. A. 68:3206-3210.

4. Gospodarowicz, D., C. R. Ill, P. J. Homsby, and G. N.Gill. 1977. Control of bovine adrenal cortical cell pro-liferation by fibroblast growth factor. Lack of effect ofepidermal growth factor. Endocrinology. 100: 1080-1089.

5. Weidman, E. R., and G. N. Gill. 1977. Differential*effects of ACTHor 8-Br-cAMP on growth and replicationin a functional adrenal tumor cell line. J. Cell. Physiol.90: 91-103.

6. Gospodarowicz, D., and H. H. Handley. 1975. Stimula-tion of division of Yl adrenal cells by a growth factorisolated from bovine pituitary glands. Endocrinology.97: 102-107.

7. Gill, G. N., and E. R. Weidman. 1977. Hormonal regula-tion of initiation of DNAsynthesis and of differentiatedfunction in Y-1 adrenal cortical cells. J. Cell. Physiol.In press.

8. Nickerson, P. A. 1975. Quantitative study of the effectof an ACTH-producing pituitary tumor on the ultra-structure of the mouse adrenal gland. Am. J. Pathol. 80:295-308.

9. Farese, R. V., and W. J. Reddy. 1963. Observations onthe interrelations between adrenal protein, RNA andDNAduring prolonged ACTHadministration. Biochim.Biophys. Acta. 76: 145-148.

10. Masui, H., and L. D. Garren. 1970. On the mechanismof action of adrenocorticotropic hormone. Stimulationof deoxyribonucleic acid polymerase and thymidine

kinase activities in adrenal glands. J. Biol. Chem. 245:2627-2632.

11. Machemer, R., and W. Oehlert. 1964. Autoradio-graphische Untersuchungen uber den physiologischenZellumsatz und die gesteigerte Zellneubildung derNebenniere der ausgewachsenen Ratte nach Behandlungmit ACTH. Endokrinologie. 46: 77-91.

12. Bury, H. P. R., and W. A. J. Crane. 1965. Effect of ageand hormonal state on the numbers of deoxyribonu-cleic acid synthesizing nuclei in rat adrenal cortex.Nature (Lond.). 205: 301-302.

13. Haeuber, H-D. 1965. Zur Regeneration des Nebennier-enrindenparenchyms beim Meerschweinchen nach sog.¾4-Resektion der Nebennierenrinde. Endokrinologie. 48:255-265.

14. Coon, H. G., and M. C. Weiss. 1969. A quantitativecomparison of formation of spontaneous and virus-pro-duced viable hybrids. Proc. Natl. Acad. Sci. U. S. A.62: 852-859.

15. Holley, R. W., and J. A. Kieman. 1974. Control of theinitiation of DNAsynthesis in 3T3 cells: Serum factors.Proc. Natl. Acad. Sci. U. S. A. 71: 2908-2911.

16. Crissman, H. A., and R. A. Tobey. 1974. Cell-cycleanalysis in 20 minutes. Science (Wash. D. C.). 184:1297-1298.

17. Holley, R. W., and J. A. Kie-man. 1974. Control of theinitiation of DNAsynthesis in 3T3 cells: Low-molecular-weight nutrients. Proc. Natl. Acad. Sci. U. S. A. 71:2942-2945.

18. Silber, R. H. 1966. Fluorimetric analysis of corticoids.Methods Biochem. Anal. 14: 63-78.

19. Bush, I. E. 1953. Species differences and other factorsinfluencing adrenocortical secretion. Ciba Found.Colloq. Endocrinol. 7: 210-232.

20. O'Hare, M. J. 1976. Monolayer cultures of normal adultrat adrenocortical cells: Steroidogenic responses tonucleotides, bacterial toxins and antimicrotubularagents. Experientia (Basel). 32: 251-253.

21. O'Hare, M. J., and A. M. Neville. 1973. The steroido-genic response of adult rat adrenocortical cells in mono-layer culture. J. Endocrinol. 56: 537-549.

22. O'Hare, M. J., and A. M. Neville. 1973. Steroid metab-olism by adult rat adrenocortical cells in monolayerculture. J. Endocrinol. 58: 447-462.

23. Kowal, J. 1969. Adrenal cells in tissue culture. III. Effectof adrenocorticotropin and 3',5'-cyclic adenosinemonophosphate on 11 ,8-hydroxylase and other steroido-genic enzymes. Biochemistry. 8: 1821-1831.

24. Dexter, R. N., L. M. Fishman, R. L. Ney, and G. W.Liddle. 1967. Inhibition of adrenal corticosteroidsynthesis by aminoglutethimide: Studies of the mech-anism of action. J. Clin. Endocrinol. Metab. 27: 473-480.

25. O'Hare, M. J., and A. M. Neville. 1973. Morphologicalresponses to corticotrophin and cyclic AMPby adultrat adrenocortical cells in monolayer culture. J. Endo-crinol. 56: 529-536.

26. Seelig, S., and G. Sayers. 1973. Isolated adrenal cortexcells: ACTHagonists, partial agonists, antagonists; cyclicAMP and corticosterone production. Arch. Biochem.Biophys. 154: 230-239.

27. Saez, J. M., A. M. Morera, A. Dazord, and P. Bataille.1974. Interactions of ACTHwith its adrenal receptors:Specific binding of ACTH1_24, its o-nitrophenyl sulfenylderivative and ACTH,124. J. Steroid Biochem. 5: 925-933.

28. Grahame-Smith, D. G., R. W. Butcher, R. L. Ney, andE. W. Sutherland. 1967. Adenosine 3',5'-monophos-

ACTHand Fibroblast Growth Factor Control of Adrenal Cell Proliferation 351

phate as the intracellular mediator of the action ofadrenocorticotropic hormone on the adrenal cortex. J.Biol. Chem. 242: 5535-5541.

29. Saruta, T., and N. M. Kaplan. 1972. Adrenocorticalsteroidogenesis: the effects of prostaglandins. J. Clin.Invest. 51: 2246-2251.

30. Haksar, A., D. V. Maudsley, and F. G. Peron. 1975.Stimulation of cyclic adenosine 3':5'-monophosphateand corticosterone formation in isolated rat adrenalcells by cholera enterotoxin. Comparison with theeffects of ACTH. Biochim. Biophys. Acta. 381: 308-323.

31. Saez, J. M., A. Dazord, A. M. Morera, and P. Bataille.1975. Interactions of adrenocorticotropic hormone with itsadrenal receptors. Degradation of ACTH1-24 andACTH11,24.J. Biol. Chem. 250: 1683-1689.

32. Wishnow, R. M., E. Lifrak, and C-C. Chen. 1976. Modeof action of Vibrio cholerae enterotoxin in culturedadrenal tumor cells. J. Infect. Dis. 133: S108-S114.

33. Gavin, J. R., III, J. Roth, D. M. Neville, Jr., P. de Meyts,and D. N. Buell. 1974. Insulin-dependent regulation ofinsulin receptor concentrations: A direct demonstra-tion in cell culture. Proc. Natl. Acad. Sci. U. S. A. 71:84-88.

34. Hinkle, P. M., and A. H. Tashjian, Jr. 1975. Thyrotropin-releasing hormone regulates the number of its own re-ceptors in the GH3 strain of pituitary cells in culture.Biochemistry. 14: 3845-3851.

35. Mukherjee, C., M. G. Caron, and R. J. Lefkowitz. 1975.Catecholamine-induced subsensitivity of adenylatecyclase associated with loss of /3-adrenergic receptorbinding sites. Proc. Natl. Acad. Sci. U. S. A. 72:1945-1949.

36. Mukheijee, C., and R. J. Lefkowitz. 1976. Desensitiza-tion of 3-adrenergic receptors by S-adrenergic agonistsin a cell-free system: Resensitization by guanosine 3'-(,3,y-imino)triphosphate and other purine nucleotides.Proc. Natl. Acad. Sci. U. S. A. 73: 1494-1498.

37. Bockaert, J., M. Hunzicker-Dunn, and L. Birnbaumer.1976. Hormone-stimulated desensitization of hormone-dependent adenylyl cyclase. J. Biol. Chem. 251: 2653-2663.

38. Hofmann, K. 1974. Relations between chemical structureand function of adrenocorticotropin and melanocyte-stimulating hormones. Handb. Physiol. 4(2)(Sect. 7. Endo-crinology.): 29-58.

39. Mukherjee, C., M. G. Caron, and R. J. Lefkowitz.1976. Regulation of adenylate cyclase coupled 38-adrenergic receptors by 3-adrenergic catecholamines.Endocrinology. 99: 347-357.

40. Jones, K. L., and D. Gospodarowicz. 1974. Biologicalactivity of a growth factor for ovarian cells. Proc. Natl.Sci. U. S. A. 71: 3372-3376.

41. Cater, D. B., and M. P. Stack-Dunne. 1955. The effectsof growth hormone and corticotrophin upon the adrenalweight and adrenocortical mitotic activity in the hypo-physectomized rat.J. Endocrinol. 12: 174-184.

42. Segal, B. M., and N. P. Christy. 1968. Potentiation ofthe biologic activity of ACTHby human plasma. A pre-liminary study.J. Clin. Endocrinol. 28: 1465-1472.

43. Engeland, W. C., J. Shinsako, and M. F. Dallman. 1975.Corticosteroids and ACTH are not required for com-pensatory adrenal growth.Am.J. Physiol. 229: 1461-1464.

44. Engeland, W. C., and M. F. Dallman. 1975. Com-pensatory adrenal growth is neurally mediated. Neuro-endocrinology. 19: 352-362.

352 P. J. Hornsby and G. N. Gill