The Prostate 48:179 ^187 (2001)

HistamineH1ReceptorActivation Inhibits theProliferationofHumanProstaticAdenocarcinoma

DU-145Cells

Sandra Valencia, Adriana HernaÂndez-Angeles, Luis-Enrique Soria-Jasso,and JoseÂ-Antonio Arias-MontanÄ o*

Departamento de Fisiologia,BiofisicayNeurociencias,Centro de Investigacionyde Estudios Avanzados,Mexico,D.F.,Mexico

BACKGROUND. Histamine stimulates cell proliferation in some tumor cell lines through theactivation of H1 receptors coupled to phosphoinositide hydrolysis. We therefore set out tostudy the presence of H1 receptors in the prostate cancer cell line DU-145 and the effect of theirstimulation on cell growth.METHODS. The presence of histamine receptors was studied by radioligand binding.Phosphoinositide hydrolysis was assessed by measuring [3H]-inositol phosphate ([3H]-IPs)accumulation and changes in the intracellular concentration of free Ca2� ([Ca2� ]i). Proli-feration was assessed by cell counting and by [3H]-thymidine incorporation.RESULTS. DU-145 cells express H1 receptors (110� 14 fmol/mg of protein) whosestimulation results in [3H]-IPs accumulation (602� 23% of basal, EC50 2.2� 0.4 mM) andcalcium mobilization (resting level 96� 5 nM, �[Ca2� ]i 517� 32 nM, EC50 6.2� 0.1 mM).Incubation with histamine (100 mM, 24 hr) resulted in a decrease in both cell number and [3H]-thymidine incorporation, blocked by the H1 antagonist mepyramine (1 mM).CONCLUSION. Histamine inhibits the proliferation of DU-145 cells through the activation ofH1 receptors coupled to phosphoinositide hydrolysis. Prostate 48:179±187, 2001.# 2001 Wiley-Liss, Inc.

KEY WORDS: prostate; proliferation; histamine; cell growth; phosphoinositide hydro-lysis; Ca2� signaling

INTRODUCTION

Histamine is closely associated with mast cells inalmost all tissues, with a long established role as amediator of in¯ammation. Other functions in whichhistamine is involved include smooth muscle contrac-tion, regulation of endothelial cell function, gastricacid secretion, hormone release and heart musclecontraction [1]. In addition, histamine is widelydistributed within the mammalian central nervoussystem where its function as a neuromodulator isstrongly supported by experimental evidence [2,3]. Onthe basis of their pharmacology and signal transduc-tion mechanisms, histamine receptors have beensubdivided into H1, H2, and H3 subtypes, coupled todifferent G proteins [3].

Growth factors are involved in the regulation ofnormal cell division and in the generation of tumorsand some of them, such as endothelin-1 and angio-tensin II, act on cell surface, G-protein-coupled re-ceptors [4]. Histamine has been shown to stimulatethe proliferation of HeLa cells [5], rat cortical astro-

Grant sponsor: CINVESTAV; Grant Sponsor: CONACYT; Grantnumber: 28276N.

The current af®liation of L. Soria-Jasso is: Programa de BiomedicinaMolecular, CICATA-IPN.

*Correspondence to: JoseÂ-Antonio Arias-MontanÄo, M.D., Ph.D.,Departamento de FisiologõÂa, BiofõÂsica y Neurociencias, Centro deInvestigacioÂn y de Estudios Avenzados, Apdo. postal 14-740, 07000MeÂxico, D.F., MeÂxico. E-mail: jaarias@®sio.cinvestav.mx

Received 16 November 2000; Accepted 13 April 2001

ß 2001Wiley-Liss, Inc.

cytes [6], and human astrocytoma U373 MG cells [7].The effect of histamine on the growth of HeLa andU373 MG cells appears to be mediated by H1 receptors,known to activate a membrane-bound, PIP2-speci®cphospholipase C (PLC) that breaks down phosphati-dylinositol 4,5-biphosphate (PIP2) into inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol (DAG)[3]. Ins(1,4,5)P3 mobilizes Ca2� ions from intracellularstores while DAG activates protein kinase C (PKC) [8].Changes in the intracellular concentration of free Ca2�

([Ca2� ]i) are thought to play a role in the cell cycle,particularly during mitosis, and phorbol esters, whichactivate PKC, are mitogenic [8,9]. Phosphoinositidehydrolysis may thus be a necessary event in themechanism of action of some growth factors.

It was recently reported that among other sub-stances, histamine was able to elicit changes in [Ca2� ]iin DU-145 cells [10], a line derived from a humanprostate adenocarcinoma [11]. Histamine could thushave a mitogenic action on DU-145 cells, and in thisstudy we set out to investigate the presence ofhistamine receptors and whether histamine was ableto stimulate cell proliferation.

MATERIALSANDMETHODS

Cell Culture

DU-145 cells were a kind gift from Dr. A. GutieÂrrez(National Cancer Institute, Mexico City, Mexico). Cellswere grown in Eagle's medium (MEM) supplementedwith 10% fetal bovine serum, penicillin (50 UI/ml) andstreptomycin (0.1 mg/ml). Cells were grown asmonolayers in either 150 cm2 ¯asks or 24-well platesin a humidi®ed atmosphere (5% CO2) at 37�C.

[3H]-MepyramineBinding toMembranesFromDU-145 Cells

DU-145 cells were broken with a Polytron (10 sec,setting 5) in 10 mM Tris-HCl buffer (pH 7.4) contain-ing 1 mM EGTA. Lysates were centrifuged at 20,000gfor 30 min and pellets were then resuspended in10 mM Tris-HCl and centrifuged again. The resultingpellet was resuspended (�1 mg protein/ml) [12] inincubation buffer (50 mM Tris-HCl, pH 7.4).

Saturation analysis of [3H]-mepyramine bindingwas carried out in 1 ml buffer containing increasingconcentrations (0.05±14 nM) of radioligand and 45±50 mg membrane protein. Equilibration was for 60 minat 30�C and was terminated by ®ltration throughWhatman GF/B glass ®ber paper, presoaked in 0.3%polyethylenimine for 2 hr, using a Brandel (Gaithers-burg, MD) cell harvester. The ®lters were washedthrice with ice-cold buffer and then transferred to vialscontaining 10 ml scintillator and allowed to stand at

room temperature for at least 2 hr before determina-tion of the tritium content. Nonspeci®c binding wasdetermined as that insensitive to inhibition by 1 mMunlabeled mepyramine.

In parallel determinations membrane aliquots wereincubated with radioligands for histamine H2 recep-tors (20 nM [3H]-tiotidine) and H3 receptors (4 nM N-a-[methyl-3H]-histamine). Nonspeci®c binding wasde®ned with 10 mM tiotidine and 1 mM thioperamide,respectively.

[3H]-Inositol Phosphate ([3H]-IPs) Accumulation

DU-145 cells were grown to near con¯uence in24-well plates, the culture medium was removed andthe cells were washed with MEM before the additionof medium containing 10% dialyzed calf serum, 10 mMmyo-inositol and 0.5 mCi/well of [3H]-inositol(0.16 mM). After 24 hr the [3H]-inositol-labeled cellswere washed with Krebs±Henseleit medium (in mM:NaCl 116, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, NaHCO3

25, CaCl2 2.5, and D-glucose 11) and incubated at 37�Cin Krebs±Henseleit buffer containing 20 mM LiCl.After 10 min histamine was added in a 10 ml volumeand incubations were continued for the requiredperiods before the addition of 250 ml of an ice-coldsolution of 10% perchloric acid, containing 1 mMEGTA and 1 mg/ml phytic acid. Plates were allowedto stand on ice for 15 min before [3H]-inositol phos-phates ([3H]-IPs) were extracted using the trioctyla-mine-freon method [13,14]. Brie¯y, aliquots (0.45 ml)were placed into plastic tubes and trioctylamine/1,1,2-trichlorotri¯uoroethane (1:1, v/v; 0.4 ml) was added;the solution was mixed thoroughly and then centri-fuged at 950g for 5 min to separate the phases.A sample (0.38 ml) of the upper phase was transferredto an insert vial, 4 ml 50 mM Tris-HCl buffer, pH 7.7,added and the mixture applied to an AG 1-X8 (formateform, 100±200 mesh; BioRad) anion-exchange column.[3H]-Inositol and [3H]-glycerophosphoinositol wereeluted with 10 ml water and 10 ml 60 mM ammoniumformate/5 mM sodium tetraborate, respectively, andtotal [3H]-IPs ([3H]-IP1� [3H]-IP2� [3H]-IP3) theneluted with 8 ml 800 mM ammonium formate/100 mM formic acid. Scintillator (10 ml) was addedto each fraction and tritium determined by liquidscintillation counting.

FluorimetricDetermination ofIntracellular Ca2� ([Ca2�]i)

DU-145 cells grown to near con¯uence werewashed with 15 ml phosphate-buffered saline (inmM: NaCl 137, KCl 2.7, Na2HPO4 8.1, and KH2PO4

1.5) containing 0.6 mM EDTA before dissociation with5 ml trypsin/EDTA (500±750 U/ml per 0.6 mM). After

180 Valencia et al.

centrifugation at 100g for 5 min the cells wereresuspended in 5 ml HEPES-buffered medium(in mM: NaCl 120, KCl 5.4, CaCl2 1.8, MgCl2 1.6,D-glucose 11, HEPES 20; pH 7.4 with NaOH) contain-ing 2 mM fura-2 acetoxymethyl ester (fura-2 AM) and5 mg/ml bovine serum albumine and then incubatedin the dark for 1 hr at 37�C. After centrifugation asabove, the cells were resuspended in HEPES-bufferedmedium, aliquoted and allowed to stand on ice untiluse.

Aliquots were diluted in 2 ml warmed medium in aplastic cuvette placed into the warmed cell of a PerkinElmer LB-50 ¯uorometer. Fluorescence was monitoredat an emission wavelength of 510 nm, with excitationwavelengths of 340 and 380 nm in a Perkin Elmer LS 50spectrophotometer. The ratio of the ¯uorescence at 340and 380 nm was converted to [Ca2� ]i as described byGrynkiewicz et al. [15].

Cell ProliferationAssay

Cell Counting. Cells were plated in 24-well culturetrays in serum- and antibiotic-supplemented medium.After 12 hr in culture, histamine was added in a 10 mlvolume and incubations were continued for a further24 hr, before dissociation with trypsin/EDTA. Aftercentrifugation (3 min, 100g) cells were resuspended inKrebs±Henseleit buffer (1 ml per well) and countedwith an hemocytometer. Viable and dead/dying cellswere distinguished using a trypan blue exclusionassay. Since no ¯oating cells were found underinspection with an inverted light microscope, thecounts of the cells attached to the bottom of the wellswere taken as measures of total cell number.

[3H]-Thymidine incorporation. Cells were grown in24-well plates in serum- and antibiotic-supplementedmedium. After 12 hr in culture, histamine was addedin a 10 ml volume and incubations were continued for afurther 24 hr with [3H]-thymidine (1 mCi per well)being present for the last 4 hr. For the determination of[3H]-thymidine incorporation into the trichloroaceticacid (TCA)-insoluble fraction, cells were washed twicewith warmed (37�C) Krebs±Henseleit medium, beforeadding 1 ml of ice-cold 5% TCA (w:v). After 20 min onice the TCA-insoluble material was washed twice withethanol before adding 1 ml of 0.1 M NaOH containing2% (w:v) Na2CO3. Plates were allowed to standovernight at room temperature. Samples were neu-tralized with 0.1 M HCl, scintillation liquid was addedand the tritium content was determined by liquidscintillation counting.

Data analysis. All data are expressed as means� S.E.M. Concentration-response curves were ®tted

by nonlinear regression to a logistic (Hill) equationusing the program Prism (GraphPad software, SanDiego, CA).

Materials. The following compounds were purchasedfrom New England Nuclear (Boston, MA): [methyl-3H]-thymidine (speci®c activity, 83 Ci/mmol), myo[3H]-inositol (22.3 Ci/mmol), [3H]-mepyramine (20 Ci/mmol), [3H]-tiotidine (89.7 Ci/mmol), and N-a-[methyl-3H]-histamine (85 Ci/mmol). Fura 2-AMwas from Sigma Chemical Co. (St Louis, MO).Mepyramine (pyrilamine maleate), histamine dihy-drochloride, thioperamide maleate and thapsigarginwere from Research Biochemicals Inc. (Natick, MA).Tiotidine was a kind gift from Dr. J. M. Young(University of Cambridge, UK).

RESULTS

Radioligand Binding

The speci®c binding of [3H]-mepyramine to mem-branes from DU-145 cells (Fig. 1) did not differsigni®cantly from binding to a single site (Hillcoef®cient, nH, 1.2� 0.2). The best-®t value for theequilibrium dissociation constant (Kd) was 1.1� 0.2 nMand that for the maximum binding 110� 14 fmol/mgof protein (combined data from three experiments).No speci®c binding for either [3H]-tiotidine (20 nM) orN-a-[methyl-3H]-histamine (4 nM) was detected.

Fig. 1. Saturation binding of [3H]-mepyramine to membranesfrom DU-145 cells. Specific receptor binding was determined asthe totalbindingminusbinding in thepresence of1mMmepyramine(nonspecific binding). Points are themeans of triplicate determina-tions from a single experiment, which was repeated twice withsimilar results.The line drawn for the specific binding is the best-fitto a hyperbola.Best-fit parameters are given in the text.

Histamine Inhibition of Cell Proliferation 181

Histamine-Induced [3H]-Inositol Phosphate([3H]-IPs) Accumulation

In [3H]-inositol-labeled DU-145 cells and in thepresence of 20 mM LiCl, histamine-stimulated [3H]-IPs accumulation was linear with time up to 60 min,the longest period studied (Fig. 2A). The response tohistamine, measured over a 30 min incubation period,was concentration-dependent (Fig. 2B), with max-imum response 602� 23% of basal (best-®t value tothe combined data from four experiments), EC50

2.2� 0.4 mM and Hill coef®cient (nH) 1.1� 0.2 (notsigni®cantly different from unity, P> 0.05, Student'st test).

Histamine-Induced Ca2� Mobilization

The resting level of intracellular Ca2� ([Ca2� ]i) incell suspensions was 96� 5 nM (23 determinations).Histamine increased [Ca2� ]i in a concentration-dependent manner (Fig. 3) with EC50 6.2� 0.1 mM,�[Ca2� ]i 517� 32 nM and nH 0.96� 0.06 (not sig-ni®cantly different from unity, P> 0.05, Student'st test).

Fig. 4 (panel A) shows that Ca2� mobilization in-duced by 100 mM histamine (mean peak 673� 61 nMfor this series of determinations, �[Ca2� ]i 557�44 nM, n� 7) was fully blocked by the selective H1

antagonist mepyramine (1 mM). In this series ofexperiments and in those related to the action ofhistamine on cell growth (see below), the concentra-tion of histamine was that estimated to yield 94±98%receptor occupancy assuming that the EC50 valuesfor histamine-induced [3H]-IPs accumulation or Ca2�

mobilization approximate the Kd of the agonist.Likewise, the concentration of the H1 antagonistmepyramine was that calculated to block over 98%of H1 receptors in the presence of a supramaximalconcentration of histamine.

The inhibition of sarcoendoplasmic Ca2� pumpsby thapsigargin (5 nM) led to an increase in [Ca2� ]i

(326� 69 nM from a resting level of 79� 3 nM; threedeterminations), which returned to levels not signi®-cantly different from basal after 12 min (Fig. 4B). Thesubsequent exposure to 100 mM histamine failed toproduce any signi®cant increase in [Ca2� ]i.

Effect ofHistamine onCell Proliferation

The pattern of growth of DU-145 cells is shown inFigure 5A. The effect of histamine on cell proliferationwas assessed either by cell counting or by determiningthe incorporation of [3H]-thymidine to cellular DNA.

After 36 hr in culture, cell density rose to 183� 28%of the plating value (Fig. 5B). In the presence ofhistamine (100 mM, present for 24 hr) cell growth was

markedly reduced (90� 5% inhibition). The inhibitoryaction of histamine on cell growth did not appear to bedue to cell damage, since no signi®cant differencein trypan-blue staining (less than 2% of cells) wasobserved between control and histamine-treated cells.

Fig. 2. Histamine-induced [3H]-IPs accumulation. (A) Timecourse of [3H]-IPs accumulation in the presence and absence of100 mM histamine. [3H]-Inositol-labeled cells were preincubated(10 min) with 20 mM LiCl and then exposed to histamine for theindicatedperiods.Values aremeans � SEMfromtriplicate determi-nations from a single experiment. The whole experiment wasrepeated twicewith similarresults.Whereno errorbars are shownthe error was within the size of the symbol. (B) Concentration-response curve. Incubation with the indicated concentrations ofhistamine was for 30 min.Values are expressed as a percentage ofbasalaccumulation (380� 20dpm)andrepresent themeans� SEMfromthe combineddata fromfourexperiments.The curvedrawnisthe best-fit line to a logistic equation.Best-fit parameters are givenin the text.

182 Valencia et al.

The incorporation of [3H]-thymidine to DU-145cells grown in serum-supplemented medium was alsoinhibited by histamine. In the presence of the agonist(100 mM, 24 hr) [3H]-thymidine incorporation wasreduced by 45� 7% (n� 4 experiments; Fig. 5C).

In agreement with an H1 receptor-mediated effect,the action of histamine on both cell growth and [3H]-thymidine incorporation was blocked by the selectiveH1 antagonist mepyramine (Fig. 5, panels B and C).

DISCUSSION

Prostate cancer is the most common type of malemalignancy and the second leading cause of malecancer-related deaths in the US [16]. Thus, much effortis being directed at understanding the cellular mech-anisms involved in the regulation of proliferation ofprostatic cancer cells. The cell line DU-145, isolatedfrom the brain metastasis of a primary prostate adeno-

Fig. 3. Histamine-induced Ca2� mobilization. Cell suspensionswere loadedwith Fura2 as described inMaterials andMethods andthen exposed to histamine. (A) Representative traces of theresponse to histamine.Traces correspond (frombottom to top) to1, 3,10, 30, and100mMhistamine, added at the time indicatedby thearrow. (B) Concentration-response curve. Values are the means� SEM (basal subtracted) from the combined data from at leastfourdeterminations.The curve drawnis thebest-fit line to a logisticequation.Best-fit parameters are given in the text.

Fig. 4. Effect of mepyramine and thapsigargin on histamine-induced Ca2� mobilization. Cell suspensions were loaded withFura2 as described in Materials andmethods and then exposed tohistamine (100 mM). (A) Antagonism by mepyramine. The H1

antagonistmepyramine (1 mM)was added 5minbefore the agonist.The trace is representative of four determinations with differentcultures. (B) Effect of thapsigargin.Cells were exposed to thapsi-gargin (5 nM) before the addition of histamine (100 mM).The traceis representative of seven determinations with differentbatches ofcells.

Histamine Inhibition of Cell Proliferation 183

carcinoma [11], has been used as a model forandrogen-independent prostatic cancer, and in thiswork we aimed to characterize the presence of hist-amine H1 receptors and the effect of their stimulationon cell proliferation.

G-protein-coupled receptors can act as growthfactors by stimulating PKC through DAG andIns(1,4,5)P3 -mediated Ca2� mobilization [4,17]. PKCappears to phosphorylate and activate the proteinRaf-1 which in turn stimulates MEK1/MEK2(mitogen-activated protein kinase (MAPK) kinases).The phosphorylation by MEK1/MEK2 is required foractivation of MAPKs, a subfamily of extracellularly-responsive or extracellular signal-regulated kinases(ERKs), the ®nal result being the phosphorylation andactivation of transcription factors and gene activation[17,18,19]. Histamine H1 receptors are coupled toIns(1,4,5)P3 and DAG formation [3] and their activa-tion stimulates cell growth in HeLa [5] and humanastrocytoma cells [7]. In line with these observations,the activity of the enzyme histidine decarboxylase,which catalyzes the formation of histamine fromhistidine, has been shown to be high in tissuesundergoing rapid growth or repair [20,21]. We reportherein that DU-145 cells express [3H]-mepyraminebinding sites whose af®nity for the radioligand (Kd

1.1� 0.2 nM) is similar to that reported for H1

receptors in mammalian tissues, including the centralnervous system, and for a number of cell lines [1]. Thelack of binding sites for radioligands selective for H2

and H3 receptors indicates that the H1 receptor isthe only subtype of histamine receptors expressed byDU-145 cells.

Fig. 5. Effect of histamine on cell proliferation. (A) Pattern ofgrowth ofDU-145 cells.Cell countingwas carried out as describedin Materials andMethods. A representative determination (n� 3 )is shown.Values aremeans � SEMof the combined values from sixreplicates foreachcondition. (B) Effectofhistamine oncellnumber.Cellswereplated and incubated for12 hr in serum-containingmed-ium, histamine (Hist, 100 mM) was then added for a further 24 hrbefore cell dissociation and counting.When required, mepyramine(Mepy,1mM)was added15minbefore theagonist.To allow forvaria-tions between experiments, data are expressed as percentage ofthe number of cells plated and represent the means � SEM fromthe combineddata from fourdifferentexperimentswith 4 ^5repli-cates for each condition. aSignificantly different (P<0.05) from theplating value; bNot significantlydifferent (P> 0.05) from theplatingvalue; cNot significantly different (P> 0.05) from control, ANOVAandposthocStudent^Newman^Keuls test. (C) Effectofhistamineon [3H]-thymidine incorporation.Cells were plated and incubatedfor12hr in serum-containingmedium,histamine (100mM)was thenadded for a further 24hrwith [3H]-thymidinebeingpresent for thelast 4 hr.When required, mepyramine (1 mM) was added 15 minbefore histamine. The incorporation of the labeled compound isexpressed as a percentage of control incorporation (23,769�585dpm/well). Values are means� SEM of the combined values fromfour experiments with six replicates for each condition.a Signifi-cantly different (P<0.05) from control, ANOVA and post hocDunnett test.

184 Valencia et al.

Our results also show that H1 receptor activationresults in robust phosphoinositide hydrolysis andCa2� mobilization. The rise in intracellular Ca2� ap-pears to be initiated by the mobilization of Ca2�

ions from intracellular stores since the response tohistamine was abolished by thapsigargin, a drug thatempties Ins(1,4,5)P3 -sensitive stores [22], althoughresearch in progress suggests that histamine-inducedchanges in intracellular Ca2� are complex and includecapacitative Ca2� in¯ux and, probably, receptor-operated Ca2� entry (L.E. Soria-Jasso and J.A. Arias-MontanÄ o, unpublished results).

Our next aim was to investigate whether histaminecould function as a growth factor for DU-145 cells.However, our results clearly indicate that rather thanpromoting it, histamine prevents cell proliferation.The inhibition by mepyramine and the lack ofexpression of H2 and H3 receptors by DU-145 cellsmakes it clear that the anti-mitogenic action ofhistamine is mediated by H1 receptors. Preliminaryexperiments were carried out with serum-deprivedcells (data not shown) and a marked decrease in bothcell number and [3H]-thymidine incorporation wasalso observed. We reasoned that histamine could beenhancing the arresting action of serum deprivationand subsequent experiments were therefore carriedout in serum-containing medium in which the inhib-itory action of histamine was also present. In ®bro-blasts grown in serum (3%)-containing mediumhistamine reduced by 45±60% the incorporation of[3H]-thymidine and such an effect was blocked by theH1 antagonist mepyramine [23]. These results aresimilar to those presented herein and show that insome cells histamine H1 receptor activation can pre-vent cell growth whereas in others (v. gr. HeLa andhuman astrocytoma cells) its action is the opposite.

The mechanism by which histamine H1 receptoractivation results in inhibition of cell growth and [3H]-thymidine incorporation to DU-145 cells remains to beestablished. However, it is tempting to relate theseactions to histamine-induced Ca2� mobilization andPKC activation by DAG and Ca2� ions. In DU-145cells long exposure (4 hr) to high concentrations ofthapsigargin (500 nM) resulted in sustained [Ca2� ]i

elevations (4-fold of basal) and within 24 hr cellsarrested in the G0-G1 compartment of the cell cycle,whereas longer incubations (� 36 hr) led to pro-grammed cell death or apoptosis [24]. A sustainedincrease in [Ca2� ]i by continuous H1 receptor activa-tion could thus prevent DU-145 cells from entering theS-phase of the proliferative cell cycle explaining theresults presented herein.

As stated above, we have found that under similarexperimental conditions to those described here forDU-145 cells, H1 receptor activation stimulates the

proliferation of astrocytoma U373 MG cells [7].Whereas the density of H1 receptors is similar in bothcell lines, the characteristics of histamine-inducedCa2� mobilization appear different. In experimentscarried out in cell suspensions, histamine evokes apeak in U373 MG cells (�[Ca2� ]i 462� 21 nM)that declines rapidly to reach a plateau (�[Ca2� ]i

107� 10 nM) [25] that is maintained for at least 60 min(the longest period studied). Under the same experi-mental conditions, in DU-145 cells the initial peak in[Ca2� ]i is followed after a few seconds by a secondpeak, similar in amplitude, that declines with a muchslower ratio to reach a plateau with a much higher[Ca2� ]i level (�[Ca2� ]i 257� 11 nM). No signi®cantdifference was found for resting [Ca2� ]i (96� 5 and108� 6 nM for DU-145 and U373 MG cells, respec-tively). Thus, although in both cell lines the continuousactivation of H1 receptors results in a sustainedincrease in [Ca2� ]i, the level reached is signi®cantlyhigher in DU-145 cells. This difference could be due tosome degree of H1-receptor desensitization inU373MG cells, to less ef®cacious mechanisms for theremoval of cytosolic Ca2� in DU-145 cells or toadditional mechanisms such as H1 receptor-operatedCa2� entry as preliminary data of our own suggest forthe latter type of cells. Differences in Ca2� home-ostasis could thus underlain the opposite effects oncell proliferation, with low [Ca2� ]i levels associatedwith cell proliferation and higher, sustained levelswith inhibition of cell growth.

Regarding the participation of PKC, in cultured®broblasts the H1 receptor-mediated inhibition of[3H]-thymidine incorporation was mimicked by thephorbol ester 12-O-tetradecanoylphorbol-13-acetate(TPA), known to activate PKC, and was preventedby PKC downregulation indicating the participation ofthis kinase [26]. In the androgen-dependent prostatecancer cell line LNCaP, PKC stimulation results inmembrane translocation of the enzyme, prolongedactivation of ERKs and apoptosis, particularly in cellsoverexpressing PKCa [27,28]. In line with the latterreport, in DU-145 cells the induction of apoptosis wasarrested by downregulation of PKC activity byphorbol ester treatment [29]. PKC stimulation couldthus be an alternative mechanism for the H1 receptor-mediated inhibition of cell proliferation found in DU-145 cells in which histamine induces DAG formationand Ca2� mobilization that would lead to PKC acti-vation. Whether apoptosis is involved in the reductionof cell number and [3H]-thymidine incorporationobserved in DU-145 cells can not be established fromour experiments. We found no signi®cant differencesin cell viability (as judged by the trypan blue exclusionassay) between control and histamine-treated cells,but these observations were carried out at a time (24 hr

Histamine Inhibition of Cell Proliferation 185

incubations) when the initial phases of the apoptoticprocess could have just begun [24]. It is also worthnoting that in contrast to other prostate carcinoma cellssuch as LNCaP, DU-145 cells do not show activation ofthe apoptotic machinery in response to hormonal/cytotoxic therapy or the apoptosis-inducer stauros-porine. This resistance appears to be due to low exp-ression levels of Bcl-2 family members, decreasedtranslocation of cytochrome c to the cytosol and dimi-nished ability of the caspase pathway to react inresponse to activation of the mitochondrial phase [30].

On the other hand, it is not clear how PKCactivation could lead to antagonistic actions on cellproliferation depending on cell type, since in astro-cytoma U373 MG cells both histamine and TPA aremitogenic and histamine-induced [3H]-thymidineincorporation is partially blocked by PKC inhibition(A. HernaÂndez-Angeles and J.A. Arias-MontanÄ o,unpublished observations). Interestingly, it has beenshown that in primary cultures of rat hepatocytes, thenerve growth factor-induced activation of the ERKscascade promotes DNA synthesis if the stimulation isacute/phasic whereas tonic receptor activation resultsin the opposite action [31]. With this in mind, wecarried out a series of experiments in which histaminewas present for 1 hr with a further incubation for 23 hrwith fresh serum-containing medium before cellcounting. Under these conditions, a similar decreasein cell number was observed in histamine-treatedcultures (data not shown), indicating that the effect ofhistamine does not depend on the length of stimula-tion, at least between 1±24 hr.

It would be of interest to know whether theinhibitory action of histamine on cell growth reportedhere for DU-145 cells is exerted on other humanprostatic cancer cell lines. In the androgen-dependentcells LnCaP histamine failed to evoke a signi®cantincrease in [Ca2� ]i. In contrast, in PC3 cells (androgen-insensitive, likewise DU-145 cells), histamine alsoincreased [Ca2� ]i (although to a lesser extent whencompared with DU-145 cells), and preliminary experi-ments show that H1 receptor activation also results in amodest, but signi®cant, decrease (15� 3% inhibition)in [3H]-thymidine incorporation. The inhibitory actionof histamine on the growth of prostatic cancer cellsappears thus to be related to the magnitude of thechange in [Ca2� ]i evoked by H1 receptor activation.

CONCLUSIONS

The data presented herein indicate that the humanprostate carcinoma DU-145 cells express signi®cantlevels of histamine H1 receptors, coupled to phos-phoinositide hydrolysis and Ca2� mobilization, andwhose activation inhibits cell proliferation. Further

research will be required to investigate the mechan-isms underlying the antimitogenic action of histamineas well as the potential use of H1 receptor agonists aspharmacological tools in the treatment of prostatecancer.

ACKNOWLEDGMENTS

This work is based on the dissertation submitted byS. Valencia to obtain the B. Sc. degree (Pharmacy) fromUniversidad Autonoma de Puebla, MeÂxico.

REFERENCES

1. Hill SJ. Distribution, properties, and functional characteristicsof three classes of histamine receptor. Pharmacol Rev1990;42:46±84.

2. Schwartz J-C, Arrang J-M, Garbarg M, Pollard H, Ruat M.Histaminergic transmission in the mammalian brain. PhysiolRev 1991;71:1±51.

3. Hill SJ, Ganellin CR, Timmerman H, Schwartz J-C, ShankleyNP, Young JM, Schunak W, Levi R, Haas HL. Classi®cation ofhistamine receptors. Pharmacol Rev 1997;49:253±278.

4. Boarder MR. A role for phospholipase D in control ofmitogenesis. Trends Pharmacol Sci 1994;15:57±62.

5. Tilly BC, Tertoolen LGJ, Remorie R, Ladoux A, Verlaan I, LaatWS, Moolenar HW. Histamine as a growth factor andchemoattractant for human carcinoma and melanoma cells:action through Ca2� -mobilizing H1 receptors. Cell Biol1990;110:1211±1215.

6. Guizzeti M, Costa LG. Inhibition of muscarinic receptor-stimulated glial cell proliferation by ethanol. J Neurochem1996;67:2236±2245.

7. HernaÂndez-Angeles A, Ortega A, Arias-MontanÄ o J-A. Hista-mine H1-receptor activation stimulates proliferation in humanastrocytoma cells [abstract]. Soc Neurosci Abstr 26:2170 (816.9).

8. Berridge M. Inositol triphosphate and calcium signalling.Nature 1993;361:315±325.

9. Nishizuka Y. Protein kinase C and lipid signaling for sustainedcellular responses. FASEB J 1995;9:484±496.

10. Wasilenko WJ, Cooper J, Palad AJ, Somers KD, Blackmore PF,Rhim JS, Wright GL Jr., Schellhammer PF. Calcium signaling inprostate cancer cells: evidence for multiple receptors andenhanced sensitivity to bombesin/GRP. Prostate 1997;30:167±173.

11. Stone KR, Mickey DD, Wunderli H, Mickey GH, Pauloson DF.Isolation of a human prostate carcinoma cell line (DU-145). Int JCancer 1978;21:274±281.

12. Lowry DH, Rosenbrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenol reagent. J Biol Chem1951;193:265±275.

13. Sharpes ES, McCarl RL. A HPLC method to measure32P incorporation into phosphorylated metabolites in culturedcells. Anal Biochem 1982;124:421±424.

14. Downes CP, Hawkins PT, Irving RF. Ins(1,3,4,5)tetrakispho-sphate and not PIP2 is the probable precursor of Ins(1,3,4)tri-sphosphate in stimulated parotid gland. Biochem J1986;238:501±506.

15. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2�

indicators with greatly improved ¯uorescence properties. J BiolChem 1985;260:3440±3450.

186 Valencia et al.

16. Lecrone V, Li W, Devoll RE, Logothetis C, Farach-Carson MC.Calcium signals in prostate cancer cells: speci®c activation bybone-matrix proteins. Cell Calcium 2000;27:35±42.

17. Gudermann T, Grosse R, Schultz G. Contribution of receptor/Gprotein signaling to cell growth and transformation. Naunyn-SchmiedebergÂs Arch Pharmacol 2000;361:345±362.

18. Egan SE, Weinberg RA. The pathway to signal achievement.Nature 1993;365:781±783.

19. Buchner K. Protein kinase C in the transduction of signalstoward and within the cell nucleus. Eur J Biochem 1995;228:211±221.

20. Watanabe T, Taguchi Y, Sasaki K, Tsuyama K, Kitamura Y.Increase in histidine decarboxylase activity in mouse skin afterapplication of the tumor promoter tetradecanoylphorbolacetate. Biochem Biophys Res Commun 1981;100:427±432.

21. Bartholeyns J, Fozard JR. Role of histamine in tumor develop-ment. Trends Pharmacol Sci 1985;6:123±125.

22. Simpson PB, Challis RAJ, Nahorski SR. Neuronal Ca2� stores:activation and function. Trends Neurosci 1995;18:299±306.

23. Johnson CL, Johnson CG, Bazan E, Garver D, Gruenstein E,Ahluwalis M. Histamine receptors in human ®broblasts:inositol phosphates, Ca2� , and cell growth. Am J Physiol1990;258:C533±C543.

24. Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs JT. The role ofcalcium, pH and cell proliferation in the programmed(apoptotic) death of androgen-independent prostatic cancercells induced by thapsigargin. Cancer Res 1994;54:6167±6175.

25. Soria-Jasso LE, Arias-MontanÄo J-A. Histamine H1-receptoractivation stimulates calcium mobilisation and [3H]-GABA

release from human astrocytoma U373MG cells. Eur J Pharma-col 1996 ;318:185±192.

26. Johnson CL, Johnson CG. Inhibition of human skin ®broblastproliferation by histamine and phorbol esters is mediated byprotein kinase C. Cell Signal 1990;2:105±113.

27. Powell TC, Brittis NJ, Stec D, Hugh H, Heston WD, Fair WR.Persistent membrane traslocation of protein kinase C alphaduring 12-O-tetradecanoylphorbol-13-acetate-induced apopto-sis of LNCaP human prostate cancer cells. Cell Growth Differ1996;7:419±428.

28. Gschwend JE, Fair WR, Powell TC. Bryostatin 1 inducesprolonged activation of extracellular regulated protein kinasesin and apoptosis of LNCaP human prostate cancer cellsoverexpressing protein kinase Ca. Mol Pharmacol 2000;57:1224±1234.

29. Rusnak JM, Lazo JS. Downregulation of protein kinase Csuppresses induction of apoptosis in human prostatic carci-noma cells. Exp Cell Res 1996;224:189±199.

30. Marcelli M, Marani M, Li X, Sturgis L, Haidacher SJ, Trial JA,Mannucci R, Nicoletti I, Denner L. Heterogeneous apoptoticresponses of prostate cancer cell lines identify an associationbetween sensitivity to staurosporine-induced apoptosis,expression of Bcl-2 family members, and caspase activation.Prostate 2000;42:260±273.

31. Tombes RM, Auer KL, Mikkelsen R, Valerie K, Wymann MP,Marshall CJ, McMahon M, Dent P. The mitogen-activatedprotein (MAP) kinase cascade can either stimulate or inhibitDNA synthesis in primary cultures of rat hepatocytes depend-ing upon whether its activation is acute/phasic or chronic.Biochem J 1998;330:1451±1460.

Histamine Inhibition of Cell Proliferation 187