Antiproliferative Effect of Spermine Depletion by N-Cyclohexyl-1,3-Diaminopropane in Human Breast Cancer Cells'

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[CANCERRESEARCH55,934-943,February15,1995J Antiproliferative Effect of Spermine Depletion by N-Cyclohexyl-1,3-diaminopropane in Human Breast Cancer Cells'

Maria Huber and Richard Poulin2

Department of Physiology. Laval University and Laboratory of Molecular Endocrinology, Centre Hospitalier de L'UniversitÃ© Laval Research Center, 2705 Laurier Boulevard, Ste. Foy. Quebec.Canada GIV 4G2

ABSTRACT vated by the c-Myc nuclear oncoprotein and could thus participate in the activation of cell cycle progression and transformation by Spermineisoftenthemostabundantpolyaminein humantumorssuchas c-Myc (7, 8). breast carcinomas. However, its specific role in tumor bioloej' is still uncer taiii, sinceinhibitors of ornithine decarboxylasesuchas a-difluoromethylo Because of the absolute polyamine requirement for cell growth, rnithine depresscell growth while leaving spenninecontent mostly wiaf interference with polyamine biosynthesis has long been considered as fected, We have assessedthe specific role of spermine in breast cancer cell a promising therapeutical approach against proliferative diseases, growth usingN-cyclohexyl-1,3-diaminoprupane(C-DAt'),a potentspermine including variousmalignancies(1â€”5,9).DFMO, a suicidesubstrate synthaseinhibitor. In ZR-75--1cells,C-DAPdecreasednetcell growth after inhibitor of ODC (1â€”4,10,11), has been the prototype tool to study l4daysby65%at5Opse,@thanIC@1,ofabout5pi@i,andwasabout1Olimes the therapeuticeffectivenessof polyaminedepletionin experimental more potent than N-(n-butyl)-1,3-diaminopropane, another spermine syn tumors (4). DFMO profoundly depletesputrescine and spermidine and thaseinhibitor. C-DAPactedasa specificinhibitor ofsperminebiosynthesis, potentlyinhibits cell growth in mostanimalcells (1â€”4).However,it since (a) it depleted spermine content while causing an equal or greater accumulationof spermidineon a molar basis,(b) it rapidly induced S is inactive in vivo in many tumor models and has had very limited adenosylmetbioninedecarboxylaseactivityand theaccumulationofits prod efficacy in clinical trials(1â€”4,6).The lack of effectivenessofDFMO uds due to reliefofspermine-dependent inhibition ofenzyme expression, and therapy in vivo hasbeen attributed in part to its up-regulatory effect on (c) exogenous spermine (1 pM) completely reversed C-DAP-induced growth polyaminetransport,which is highly activein tumor cells andallows inhibition. C-DAPandrelatedcompoundswereaccumulated,atleastin part, the compensatory uptake of exogenous polyamines (12â€”14).More through a mechanismdistinctfrom the polyaminetraitsport system,while over, since ODC has one of the shortesthalf-lives known among alsoMockingputrescineandspermidineuptakewith variom potencies.Re eukaryotic proteins (1â€”4),efficient suppression of its activity needs versibility of C-DAP-inducedgrowthinhibition by exogenoussperminewas the maintenance of high steady-state drug levels (1, 4). progressivelyâ€˜ostonprolongedtreatment,in associationwithmarked mor phologicalchanges.In 4 different human breastcancercell lines(ZR-75â€”1, Furthermore, DFMO has little effect on spermine content, since (a) T47-D,MCF-7,andMDA-MB-231),relativegrowthsensitivitytoC-DAPwas the inhibition of AdoMetDC expression by spermidine (15â€”17)is inverselyrelatedto the extentofspermidineaccumulationcausedbysperm relieved by treatment with DFMO, thus increasing decAdoMet for iiie synthase inhibition, suggesting that spermidine overaccumulation can mation and conversion of spermidine to spermine (Fig. 1A, 2); and (b) functionally replace spermine. Interestingly, C-DAP strongly potentiated spermine turnover is normally so low that its content decreasesonly growth inhibition causedbya.difluoromethylornithineIn all cell linestested through dilution by cell division, which is itself considerably reduced bypreventingconversionofresidualspermidinetospermine,indicatingthat by DFMO (1). It has thus been suggested that specific inhibitors of spenninesynthesislimits a-difiuoromethylornithineaction and that under spermidine and spermine synthases (Fig. 1A, 3 and 4) might some Critical threshold, spermidine cannot fulfill cellular needsfor spermine. Thus, sperinine plays specific and important Ã±mction.sin breast tumor facilitate polyamine depletion because these enzymes have a slow growth,and sperininesynthaseinhibitorscouldmarkedlyimprovethe ther turnover and their high expression levels are not subject to acute apeutic effectiveness of existing polyamine depletion strategies, especially in feedback regulation (1â€”4).Thus, combining such drugs with spermine.rkh tumors. DFMO to achieve more complete polyamine depletion might improve the outcome of antipolyamine therapy, although this INTRODUCTION possibility has not been tested thoroughly. While putrescine and spermidine are known to play specific roles, The essential role of the diamine putrescine and the polyamines suchas in volume regulation(18) and the maturationof eukaryotic spermidine and spermine for eukaryotic cell growth is well estab translation initiation factor eIF-5A (19, 20), respectively, the exact lished (1â€”4).A dramatic elevation of polyamine biosynthesis and functionof sperminebiosynthesishasthusfar beendifficult to iden transport is characteristically associatedwith the onset of rapid growth tify. Because spermine is rarely found in prokaryotes but widespread and cell transformation (1â€”5).Theimportance of an increased rate of in eukaryotes, its role could be more specifically related to chromatin polyamine biosynthesis in the malignant process has been demon structure (21, 22) or to the regulation of mitochondrial Ca2@transport strated by the induction of tumorigenicity on transfection of normal (23). Previous work with spermine synthaseinhibitors such asS-meth rodent cell lines with a vector conferring constitutive expression of yl-5'-methylthioadenosine (24, 25) or S-adenosyl-1,12-diamino-3- ODC3 (5, 6), which is one of the key regulatorystepsin polyamine thio-9-azadodecane (26) has shown that spermine content can be biosynthesis (Fig. 1A, 1). Moreover, ODC is directly trans-acti reduced profoundly without obvious effects on growth and viability. However, interpretingthe effect of these inhibitors on polyamine Received7/1/94;accepted12/14/94. metabolism has been hampered by the limited specificity of the The costsof publicationof this articleweredefrayedin part by thepaymentof page charges.Thisarticle mustthereforebeherebymarkedadvertisementinaccordancewith former inhibitor(24) andby enzymaticdegradationof the latter (26). 18 U.S.C. Section 1734 solely to indicatethis fact. More recently,N-alkylated derivativesof 1,3-diaminopropanesuchas I This research was supported by grants from the Cancer Research Society, Inc., the B-DAP (27, 28) and C-DAP (29, 30) have been shown to combine Fondsde Ia Rechercheen Santedu QuÃ©bec,andEndorecherche,Inc.M. H. is supported by La Fondationde l'UniversitÃ©Laval. high specificity and potency as competitive inhibitors of spermine 2 To whom requests for reprints should be addressed. synthase (Fig. 1, A and B), with K1 values of about 12 and 5 nr@i, 3 The abbreviations used are: ODC, omithine decarboxylase (EC 4.1.1.17); DFMO, (D,L)-a-ditluoromethylornithine;decAdoMet,decarboxylatedS-adenosylmethionine;Ac decAdoMet, decarboxylatedN-acetyl,S-adenosyl-i-methionine;AdoMet, S-adenosyl-i N-(n-butyl)-l,3-diaminopropane;C-DAY,N-cyclohexyl-l,3-diaminopropane;FBS,fetal methionine;AdoMetDC,S-adenosyl-L-methioninedecarboxylase(EC4.1.1.50); B-DAY, bovineserum;IMEM, improvedMEM; P-DAY,N-(n-propyl)-l,3-diaminopropane. 934

Ado MATERIALS AND METHODS A Materials. L-[1-'4C]Ornithine (58 Ci/mol), S-adenosyl-L-[carboxyl CH@ 14C]methionine (47.8 Ci/mol), [2,3-3H]putrescine dihydrochloride (4.10 X i0@ NH3+/L.@%.@ NH3@ AdoMet Ci/mol), and [1,8-3H]spermidine trihydrochloride (1.50 X iO@Ci/mol) were Omithine obtained from DuPont New England Nuclear (Lachine, QuÃ©bec,Canada). C-DAP andP-DAYwerefrom Aldrich. B-DAY dihydrochioride,synthesized Ado LI asdescribed(27),waskindly providedby Dr. J. K. Coward(Departmentsof ChemistryandMedicinalChemistry,Universityof Michigan,Ann Arbor, MI). NH3@'@!.â€•@â€•NH3@ @ CH@â€” Ac-decAdoMetwas obtainedfrom Dr. A. E. Pegg(Departmentsof Cellular Putrescine andMolecularPhysiologyandPharmacology,PennStateUniversityCollege @.4 decAdoMet of Medicine, Hershey, PA). DFMO was a generous gift from the Marion MTA Merrell Dow Research Institute (Cincinnati, OH). FBS was obtained from @ NH3@ Hyclone(Logan,UT). Growth mediaandsupplementswerepurchasedfrom Sigma Chemical Co. Spermidine Cell Culture. ZR-75-l, MCF-7, T47-D, andMDA-MB-231 humancell MTA m $4 lines were obtainedfrom the AmericanType Culture Collection(Rockville, MD). ZR-75â€”1cellsweremaintainedinphenolred-freeRPMI1640contain @ NH@'@â€•.'@ NH3@ ing 10% (v/v) FBS and supplemented as described [MEZR medium (38)], with weekly passages.Forthe simultaneouscomparisonof multiple cell lines,the Spermine growthmediumusedwasphenolred-freeRichter'sIMEM without putrescine (39) and supplemented with 2 mM L-glutamine, 15 mM HEPES, 5% FBS, and antibiotics (IMEM@ medium). Cell lines were adapted to this medium for at B â€˜--NH@(@,' least 2 weeks prior to experiments. Experimental media were supplemented with aminoguanidine (1 mM) to prevent polyamine degradation by serum N-(n-butyl)-1 ,3-dlamlnopropane (B-DAP) amine oxidases (26). CellGrowthExperiments.ZR-75â€”1cellswereseededin24-wellplatesat a densityof 2 X i04 cells/well in MEZR medium.Various treatmentswith enzymeinhibitors were initiated 2 daysafter plating, and fresh mediawere N-(cyclohexyl}-1 ,3-dlamlnopropane (C-DAP) addedeveryotherday.At the indicatedtimes,total cellular DNA wascob rimetrically determined with 3,5-diaminobenzoic acid (40). For more extended Fig. 1. (A ) Pathway of de novo polyamine biosynthesis in mammalian cells. (B) periodsof incubationwith the drug, control and C-DAP-treatedcells were Structure of B-DAY and C-DAY. The enzymatic reactions shown are: I, ODC; 2, harvested after 5 to 7 days, replated at the same starting density with the AdoMet/DC; 3, spermidine synthase; 4, spermine synthase; MTA, 5'-methylthioad appropriate additions, and further incubated for the period indicated. For enosine. simultaneous comparison of inhibitor effects, ZR-75â€”1and T47D cells were platedin IMEM@mediumin 24-well platesat 2 X i0@cells/well,whereas MCF-7andMDA-MB-231cellswereseededat1 X i0@cells/well.Incubation respectively, for the partlypurifiedratbrainenzyme (27, 29). These periods were adjusted to the respective generation time of the cell lines used to inhibitors thus have considerable potential for elucidating the specific allow at least3 to 4 populationdoublings,exceptin ZR-75â€”lcells,which had function of sperminebiosynthesisin eukaryoticcells. Initial studies the longestgenerationtime. Determination of Polyamine and Nucleoside Contents. ZR-75â€”l cells with B-DAP have failed to demonstrate a growth-inhibitory action in wereplatedin 100-mmPetridishesat5 X l0@cells/dishin MEZR mediumand mammalian cells (27), which was attributed,as for other spermine incubatedunder the various conditions describedin the text, with fresh synthaseinhibitors, to the compensatoryeffect of spermidineaccu mediumbeing replacedeveryotherday. For simultaneousdeterminationsof mulation caused by the blockade in spermine biosynthesis (25â€”28). polyamine contents, ZR-75â€”1andT47-D cells were plated at 5 X liP cells/ However, preliminary evidence was shown that chronic incubation dish, whereas MDA-MB-231 and MCF-7 cells were plated at 1 X i0@ and with B-DAP might depress cell growth after 9â€”10generations in 2.5 X 10@cells/dish,respectively, in IMEM@medium. At the indicated times, mouse leukemia cells (28). medium was removed,cell monolayerswere rinsed twice with 10 ml of ice-cold Ca2@/Mg@@-freePBS(2.7 mM KC1-l.5 mM KH2PO4-8.l mM Polyamines play important functions in mammary gland biology Na2HPO4-137mMNaCl), andharvestedbycentrifugation(2000X g for 90 s (31). Accordingly, breast cancer cell proliferation is exquisitely sen at 4Â°C)followinga 5â€”7-mmincubationwith bovine trypsin/EDTA solution sitive to polyamine depletion by DFMO (32â€”35)or treatment with (0.05:0.02%, w/v) in HBSS (38). Cell pellets were resuspended in 300â€”500 @l antitumor polyamine analogues (36). Moreover, spermine has been of 10%(w/v) trichloroaceticacidandstoredat â€”20Â°Cuntilfurther analysis. suggested to play a specific role in the mitogenic action of estrogens The trichloroaceticacid-insolublepelletwassolubilizedin 300â€”500@lof1 N in targetbreasttumors(37). In order to examinethe role of the high NaOH andusedto determineproteincontent(41) with BSA (fraction V) as sperminepool presentin tumorssuchas breastcarcinomas,we have standard. Polyamine content was determined by ion pair reverse-phase HPLC with fluorometricdetectionafterpostcolumnderivatizationwith o-phthalalde investigatedthe effect of sperminesynthaseinhibitorson growth and hydeessentiallyasdescribed(26),exceptthattheconcentrationofmethanolin polyamine metabolism in various humanbreastcancercell lines. We buffer B was increased2-fold.Nucleosidesweremeasuredbyparallelmoni arereportingthatspecificsperminedepletioncan readily andstrongly toring of A,@4of the column effluent prior to amine derivatization (26). antagonizebreastcancercell growth. In a given cell line, the potency Determinaflon of ODC and AdoMetDC ActivIty. ZR-75-1 cells were of the antiproliferative action of C-DAP, the most potent spermine platedin 100-mmculturedishesat5 X 10@cells/dishin MEZR medium.Two synthase inhibitor found to date, depends on the extent of spermidine dayslater,treatmentwasinitiatedby theadditionof freshmediumcontaining 50 p@MC-DAYor vehicle(0.1 [email protected]).After 24 or 48 h of incubation,cell accumulation resulting from the blockade in spermine synthesis. In monolayers were rinsed once with Ca2@/Mg2@-freePBS and harvested as terestingly, spermine depletion by C-DAP markedly potentiates above.Cellswerewashedoncewith 10ml PBS,resuspendedinhomogeneiza growth inhibition by DFMO in all cell linesexamined,and is accom tion buffer (50 mM Tris-HCI, pH 7.5-0.1 mM EDTA-5 m@iDTF) and frozen at panied by marked morphological changes which coincide with a â€”80Â°Cuntilenzymeassays.ODCandAdoMetDCactivitiesweredetermined decreasedreversibilityof growthinhibitionby exogenouspolyamines. from the enzymatic release of â€˜4C02from L-[l-'4C]ornithine and S-adenosyl 935

10 10 RESULTS -0-Confrol A B -.-+ 5 @tMC-DAP --+ 5 @tM C-DAP 8 Effect of Spermine Synthase Inhibitors on ZR-75--1 Cell Pro 8 -D-+ 50 @sMC-DAP -iJ-+ 50 @tMC-DAP @ DAP @@+1@,::AP liferation. We initially investigated the effect of spermine depletion 6 on the growth of ZR-75â€”1 cells, in which spermine represents 90% of I thepolyaminepool (seebelow),usingthe2 potentsperminesynthase 4 inhibitors, B-DAP and C-DAP. As shown in Fig. 2, A and C, both @ inhibitors strongly depressedZR-75â€”1cellgrowth, C-DAP being the 2 2 most active compound in this respect. The effect of C-DAP was nearly Spermine-0-ControlNo Spermine-o-controi + 1 @tM maximal at 50 p@M,witha 63 Â±5% (n = 6) decrease in net growth 0 0 after a 14-day incubation. A 32% decreasein cell proliferation was C D -.-+ 20 @&MB-DAP -â€¢--+ 20 @tM8-DAP alreadyobservedafter the sameperiod at 5 @LMC-DAP.By compar 8 8 â€”D--+5O@tMB-DAP â€”O--+5O@tMB-DAP D ison, 50 @.LMB-DAP decreased net growth by only 23 Â± 3%, although @JDA@@ 6 > it was as effective as C-DAP (P > 0.50) at 200 ELM. Concentration @ dependence studies yielded IC50 values of 7.5 Â± 2.0 and 100 Â± 18 ,.LM @ 4 for C-DAP and B-DAP, respectively,after a 14-day incubation(re

â€˜@ salts not shown). Growth inhibition by either C-DAP (up to 100 @M) 2 or B-DAP (up to 50 ELM)was completely reversed by the concomitant 2 No Spermine-0-Control + i @MSpermine addition of 1 p.M spermine,indicating that the effect of both com 0 0 poundswas specificallyrelatedto their action as sperminesynthase 2 4 6 8 10 12 14 24 6 8 10 12 14 Time (d) Time (d) inhibitors (Fig. 2, B and D). The antiproliferative effect of the highest B-DAP concentration used (200 g.LM)wasonly partly counteracted by Fig.2. Effectof C-DAYandB-DAY on ZR-75â€”1breastcancercellproliferation.Cells spermine, most likely due to competition by B-DAP for spermine were incubatedwith the indicatedconcentrationof C-DAY (A and B) or B-DAY (C and D) in the presence(B andD) or absence(A and C) of 1 p.t.@spermine,andharvestedafter uptake (see below). 2,5,10,and14days(oftreatmentfordeterminationofDNAcontentperculture.Points, Althoughexogenoussperminefully preventedZR-75â€”1cellgrowth meanfrom triplicateculturesfrom a typical experiment;bars,SD. Whereno error bars inhibition when added simultaneously with C-DAP (compare Fig. are represented, the symbol size exceeds the SD value. 2B), this ability was strongly impaired after prior incubation with the drug (Fig. 3). Spermidine and putrescine were even less effective than spermine in restoring growth in cells pretreated with C-DAP, and L-[carboxyl-'4C]methionine, respectively (42). Protein was measured in the sperminedepletionby B-DAP led to a similar lossof rescuingeffect enzyme extracts (43) using BSA (fraction V) as standard. of exogenouspolyamines(data not shown). This decreasedrevers Determination of PolyamineUptakeActivity. ZR-75â€”1cellswereplated ibiity of growth inhibition was associated with the progressive ap in 24-well platesat 3 X i04 cells/well in MEZR medium.After 6 days, pearanceof striking morphological changes(Fig. 4), which were fully mediumwascarefullyaspiratedandreplacedwith400 @dofserum-freeRPM! developed after a 21-day incubation. The most characteristic feature 1640(otherwisesupplementedasabove)containingeither 3.0 @M[3H]pu was a distorted, fusiform contour associated with a more abundant and trescine or 1.0 ,LM [3H]spermidine (at final specific activities of 171 and 22.4 Cl/mob,respectively)inthepresenceofincreasingconcentrationsofcompet flattenedcytoplasm.AlthoughC-DAP still allowed slow cell growth, itor (1â€”1000 @.LM).After a 20-mm incubation at 37Â°C (i.e., conditions under coloniesformedin its presenceweresignificantlyreducedin size and whichuptakewaslinearfor bothsubstrates4),substratesolutionwasremoved, cells exhibited long, irregular cytoplasmic processes (Fig. 4, B and D), and cell monolayers were washed twice with 1 ml of ice-cold serum-free RPM! in contrastwith the smooth,roundedmorphologyof controlcolonies 1640containing20 mMunlabeledputrescineor 1 m@iunlabeledspermidine (Fig. 4, A and C). The frequencyof bi- and multinucleatedcellswas (for therespective3H-labeledsubstrate),andthenwith 1 ml of PBS.After the lastrinsing,200 @.dof1 NNaOHwasaddedto thewell, andcellularmaterial washomogeneizedbyheatingfor30 mm at 60Â°C.Followingneutralization 8 with 200pi of 1NHG, a 250-pi aliquotof thehomogenatewastransferredto -0-Control 10 ml of scintillation cocktail for radioactivity determination.Nonspecific -â€¢-+i @tMSpm polyamine binding was determined similarly after incubating parallel cell cultures for 10 s with ice-cold uptake mixture. IC50values for uptake inhibition . -D--+50j.tMC-DAP werecalculatedbyiterativesigmoidcurvefitting usingtheSigmaPlotsoftware -U+50j.tMC-DAP program(JandelScientific,SanRafael,CA). K1wasthenestimatedfromthese + I jiM Spm valuesusingthe Cheng-Prusoffequation(44). Morphological Studies. ZR-75â€”lcells were seededin T75plastic flasks at 1 X 106 cells/flask in MEZR medium. C-DAP (50 ,&M) or vehicle (0.1 mM HCI) wasadded2 daysafterplating,andincubationfor7 or 14 days(with 2 replatingafter the first 7 daysin the latter case).Cells were thenseededin 6-well plates (9.62 cm2/well) at 5 X i04 cells/well, and after a further 7-day incubation period, fixed in situ and stained with hematoxylin and eosin for microscopicexamination(45). 0 Statistical Analysis. The statisticalsignificanceof the differencesob 0 2 4 6 8 10 12 served was determined using two-sample t tests (P < 0.05) assuming equal Time (d) variance. Unless otherwise indicated, results are expressed as the mean Â±SD Fig. 3. Decreasedreversibilityof C-DAY-induced growth inhibition in ZR-75â€”lcells of determinations from triplicate experiments. afterpretreatmentwiththe inhibitor. Cellswereculturedfor 6 days(in the presence(0 andI) or absence(0 and â€¢)of50 @MC-DAY,harvested,and treatmentwas continued foranother4-,8-,or li-day periodafterreplatingcellsatthesameinitialdensityforthe 4 R. Poulin and M. Lessard, Physicochemical parameters and inorganic cation depend time courseshown,in the presence(solid)or absence(open)of 1 ps.ispermine(Spm). enceof polyaminetransportin ZR-75â€”1humanbreastcancercells,manuscriptin Points, mean of triplicate determinations from a representative experiment; the symbol preparation. sizeexceedstheSD of DNA measurements. 936

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Fig. 4. Morphologicalchangesinducedby C-DAY in ZR-75â€”1breastcancercells. Cells were incubatedfor 14 (A andB) or 21 (C andD) daysin the presence(B and D) or absence (A and C) of 50 pi@C-DAY priorto fixationandstainingwith hematoxylinandeosin as described in â€œMaterialsandMethods.â€•AandB, X 100;C andD, X25. Arrow, a multinucleated cell.

also clearly increased by C-DAP, as well as the incidence and size of spermidine, without substantially altering total polyamine levels (Ta intracytoplasmic vacuoles, the latter reflecting increased secretory ble 1). Increasing C-DAP concentration from 50 to 100 @iMledto a activity(46). roughly proportional increase in drug accumulation but only margin Effect of C-DAP on Polyamine Pools in ZR-75--1 Cells. We allyaffectedthedegreeofsperminedepletionorspermidineaccumu further assessedthespecificity of action of C-DAP by determining the lation. Thus, the saturation of the effect of C-DAY on cell growth changesin polyaminecontentsinducedby the drug in ZR-75â€”1cells. observed at 50 @LM(compareFig. 2, A and B) paralleled its action on C-DAP acted as expected from a specific spermine synthaseinhibitor, polyaminebiosynthesis(Table 1). causing a rapid depletion of spermine which was nearly maximal after Quite notably, spermine synthaseinhibition led to the accumulation 5 days, as well as the dramatic (up to 14-fold) accumulation of of both decAdoMet and its acetylated metabolite Ac-decAdoMet 937

cellsIncubation Table 1Effect of C-DAP on polyamineand decAdoMetcontentsin ZR-75-1 breast cancer protein)â€•SpdSpm contents(nmol/mg

+ periodAc-decAdoMet5 (days)TreatmentIntracellular Spd+ 5pm C-DAYdecAdoMet Â±0.1 Â±0.9 16.0Â±0.9 5 +50pi@iC-DAY 87,11b 7.3Â±0.7b 16.1Â±1.0 6.9Â±0.3 3.2Â±o.l@' 0.1â€•95Control + 100 @MC-DAY0.8 10.3 Â±0.2â€•15.2 7.9 Â±0.1â€• 18.3 Â±0.2'@ 16.5 Â±0.3<0.02 4.4 Â± Â±0.2 Â±0.6 18.3Â±0.7 9 + 50 gLM C-DAY 14.2Â±15b 6.9Â±0.2â€• 21.1Â±1.5 7.1Â±0.4 2.6 Â±01b 0.1â€•129Control + 100 @AMC-DAY1.0 15.5 Â±0.8â€•17.3 5.9Â±0.3â€• 21.3Â±0.8 15.9Â±0.5<0.02 3.1 Â± Â±1.9 14.9 Â±1.9 @@ 12 +50 @LMC-DAY 13.4 Â±22b 04b 18.9 Â±2.3 8.1 Â±0.3 2.9 Â±0.2â€• 12Control +100j@MC-DAY<0.05 13.9Â±0.3â€•14.9 4.5 Â±01b 18.4 Â±0.3 20.2 Â±0.3<0.02 3.4Â± a Putrescinewas undetectablein these experiments(<0.05 nmol/mg protein);Spd, spermidine;5pm, spermine.The sum of cellularcontents in the nucleosidesdecAdoMet and its acetylatedmetabolite(Ac-decAdoMet)representsthetotal numberof equivalentsofaccumulatedproductsof AdoMetDCactivity. Dataareexpressedasthe meanÂ±SDof triplicate determinationsfroma representativeexperiment. b Significantly different from control (P < 0.01). C Significantly different from control (P < 0.05).

(Table 1), which were both undetectable in control cells, while Although C-DAP uptake was not significantly affected by spermine having little effect on AdoMet content (data not shown). As seen at the concentrations used, C-DAP, as well as B-DAP and its lower in Table 2, C-DAP rapidly increased AdoMetDC activity in ZR homologueP-DAP (27), a weak sperminesynthaseinhibitor(datanot 75â€”1cells, leading to a 2.1- and 2.9-fold induction of enzyme shown;Ref. 27), can indeedcompetewith polyamineuptake(Fig. 6). activity after 24 and 48 h, respectively. On the other hand, ODC The polyamine transport system in ZR-75.-1 cells is shared by putres activity was essentially unaffected by C-DAP. Thus, the accumu cine, spermidine,andspermine,with apparentKmof 3.5, 0.6, and0.5 lation of decAdoMet and Ac-decAdoMet in C-DAP-treated cells ELM, respectively.5 B-DAP, P-DAP, and C-DAP blocked specific was likely favored by the induction of AdoMetDC due to the putrescine uptake with apparentK1values of 8 Â±1, 41 Â±3, and partial relief of the strong repression of AdoMetDC expression 181 Â±20 @.LM(n 3), respectively. In agreement with the higher exerted by spermine (15â€”17). affinity of spermidine for the transport system, the N-alkyl-1,3-dia Uptake of C-DAP and Other N-Alkyl-1,3-diaminopropane De minopropanederivativeswere lesseffective inhibitorsof spermidine rivatives in ZR-75â€”1 Cells. Since N-alkyl-1,3-diaminopropane de uptake, B-DAP being again the most potent competitor (K1 = 22 Â±5 rivatives are spermidine homologues, they might compete with the p.M). The higher affinity of B-DAP for the polyamine carrier might in uptake of exogenous polyamines through the high affinity polyamine fact account for the inability of spermine to fully reverse growth @ transport system present in ZR-75â€”1 cells.4' In order to assess inhibition caused by high concentrations of this inhibitor (compare whether spermine reversed C-DAP-induced growth inhibition by Fig. 2D). mere competition for cellular uptake, C-DAP accumulation and con Differential Effect of C-DAP and DFMO on Cell Growth in comitant changes in polyamine contents were determined in the Various Breast Cancer Cell Lines. We next compared the effect of presenceor absenceof exogenousspermine (Fig. 5). Maximal C-DAP sperminedepletionon the growth of 4 different humanbreastcancer accumulation was already detected after a 12-h incubation, but its cell lines, namely the estrogen-responsive ZR-75â€”1,T47-D, and intracellular level slowly declined thereafter. No evidence was ob MCF-7 cells, and estrogen-independent MDA-MB-231 cells. We also tamed for an o-phthalaldehyde-reactive metabolite of C-DAP to ac comparedthe relative ability of DFMO and C-DAP, or the combina count for this time-dependent decrease in drug content (data not tion thereof, to affect growth in these cell lines. In contrastwith shown), although loss of the primary amino group would have pre ZR-75â€”1cells,which underwent a 60% reduction in net cell growth vented detection of drug metabolites with the method used. On the as early as 8 daysafter C-DAP addition(50 @.tM),thisparameterwas other hand, spermine depletion and the parallel buildup in spermidine less readily affected in the 3 other breast cancer cell lines (Fig. 7). contents occurred progressively after C-DAP addition, resulting in an Whereas C-DAP maximally inhibited cell proliferation by 25 and approximately 50% depletion of cellular spermine (â€”9.5 Â± 0.8 33% in MDA-MB-231 (t = 8 days) and T47-D cells (t = 10 days), nmol/mgprotein)after48 h, which exactly mirroredtheaccumulation respectively, MCF-7 cell growth (t = 7 days) was virtually unaffected of spermidine (+9.6 Â±1.0 nmol/mg protein). The progressive action by C-DAP. MCF-7 cells still displayed a complete growth insensitiv of C-DAP on polyamine pools was also reflected by the gradual ity to C-DAP even after 2 passages(i.e., a 14-day incubation) in the increase in total decAdoMet (decAdoMet + Ac-decAdoMet) content presence of the inhibitor, although growth inhibition of T47-D and (Fig. 5). Exogenous spermine (1 @.tM)restoredspermine content to MDA-MB-231 cells increased to that observed in ZR-75--1 cells control levels (Fig. SC) while completely preventing decAdoMet and spermidine accumulation. Spermine addition did not interfere with net Table2 Effectof C-DAPon ODC andAdoMetDCactivitiesin ZR.75-I C-DAP uptake, and reciprocally, 50 (Fig. 5) or as much as 100 (not breast cancer cells shown) @.LMC-DAPdid not decreasethe accumulation of exogenous Enzyme activity spermine. Thus, the reversal of C-DAP-induced growth inhibition (nmol/hlmg protein)â€• by spermine was clearly due to the restoration of spermine content Time (h)TreatmentODCAdoMetDC0 rather than to competition for drug uptake. Moreover, decAdoMet 0.424 Control0.49 Â±0.024.1 Â± accumulation induced by C-DAP was closely associated with Control decreases in spermine content and not from a direct effect of the 0.1â€•4824 + 50 @AMC-DAY0.63Â±0.100.68 Â±0.053.7Â±0.4 8.0Â± inhibitor on AdoMetDC activity. Control Â±0.01 Â±0.1 2.0â€•a48 +50 @LMC-DAY0.40 0.53Â±0.033.9 11.4Â±

5 M. Lessard and R. Poulin, Hormonal and feedback regulation of the polyamine Mean Â±SDof triplicate samples. transport system in human breast cancer cells, manuscript in preparation. b Significantly different from control (P <0.01). 938

Fig.5. Early effectof C-DAYon polyamine, decAtktMet,andAc-decAdoMetcontentsin ZR 75-.1 human breast cancer cells. C-DAP(50 pj@i)or C vehiclc(O.lmaiHQ)wasaddedattiniezerointhe @ presenosorabsenceof 1 @sispermine(5pm)and C cellswereharvestedafter12(A),24 (B), and48 (C) h ofincubation for determinationof polyamine andnucleosidecontents.Colwnns,meanof tripli E.E. cate samplesfrom a representativeexperiment; bars,SD.

which exhibit the highest steady-state putrescine and spermidine pools .@100 along with the lowest spermine content in the series examined, were h80 the least affected by C-DAY, while the opposite was found for ZR-75â€”1cells. Taken together, these results strongly suggested that @g60 the differential growth sensitivity to spermine depletion found among breast cancer cell lines was inversely related to their ability to accu mulate spermidine in amounts exceeding the absolute decrease in spermine-associated cationic charges. We finally assessed the mechanism responsible for the potentiation of DFMO-induced growth inhibition by C-DAP in MCF-7 cells, â€˜o@@110 100 1000 @0@1 10 100 1000 Inhibitor (pM) Inhibitor(pM) which are growth insensitive to C-DAP alone (compare Fig. 7D). As expected, a 7-day incubation with DFMO caused a virtually complete Fig.6.Inhibitionof[3Hjputrescine(A)and[3H]spermidineuptake(B)bytheN-alkyl depletion in putrescine and spermidine as a result of ODC inhibition, 1,3-diaminopropanederivativesB-DAY(â€¢),C-DAY(1), and P-DAY(0) in ZR-75-1 human breast cancer cells. Points, mean of triplicate samples; bars, SD. while leaving the spermine pool unaffected (Table 3). On the other hand, C-DAP depleted spermine to nearly the same extent either in the presence or absence of DFMO. Moreover, as shown by the accumu under such conditions (data not shown). On the other hand, DFMO lation of spermidine in cells treated with both DFMO and C-DAP, the strongly inhibited growth in all 4 breast cancer ceillines, in agreement stability of spermine content in DFMO-treated MCF-7 cells was due with previous reports (32â€”35).Interestingly,the addition of as low a to the quantitative conversion of residual spermidine to spermine and concentration as 10 pM C-DAP strongly enhanced the antiprolifera tive effect of DFMO in all cases, including MCF-7 cells (Fig. 7). Thus, differences in growth sensitivity to C-DAP between the various 10 breast cancer cell lines were largely attenuated on a concomitant inhibition of ODC activity. We nextevaluatedthecharacteristicsofpolyaminemetabolismin these cell lines which might underlie their differential growth sensi tivity to C-DAP. For an identical treatment duration (7 days), the extent of spermine depletion caused by C-DAP in a given cell line (Fig. 8) was not correlated with the degree of growth inhibition. In I fact, the most modest spermine depletion (-60 Â±5%) was observed in ZR-75-1 cells, whereas spermine content had declined by 77 Â±5, 83 Â±6, and 76 Â±5% in T47-D, MDA-MB-231, and MCF-7 cells, 7 respectively, suggesting that spermine synthase inhibition by C-DAP was highly effective in all cell lines. The lower reduction in spermine 6 content observed in ZR-75â€”1cells could largely owe to the fact that spermine levels decrease almost solely through dilution by cell divi 4 sion due to the low turnover of this polyamine (1, 47). Hence, the degree of spermine depletion by C-DAP should increase with the rate of cell growth. On the other hand, the extent of spermidine accumu z lation due to spermine synthase inhibition markedly varied among the cell lines. When expressed as the number of equivalent polyamine borne positive charges, the highest increase in total polyamines in 2 4 6 81012140 2 4 6 8 duced by C-DAP (47 Â±6%) was found in MCF-7 cells (Fig. 8D), as Time(dl Time(d) compared with 24 Â±4 and 27 Â±3% in T47-D and MDA-MB-231 Fig. 7. Effect of C-DAY and DFMO on cell growth in 4 different human breast cancer cells, respectively (Fig. 8, B and C), while virtually no increase cell lines. MDA-MB-231 (A), T47-D (B), ZR-75-.1 (C), and MCF-7 (D) were incubated (3 Â±2%) was observed in ZR-75â€”1cells (Fig. 8A; Table 1). The for the indicated time with either 10 (0) or 50 (Lx)psi C-DAY, 1 ms@iDFMO(â€¢),orthe respective combinations thereof ($ and A). Control cells (0) received the vehicle (0.1 inst relative growth sensitivity to C-DAP also paralleled the spermidine: HQ) only. Points, mean (n 3) of one representative experiment performed with all 4 spermine ratio prevailing in a given cell line. Thus, MCF-7 cells, cell lines in parallel; bars, SD. 939

pane derivatives (29), its biological activity has thus far been assessed

C only in vivo in normal rat tissues (30). Chronic use of this drug 0 extensively depletes spermine in organs such as the ventral prostate o@ Â°E15 and liver, with no demonstrable symptoms of toxicity or growth deficit (30). The present report provides the first demonstration of a @ E io rapid and specific inhibitory effect of C-DAY on tumor cell growth. 0.5. U As shown here, human breast carcinoma cells are particularly suitable

C models to evaluate the biological functions of spermine because of their characteristically high and low spermine and spermidine pools, respectively, and their marked sensitivity to growth inhibition by

C DFMO (32â€”35)or polyamine analogues (36). 0 Our data show that C-DAP strongly inhibits ZR-75â€”1humanbreast o@ U E 15 cancer cell growth, with a more than 10-fold greater potency than B-DAP, the only other N-alkyl-1,3-diaminopropane derivative thus :@E 10 far studied as a spermine synthase inhibitor (27, 28). Inhibition of cell 0,5. U proliferation by C-DAP results from depletion of intracellular sperm C me, as shown by the complete reversal of its effect on growth and polyamine pools by coincubation with spermine. The specificity of Control + C-DAP Control + C-DAP action of C-DAP is also illustrated by the fact that the extent of growth Fig. 8. Effect of C-DAP on polyamine contents in 4 different human breast cancer cell inhibition and changes in polyamine pool patterns were essentially lines. ZR-75â€”t (A), T47-D (B), MDA-MB-231 (C), and MCF-7 (D) were grown in independent of C-DAP concentration between 10 and 100 @Mdespite IMEM+ 5% FBS for 7 days in the presenceor absenceof 50 @MC-DAYpriorto harvesting for determination of polyamine and C-DAP contents, as described in the text. a proportional increase in drug uptake. In ZR-75â€”1cells, the extent of Columns, mean of analysesof triplicate samples from one representativeexperiment spermine depletion obtained with C-DAY was most likely limited by performed with all 4 cell lines in parallel; bars, SD. The percentage of increase in total the almost complete blockade of cell division eventually reached. This polyamine-bome positive charges [i@(+)]resulting from treatment with C-DAY(relative to control) is indicated for each cell line; spermidine and spermine bear 3 and 4 positive interpretation is consistent with the observation that spermine turn charges/mol, respectively, at physiological pH. over is usually very low in mammalian cells (1, 47). Spermine turnover can occur in principle through the consecutive action of not simply to the lack of dilution of the preexisting spermine pool in spermidine/spermine N'-acetyltransferase and FAD-dependent poly growth-arrested cells. These data clearly show that DFMO sensitizes amine oxidase (47, 49). We have obtained preliminary evidence that MCF-7 cell growth to the effect of spermine depletion by largely spermidine/spermineN'-acetyltransferase is weakly induced at high preventing the compensatory spermidine overaccumulation caused concentrations of P-DAP and B-DAP (data not shown), in agreement by C-DAP. Interestingly, total decAdoMet accumulation (dcc with a recent report (28). C-DAY, however, did not induce significant AdoMet + Ac-decAdoMet) observed in DFMO-treated cells was formation of N' -acetyispermidine or N' -acetylspermine, even at the increased 2.2-fold on further addition of C-DAP (Table 3), while maximal concentration used (100 p.M; Table 1). Thus, acetylation C-DAP caused a 10-fold lower accumulation of these nucleosides appears to play only a minor role, if any, in the depletion of spermine than DFMO when either agent was used alone. These results suggest caused by C-DAP. that the residual spermine pool still present in DFMO-treated cells is Previous studies with N-alkyl-1,3-diaminopropane derivatives had highly effective in repressing AdoMetDC synthesis (48), and that the not addressed the mechanism responsible for their cellular uptake. The overcompensatory increase in spermidine synthesis caused by present evidence clearly shows that P-DAP, B-DAP, and C-DAP all spermine depletion in MCF-7 cells is likely favored by the resulting competitively inhibit putrescine and spermidine transport in ZR-75â€”1 enhancement in decAdoMet formation. cells, albeit with different potencies. The lower affinity of C-DAP for the polyamine transport system, as compared with P-DAP and B- DISCUSSION DAP, may owe to the lesser structural analogy with spermidine conferred by the cyclohexyl group. If the polyamine transport system Although C-DAP has been singled out as the most potent spermine is the sole mechanism used for C-DAY uptake in ZR-75â€”1cells, as synthase inhibitor in an extensive series of N-alkyl-1,3-diaminopro little as 1 p.Mspermine, which has a Km comparable to spermidine for

Table 3 Effect of a 7-dav treatment with DFMO andC-DAP on polyaminecellsIntracellular and nucleoside contents in MCF-7 breast cancer content (nmol/mg protein)â€•+

+ComponentControl+ 1 mM DFMO C-DAYPolyamines 1 msi DFMO + 50 @tstC-DAY50 ps.i

002b.d.e<[email protected] Â±0.13<002b.c 0.15 Â± Â±0.07 Â±[email protected] 22.00 Â±0â€¢%b4e Â±[email protected] Â±0.02â€•C-DAP0.54Spermine8.01 5.95 Â±0.250.16 4.98 Â±0.91 1.10 Â±0@@b.d6.19 1.33 O.01cNucleosidesAdoMet1.25 Â±0.041.79 Â±

001b,cdecAdoMet<0.010.73 Â±0.080.69 Â±0.10â€• 1.12 Â±0.08â€•@0.55 Â± Â±Ac-decAdoMet<0.010.95 Â±014b 0.06 Â±0.0l@@@1e1.59 0@b,c.ddecAdoMet Â±018b.c o.o@Â±001b,d.e2.11 Â± + Ac-decAdoMet<0.011.68 Â±[email protected] 0.14 Â±o.o1@@3.70 Â±

a Mean Â± SD of triplicate samples. b Significantly different (P < 0.01) from control. C Significantly different (P < 0.01) from C-DAY treated groups. d Significantly different (P < 0.01) from DFMO-treated groups. e Significantly different (P < 0.01) from DFMO/C-DAY-treated groups. 940

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. ROLE OF SPERMINE IN BREAST CANCER the polyamine transport system in these cells,5 should have prevented polyamine depletion experiments in intact cells. For most biochemical about 90% of C-DAP accumulation at a 50 ,.tM drug concentration activities of polyamines, spermine has proved to be a far more potent (compare Fig. 6B). Since spermine did not interfere with C-DAP effector than spermidine on a molar basis, presumably because of its uptake under those conditions (compare Fig. 5), the drug thus appears higher charge. However, since high spermidine concentrations can to be accumulated mainly through a mechanism distinct from the almost invariably substitute for spermine in inducing these effects, polyamine uptake system. Similar findings have been reported for spermine might not be absolutely required for most cellular functions, B-DAP (28). Whether N-alkyl-1,3-diaminopropane derivatives are provided that the spermidine pool is sufficiently high. Indeed, even transported by a saturable membrane transport agency or by passive elevated putrescine contents can sustain growth for several genera diffusion due to their more hydrophobic character remains to be tions in cells treated with an AdoMetDC inhibitor to prevent spermi determined. dine and spermine biosynthesis (18, 19), until spermidine becomes The presentdata demonstratethat sperminebiosynthesis can play limiting for the hypusine modification of eIF-5A (19, 20). Thus, as vital functions in human breast cancer cells. However, its relative suggested previously (25â€”28),spermidine can probably fulfill many importance in supporting exponential growth is largely dependent on of the functions normally played by spermine, albeit less efficiently on the concurrent rate of spermidine biosynthesis. When used alone, a molar basis. However, when biosynthesis of putrescine, spermidine, C-DAP affected cell proliferation inversely with the absolute spermi and spermine was simultaneously prevented by cotreatment with dine content and the spermidine/spermine ratio prevailing under DFMO and C-DAP, a markedenhancementof growth inhibitionwas steady-state exponential growth conditions in a given cell line. Since observed in all breast cancer cell lines examined. Therefore, restrain N-alkyl-1,3-diaminopropane derivatives are competitive inhibitors of ing the compensatory spermidine accumulation caused by spermine spermine synthase relative to spermidine (27), it might be argued that synthase inhibition unmasks an essential function of spermine in the extent of spermine depletion by these drugs is limited by the breast cancer cells. These data also show that conversion of residual concomitant spermidine accumulation. This would be consistent with spermidine to spermine in DFMO-treated cells clearly limits the the fact that exogenous spermidine was also effective in reversing antiproliferative effect of ODC inhibition. growth inhibition by C-DAP or B-DAP in susceptible breast cancer It is interesting that growth inhibition by C-DAP was poorly re cell lines (data not shown). Thus, even in MCF-7 cells which show no stored by subsequent spermine addition and led to unique, striking growth sensitivity to C-DAP used alone, complete spermine depletion morphological changes, suggesting that biological effects of pro may not be possible because of mutual competition between longed spermine depletion may be partly irreversible. Since growth spermidine and C-DAP for spermine synthase binding. was fully restored by polyamines in DFMO-treated cells, even on Although growth-arrested ZR-75--1 cells accumulated C-DAP to a prolonged incubation with the inhibitor, and did not cause similar greater extent than did other breast cancer cell lines during long-term morphological changes (data not shown), spermine deficiency appears incubations (Fig. 8; compare Tables 1 and 3) on a total protein per cell to be specifically responsible for these defects. Abortive colony for basis, this could not account for the differential effect of the drug for mation and an increased incidence of multinucleation in C-DAY the following reasons: (a) growth could be fully maintained by treated cells could result from a disruptive effect of spermine deple exogenous spermine (Fig. 2) without preventing drug uptake (Fig. 5), tion on cytoskeletal organization and cytokinesis (51, 52). On the indicating that the antiproliferative effect C-DAP is solely due to other hand, increased multinucleation and cytoplasmic vacuolation, as spermine synthase inhibition; (b) no further effect on ZR-75â€”1cell well as the formation of cytoplasmic processes are also observed in growth was observed at concentrations of C-DAP exceeding 50 p.M breast cancer cells undergoing differentiation induced by phorbol (Fig. 2), although drug accumulation was proportionately higher (Ta esters (46, 53) or by ligands of the erbB-2lneu oncoreceptor (53, 54). ble 1); and(c) the relativedegreeof sperminedepletionwas higherin A specific role of sperminein cell differentiationhas been postulated MDA-MB-231, T47-D, and MCF-7 cells than in ZR-75â€”1cells, and during spermiogenesis (55) and intestinal cell maturation (56). A thus, optimal inhibition of spermine synthase could be obtained at detailed characterization of the effect of spermine depletion on cy lower intracellulardruglevels accordingto the cell line considered.In toskeletal organization, differentiation markers, and other potential fact, the biphasic kinetics of C-DAP accumulation in ZR-75â€”1cells sites of action such as chromatin organization (21, 22, 28, 57) is in (Fig. 5) early after drug addition clearly indicate that the intracellular progress to better delineate spermine functions. level of inhibitoris activatelyregulatedandis similarto thatobserved The imbalance in AdoMet and decAdoMet levels resulting from in the other cell lines when ZR-75â€”1cellsare entering exponential spermidine depletion by DFMO has been suggested to contribute to its growth (e.g., 48 h after drug addition). These observations suggest growth-inhibitory effect (58, 59). Indeed, high decAdoMet concen that C-DAP accumulationmight increase secondarily as a result of trations interfere in vitro with DNA methylation (59) and histone growth inhibition, e.g., in ZR-75â€”1cells, and that an optimal acetylation (58). However, no correlation was found between dcc biological effect was in fact obtainedat lower intracellulardruglevels AdoMet accumulation and the extent of growth inhibition by C-DAY in all cell lines. among the cell lines examined (data not shown). It is interesting that It is noteworthy that the differential ability of C-DAP to inhibit the amounts of Ac-decAdoMet measured here in polyamine-depleted growth among the cell lines tested did not depend on the absolute breast cancer cells considerably exceed those reported previously in extent of spermine depletion. In fact, as little as 1.5 nmol of sperm other cell types (58). Very little is known on the occurrence of this inc/mg protein (a 80% reduction in the case of MCF-7 cells) still metabolite in mammalian cells. Ac-decAdoMet is formed through the allowed a normal growth rate, as long as spermidine accumulation activity of a nuclear acetylase closely related to histone acetylase, and overcompensated spermine depletion on the basis of total polyamine decAdoMet is a potent competitive inhibitor of histone acetylation borne positive charges. As pointed out above (compare â€œIntroduc (58). Thus, high rates of Ac-decAdoMet synthesis in spermine-de tionâ€•),only a few specific cellular functions are well established for pleted, DFMO-treated breast cancer cells could reflect an increasing polyamines. Their involvement in numerous aspects of cell biology, interference with histone acetylation, a possibility which clearly including nucleic acid conformation and dynamics (21, 22), protein deserves further investigation. synthesis (1, 21), and membrane stability and fusion (50) has been Our observation that C-DAP potentiated growth inhibition mostly inferred from studies with isolated in vitro systems, and the brought about by DFMO in all human breast cancer cell lines piciotropic nature of these effects complicates the interpretation of examined may have important therapeutic implications. As noted 941

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1995 American Association for Cancer Research. ROLE OF SPERMINEIN BREASTCANCER above, these results are consistent with the hypothesis that the lack 14.Ask,A., Persson,L,andHeby,0. IncreasedsurvivalofL1210leukcnsicmiceby of spermine depletion by DFMO contributes to the reduced but preventionoftheutilizationofextracellularpolyamines.Studiesusingapolyamine uptake mutant, antibiotics and of a polyamine-deficient diet. Cancer Len., 66: 29â€”34, significant growth potential of DFMO-treated cells, and support 1992. the notion that spermine biosynthesis plays a specific role in 15. Madhubala,R., Secrist,J. A., III, and Pegg,A. E. Effect of inhibitorsof S-adenosyl normal cellular functions that can be fulfilled only by an overcom methioninedecarboxylaseonthecontentsofomithinedecarboxylaseandS-adenosyl methio-nine decarboxylase in L1210 cells. Biochem. J., 254: 45â€”50,1988. pensatory accumulation of spermidine. These findings open inter 16.Autelli,R.,Stjemborg,Khomutov,A.,Khomutov,R.M.,andPersson,LRegulation esting perspectives for improving antipolyamine therapeutic strat of S-adenosyimethioninedecarboxylaseinL1210leukemiacells:studiesusingan egies against spermine-rich tumors such as breast carcinomas. A irreversible inhibitor of the enzyme. Eur. J. Biochem., 196: 551â€”556,1991. 17. Shantz,L M., HoIm, I., Jtnne, 0. A., and Pegg,A. E. Regulationof S-adenosylme large fraction of total polyamine biosynthesis in such tumors is thioninedecarboxylaseactivityby alterationin the intracellularpolyasninecontent. diverted to spermine formation, which can be chiefly considered as Biochem.J., 288: 511â€”518,1992. a terminal product becauseof its slow turnover. The potency and 18. Poulin, R., Wechter, R. S., and Pegg,A. E. An early enlargementof the putrescine pool is requiredfor growthin L1210mouseleukemiacellsunderhypoosmoticstress. specificity of C-DAP as a spermine synthase inhibitor is compa J. Biol. Chem., 266: 6142â€”6151,1991. rable with that of S-adenosyl-1,12-diamino-3-thio-9-azadodecane, 19.Byers,1. L, Ganem,B.,andPegg,A.E. CytostasisinducedinL1210murine leukemia cells by the S-adenosyl-L-methionine decarboxylase inhibitor 5'-{[Z)-4- but use of the latter has been limited by its rapid oxidation by aminobutenyljmethyl-amino}-5'-deoxyadenosinemaybeduetohypusinedepletion. serum amine oxidases (26) and by the complexity of its synthesis. Biochem.J.,287: 717â€”724,1992. Moreover, the latter compound inhibits spermine synthase by 20. Park,M. H., Wolff, E. C., and Folk, J. E. Is hypusineessentialfor eukaryoticcell proliferation?TrendsBiochem.Sci., 18: 475â€”479,1993. competition with decAdoMet (26), which may limit its efficacy 21. Marton, L J., and Morris, D. R. Molecular andcellular functionsof the polyamines. due to the large decAdoMet accumulationresulting from spermine in: P. P. McCann, A E. Pegg, and A. Sjoerdsma (eds.), Inhibition of Polyamine depletion. On the other hand, C-DAP is an inexpensive compound Metabolism:Biological Significanceand Basis for New Therapies,pp. 79-105. Orlando,FL: AcademicPress,1987. which profoundly and specifically depletes spermine both in vivo 22. Balasundaram,D., andTyagi, A. K. Polyamine-DNA nexus:structuralramifications (30) and in vitro (this study) at remarkably low concentrations. andbiological implications.Mol. Cell. Biochem.,1(X):129â€”140,1991. 23. Lenzen,S.,MOnster,W.,andRustebeck,1.Dualeffectof spermineonmitochondrial Moreover, its chronic use in rats displays a notable lack of toxicity Ce2@transport. Biochem. J., 286: 597-602, 1992. (50% lethal dose, >0.5 glkg) (30). As an extensively tested anti 24. Pegg,A. E., Coward,J. K., Talekar,R. R., andSecrist,J. A., III. Effectsof certain polyamine drug in clinical trials, DFMO also has rather benign and 5'-substitutedadenosinesonpolyaminesynthesis:selectiveinhibitors of spermine synthase. Biochemistry, 25: 4091â€”4097,1986. fully reversible side effects (1, 3, 4, 9). Whether a more extensive 25. Pegg,A. E., and Coward, J. K. Growth of mammaliancells in the absenceof polyamine depletion and better antitumor efficacy could be spermine.Biochem.Biophys.Rca.Conunun.,133: 82â€”89,1985. achieved with the combination of DFMO and a spermine synthase 26. Pegg,A. E., Wechter,R., Poulin, R., Woster,P. M., and Coward,J. K. Effect of S-adenosyl-1,12-diamino-3-thio-9-azadodecane,amultisubstrateadductinhibitor of inhibitor such as C-DAP without adverse cytotoxicity remains to sperminesynthase,onpolyaminemetabolisminmammaliancells.Biochemistry,28: be demonstrated but clearly warrants further study. 8446â€”8453,1989. 27. Baillon, J. 0., Kolb, M., and Mamont, P. S. Inhibition of mammalianspermine synthasebyN-allcylated1,3-diaminopropanederivativesin vitro andin culturedrat hepatomacells.Eur.J. Biochem.,179: 17â€”21,1989. ACKNOWLEDGMENTS 28. Pegg,A. E., andCoward,J. K. 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Maria Huber and Richard Poulin

Cancer Res 1995;55:934-943.

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