J. Biochem. 118, 802-809 (1995)

Transfection of Human Melanoma Cells with Type I Interleukin-1(IL-1) Receptor cDNA Rendered Them IL-1-Responsive and Revealedthe Importance of ODC Activity Down-Regulation in IL-1-InducedGrowth Inhibition1

De Yang,* Hidetoshi Hayashi,* Yoshio Hiyama,f Takemasa Takii,* and Kikuo Onozaki*'2

'Department of Hygienic Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori,Mizuho-ku, Nagoya 467; and 1Tokyo R&D Center, Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134

Received for publication, May 31, 1995

A human type I IL-1 receptor expression plasmid has been constructed and used to transfecthuman melanoma cells (A375-5), which have been shown to be unresponsive to the anti-proliferative effect of IL-1. Five stable transfectant cell lines have been established, ofwhich three are sensitive and the other two resistant to the anti-proliferative effect of IL-1.All the transfectant cell lines, but not progenitor A375-5 cells, expressed functional type IIL-1 receptors and could produce IL-6 in response to IL-1, suggesting that the unrespon-siveness of A375-5 melanoma cells is exactly due to an IL-1 receptor deficiency. The threeIL-1-sensitive stable transfectant cell lines expressed much more type I IL-1 receptor thanthe IL-1-sensitive A375-6 cells, thus they are expected to be useful for investigating thesignal transduction pathway of IL-1-induced growth inhibition in melanoma cells. The tworesistant cell lines produced comparable amounts of IL-6 in response to IL-1, as sensitivecell lines did, indicating that IL-6 induction does not play a major role in IL-1-inducedgrowth inhibition in these melanoma cells. The possibility of an IL-6 receptor and/or IL-6receptor signaling deficiency was ruled out as the IL-1-sensitive and -resistant trans-fectants responded similarly to a high dose of exogenous human recombinant IL-6.Examination of the ornithine decarboxylase (ODC) activity of recombinant human IL-1 atreated cells showed that all the sensitive but none of the resistant cell lines coulddown-regulate their ODC activity. These results suggest that IL-1-induced ODC activitydown-regulation is an important step in the signal transduction pathway of IL-1-inducedgrowth inhibition of melanoma cells.

Key words: A375 melanoma cells, IL-1, IL-1 receptor, IL-6, ODC.

Interleukin 1, a 17-kDa cytokine produced predominantly endothelial cells (13).by activated monocytes and macrophages, participates in Two agonist species of the IL-1 family, IL-l<z andIL-1/J,many host reactions including immune response, Mamma- although encoded by different genes and sharing only 26%tion, hematopoiesis, and homeostasis, and is implicated in amino acid sequence homology (14), can bind to the samediverse human diseases (1, 2). In vitro, IL-1 regulates cell cell surface receptors and mediate similar biological actionsgrowth either positively or negatively. IL-1 stimulates the (15). Two types of IL-1 receptors have until now beenproliferation of fibroblasts (3), mesangial cells (4), B cells identified: type I IL-1 receptor (IL-1RI) is an 80-kDa(5), NK-like cells (6), thymocytes (7), and a human glycoprotein preferentially expressed on fibroblasts and Tglioblastoma cell line (8). In contrast, IL-1 inhibits the cells (16, 17), whereas type IIIL-1R (IL-1RII) isa60-kDagrowth of a number of malignant and normal cell types such glycoprotein primarily present on B cells, macrophages andas a human melanoma cell line (9), several human breast neutrophils (18). Transduction of the IL-1 signal intocarcinoma cell lines (10), a murine myeloid leukemia cell responsive cells is mediated by IL-1RI as recent studiesline (11), rat pancreatic Langerhans cells (12), and human showed that only IL-1RI can transduce the IL-1 signal (19,• The present work was supported by grants from the Ministry of 2 0 \ T h e Pleiotrophic actions of IL-1 suggest that thisEducation, Science and Culture of Japan, and the Fujisawa Founda- cytokine may have different intracellular signal transduc-tion. tion pathways with respect to different target cells. In fact,2 To whom correspondence should be addressed. Tel: +81-52-836- IL-1 has been reported to evoke protein kinase C in T cells,3419, Fax: +81-52-836-3419 mesangial cells and mouse fibroblasts (21-23), to activateAbbreviations: IL-1, interleukin-1; IL-6, interleukin-6; IL-1RI, type CAMP-dependent protein kinase in human foreskin fibro-I IL-1 receptor; rhIL-la, recombinant human IL-1-alpha; rhIL-6, • , , i n _^n r nr\ n in I o/i oc\ t • A J-U

u- tu TT a nnn tu- J u i c>r>o t L \ blasts and pre-rs cell line 70 L 6 (24, 25), to induce therecombinant human IL-6; ODC, ornithine decarboxylase; FBS, fetal . . . . . . . . . _„_ „ ,bovine serum; HEPES, iV-2-hydroxyethylpiPerazine-iV'-2-ethane- activation of protein tyrosine kinase m A375-6 humansulfonic acid; PMA, phorbol 12-myristate 13-acetate; MTT, 3-(4,5- melanoma cells, human fibroblasts, and mouse T cells (26,dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. 27), and to down-regulate protein phosphatase activity in

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human fibroblasts (28, 29).We first reported that IL-1 has an anti-proliferateve

effect on a human melanoma cell line, A375-6 (9), and thatthis anti-proliferative effect is mediated through IL-1R(30). Subsequently, IL-1 was shown to induce down-regu-lation of ornithine decarboxylase (ODC) activity (6) andsecretion of IL-6 (31), both of which are considered to beinvolved in the anti-proliferative effect of IL-1 on A375-6cells. We also found out that IL-1 treatment caused thegrowth of A375-6 cells to be arrested in the Go/G].phase ofcell cycle (32). Quite recently, Rangnekar et al. document-ed that IL-1 treatment induced the expression of a compos-ite set of early genes characterized by the specific inductionof melanoma growth-stimulating activity in A375-6 cells(33, 34). Despite all these endeavors, the immediatemolecular changes of A375 on IL-1 binding have beenlargely unexplored as, to some extent, studies on theseproblems may be hampered by the fact that A375-6 cellsexpress only a limited number of IL-1RI (30). What is alsounclear is the precise biochemical pathway connectingreceptor binding to the final outcome, i.e. growth arrest ofA375 melanoma cells, particularly what intracellularchanges are strictly associated with the anti-proliferativeeffect of IL-1. On the other hand, A375-5, a twin subcloneof A375-6, is known to be unresponsive to the IL-1anti-proliferative effect (6, 9, 30), but the exact reason(s)for the IL-1-unresponsiveness of A375-5 cells has not yetbeen clarified, although it is speculated to be probably dueto an IL-1R deficiency as no receptor was identified onScatchard plot analysis (30) and only a very low level ofIL-1R mRNA was detected on PCR (40). In the presentstudy, we transfected A375-5 cells with human IL-1RI(ML-1RI) cDNA with two primary purposes in mind: (i) toobtain stable transfectants with high expression of IL-1RI,and (ii) to determine whether or not the unresponsivenessof A375-5 to the action of IL-1 is actually due to an IL-1RIdeficiency.

MATERIALS AND METHODS

Reagents and Cell Lines—RPMI 1640 was purchasedfrom Sigma (St. Louis, MO, USA). D-MEM/F-12 andG418 were obtained from Gibco (Grand Island, NY, USA).Fetal bovine serum (FBS) was purchased from Bocknek(Toronto, Canada). Human recombinant IL-1<* (hrlL-lor,2 X 107 U/mg) was provided by Dr. M. Yamada (Dainippon,Osaka). Human recombinant IL-6 was provided by Dr. Y.Akiyama (Ajinomoto, Yokohama). A Bolton-Hunter Re-agent [125I] Kit (2,000-4,000 Ci/mmol) was purchasedfrom ICN Radiochemicals (Costa Mota, CA, USA). DL-[1-14C] Ornithine hydrochloride (56 mCi/mmol) was pur-chased from Amersham (Buckinghamshire, UK). TheA375-5 and A375-6 melanoma cell lines (9) were routinelypassaged in culture medium (RPMI 1640 supplementedwith 100 U/ml of penicillin, 100 //g/ml of streptomycin, 15mM HEPES, and 5% FBS) in our laboratory. MH60.BSF2,an IL-6-dependent murine hybridoma clone, was kindlysupplied by Dr. T. Hirano (Osaka University, Osaka), andpassaged in RPMI-1640 supplemented with 10% FBS and 1U/ml human recombinant IL-6.

Construction of ML-1RI Expression Vector and Trans-fection—Human IL-1RI cDNA was obtained from PMA-stimulated human peripheral mononuclear cells by reverse

transcriptase PCR and cloned into pBluescript II sk( —) tocreate pBluescript-hlL-lRI. The cloned hIL-lRI cDNA wasconfirmed by sequencing to be a 1,800-base fragmentidentical to the sequence comprising bases 264 to 2064 ofthe reported hIL-lRI cDNA (17), which covers the 45-basenucleotide upstream of the authentic initiation codon, ATG,the full length of the hIL- 1RI coding region from ATG to thestop codon, TAG, and the 38-base nucleotide downstreamof TAG. A Xhol/Notl-digested fragment derived frompBluescript-hIL- 1RI containing the full length of the clonedhIL- 1RI cDNA was treated with Klenow fragment and thenligated with the Klenow fragment-treated large part ofHindlll-digested pko-neo (35) using a ligation kit (Takara,Otsu). After transformation, the resultant colonies wereanalyzed for the correct orientation creating pSV-hlL-lRI.Pure pSV-hIL- 1RI plasmids were prepared by two cycles ofCsCl ultracentrifugation. Transfection was performedaccording to the modified calcium phosphate-mediatedmethod reported by Chen and Okayama (36) using thepSV-hlL-lRI and pSVneo plasmids. Forty-eight hoursafter transfection, A375-5 cells were detached and replatedon fresh Ham/F-12 medium supplemented with 10% FBSand 0.6 mg/ml of G418 until colonies appeared. Individualcolonies were then transferred to new dishes and propagat-ed until enough cells were obtained for IL-RI expressionanalysis by flow cytometry after staining with mouse anti-ML-1RI monoclonal antibody (Genzyme, MA, USA) andFITC-conjugated goat anti-mouse IgG (Becton-Dickinson,CA, USA). Colonies exhibiting relatively high humanIL-1RI expression were subcloned by limiting dilution toestablish stable transfectants. Finally, stable transfectantswere accomodated to culture medium and then subjected todetailed characterization.

Total RNA Extraction and Northern Hybridization—Total RNA was extracted from semi-confluent cultures ofA375 and transfectant cells according to Chomczynski andSacchi (37). After size-fractionation on an agarose-form-aldehyde gel and transfer to a nitrocellulose filter, thespecific mRNA on the filter was detected by hybridizationwith a 32P-labeled cDNA probe at 42°C for about 17 h inhybridization buffer comprising 50% formamide, 5 X SSPE(1X SSPE: 0.15 M NaCl, 10 mM NaH2PO4,10 mM EDTA,pH7.4), 5xDenhardts solution, 1% SDS, and 100^g/mldenatured salmon sperm DNA. The following probes wereused: a 473-base cDNA fragment encoding extracellularregion of human IL-1RI (bpl98-670) derived from TIG-1,a human lung fibroblast cell line (38); a iVotl/X/ioI-digest-ed 1,850-base fragment of pBluescript-hlL-lRI, corre-sponding to the total length of hIL-lRI cDNA; and aPs£l-digested 1,300-base fragment of human glyceralde-hydephosphate dehydrogenase (GAPDH) (38). The probeswere labeled by random priming (Multi Prime DNALabeling Kit, Amersham, UK). After hybridization, thefilters were washed twice at room temperature in 2 X SSC(1 x SSC: 0.15 M NaCl, 15 mM sodium citrate, pH 7.0) for30min, followed, if necessary, by washing in 0.2 X SSCuntil a reasonably low background was obtained. The filterswere autoradiographed using a Bio-image analyzer (FujiBAS 2000, Tokyo).

Assay for Proliferation—A375 and transfectant cellswere detached with 0.02% EDTA-PBS. After washing withculture medium, the cell suspensions (4X104 cells/ml)were distributed into 96-well flat-bottomed plates (Falcon,

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Lincoln, NJ, USA) at 0.1 ml/well, and then subjected toincubation for 24 h at 37°C under humidified air containing5% CO2. Thereafter, 0.1 ml aliquots of culture medium inthe absence or presence of different concentrations ofrhIL-lar or rhIL-6 were added in triplicate, and the cellswere cultured for another 72 h. In some experiments,putrescine was added simultaneously with rhIL-la to afinal concentration of 2 mM. After staining with crystalviolet and solubilizing the dye-stained cells with 0.1% SDS(39), the dye uptake was measured as the absorbance at595 nm with an ELISA reader (Bio-Rad, Richmond, CA,USA). The percentage of cell growth was calculated accord-ing to the following formula:

% cell growth=Mean ABS595 of cells cultured in medium alone

or with rhIL- a 1Mean ABS59s of cells cultured in medium alone

X100

Examination of IL-6 Production—A375 and stable trans-fectant cells were seeded into a 24-well flat-bottomedplate (Falcon 3047) at 5X105 cells/well in 2 ml culturemedium. After incubation overnight at 37°C in a CO2

incubator, the medium was replaced with 1.5 ml/well ofprewarmed fresh culture medium in the presence orabsence of lOOU/ml rhIL-la, followed by incubationunder the same conditions for 24 h. IL-6 secreted into theculture supernatant was quantitated by the use of an IL-6-dependent mouse hybridoma cell line, MH60.BSF2, aspreviously described (40). Recombinant hIL-6 was used asthe standard. The IL-6 concentration was calculated fromthe dilution of samples equivalent to the concentration ofstandard rhIL-6 which caused 50% of the maximal growthresponse of MH60.BSF2.

Assay for ODC Activity—A375 and transfectant cellswere detached with 0.02% EDTA-PBS. After washing withculture medium, 106 cells of each cell line was seeded intoFalcon 3003 dishes in 10 ml culture medium, and incubatedat 37°C for 24 h under humidified air containing 5% CO2,followed by the addition of 0.1 ml culture medium alone(control) or with 1,000 units rhlL-lar. After incubation foranother 24 h under the same conditions, the cells werecollected by trypsinization, washed twice with ice-cold TEDbuffer (25 mM Tris, 0.15 mM EDTA, 2.5 mM DTT, pH7.5) containing 0.15 M NaCl, and resuspended in 0.15 mlTED buffer, followed by sonication for 30 s on ice with ahandy sonicator (Tomy Seiko, Tokyo) and centrifugationfor 5 min at 10,000 X g in a chilled microfuge. The proteincontent of the supernatant was determined using a Bio-Radprotein assay kit with bovine serum albumin as the stan-dard. ODC activity in the supernatant was immediatelymeasured by the method of Seely and Pegg (41) withmodifications. The enzyme reaction mixture without asample, consisting of 100 n\ TED buffer, 25 //I of 0.8 mMpyridoxal phosphate, 15 /^l of 8 mM L-ornithine, and 10 //Iof DL- [l-14C]ornithine, was prepared in advance. After theaddition of 100 //I of TED buffer (blank) or supernatant, themixture was incubated at 37°C for 30 min with shaking, 0.5ml of 0.5 N HC1 was added to stop the reaction, and thenincubation was performed for another 2 h under the sameconditions. ODC activity was determined as the release of[I4C]O2 (dpm/h/mg of protein), which was collected on a 4cm2 filter paper soaked with 0.5 N NaOH, and measuredwith a fluid scintillation counter (LSC-1000; Aloka,

Tokyo). Percentage inhibition of ODC activity was calcu-lated with the following formula:

% inhibition^ 1 - S * " - " " n p ! b " n t X 100D P M D P M

125I-Labeling of rhIL-la and Receptor Binding Assay—Labeling of rhIL-la with 125I was performed using aBolton-Hunter Reagent Kit according to the manufac-turer's instructions. The specific radioactivity of 125I-rhIL-lar obtained was 10" cpm/mg. Receptor binding wascarried out as described previously (30). Briefly, aliquots of5xlO5 cells were incubated at 4°C for 1.5 h in a totalvolume of 0.2 ml RPMI1640 containing 1 mg/ml BSA, andvarious concentrations (0.005-4 nM) of '"I-rhlL-la in theabsence or presence of 200-fold unlabeled rhlL-la (1-800nM). The free and bound radioactivity was separated by thebinding oil column method and measured with a y-counter(ARC-500; Aloka, Tokyo). The receptor number anddissociation constant were determined by Scatchard plotanalysis.

RESULTS

Construction of a hIL-lRI Expression Vector, Transfec-tion, and Establishment of Stable Transfectant CellLines—Cloning of hIL-lRI cDNA into pko-neo producedthe expression vector, pSV-hlL-lRI (Fig. 1), in whichhuman type IIL-1 receptor gene is under the control of aSV40 transcription unit consisting of the early promoter,the small-t intron and the polyadenylic acid addition signal.The ability of pSV-hlL-lRI to transfect mammalian cellswas confirmed as transfection of CHO cells with pSV-hlL-1RI plasmids resulted in the production of cell linesexpressing about 105-106 hIL-1 binding sites per cell (datanot shown). Cotransfection of 5 X 105 A375-5 cells with 25/xg pSV-hlL-lRI and 1 //g pSVneo plasmids followed byselection against G418 for 2 weeks resulted in the forma-tion of 12 G418-resistant colonies. Analysis of the 12colonies by flow cytometry using mouse monoclonal anti-body against hIL-lRI showed that 5 colonies (C2, C6, C12,

pSU-hlL-1 Rl6690 bp

SU40 small T intron

SU4B polyfl

BamHI 1217

BamH\ 227B

Sal\ 3448 BamHi 3179

Fig. 1. Schematic representation of pSV-hlL-lRI. See "RESULTS" for description.

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CNl, and CN7) expressed relatively high WL-1RI. There-fore, these five colonies were subcloned by limiting dilutionfor the establishment of stable transfectant cell lines. Fivestable transfectant cell lines (C2-1, C6-3, C12-4, CN1A1,and CN7) were finally established and subjected to detailedcharacterization.

IL-1RI mRNA Expression of Stable Transfectant CellLines—The hIL-lRI mRNA levels of stable transfectantcell lines were examined by Northern hybridization with a473-base cDNA fragment of ML-1RI (Fig. 2). A375-5 hadno, while A375-6 expressed a detectable level of, IL-1RImRNA. In the five stable transfectants, the IL-1RI mRNAexpression was about 2.5-fold for C2-1, C6-3 and CN1A1,1.6-fold for C12-4, and 1.2-fold for CN7, as compared withthe IL-1RI mRNA expression of A375-6. Similar resultswere obtained when the full hIL- 1RI cDNA was used as aprobe. In a separate experiment, pSVneo-transfectedA375-5 cells did not show a detectable IL-1RI mRNAsignal, suggesting that the hIL-lRI mRNA detected in thestable transfectants was due to the transfection of pSV-HL-1RI not pSVneo (data not shown).

Response of Stable Transfectants to the Anti-Prolifer-ative Effect of IL-1—The dose-dependent response of A375and transfectant cells to the anti-proliferative effect ofrhlL-ltf was investigated and the results are shown in Fig.3. Among the five stable transfectant cell lines, the growthof C2-1, C6-3 and C12-4 was dose-dependently suppressedby rhIL-lar. In contrast, rhIL-la' could not suppress thegrowth of CNl Al or CN7, even at a concentration as high as1,000 U/ml, although they expressed IL-1RI (Fig. 2). The

SampleA375-5 C2-1 C12-4 CN7

A375-6 C6-3 CN1A1

IL-1 R ->-

GAPDH >• §§••#••Fig. 2. IL-1RI mRNA expression of A375 and stable trans-fectants. Bottom: Northern hybridization with a 473-base humanIL-1RI cDNA fragment or a 1,300-base human GAPDH cDNAfragment using the same filter. Top: The mRNA level (arbitraryunits) of each cell line after normalization with GAPDH mRNA.Similar data were obtained when the full length human IL-1RI cDNAwas used as a probe.

finding that A375-6 was sensitive to but A375-5 did notrespond to IL-1 (Fig. 3) is in accord with previous reports(9, 30, 31). The growth characteristics of the stable trans-fectants were identical to those of progenitor A375-5 cells,except those of C12-4, whose growth was quite slow as thecontrol ABS595 of C12-4 was much lower than those of theother cells (0.61±0.08 versus 1.15±0.16).

IL-1RI Number and Affinity of Stable Transfectants—Although all five stable transfectants expressed IL-1RImRNA (Fig. 2), CN1A1 and CN7 cells failed to respond tothe anti-proliferative effect of rhlL-ltf (Fig. 3). This mightbe merely due to that CN1A1 and CN7 cells either exhibit-ed no IL-1RI expression at the protein level and/or ex-pressed a receptor with very low affinity. To clarify thispoint, receptor binding experiments with i25I-labeledrhIL-la were performed. Figure 4 shows the equilibriumbinding results for C2-1 cells. Specific binding increasedwith 125I-rhIL-lar concentration, and plateaued at morethan 1 nM. Scatchard plotting indicated that C2-1 express-ed an average of approximately 1,500 receptors/cell with aKa of about 56 pM. The results of receptor binding analysisof the other transfectant and A375 cells are summarized inTable I. A375-6 had only 150 receptors/cell, whereasA375-5 had no receptors. All the stable transfectant cellshad IL- 1RI on their surface albeit that the receptor numberranged from 430 to 1,500 per cell. The Ka value rangedfrom 36 to 80 pM. The affinity of IL-1R on stable trans-fectant cells is in good agreement with that reported for thetype I human IL-1 receptor {17).

IL-1-Induced IL-6 Production by Stable Transfectants—IL-1 can induce IL-6 production by a variety of cell types,and IL-1-induced IL-6 production has been proposed topartially contribute to the anti-proliferative effect of IL-1on A375-6 cells (31). Whether the resistance of CNl Al andCN7 to the anti-proliferative effect of rhlL-la- is due to alack of IL-1-induced IL-6 production was investigated.Figure 5 shows that all the stable transfectants could

5 100oO)— 80ao

60

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Concentration of rhlL-1a (U/ml)

Fig. 3. Response of stable transfectants to the anti-prolifer-ative effect ofrhIL-la. A375 and transfectant cells were cultured intriplicate wells for 72 h in the absence or presence of variousconcentrations of rhlL-lo-, as specified. Cell growth was determinedby crystal violet staining. Percentage of cell growth was calculated asdescribed under "MATERIALS AND METHODS." Standard devia-tions are not shown in the figure for clarity, but they were not morethan 6%. The results are representative of four experiments.

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secrete IL-6 into the culture supernatant upon rhlL-lo-stimulation. Moreover, CN1A1 and CN7 cells producedmuch more rhIL-la-induced IL-6 than C12-4, one of thesensitive transfectants. Although the data are not present-ed, A375-6 cells produced approximately 0.3 ng/ml of IL-6into the culture supernatant under the same conditions.Neutralization experiments using anti-hIL-6 antiserumconfirmed that almost 90% of the MH60.BSF2-stimulatingactivity in the culture supernatant was due to authenticIL-6 (data not shown). These results demonstrate that thefailure of IL-1 to suppress the growth of CN1A1 and CN7is not due to a lack of IL-6 induction in these cells.

Response of Stable Transfectants to rhIL-6—AlthoughCN1A1 and CN7 can produce IL-6 in response to rhlL-latreatment, whether they have deficiencies of IL-6R and/orIL-6R-mediated signaling transduction is unknown. As itwas reported previously (32) that the growth of progenitorA375-5 cells can be partially inhibited by high doses of

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Fig. 4. Equilibrium binding of C2-1 to I25I-labeled rhIL-la.Aliquots of 5 X 10s cells were incubated for 1.5 h at 4'C with variousconcentrations of 125I-labeled rhIL-l<* in the absence (for totalbinding, solid triangles) or presence (for nonspecific binding, opencircles) of 200-fold cold rhlL-la. Specific binding (solid circles) wascalculated by subtracting nonspecific binding from the correspondingtotal binding. The insert shows Scatchard plotting of the binding data.The x-axis intersection gave the extrapolated number of IL-1 recep-tors per cell. The apparent dissociation constant was obtained bydividing the number of receptors per cell by the bound/free ratio atthe y-axis intersection.

TABLE I. IL-1R numbers and K^ of A375 and transfectants.Equilibrium binding was performed as described under "MATE-RIALS AND METHODS." The IL-1 receptor numbers and dissocia-tion constants (iCi) were calculated as described in the legend to Fig.4.

Cell lineA375-6A375-5C2-1C6-3C12-4CN1A1CN7

IL-1R number/cell150

Undetectable8

1,5001,460

530430500

iC (pM)80—5658743653

rhIL-6, we examined whether CN1A1 and CN7 could alsorespond to a high dosage of exogenous rhIL-6. ExogenousrhIL-6 at a concentration of 100 ng/ml suppressed thegrowth of all transfectants (both IL-1-sensitive and-resistant) as well as progenitor A375-5 cells, indicatingthat no IL-6R and/or IL-6R-mediated signaling deficiencyexists in CN1A1 and CN7 cells. However, rhIL-6 at 10 ng/ml or lower did not suppress the growth of these cells.

Alteration of ODC Activity of Stable Transfectant Cellsupon IL-1 Treatment—Down-regulation of ODC activityhas been proposed to participate in the anti-proliferativeeffect of IL-1 on A375 melanoma cells (6). We wonderedwhether the resistance of CN1A1 and CN7 is due to the

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Fig. 5. IL-6 production of stable transfectants. Aliquots of 5x105 cells of the cell lines were seeded into the wells of a 24-wellflat-bottomed plate, and incubated overnight in a CO2 incubator,followed by a change to 1.5 ml/well of prewarmed fresh culturemedium in the presence (dotted bars) or absence (closed bars) of 100U/ml of rhlL-la. After 24 h culture, IL-6 secreted into the culturesupernatant was quantitated by means of IL-6 assaying using rhIL-6as a standard. The values represent means ±SD for triplicate cul-tures. Similar results were obtained in three separate experiments.

% inhibition of ODC activity

A375-6-

A375-5 -

C2-1-

C6-3-

C12-4-

CN1A1 4

CN7-J

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"There was no specific binding.

Fig. 6. Alteration of ODC activity of stable transfectants uponrhIL-la treatment. A375 and transfectant cell monolayers werecultured in the presence or absence of 100 U/ml rhIL-lar for 24 h ina C02 incubator. Subsequently, their ODC activity was measured andthe percentage of inhibition calculated as described under "MATE-RIALS AND METHODS." A375-6 was used as a positive control. Theresults are representative of two experiments.

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Fig. 7. Effect of putrescine on the anti-proliferative action ofrhIL-la on IL-1-sensitive transfectants. Transfectant cells intriplicate wells were cultured for 72 h in the presence of variousconcentrations of rhlL-lar with or without 2 mM putrescine (put).Cell growth was determined by crystal violet staining. Percentage ofcell growth was calculated as described under "MATERIALS ANDMETHODS." Error bars are not shown in the figure for clarity, butthey were not more than 10%. Putrescine alone did not have muchinfluence on the growth of these cells. The results are representativeof two experiments.

failure of down-regulation of ODC activity upon rhIL-latreatment. Therefore, the ODC activity of stable trans-fectants upon rhIL-l<z treatment was measured and theresults are presented in Fig. 6. Treatment with 100 U/mlof rhIL-1 a for 24 h inhibited 70 to 80% of the ODC activityin A375-6, C2-1, C6-3, and C12-4 cells, but had no effect onthe ODC activity in the two IL-1-resistant transfectant celllines, CN1A1 and CN7. The ODC activity of A375-5 did notchange.

Effect of Putrescine on the Anti-Proliferative Action ofIL-1 in IL-1-Sensitive Transfectant Cells—To furtherconfirm the involvement of ODC activity down-regulationin IL-1-induced growth inhibition of A375 melanoma cells,we investigated whether IL-1-induced growth inhibition ofIL-1-sensitive transfectants could be reversed by thesimultaneous addition of putrescine, a physiological prod-uct of the ODC action. The results in Fig. 7 show that at lowrhIL-la concentrations ( < 1 U/ml), 2 mM putrescine wasable to overcome the anti-proliferative action in all thethree IL-1-sensitive transfectants. However, 2 mM putre-scine could only reverse about half the rhIL- la -inducedgrowth inhibition at concentrations higher than 1 U/ml.Putrescine at 2 mM did not influence the growth of anytransfectants examined. Putrescine at concentrationshigher than 2 mM was also tried to see whether or not itcould overcome the greater growth inhibition caused byhigh doses of rhIL-1 a, but this turned out to be difficult ashigher concentrations of putrescine themselves hamperedcell growth (data not shown).

DISCUSSION

Among a number of normal or malignant cell types whose

growth can be suppressed by IL-1 (9-13), melanoma cells,especially A375-6 melanoma cell line, are widely used toelucidate the signal transduction pathway of IL-1-inducedgrowth inhibition (6, 9, 26, 31-34). To obtain a fullunderstanding of the signal transduction leading to growthinhibition, it is apparently important to determine how theinformation of IL-1R occupancy is transduced into down-stream changes leading to growth inhibition. Two types ofIL- 1R, which do not share any similarity with the signalingmechanisms of any of the major cytokine receptor families(42, 43), are members of the immunoglobulin superfamily(16-18). By analogy with the signaling mechanisms ofother members of the immunoglobulin family such as T andB cell antigen receptors (44), it is possible to hypothesizethat accessory molecule (s) might exist in the membraneand/or cytoplasm of A375 cells, which may associate withIL-1R and couple the information of receptor occupancy tothe corresponding downstream intracellular changes, lead-ing to the final outcome of growth inhibition. Unluckily, thefact that the widely used IL-1-sensitive A375 melanomacell line, A375-6, has an average of only 100 to 200 IL-1Rper cell (30) (also see Table I in the present paper) hampersattempts to confirm the above hypothesis by methodologiessuch as coimmunoprecipitation. We therefore constructedan IL-1RI expression plasmid, pSV-hlL-lRI, and trans-fected it into A375-5, an IL-1-unresponsive melanoma line(6, 9, 30), to establish stable IL-1-sensitive transfectantcell lines expressing more IL-1RI than A375-6. Threetransfectant cell lines (C2-1, C6-3, and C12-4) wereobtained that, similar to A375-6, are sensitive to the anti-proliferative effect of IL-1 (Fig. 3). In addition, these celllines express 3.5- to 10-fold IL-1R compared to A375-6 asmeasured by receptor binding and Scatchard plot analysis(Fig. 4 and Table I). The similarity of C2-1, C6-3, andC12-4 to A375-6 was further supported by the results that,upon rhIL-lar treatment, all of them produced IL-6 (Fig. 5)and down-regulated their ODC activity (Fig. 6). Why thesethree cell lines are more sensitive than A375-6 to rhlL-lff-induced growth inhibition (Fig. 3) is not known, but the factthat they express more IL-1R (Table I) may provide apossible explanation. Why C12-4 cells seem extremelysensitive may partly be due to their slow growth. It is hopedthat these cell lines will facilitate exploration of the signaltransduction pathway of IL-1-induced growth inhibition inmelanoma cells.

Quite unexpected was the simultaneous finding of twoIL-1-resistant transfectant cell lines, CN1A1 and CN7(Fig. 3). As they expressed functional IL-1RI (Fig. 2 andTable I), it was speculated that their resistance to IL-1 isdue to a deficiency of some postreceptor event(s) which iscritical for IL-1-induced growth inhibition of A375 mela-noma cells. To the best of our knowledge, down-regulationof ODC activity (6), IL-6 induction (31), superoxideradicals (9, 46), and induction of gro gene (coding formelanoma growth-stimulating activity) expression (33)have been supposed to be involved in or associated with theanti-proliferative effect of IL-1 on melanoma cells. As IL-1-induced gro expression has been confirmed in cells whosegrowth could not be inhibited by IL-1 (26, 33, 34, 45), it isless likely that IL-1-induced gro gene expression is essen-tial for the anti-proliferative effect of IL-1. Althoughsuperoxide radicals can kill A375 cells (our unpublishedobservations) and overexpression of mitochondrial super-

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oxide dismutase can prevent A375 cells from being inhib-ited by IL-1 (46), no experimental data have shown thatA375 cells can produce superoxide radicals in response toIL-1. Therefore, we focused on whether a deficiency of IL-6induction and/or ODC down-regulation exists in the twoIL-1-resistant transfectant cell lines. As shown by Fig. 5,similar to the IL-1-sensitive cell lines, CN1A1 and CN7produced considerable amounts of IL-6 in response torliLL-la. It may still be argued that these two cell linesmight lack IL-6 receptors or have a deficiency in the IL-6signal transduction pathway, but that high dosages ofexogenous rhIL-6 inhibited the growth of all the trans-fectant cell lines as well as progenitor A375-5 cells to thesame extent rules out this possibility. IL-Iff-induced IL-6failed to suppress the growth of CN1A1 and CN7, whileexogenous rhIL-6 could do so, because IL-1-induced IL-6was present at only 1 ng/ml or so, a concentration far frombeing able to suppress the growth of A375 melanoma cells.Previously we reported that there are two pathways for theanti-proliferative effect of IL-1 on A375-6 cells, one isdependent on IL-6 induction and another is independent onIL-6 induction (31). Therefore, the IL-6-independentpathway seems to be predominant in these transfectantcells. Collectively, our data suggest that IL-1-induced IL-6production may play a synergistic role in IL-1-inducedgrowth inhibition of A375 cells, but it is unlikely to be anindispensable prerequisite for IL-1-induced growth inhibi-tion of A375 cells. The finding of two transfectants (CN1A1and CN7) responsive to IL-1-induced IL-6 production butresistant to IL-1-induced growth inhibition also suggeststhat the mechanisms involved in IL-1-induced IL-6 pro-duction and growth inhibition are different in A375 mela-noma cells. When the alteration of ODC activity in the 5transfectant cell lines was examined, it was observed thattreatment with rhIL-la for 24 h down-regulated the ODCactivity in only IL-1-sensitive, not-resistant, cell lines,indicating the resistance of CN1A1 or CN7 is due to adeficiency of one or some unknown step(s) in the signaltransduction pathway from the point right after receptoroccupancy to where ODC activity is down-regulated. It iscurrently unknown which step is deficient and responsiblefor the failure of IL-1-induced ODC activity down-regu-lation in CN1A1 and CN7 cells. The importance of ODCactivity down-regulation in IL-1-induced growth inhibitionwas further supported by that putrescine, a direct productof the ODC reaction and a precursor of polyamines, over-came most, although not all, of the rhIL- la -inducedgrowth inhibition of all the IL-1-sensitive transfectantsobtained (Fig. 7). The failure of 2mM putrescine tocompletely reverse the growth inhibition caused by highdoses of rhIL-la has a number of possible explanations.The simplest one is that 2 mM putrescine was not enough.However, this possibility is hard to confirm as putrescine atconcentrations higher than 2 mM itself showed toxicitytoward cultured cells. The second one is that IL-1 mightconcomitantly influence other enzyme(s) in the biosynthe-sis of polyamines downstream to putrescine formation. Thelast possibility might be that besides ODC activity down-regulation, other factor(s) or pathway(s) are also involvedin IL-1-induced growth inhibition of A375 melanoma cells.Nevertheless, these results combined with the previousreport (6) suggest that down-regulation of ODC activity isa quite important, if not the sole, event in the signal

transduction cascade of IL-1-induced growth inhibition ofA375 melanoma cells.

Identification of ODC activity down-regulation as anessential step in the IL-1-induced growth inhibition inmelanoma cells is of importance. Current work is concen-trated on how IL-1 down-modulates ODC activity in thesetransfectants. We have also noticed that NF-kB (26, 47)and protein kinases (21-28, 48) have been reported toparticipate in IL-1 signaling in cells whose growth can beeither promoted or suppressed. Determination of whetherthey also take part in IL-1-induced down-regulation ofODC activity in A375 melanoma cells awaits furtherinvestigations.

We are grateful to Mr. Yoshitaka Inukai for his excellent help inpreparing this manuscript.

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