Regulation of ornithine decarboxylase in B16 mouse melanoma cells:synergistic activation of melanogenesis by KMSH and ornithine

decarboxylase inhibition

J. Sanchez Mas, M. Mart|nez-Esparza, C.M. Bastida, F. Solano, R. Pen¬a¢el,J.C. Garc|a-Borron *

Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Apto 4021, Campus Espinardo,30100 Murcia, Spain

Received 14 June 2001; received in revised form 17 September 2001; accepted 2 October 2001

Abstract

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in the biosynthesis of polyamines, a family of cationiccompounds required for optimal cell proliferation and differentiation. Within mammalian melanocytes, the expression ofgenes regulating cell growth and/or differentiation can be controlled by K-melanocyte-stimulating hormone (KMSH) andother melanogenesis modulating agents. In the B16 mouse melanoma model, KMSH stimulates melanogenesis byupmodulation of tyrosinase (tyr) activity, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibitsmelanin synthesis. Therefore, we analyzed the regulation of ODC by these agents, as related to changes in the melanogenicpathway. Treatment of B16 cells with TPA or KMSH rapidly stimulated ODC activity. The effect was stronger for TPA andappeared mainly posttranslational. Irreversible inhibition of ODC with the active site-directed inhibitor K-difluoromethyl-ornithine (DFMO) did not block TPA-mediated inhibition of tyr. Conversely, prolonged treatment of B16 cells with DFMOstimulated tyr activity by a posttranslational mechanism, probably requiring polyamine depletion. Combination treatmentwith KMSH and DFMO synergistically activated tyr. Therefore, ODC induction is not involved in the melanogenic responseof B16 cells to KMSH. Rather, increased intracellular concentrations of polyamines following ODC induction mightconstitute a feedback mechanism to limit melanogenesis activation by KMSH. ß 2002 Elsevier Science B.V. All rightsreserved.

Keywords: Melanoma cell ; Tyrosinase; Ornithine decarboxylase; K-Di£uoromethylornithine; Polyamine; Regulation of melanogenesis

1. Introduction

Mammalian melanogenesis is a complex pathwayrestricted to specialized cells, the melanocytes, andleading to the formation of colored melanin poly-mers from the amino acid L-tyrosine. Melanin syn-thesis is catalyzed by at least three enzymatic pro-teins, tyrosinase (tyr, monophenol dihydroxyphenyl-alanine:oxygen oxidoreductase, EC 1.14.18.1) and

0167-4889 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 7 - 4 8 8 9 ( 0 1 ) 0 0 1 6 5 - 3

Abbreviations: Dct, dopachrome tautomerase; DFMO, K-di-£uoromethylornithine; L-DOPA, L-3,4-dihydroxyphenylalanine;DHICA, 5,6-dihydroxyindole-2-carboxylic acid; ODC, ornithinedecarboxylase; TPA, 12-O-tetradecanoylphorbol 13-acetate; tyr,tyrosinase; tyrp, tyrosinase-related protein

* Corresponding author. Fax: +34-968-83-09-50.E-mail address: gborron@um.es (J.C. Garc|a-Borron).

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the tyr-related proteins (tyrps) 1 and 2. Tyr catalyzesthe rate-limiting generation of L-dopaquinone fromL-tyrosine and is also able to oxidize L-3,4-dihydrox-yphenylalanine (L-DOPA) to L-dopaquinone [1].L-Dopaquinone evolves spontaneously into a semi-stable intermediate, L-dopachrome, which is con-verted into 5,6-dihydroxyindole-2-carboxylic acid(DHICA) by dopachrome tautomerase (Dct, tyrp2)[2]. Finally, mouse tyrp1 [3,4] and human tyr [5] areable to oxidize DHICA to the corresponding 5,6-in-dolequinone-2-carboxylic acid, thus promoting its in-corporation into eumelanin.

In mammalian melanocytes, the enzymatic activityof the rate-limiting tyr is regulated by a variety ofextracellular signals. Physiologic melanogenic stimuliinclude UV radiation and K-melanocyte-stimulatinghormone (KMSH) or other peptide hormones, collec-tively termed melanocortins [6]. KMSH stimulatesthe di¡erentiation and/or proliferation of mammali-an melanocytes [7], by binding to the melanocortin 1receptor (MC1R) and triggering the cAMP signalingpathway [8]. The molecular mechanisms of the stim-ulation of melanogenesis by KMSH have been inten-sively investigated using mouse melanoma cells as amodel [9^11]. In these cells, activation of the cAMPpathway causes a strong transcriptional induction ofthe tyr gene, mainly through a robust induction ofthe microphthalmia (Mitf) transcription factor [12^14]. Conversely, the hormonal e¡ects on tyrp1 andDct gene expression and on the corresponding enzy-matic activities are modest [11,15]. Other signalingpathways are also involved in the control of melano-genesis. For instance, protein kinase C (PKC) hasbeen reported to strongly activate melanogenesis inhuman melanocytes [16] but to decrease Mitf expres-sion and tyr activity in B16 mouse melanoma cells[17]. Similarly, activation of the MAPK pathway ap-pears to trigger an inhibition of melanogenesis withinB16 cells [18]. Therefore, multiple signaling pathwayscontribute to the control of melanogenesis, whosecomplexity could be increased by a possible cross-talk between them.

The polyamines putrescine, spermine and spermi-dine are ubiquitous low molecular weight cationiccompounds required for optimal cell proliferationand/or di¡erentiation [19]. Ornithine decarboxylase(ODC) is the key, rate-limiting enzyme in polyamine

biosynthesis, and catalyzes the decarboxylation ofL-ornithine to yield putrescine. In keeping with itsimportant role in the regulation of cellular prolifer-ation and di¡erentiation, ODC gene expression andenzyme activity are tightly controlled by a variety ofextracellular signaling molecules in eukaryotic cells[20]. Regulatory mechanisms include the modulationof ODC mRNA levels by transcriptional and post-transcriptional e¡ects, the regulation of its rate oftranslation [21,22], covalent posttranslational modi¢-cation of the enzyme, and changes in the extremelyshort half-life of the protein [23] or in the levels ofe¡ector proteins able to modulate ODC activitythrough speci¢c interactions [24,25].

Several observations suggest that ODC and poly-amines might be involved in various aspects ofskin biology, including the regulation of melanogen-esis. UV radiation induces ODC activity in the skin[26]. Inhibition of ODC with the irreversible inhibi-tor K-di£uoromethylornithine (DFMO) enhances themelanogenic activity of mouse melanoma cells [27],and spermine, at concentrations in the low milli-molar range, inhibits mouse melanoma tyr [28]. Inthe light of these observations, it is surprisingthat little attention has been paid to the regulationof ODC activity in mammalian melanocytes andmelanoma cells, as related to the control of pigmen-tation.

Here we report a study on the regulation of ODC,at the gene expression, protein and enzyme activitylevels, in the well established B16 mouse melanomamodel. We have also analyzed its possible involve-ment in the control of the rate of the melanogenicpathway. Our results show that ODC mRNA andprotein levels, as well as ODC enzymatic activityare upregulated by 12-O-tetradecanoylphorbol 13-acetate (TPA), although to di¡erent extents. Con-versely, KMSH moderately and transiently downre-gulates ODC mRNA, but has no detectable e¡ect onODC protein levels, and triggers a modest activationof its enzymatic activity. Therefore, within B16 mel-anoma cells the regulation of ODC is complex andapparently involves transcriptional, translational andposttranslational e¡ects. Moreover, polyamine deple-tion increases tyr activity by a posttranslationalmechanism that synergistically potentiates the stim-ulatory action of KMSH.

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2. Materials and methods

2.1. Reagents

The radioactive substrates L-[3,5-3H]tyrosine andL-[1-14C]ornithine were from Amersham PharmaciaBiotech (Little Chalfont, Buckinghamshire, UK)and Moravek Biochemicals (Brea, CA, USA), respec-tively. L-Tyrosine, dithiothreitol, pyridoxal phos-phate, Igepal CA-630, BSA, L-DOPA, phenylmethyl-sulfonyl £uoride (PMSF), the superpotent KMSHanalogue [Nle4, D-Phe7] KMSH (from now on termedKMSH), synthetic melanin and bicinchoninic acidwere from Sigma (St. Louis, MO, USA), DFMOwas obtained from Ilex Oncology (San Antonio,TX, USA). Reagents and plasticware for cell culturewere from Nunc (Roskilde, Denmark) or Gibco(Gaithersburg, MD, USA). Other reagents werefrom Merck (Darmstadt, Germany) or Prolabo (Bar-celona, Spain).

2.2. Cell culture and treatments

B16 mouse melanoma cells were cultured in MEMsupplemented with 10% FCS, 2 mM glutamine, 100U/ml penicillin and 100 WU/ml streptomycin. KMSH,TPA and DFMO were used at 1037 M, 25 ng/ml and0.1 mM respectively. Cultures were serum-deprived24 h before addition of the agents. Cells were har-vested with trypsin, washed with PBS, and solubi-lized in 10 mM sodium phosphate, pH 6.8, 1% IgepalCA-630, 0.1 mM EDTA, and 0.1 mM PMSF. Theextracts were centrifuged (20 000Ug, 30 min) beforeenzyme activity assays.

2.3. Enzyme activity determinations

Tyrosine hydroxylase activity was determined by aradiometric method [29] and DOPA oxidase activitywas measured spectrophotometrically, in the pres-ence of 3-methyl-2-benzothiazolinone hydrazone[30]. ODC activity was monitored by a radiometricprocedure based on the determination of 14CO2 re-lease from L-[1-14C]ornithine, as described elsewhere[31].

2.4. Western blot

Western blotting was performed as described [5],using the speci¢c anti-tyr polyclonal antiserumKPEP7 (a gift from Prof. V. Hearing, NIH, Bethes-da, MD, USA) or a commercial monoclonal anti-ODC antibody (Sigma) as primary antibodies. Cellswere solubilized as for enzyme activity measure-ments, and equal amounts of protein were loadedin each lane of 12% SDS^PAGE gels. Transfer wascarried out in a semi-dry unit from Bio-Rad (Her-cules, CA, USA) and checked by Coomassie bluestaining of the gel. Before blocking, the lower por-tion of the membrane was cut and stained for totalprotein with Amido black to ascertain comparableloading and transfer. Staining of immunoreactivebands was done with a chemiluminescent substratefrom Amersham Pharmacia. Quanti¢cation of theblots was performed in a Gel Doc system (Bio-Rad) using the Multi-Analyst software.

2.5. Northern blot

Total RNA was isolated with guanidinium thio-cyanate, denatured with glyoxal and DMSO, electro-phoresed on 1.5% agarose gels in 10 mM sodiumphosphate bu¡er, pH 7.0, and transferred to Hybondnylon membrane. Prehybridization and hybridizationwere performed as described [32]. The random prim-ing 32P-labeled probes used were: tyr : 1.6 kb EcoRIfragment from plasmid pMEL34 [33]; ODC : 550 bpPCR fragment (primers GGATTTGACTGTGCA-AGC and GAGTCTGATGGGAAGTAC); Mitf :1.3 kb PCR fragment (primers AAGTGGTCTGC-GGTGTCTCC and AAGGCAGGCTCGCTAAC-ACG).

PCR fragments were generated using cDNA fromB16 melanocytes as target. Their identity was ascer-tained by restriction mapping. Signal intensities werequanti¢ed by phosphorimaging and normalized tothe GAPDH signal.

2.6. Other procedures

Protein was determined by the bicinchoninic acidmethod using BSA as standard. Data were statisti-cally analyzed by Student's t-test, using the Graph-

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Pad Prism software, and a signi¢cant level of di¡er-ence was set at P6 0.05.

3. Results

3.1. Regulation of ODC gene expression in B16melanocytes

Since ODC behaves as an early gene in many cel-lular systems, we analyzed ODC mRNA levels in B16cells treated with KMSH or TPA for short times,ranging from 1 to 16 h. The response of ODCmRNA levels to KMSH was biphasic (Fig. 1A). Arapid and transient decrease, with maximal inhibitionof about 30%, was followed by an increase in ODCmRNA, up to 170% of control levels 8 h after addi-tion of KMSH. The e¡ect was most likely cAMP-dependent, since it could be mimicked by forskolin

(not shown). On the other hand, treatment with TPAsigni¢cantly upregulated ODC mRNA (Fig. 1B). Themaximal stimulation achieved by TPA was approx.2-fold, after 8 h of treatment. However, in this case,ODC mRNA induction was not preceded by an ini-tial and transient downregulation, as observed forKMSH.

3.2. Regulation of ODC activity and protein levels

The activating e¡ect of TPA on ODC enzymaticactivity was much stronger than the induction ofODC gene expression (Fig. 2A). It was also faster,since the maximal induction was reached 4 h (insteadof 8 h) after addition of the phorbol ester to theculture medium. The stimulation of ODC enzymaticactivity was also much higher than the increase inprotein abundance, as detected by Western blot(Fig. 2B). In cells treated with KMSH, and in spite

Fig. 2. TPA induction of ODC enzymatic activity and proteinin B16 cells. (A) Strong induction of ODC activity by TPA.Cells treated as in Fig. 1 were trypsinized and solubilized forenzyme activity measurements at the times shown. Results(mean þ S.D., n = 4) are expressed as % speci¢c ODC activityrelative to controls harvested at the same time as the experi-mental points. (B) Western blot analysis of ODC proteinchanges in TPA-treated cells. Cells were harvested at the timesshown, and ODC was detected by Western blot using a speci¢cmonoclonal antibody. Similar trends were obtained in two dif-ferent experiments. Note that the increase in protein abundanceis much lower than the stimulation of ODC enzymatic activity.

Fig. 1. Regulation of ODC mRNA levels by KMSH and TPA.Semicon£uent B16 mouse melanoma cells were serum-deprived24 h before addition of KMSH (1037 M, ¢nal concentration,A) or TPA (25 ng/ml, B). Cells were lysed with guanidiniumthiocyanate at the times shown, and total RNA was extractedand analyzed by Northern blot. For each panel, a representa-tive blot is shown on the left and the kinetics of the relativeODC mRNA changes, corrected for loading with a GAPDHprobe and quanti¢ed by phosphorimaging, on the right (shownas mean þ S.D., nv3). C stands for control, untreated cells. Inthe experiment described in A, two independent controls werecollected along with the 1 h and 16 h time points, to correctfor possible changes in the basal expression of the ODC gene.Since no signi¢cant variations were found, the 16 h control wasomitted in the experiment shown in B.

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of the transient downregulation of ODC mRNA lev-els, ODC enzymatic activity increased rapidly andmoderately, up to 2-fold (Fig. 3A). This increasewas, however, small as compared to the potent in-duction achieved by TPA. Consistent with a limitede¡ect on enzymatic activity, changes in ODC proteinlevels were minor, as detected by Western blot ofextracts from cells treated with the hormone (Fig.3B).

3.3. Inhibition of ODC with DFMO does not blocktyr downregulation by TPA, and synergisticallypotentiates tyr activation by KMSH

Since both TPA and KMSH signi¢cantly activatedODC activity in B16 cells, we next examined whethera blockade of this activation would interfere witheither the inhibition of tyr activity mediated byTPA or its activation in response to KMSH. Thisstudy was performed by treating B16 melanoma cellswith TPA or KMSH in the presence of DFMO, astrong, speci¢c and irreversible inhibitor of ODC.

As previously reported by others [27], treatment ofB16 melanocytes with DFMO caused a slow activa-tion of both the tyrosine hydroxylase and DOPAoxidase activities of tyr (Fig. 4A). Activation wasstatistically signi¢cant only after a 48 h treatment,and similar in extent and kinetics for the two enzy-matic activities. Tyr activation by DFMO occurredwith a smaller increase in tyr mRNA (1.5-fold), butwithout changes in enzyme abundance, as detectedby Western blot with the speci¢c antibody KPEP7(Fig. 4B,C). Therefore, tyr activation by DFMOtreatment is most likely carried out at the posttrans-lational level.

Fig. 4. Activation of tyr in B16 melanoma cells treated withDFMO. (A) Time course of induction of the tyrosine hydroxy-lase (closed bars) and the DOPA oxidase (open bars) activitiesof tyr. Results are the mean þ S.D. for triplicate experiments.Basal levels of tyrosine hydroxylase and DOPA oxidase activ-ities were 138 þ 22 WU/mg protein and 9.5 þ 2.4 mU/mg protein,respectively. (B) Northern blot analysis of tyr mRNA changesafter DFMO treatment (72 h). A small increase in tyr mRNAabundance was noticed in two independent experiments.(C) Lack of upregulation of tyr protein in cells treated withDFMO. B16 cells were grown in the presence of DFMO for72 h and analyzed by Western blot using the speci¢c antiserumKPEP7. Similar trends were obtained in two experiments.

Fig. 3. KMSH activation of ODC without a parallel change inprotein abundance. (A) Time course of ODC activity changes.Cells were treated with 1037 M KMSH for the times shown.Results (mean þ S.D., n = 4) are expressed as % ODC with re-spect to control cultures. (B) Lack of signi¢cant modulation ofODC protein. Cells were solubilized and their ODC contentswere analyzed by Western blot. Similar trends were obtained intwo di¡erent experiments.

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In cells treated simultaneously with DFMO andTPA, tyr activity levels were smaller than in controlcells, even after a 72 h treatment (Fig. 5A). Inhibi-tion of tyr activity by TPA was quantitatively similarin the presence or absence of DFMO (when com-pared to DFMO-treated cells or untreated controls,respectively). Conversely, inhibition of ODC with

DFMO potentiated synergistically the stimulatoryaction of KMSH on tyr. After a 48 h treatment,the combination treatment caused activations higherthan 400%, whereas KMSH only achieved a 2-foldactivation and the e¡ect of DFMO was still not evi-dent (Fig. 5B). Longer treatments could not beachieved since they proved to be toxic to B16 cells.Extensive cell death was observed at 72 h in thepresence of both KMSH and DFMO, but not ofeither agent acting independently. This might bethe result of the potent synergistic induction of tyr,since melanogenic intermediates are highly cytotoxicspecies [34], owing to their ability to react with pro-teins [35] and nucleic acids [36].

Northern blot analysis of tyr mRNA abundancerevealed that the increases mediated by DFMO and

Fig. 6. Lack of synergistic upregulation of tyr mRNA by com-bined treatment with KMSH and DFMO. (A) Northern blotanalysis of tyr mRNA abundance in B16 cells treated withDFMO and/or KMSH. Cells were treated as indicated, for 48 h,before analysis of tyr mRNA abundance by Northern blot. Theexperiment was repeated twice with similar results. Quanti¢ca-tion of the blots is shown on the right (shown as relative levelsof tyr mRNA, mean þ range for two independent experiments,and normalized to the GAPDH mRNA signal). (B) MitfmRNA is not upregulated by DFMO. B16 melanoma cellswere treated (2 h) with DFMO and/or KMSH. Total RNA wasextracted and analyzed for Mitf mRNA levels by Northernblot. A representative blot is shown (left), and the histogramon the right shows the results of the quantitative analysis of thesignals, presented as in A.

Fig. 5. Failure of DFMO to prevent TPA-mediated tyr activitydownregulation, and synergistic stimulation of tyr in the pres-ence of KMSH. (A) Kinetics of tyrosine hydroxylase activitychanges in B16 cells treated with DFMO (open bars), TPA(hatched bars) or both agents simultaneously (closed bars). Re-sults are the mean þ S.D. for triplicate experiments, and are ex-pressed as % activity with respect to controls harvested simulta-neously. (B) Synergistic activation of tyr by DFMO andKMSH. Cells were treated for the times shown with DFMOalone (open bar), KMSH (hatched bars), or both agents (closedbars). Results are expressed as in A.

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KMSH were additive, rather than synergistic. More-over, DFMO by itself had little, if any, e¡ect on Mitfgene expression, and failed to increase the inductionof Mitf gene expression by KMSH (Fig. 6).

4. Discussion

ODC is the rate-limiting enzyme in the biosynthe-sis of polyamines, a group of small aliphatic cationicmolecules known to play a key role in the regulationof cell proliferation and di¡erentiation [19]. ODC israpidly and transiently induced by a variety of mito-genic signals [20]. Within the skin, overexpression ofODC induces tumor promotion in mice [37] andtreatment of keratinocytes with the tumor promoterTPA also increases ODC activity [38]. Therefore,ODC and the polyamines are certainly involved inthe control of the proliferation of skin cells. More-over, several observations also suggest a possible in-volvement of polyamines in the regulation of thedi¡erentiation of skin cells, including melanocytes[26^28]. However, neither the regulation of ODC ac-tivity in melanocytes or melanoma cells, nor the pos-sible connections between ODC activity changes andtheir melanogenic status, have been studied in detail.Using the well established B16 mouse melanomamodel, we have analyzed the regulation of ODC ac-tivity by TPA, an agent known to decrease the mel-anogenic activity of B16 cells [17], and by KMSH, apotent inducer of melanogenesis. For this purpose,the levels of ODC mRNA, protein and the enzymaticactivity were analyzed in kinetic experiments.

In many cellular systems treated with ODC-stim-ulating agents, upregulation of ODC mRNA issmaller than the increases in ODC protein and/oractivity [39]. In B16 melanocytes treated with TPA,a very strong increase in ODC activity was observed.However, only a modest upmodulation of ODCmRNA (2-fold), and a comparable elevation of pro-tein levels was associated to this much larger stimu-lation of enzyme activity. Moreover, maximal activa-tion of ODC occurred at 4 h, thus precedingmaximal induction of mRNA. In addition, 2 h afteraddition of TPA to the culture medium ODC proteinlevels were already higher than those of the controls,whereas ODC mRNA was not signi¢cantly di¡erentin control and TPA-treated cells. Thus, as in other

cell types, TPA regulates ODC activity in B16 mela-noma cells mainly by posttranscriptional and/orposttranslational mechanisms, probably involvingPKC activation.

When compared to the potent induction of ODCactivity by TPA, the e¡ects of KMSH were relativelyminor. In B16 cells treated with the melanocortin, amoderate elevation of ODC enzymatic activity pro-ceeded without noticeable changes in protein levels.Moreover, at short treatment times where ODC ac-tivation was maximal, ODC mRNA levels appearedslightly decreased. The e¡ects of KMSH are mostlikely mediated by cAMP, since (i) forskolin wasable to mimic the changes in ODC mRNA levels(results not shown), and (ii) in other cell types andtissues inhibition of protein kinase A abolishes theinduction of ODC elicited by peptidic hormones [31].Concerning the mechanism of activation, comparisonof the changes in ODC mRNA, protein and enzy-matic activity clearly shows that induction of ODCactivity is again most likely exerted at a posttransla-tional level. However, our data do not allow to iden-tify which one of the possible posttranslationalevents known to be involved in the regulation ofODC activity is the actual target of the hormone.An interesting possibility is a downregulation of theantizyme, a protein essential for ODC regulation[25]. In keeping with this possibility, the antizymeis expressed by human melanoma cells, and its intra-cellular concentration is regulated by extracellularsignaling molecules [40]. Moreover, the possibilityexists that phosphorylation of ODC may increasethe catalytic e¤ciency of the enzyme [41].

We also analyzed the possible involvement ofODC activity in the regulation of melanogenesis inB16 cells. Sustained inhibition of ODC with the irre-versible and speci¢c inhibitor DFMO caused a slowactivation of tyr. This activation occurred with asmall increase in tyr mRNA, but without changesin protein abundance. The modest increase in tyrmRNA might not be related to a stimulation of tyrgene transcription, since DFMO had little, if any,e¡ect on the expression of Mitf, the key transcrip-tional activator of tyr gene expression. Alternatively,polyamine depletion following prolonged ODC inhi-bition might lead to a stabilization of tyr mRNA,thus increasing its levels. The ability of polyaminesto interact with nucleic acids is well documented and

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polyamine depletion has been shown to increase thehalf-life of certain mRNA species, leading to parallelchanges in their abundance [42,43]. A possible e¡ectof polyamines in the stability of mRNAs for severalmelanocyte di¡erentiation markers will be analyzedin the near future. In any case, the ¢nding that tyractivity is stimulated by polyamine depletion withoutchanges in tyr protein contents indicates that, againin this case, the mechanism of activation is clearlyposttranslational, and probably involves the reversalof tyr inhibition by polyamines [28]. Accordingly, thee¡ect of DFMO is slow, and a 48^72 h treatment ofmelanocytes is needed to observe a clear-cut activa-tion of tyr. This time is likely required to deplete theintracellular polyamine pool of B16 cells, under con-ditions of inhibited synthesis of new polyamines [27].

Consistent with a posttranslational activation oftyr, treatment of B16 cells with DFMO synergistical-ly enhanced tyr stimulation by KMSH. In mousemelanoma cells, the hormone is known to increasethe intracellular levels of the protein [10,11]. There-fore, combined treatment of melanocytes withKMSH and DFMO would result in higher levels ofenzyme, with higher speci¢c activity, thus accountingfor the synergistic activation observed in our study.It is worth noting that the combination treatmentwas extremely cytotoxic at incubation times longerthan 48 h with extensive cell death being observedat 72 h. A similar observation has already been re-ported by others, using DFMO chemically coupledto KMSH fragments [44]. Conceivably, a limited celldeath may also occur at 48 h, a¡ecting preferentiallyhigh tyr activity cells. Should this be the case, themeasured synergistic e¡ect may re£ect the behaviorof the lower tyr activity cell population. Therefore,the actual synergistic potentiation of KMSH melano-genic action by DFMO might be even higher thanthe one reported above.

In summary, prolonged inhibition of ODC activityin B16 mouse melanoma cells causes a posttransla-tional activation of tyr. It can be hypothesized thatthe treatments leading to ODC activation could de-crease tyr activity, whereas inhibition of ODC lead-ing to a decreased intracellular polyamine poolwould exert a melanogenic e¡ect. Moreover, sincethis melanogenic e¡ect is posttranslational in nature,ODC inhibition might act synergistically with treat-ments increasing the intracellular levels of the tyr

protein. The physiological relevance of these ¢nd-ings, as related to the control of mammalian pigmen-tation, is, at present, di¤cult to evaluate. If theKMSH-triggered activation of ODC results in an in-creased intracellular polyamine concentration, thismight constitute a negative feedback mechanism tomoderate the melanogenic action of the hormone.This e¡ect could be ampli¢ed if polyamines tend toconcentrate in certain subcellular compartments,such as the melanosomes, by virtue of their positivecharge. A higher concentration of polyamines in themelanosome is likely, since a variety of positivelycharged molecules have been shown to accumulatein melanized tissues by binding to the negativelycharged melanin polymer [45]. Moreover, negativefeedback mechanisms acting on the melanogenicpathway appear quite common. For instance, UVlight increases the production of TNFK in the skin[46], and this cytokine acts as a potent inhibitor oftyrosinase in both human [47] and mouse [48] mela-nocytes and melanoma cells. Moreover, H2O2, aproduct of the melanogenic pathway, also inhibitstyr activity and gene expression [49]. These negativeregulatory mechanisms are usually interpreted asmelanocyte-speci¢c defensive processes, that wouldlimit the cytotoxicity inherent to an excessive rateof melanogenesis [47]. However, a ¢nal assessmentof the role of ODC in the modulation of the mela-nogenic e¡ect of KMSH will only be possible afterthe measurement of polyamines levels in di¡erentsubcellular compartments of control and hormone-treated cells.

Acknowledgements

Supported by grant PM99-0138 from the Comi-sion Interministerial de Ciencia y Tecnolog|a (CI-CYT). J.S.M. and M.M.-E. are recipients of fellow-ships from the AECC and from CajaMurcia,respectively.

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