Inhibitors of Polyamine Biosynthesis Block Tumor Necrosis ... · phages by TNF. The regulatory role...

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[CANCER RESEARCH 52, 1891-1894. April 1, 1992] Inhibitors of Polyamine Biosynthesis Block Tumor Necrosis Factor-induced Activation of Macrophages1 Leszek Kaczmarek, Bozena Kaminska, Luigi Messina, Giuseppina Spampinato, Antonio Arcidiacono, Lucia Malaguarnera, and Angelo Messina2 .\encki Institute of Experimental Biology, 02-093 Harsa*: Pasteara } Poland ¡L.K., B. K.]; and Institute of General Pathology, University of Catania, Via Androne 83, 95124 Catania, Italy ¡L.A.. B. A'., L. Me.. G. S., A. A., L. Ma., A. M.] ABSTRACT The activation of polyamine biosynthesis, dependent on increased gene expression of ornithine decarboxylase, has been found to play an impor tant role in the control of cell proliferation and differentiation. In this report it has been found that accumulation of ornithine decarboxylase mRNA also follows stimulation of human monocytes/macrophages by tumor necrosis factor. Human recombinant tumor necrosis factor (100 units/ml) also evoked an enhanced respiratory burst of macrophages. The respiratory burst response was inhibited in a dose-dependent manner with difluoromethylornithine, an inhibitor of ornithine decarboxylase, and methylglyoxal-bis(guanylhydrazone), an inhibitor of the formation of spermidine and spermine. The data presented in this paper suggest that polyamines may play a functional role in tumor necrosis factor-driven macrophage activation, and they are discussed in the context of their possible use as inhibitors of polyamine metabolism in tumor chemotherapy. INTRODUCTION Many of the observed biological responses to invasive stimuli are mediated by host-secreted cytokines, in particular, the se cretory products of activated macrophages. One such cytokine is TNF,1 also known as cachectin. TNF was originally described by Carswell et al. (1) as an agent found in the serum of Bacillus Calmette-Guérin-immunized mice challenged with endotoxin. The role of TNF was presumed to be the killing of tumor cells (1). Although the initial interest focused on the cytotoxic effect of TNF, recent evidence has demonstrated that TNF must be considered as a compound that can stimulate a variety of cellular activities associated with an immune response (2), including augmenting inflammatory reaction by increasing the phagocytic and cytotoxic functions of neutrophils (3) and stim ulating the release of Superoxide anión (Or) and hydrogen peroxide (H^O?) (4-6). These reactive oxygen intermediates are used by macrophages to mediate host defenses against bacterial and fungal infection (7). In response to various stimulants like IFN-7 and LPS they produce oxygen-derived metabolic prod ucts that destroy target cells (5, 8). TNF can be listed among the macrophage-derived cytokines and it can act in an autocrine fashion, activating mouse resting macrophages and enhancing their cytotoxic potential in vitro (9). However, little is known about the mechanism of activation and regulation of macro phages by TNF. The regulatory role of polyamines (putrescine. Received 9/17/91; accepted 1/24/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This study was supported by grants from Ministero dell'Università e Ricerca Scientifica e Tecnologia (40cc), Associazione Italiana Ricerca sul Cancro (AIRC). Consiglio Nazionale delle Ricerche (CNR) Grant 89.03992.CT04. and the Polish Academy of Science (CPBP 04.01). 2 To whom requests for reprints should be addressed. 'The abbreviations used are: TNF, tumor necrosis factor; ODC, ornithine decarboxylase; IFN->. -y-interferon; LPS. lipopolysaccharide; DFMO, difluoro methylornithine: MGBG. methylglyoxal-bis(guanylhydrazone); PBS. Ca2+-Mg2*- free phosphate-buffered saline: SSC, standard saline-citrate (sodium chloride 0.15 M. sodium citrate 0.015 M. pH 7): PMA, phorbol myristate acetate. spermidine, and spermine) in cell proliferation, differentiation, and tumor transformation has been repeatedly documented using different methods (10, 12). In particular, functional stud ies on polyamines greatly benefited from the use of specific inhibitors such as DFMO or MGBG. DFMO specifically and irreversibly inhibits ODC while MGBG, inhibiting S-adenosylmethionine decarboxylase, blocks the formation of spermidine and spermine (13, 14). ODC converts ornithine into putrescine; thus it is a key and rate-limiting enzyme in the biosynthesis of polyamines. Inhi bition of polyamine biosynthesis by DFMO has been shown to retard tumor growth and metastasis in a variety of different tumor models (14, 15). However, little is known regarding the effect of polyamine depletion on the function of the various tumoricidal effector cells within the immune system. It was shown that LPS and different immunoadjuvants are able to induce ODC in macrophage-like cell lines (16, 17). We have observed an induction of ODC gene expression following acti vation of human and mouse macrophages by bacterial lipopoly saccharide and IFN--y (18). Bowlin et al. (19, 20) demonstrated that following 6 days of DFMO treatment tumoricidal macro phage activity was not impaired. On the other hand, Kierszen- baum et al. (21) showed that the inhibition of ODC activity in macrophages by either DFMO or other specific ODC inhibitors blocks the capacity of macrophages to kill phagocytized para sites. Additionally, Prosser and Wahl (22) demonstrated that DFMO inhibited collagenase production when added to LPS- treated macrophages. In both cases this inhibitory effect was reversed by exogenous putrescine. Recently we have observed that inhibitors of ODC and other enzymes engaged in polyam ine biosynthesis have blocked the functional activation of hu man monocytes/macrophages activated by LPS and IFN-7.4 In the present paper we have demonstrated the ability of TNF to stimulate the expression of ODC in human monocytes and macrophages as well as the importance of polyamines in the functional activation of macrophages, in fact using the antisense oligodeoxynucleotide targeted against ODC mRNA we can directly correlate down-regulation of ODC expression and bi ological effect. MATERIALS AND METHODS Cells and Culture Conditions. Fresh human buffy coats were obtained from a blood bank and. after 2-fold dilution with PBS supplemented with 2.5 mivi EDTA, were layered on the Ficoll-Hypaque (Pharmacia) gradient. After 40 min centrifugaron at 400 x g the interphase of peripheral blood mononuclear cells was collected, washed twice with PBS, and placed in plastic tissue culture flasks at the concentration of 1-2 x 10* cells/cm2 surface area. The cells were cultured in Iscove medium supplemented with 10% fetal calf serum, 2 IÃOEIM glutamine, and 50 Mg/ml gentamicin. After 3-4 h of incubation at 37°Cin an atmos phere of 5% CO2 humidified air, the nonadherent cells were washed out using PBS with the addition of Ca2+ and Mg2+. The adherent cells * Manuscript in preparation. 1891 on April 2, 2021. © 1992 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Transcript of Inhibitors of Polyamine Biosynthesis Block Tumor Necrosis ... · phages by TNF. The regulatory role...

  • [CANCER RESEARCH 52, 1891-1894. April 1, 1992]

    Inhibitors of Polyamine Biosynthesis Block Tumor Necrosis Factor-inducedActivation of Macrophages1

    Leszek Kaczmarek, Bozena Kaminska, Luigi Messina, Giuseppina Spampinato, Antonio Arcidiacono,Lucia Malaguarnera, and Angelo Messina2

    .\encki Institute of Experimental Biology, 02-093 H arsa*: Pasteara } Poland ¡L.K., B. K.]; and Institute of General Pathology, University of Catania, Via Androne 83,95124 Catania, Italy ¡L.A.. B. A'., L. Me.. G. S., A. A., L. Ma., A. M.]

    ABSTRACT

    The activation of polyamine biosynthesis, dependent on increased geneexpression of ornithine decarboxylase, has been found to play an important role in the control of cell proliferation and differentiation. In thisreport it has been found that accumulation of ornithine decarboxylasemRNA also follows stimulation of human monocytes/macrophages bytumor necrosis factor. Human recombinant tumor necrosis factor (100units/ml) also evoked an enhanced respiratory burst of macrophages. Therespiratory burst response was inhibited in a dose-dependent manner

    with difluoromethylornithine, an inhibitor of ornithine decarboxylase,and methylglyoxal-bis(guanylhydrazone), an inhibitor of the formation of

    spermidine and spermine. The data presented in this paper suggest thatpolyamines may play a functional role in tumor necrosis factor-driven

    macrophage activation, and they are discussed in the context of theirpossible use as inhibitors of polyamine metabolism in tumorchemotherapy.

    INTRODUCTION

    Many of the observed biological responses to invasive stimuliare mediated by host-secreted cytokines, in particular, the secretory products of activated macrophages. One such cytokineis TNF,1 also known as cachectin. TNF was originally described

    by Carswell et al. (1) as an agent found in the serum of BacillusCalmette-Guérin-immunized mice challenged with endotoxin.

    The role of TNF was presumed to be the killing of tumor cells(1). Although the initial interest focused on the cytotoxic effectof TNF, recent evidence has demonstrated that TNF must beconsidered as a compound that can stimulate a variety ofcellular activities associated with an immune response (2),including augmenting inflammatory reaction by increasing thephagocytic and cytotoxic functions of neutrophils (3) and stimulating the release of Superoxide anión (Or) and hydrogenperoxide (H^O?) (4-6). These reactive oxygen intermediates areused by macrophages to mediate host defenses against bacterialand fungal infection (7). In response to various stimulants likeIFN-7 and LPS they produce oxygen-derived metabolic products that destroy target cells (5, 8). TNF can be listed amongthe macrophage-derived cytokines and it can act in an autocrinefashion, activating mouse resting macrophages and enhancingtheir cytotoxic potential in vitro (9). However, little is knownabout the mechanism of activation and regulation of macrophages by TNF. The regulatory role of polyamines (putrescine.

    Received 9/17/91; accepted 1/24/92.The costs of publication of this article were defrayed in part by the payment

    of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    ' This study was supported by grants from Ministero dell'Università e RicercaScientifica e Tecnologia (40cc), Associazione Italiana Ricerca sul Cancro (AIRC).Consiglio Nazionale delle Ricerche (CNR) Grant 89.03992.CT04. and the PolishAcademy of Science (CPBP 04.01).

    2To whom requests for reprints should be addressed.'The abbreviations used are: TNF, tumor necrosis factor; ODC, ornithine

    decarboxylase; IFN->. -y-interferon; LPS. lipopolysaccharide; DFMO, difluoromethylornithine: MGBG. methylglyoxal-bis(guanylhydrazone); PBS. Ca2+-Mg2*-free phosphate-buffered saline: SSC, standard saline-citrate (sodium chloride 0.15M. sodium citrate 0.015 M. pH 7): PMA, phorbol myristate acetate.

    spermidine, and spermine) in cell proliferation, differentiation,and tumor transformation has been repeatedly documentedusing different methods (10, 12). In particular, functional studies on polyamines greatly benefited from the use of specificinhibitors such as DFMO or MGBG.

    DFMO specifically and irreversibly inhibits ODC whileMGBG, inhibiting S-adenosylmethionine decarboxylase,

    blocks the formation of spermidine and spermine (13, 14).ODC converts ornithine into putrescine; thus it is a key andrate-limiting enzyme in the biosynthesis of polyamines. Inhibition of polyamine biosynthesis by DFMO has been shown toretard tumor growth and metastasis in a variety of differenttumor models (14, 15). However, little is known regarding theeffect of polyamine depletion on the function of the varioustumoricidal effector cells within the immune system. It wasshown that LPS and different immunoadjuvants are able toinduce ODC in macrophage-like cell lines (16, 17). We haveobserved an induction of ODC gene expression following activation of human and mouse macrophages by bacterial lipopolysaccharide and IFN--y (18). Bowlin et al. (19, 20) demonstrated

    that following 6 days of DFMO treatment tumoricidal macrophage activity was not impaired. On the other hand, Kierszen-baum et al. (21) showed that the inhibition of ODC activity inmacrophages by either DFMO or other specific ODC inhibitorsblocks the capacity of macrophages to kill phagocytized parasites. Additionally, Prosser and Wahl (22) demonstrated thatDFMO inhibited collagenase production when added to LPS-treated macrophages. In both cases this inhibitory effect wasreversed by exogenous putrescine. Recently we have observedthat inhibitors of ODC and other enzymes engaged in polyamine biosynthesis have blocked the functional activation of human monocytes/macrophages activated by LPS and IFN-7.4 In

    the present paper we have demonstrated the ability of TNF tostimulate the expression of ODC in human monocytes andmacrophages as well as the importance of polyamines in thefunctional activation of macrophages, in fact using the antisenseoligodeoxynucleotide targeted against ODC mRNA we candirectly correlate down-regulation of ODC expression and biological effect.

    MATERIALS AND METHODS

    Cells and Culture Conditions. Fresh human buffy coats were obtainedfrom a blood bank and. after 2-fold dilution with PBS supplementedwith 2.5 mivi EDTA, were layered on the Ficoll-Hypaque (Pharmacia)gradient. After 40 min centrifugaron at 400 x g the interphase ofperipheral blood mononuclear cells was collected, washed twice withPBS, and placed in plastic tissue culture flasks at the concentration of1-2 x 10* cells/cm2 surface area. The cells were cultured in Iscove

    medium supplemented with 10% fetal calf serum, 2 IÌIMglutamine, and50 Mg/ml gentamicin. After 3-4 h of incubation at 37°Cin an atmos

    phere of 5% CO2 humidified air, the nonadherent cells were washedout using PBS with the addition of Ca2+ and Mg2+. The adherent cells

    * Manuscript in preparation.

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  • POLYAMINE BIOSYNTHESIS INHIBITORS BLOCK MACROPHAGE ACTIVATION

    (monocytes) were next cultured overnight or for 7-10 days dependingon experimental design. In the experiments for testing the effect ofpolyamine inhibitors the last 48 h of culture were carried out in mediumwithout serum (23). Then the adherent cells were washed once withPBS, supplemented with fresh medium without serum, and treated for24 h with 100 units/ml TNF together with polyamine inhibitors DFMOor MGBG, at different concentrations, and/or 10 MMspermine. In theexperiments with oligodeoxynucleotides (a kind gift of Dr. Ada Sacchi,Istituto Regina Elena, Rome, Italy), human macrophages, cultured for7 days, were washed once with PBS, supplemented with fresh mediumcontaining 20 ng/ml ODC sense (5' ATG AAG TTT GGT AAT GAAGAG 3') or antisense (5' CTC TTC TTA CCA AAG TTC ATC 3')oligodeoxynucleotides and incubated for 6 h at 37°Cin an atmosphere

    of 5% CO¡humidified air. After 6 h the incubation with oligodeoxynucleotides was stopped by one washing with PBS and the adherent cellswere supplemented with fresh medium with the addition of 100 units/ml TNF and incubated again for 24 h.

    RNA Extraction and Hybridization. Following culture, the adherentcells were washed twice with 0.9% NaCI and then guanidium lyseddirectly in the culture flasks for RNA extraction using the procedureof Chomczynsky and Sacchi (24). Fifteen ng of total cellular RNA ofeach sample (as measured by A260 absorption) next underwent electro-phoresis in 1.2% agarose-2.2 M formaldehyde in the presence of 0.5 Mgethidium bromide added directly to the sample. After electrophoresisthe gel was capillary blotted onto nitrocellulose. Prehybridization andhybridization were carried out at 42'C in a mixture containing 50%formamide, 5x SSC, 5x Denhardt's solution. 20 mMsodium phosphate

    buffer (pH 6.5), and 100 ¿igdenatured, sheared salmon sperm DNAper ml. In the case of hybridization, the mixture was supplementedwith 10% dextran sulfate and denatured probe. Human ODC probe(18) was labeled by the random primer method to a mean specificactivity of 0.5-1.3 x 10* cpm/^g DNA. Membranes were hybridizedfor 18-24 h and then were washed twice with a mixture containing 2xSSC and 0.2% sodium dodecyl sulfate for 5 min each time at roomtemperature and 3 times with a mixture of 0.1 x SSC and 0.1% sodiumdodecyl sulfate for 30 min; each washing was at 52°C.Membranes wereexposed for 24-72 h at -70°Cusing intensifying screens.

    Measurement of the Respiratory Burst Activity with Superoxide Assay. Superoxide anión (O:~) release by monocytes and macrophages

    was assessed by spectrophotometric measurement of ferrocytochromec reduction, slightly modified from the procedure originally describedby Babior et al. (25). Monocytes/macrophages were cultured in 96-wellplates for the desired period of time. Adherent macrophages werewashed once with PBS and incubated with 0.2 ml of reaction mixtureconsisting of Krebs-Ringer phosphate buffer containing 80 n\i cyto-chrome c (Sigma), 2 IJIMNaNj, and 100 ng/ml PMA (Sigma). After 1h of incubation the supernatants were collected and used to assay theamount of reduced cytochrome c, determining the difference in absorb-ance at 550-468 nm. The O2~release was calculated using a coefficient

    of 0.0245 (the extinction coefficient ^mol/liter of cytochrome c determined at 550-468 nm) and expressed as nmol O2~/mg cell protein.

    Cell protein was assessed by the method of Lowry et al. (26). Allexperiments were carried out in triplicate. The results are expressed asmean ±SD. The significance of the effects was calculated using Student's / test.

    RESULTS

    In the first experiment we decided to study the effect of TNFon ODC mRNA levels in human monocytes of peripheral bloodcultured for 7 days in order to mature into macrophages. Fig.\A shows that the ODC mRNA levels were significantly induced at 2 h after stimulation with TNF and decreased thereafter. The data presented in Fig. \B are intended to serve as acontrol assuring that essentially the same amounts of RNAwere electrophoresed in each lane. We also observed a similarpattern of ODC mRNA accumulation after the treatment withTNF in cells cultured for other times (including 1-day mono-

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    «MK •¿�*-2.6KB

    Fig. 1. Induction of ODC mRNA in mature human macrophages by humantumor necrosis factor n. Human monoc> tes/macrophages from peripheral bloodwere cultured under standard conditions for 7 days and than treated with TNF a(100 units/ml). The total RNA was extracted from untreated and TNF-treatedcells and processed as described in "Materials and Methods." A, accumulation ofODC mRNA (2.6 kb ). Line I. untreated cells; Lines 2-5, TNF-treated for 0.5, 2.8. 24 h. B, quantification and integrity of each RNA sample confirmed by analysison agarose gels stained with ethidium bromide. For hybridization, filters wereused only when RNA levels (represented by 28S and 18S rRNA) were essentiallythe same for all lanes.

    cytes and 3-day maturing cells, not shown).For the functional studies human macrophages obtained from

    peripheral blood monocytes cultured for 7-10 days were used.The influence of DMFO and MGBG treatment on TNF-in-duced ability of those cells to phorbol ester-triggered respiratoryburst was measured. Fig. 2 documents the inhibitory effect ofDFMO on TNF-induced respiratory burst. DFMO inhibitedO2~ release from TNF treated cells in a dose-dependent manner

    in the absence of serum (the statistical significance of the effectswas P< 0.002). In the presence of serum DFMO did not affectthe level of Superoxide anión production even after 48 h pretreatment with 1 mM DFMO (not shown). Similarly, MGBGsignificantly blocked Superoxide anión production induced byTNF (Fig. 3, again, the statistical significance of the effects wasP < 0.002). The effects of MGBG cannot be explained by itscytotoxicity or nonspecific effects, as it was reversed by 10 MMspermine (Fig. 4). We observed that 10 MMspermine alone didnot activate otherwise nonstimulated macrophages and it didnot have additive effects with TNF on Superoxide release (Fig.4). The spermine experiment was carried out in the absence ofserum because exogenous polyamines exert a cytotoxic effect

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  • POLYAMINE BIOSYNTHESIS INHIBITORS BLOCK MACROPHAGE ACTIVATION

    0 2,5 5 10

    OIMO concentration in milFig. 2. Inhibitory effect of DFMO on TNF-indueed respiratory burst. Human

    monocytes/macrophages from peripheral blood were preincubated for 7-10 days.For the last 48 h mature macrophages were cultured in medium without serumand were treated with either culture medium alone or human tumor necrosisfactor (100 units/ml) alone or TNF-u together with various concentrations ofDFMO. After the treatment period, the respiratory burst measured by cytochromec reduction in response to stimulation with PMA (10 ng/ml). All experimentswere carried out in triplicate. •¿�-PMA; B. +PMA; 2 + PMA + TNF.Columns, means from .1experiments: SD are also shown.

    100

    MGBG concentration in jiMFig. 3. Dose-dependent inhibition of TNF-induced respiratory burst by MGBG.

    Seven-10-day macrophages were cultured in standard medium containing serum.Pretrealment conditions and data presentation as for Fig. I except that MGBGwas used. •¿�-PMA: H. +PMA; B. + PMA + TNF.

    under standard culture conditions including the use of fetal calfserum as a medium component.

    We have to stress that the effects of all the drugs used inthese studies cannot be explained by their cytotoxic activity, aseven drug-treated cells responded to PMA stimulation in allcases, similar to the control cells.

    Fig. 5 shows the inhibitory effect of ODC antisense oligo-deoxynucleotide on TNF-induced O; release.

    DISCUSSION

    The intracellular availability of ODC is controlled on multiple levels, including transcription, translation, protein stability,and endogenous inhibition of the enzymatic activity (10, 11).Rapid accumulation of ODC mRNA has been associated witha stimulation of a plethora of physiological responses, suggesting that ODC genes belong to a group of immediate-early genessuch as c-fos and c-myc protooncogenes and may be involvedin the phenomenon of cell activation (12). In fact, activation ofODC expression has been repeatedly linked to the stimulationof cell proliferation and induction of differentiation.

    Recently, we have also shown ODC mRNA accumulation interminally differentiated monocytes and macrophages afterstimulation of these cells with LPS and IFN--y (18). Some

    findings demonstrated that the ODC pathway is an importantintracellular component in the sequence of events that lead tofunctional activation of macrophages (21, 22). The objective ofour investigation was to study whether polyamines could alsobe involved in TNF-driven macrophage activation. Our initialexperiments showed that TNF augmented the ODC mRNAlevel in mature macrophages (severalfold). In other experimentswe found that this was also true for 1-day cultured humanmonocytes and for the mononuclear phagocytes cultured for 3days (data not shown). Moreover, functional activation of macrophages by TNF, namely Superoxide anión production, wasstrongly inhibited by both DFMO and MGBG. These effectscannot be explained by the cytotoxic effects of either drug,inasmuch as both control and inhibitor-treated cells (at allconcentrations tested) responded very well to PMA stimulation.The range of concentrations we used in these studies are wellwithin the range used by different authors studying differentcell types. In our experiments DFMO exerted an inhibitoryeffect only in the absence of serum. We did not observe an

    o 4n.

    î,

    o

    i

    Nihil 25 |IM MGBG MGBG+Spermii» 10 UM Spermin»

    Fig. 4. Reversal of MGBG inhibition of TNF-induced respiratory burst by 10(iM sperminc. Pretrealment conditions as for Fig. 1 including the lack of serumin culture medium. Data presentation as for Figs. 1 and 2. •¿�-PMA: B. +PMA;SS, + PMA + TNF

    Untreated TNF-treated

    Fig. 5. The inhibitory effect on O; production induced by oligodeoxinuclcotideODC-antisense. Human macrophages cultured for 7 days were pretrcated with20 ng/ml ODC sense or antisense oligodeoxynucleotides for 6 h and then wereactivated with 100 units/ml TNF for 24 h and tested for Or production as inFig. 2. •¿�.nihil: BS.ODC-sense: B. ODC-antisense.

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    inhibitory effect of DFMO in the presence of serum.This can be explained by the fact that DFMO treatment can

    be overcome by compensatory phenomena that neutralize theeffect of the inhibitor (14). These include the increased uptakeof polyamines from the environment (27, 28) and the interconversion of higher polyamines to lower ones due to the action ofpolyamine oxidases, which occur in the serum (23, 29). MGBGalso inhibited Superoxide aniónproduction in a dose-dependentmanner. The inhibitory effect of MGBG was reversible byspermine. However, we found that MGBG was more potentthan DFMO in blocking the physiological functions of macrophages. It is known that the spermine content is not greatlyaffected by the application of DFMO; however, DFMO produces a virtually complete reduction of putrescine. Our resultssuggest that inhibitors of other steps in polyamine productionsuch as MGBG leading to increased spermine depletion have astronger effect on the cell function than DFMO. This findingsuggests that MGBG also affects the interconversion pathwaysof polyamines and produces stronger depletion of intracellularpolyamines than DFMO. Furthermore, the involvement ofpolyamines on TNF-induced respiratory burst is confirmed bythe evidence that the antisense oligodeoxynucleotide targetedagainst ODC mRNA, blocking the synthesis of the key andrate-limiting enzyme of the biosynthesis of polyamines, inhibitsOi~ release from TNF-treated macrophages. In conclusion, the

    data presented in this paper further strengthen the fact thatODC and polyamines play an important role in macrophageactivation. It seems of particular interest that this is also truefor TNF-driven functional activation of mononuclear phagocytes. It has been repeatedly shown that the activity of ODC iscritical for cell proliferation (10, 11). In consequence, it hasbeen proposed that inhibitors of polyamine formation can beuseful in tumor chemotherapy. Despite some promising resultsin experimental models, clinical trials of DFMO or MGBGwere not successful (14).

    However, results presented here raise a doubt whether treatment with polyamine inhibitors would be beneficial because itmay produce adverse effects on potentially important tumori-cidal effector cell activity. Our results, demonstrating that inhibitors of polyamine biosynthesis block the respiratory burstevoked by TNF, show an example of impairment of an important tumoricidal function of the immune system as the consequence of polyamine depletion.

    ACKNOWLEDGMENTS

    The authors thank Professor B. Cassia from Servizio di Immunoe-matologia e Trasfusionale, I SI 34, Ospedale Garibaldi, Catania, Italy,for preparation of buffy coats.

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  • 1992;52:1891-1894. Cancer Res Leszek Kaczmarek, Bozena Kaminska, Luigi Messina, et al. Factor-induced Activation of MacrophagesInhibitors of Polyamine Biosynthesis Block Tumor Necrosis

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