correlations between polyamine analogue-induced increases...

[CANCER RESEARCH 51. 3715-3720, July 15, 1991]

Correlations between Polyamine Analogue-induced Increases in Spermidine/Spermine A^-Acetyltransferase Activity, Polyamine Pool Depletion,and Growth Inhibition in Human Melanoma Cell Lines1

Carl W. Porter,2 Barbara Ganis, Paul R. Libby, and Raymond J. Bergeron

Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263 [B. G., P. R. L., C. W. P.], and Department of Medicinal Chemistry,J. Hillis Miller Health Center, university of Florida, Gainesville, Florida ÃŒ2610[R. J. B.]

ABSTRACT

The poh amino analogue, /V'tA^-bisÃethyOspermine (BESPM), isknown to suppress ornithine and 5-adenosylmethionine decarboxylaselevels, deplete intracellular polyamine pools, and inhibit cell growth.Among human melanoma cell lines, MALME-3 cells were found to betypically sensitive to the antiproliferative activity of the BESPM, whereasI <)\ cells were atypically insensitive to the analogue. A comparison ofpolyamine-related parameters revealed that the most differentially altered activity between the 2 BESPM-treated cell lines was that ofspermidine/spermine W-acetyltransferase (SSAT), which increased from50 pmol/min/mg to greater than 10,000 pmol/min/mg in MALME-3 cellsand from 16 pmol/min/mg to only 120 pmol/min/mg in LOX cells over48 h. The basis for the large difference seems to be related to increasedenzyme synthesis in both cell lines coupled with differences in prolongation of SSAT half-life (>12 h in MALME-3 cells versus 1.6 h in LOXcells) after BESPM treatment. In MALME-3 cells, SSAT accumulationwas found to be differentially modulated by the BESPM homologues,A",A"'-bis-(ethyl)norspermine and A",A"4-bis-(ethyl)homospermine,which were 5-fold more and 9-fold less effective, respectively, thanBESPM in increasing SSAT but similar in analogue uptake and effectson polyamine biosynthesis and cell growth inhibition. Treatment ofMALME-3 cells with BESPM resulted in an accumulation of ,V-;n:rlyl-spermidine in cells and the enhanced excretion of putrescine, spermidine,and /V-acetylspermidine into the medium. The relationship between SSATinduction and growth sensitivity was deduced to be a possible function ofincreased excretion of acetylated polyamines leading to enhanced polyamine pool depletion. The data suggest that, in cell types in which it occurs,unusually high increases in SSAT activity may serve as a determinant ofgrowth sensitivity to bis-ethyl spermine analogues or, alternatively, as atarget for appropriately designed chemotherapeutic strategies.

INTRODUCTION

The association of increased polyamine biosynthesis withgrowth-promoting stimuli and carcinogenesis has led to thedevelopment of inhibitors of all 4 of the biosynthetic enzymesâ€”most notably, the 2 lead-in enzymes, ODC' and AdoMetDC.

This approach has been confounded, however, by compensatoryresponses mediated by the rapid turnover of the target enzymesand by sensitive regulatory mechanisms inherent in the pathway(reviewed in rÃ©f.1). As an alternative to the development ofenzyme inhibitors, we have focused on the identification ofpolyamine analogues that interfere with the polyamine pathwayby regulating synthesis of the rate-limiting biosynthetic en-

Received6/29/90;accepted5/1/91.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.

1This work has been supported in part by Grants CA-51524, CA-22153, CA-37606, and CA-24538 from the National Cancer Institute, and by a 1989 grantfrom the Buffalo Foundation (Buffalo. NY).

1To whom requests for reprints should be addressed, at Roswell Park Cancer

Institute. Grace Cancer Drug Center. Elm and Carlton Streets, Buffalo, NY14263.

1The abbreviations used are: ODC. ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase, BEHSPM, A'l,A'u-bis(ethyl)homospermine;BENSPM, A",A"'-bis(ethyl)norspermine; BESPM, /V',A"M>is(ethyl)spermine;SSAT, spemidine/spermine-iV'-acetyl-transferase; T,/;, half-life.

zymesâ€”ODC and AdoMetDC (1, 2). The strategy exploitstheir high sensitivity to synthetic control by intracellularpolyamine pools. Thus, selected polyamine analogues canachieve rapid suppression of both enzyme activities leading toa near-total depletion of intracellular polyamine pools (3).Furthermore, the analogues prevent compensatory increases inother polyamine enzyme activities that typically accompany theuse of enzyme inhibitors (4, 5). The cellular effects, theirunderlying mechanisms, and the antitumor activity of many ofthese analogues have been extensively studied in the LI210leukemia (6-13) and certain solid tumor cell lines (14-16).

From among a relatively large series of spermidine andspermine analogues (7-9), the bis-ethyl derivatives of spermineand certain of its homologues (Fig. 1) have emerged as the mostpromising antiproliferative agents directed at polyamine biosynthesis (8). Evaluation of several of these analogues in theNational Cancer Institute in vitro human tumor screening panelrevealed high sensitivity in 6 of 9 human non-small cell lungtumor lines [in agreement with previous findings by Casero etal. (14)] and in all of 5 human melanoma lines (17). In anattempt to identify novel antiproliferative mechanisms amongmelanoma cell lines, we have compared the cellular responsesto bis-ethyl analogues of spermine in the typically sensitive(MALME-3) and atypically insensitive (LOX) human melanoma cell lines. The results indicate that the sensitivity of theMALME-3 cells may be related to a major perturbation of

polyamine homeostasis mediated by an unusually large induction in SSAT activity, which does not occur in the LOX cells.A nearly identical effect has been recently noted in a large celllung carcinoma line (15), which again seems to correlate withthe growth sensitivity to the analogues. Depending on theprevalence of this unusual response among other human melanoma and large cell lung carcinoma or even unrelated tumorcell lines, these findings may have relevant implications in thedevelopment of treatment strategies for selected human cancers.

MATERIALS AND METHODS

The spermine analogue BESPM and its homologues (Fig. 1),BENSPM and BEHSPM, were synthesized according to publishedprocedures (6, 18). The human melanoma cell lines, MALME-3 andLOX, were generously provided by Dr. Robert Shumaker and colleagues of the National Cancer Institute Tumor Testing Laboratory(Frederick, MD).

Cell Culture. Human melanoma cells were maintained as monolayercultures growing in RPMI 1640 containing 2% jV-2-hydroxypiperazine-/V2-ethanesulfonic acid/3-Ar-morpholinopropanesulfonic acid (pH 7.4)

as a buffer, I imi aminoguanidine as an inhibitor of serum oxidase,and 10% NuSerum as a semi-defined serum substitute (CollaborativeResearch Products, Bedford, MA). For growth studies, cells were seededat 2 x IO5cells per T-75 flask and incubated for 24 h before treatment

with analogues was begun. Treatment was continued for up to four celldoublings without medium change. This amounted to approximately 4days for LOX cells and 8 days for MALME-3 cells. Control and treated

3715

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MELANOMA SENSITIVITY TO POLYAMINE ANALOGUES

BISIETHYLISPERMINEANALOGS

CH3CH2NH(CH2)nNH(CH2)nNH(CH2)J1NHCH2CH3

l l l

BEHSPMBESPM

Fig. I. Structural representations of the SPM analogues. BESPM. BENSPM,and BEHSPM.

cell cultures were trypsinized. and cell suspensions were prepared forgrowth determinations and biochemical assays. Growth was determinedusing a Coulter Model ZM electronic cell counter (Coulter Electronics,Hialcah, FL) and biochemical assays were performed as describedbelow.

Knzyme Assays. SSAT activity was measured using a cell extractobtained by sonication of treated or control cells in 5 mM jV-2-hydrox-ypiperazine-,V:-ethanesulfonic acid (pH 7.2) containing 1 mM dithio-thrcitol. The cytosolic extract resulting from a 1-h centrifugation at35,000 rpm in a Spinco 40 rotor was used as the source of enzyme.SSAT activity was determined according to the method of Libby et al.(12). It should be noted that in untreated cells in particular, the assayalso measures other enzyme activities capable of acetylating spermidine.Using SSAT-specific antiserum provided by Dr. A. E. Pegg (Pennsylvania State University College of Medicine, Hershey, PA), it was foundthat authentic (immunoprecipitable) SSAT typically accounted for approximately 21% of the assay-detectable activity under basal conditionsin MALME-3 cells and greater than 96% after BESPM treatment.Data are reported as total SSAT activity. Similar results were seen withSSAT from LOX cells.

The extract for assaying polyamine biosynthetic decarboxylase activities was prepared by sonicating control and treated cell pellets in 10mM Tris/HCI (pH 7.2) containing 0.5 mM EDTA, 5 mM dithiothreitol,and 50 /JMpyridoxal phosphate followed by centrifugation at 10,000 xg for 20 min. Ornithine and S-adenosylmethionine decarboxylase activities were determined according to the method of Pegg and Seely (19)and Pegg and Poso (20). respectively, based on quantitation of radio-labeled CO: released from l-[14Cj-labeled ornithine or S-

adenosylmethionine.All enzyme activities were expressed on the basis of mg protein as

determined by the method of Bradford (21) using a commercial kit(Bio-Rad Laboratories, Richmond, CA).

Polyamine Pools. Samples of I x IO7control and treated cells were

taken for polyamine determinations and extracted with 0.6 Mperchloricacid. The extract was analyzed by high pressure liquid chromatographyusing a modification of the methodology of Kabra et al. (22), in whichmethanol was substituted for potentially toxic acetonitrile as describedby Chang et al. (23). Polyamine and polyamine analogue levels werethen calculated based upon internal standard (1,7-diaminohexane) levels and external standard curves run within 48 h of the samplechromatograms.

For experiments in which polyamine content of cells was comparedwith that of medium, LOX and MALME-3 cell cultures were treatedfor up to 48 h with 10 JIMBESPM. Cells were harvested and processedas above. Media samples were treated with 50% trichloroacetic acid togive a final 10% concentration. After centrifugation at 15,000 x g for10 min, the supernatant was filtered through 0.22-^m filters, extractedwith several volumes of ether, lyophilized. and resuspended in 10%trichloroacetic acid for polyamine determinations.

Using this high-pressure liquid Chromatographie methodology, itwas not possible to distinguish A"-acetylspermidine from /V-acetyl-

spermidine. The latter was accomplished in representative samples bythin layer (silica plate) chromatography (24) after dansylation (22). Theproportion of the A'1- to JV8-acetylspermidÂ¡newas then estimated for

representative samples.

SSAT Ti/2 Determination. Cells were pretreated for 24 h with 10 MManalogue and then with 50 Â¿ig/mlcycloheximide in the continuedpresence of the analogue for periods up to 12 h at various times.Cycloheximide-treated and untreated cultures were harvested and processed for SSAT measurements.

RESULTS

As shown in Fig. 2, the MALME-3 human melanoma cellsare much more sensitive to the growth-inhibitory effects ofBESPM than the LOX cells. In addition, MALME-3 cells giveindication of a cytotoxic response at 10 UM analogue since aslight but consistent decrease in cell number was observedbetween cell doublings 2 and 4. The relative insensitivity ofLOX cells to BESPM is not likely to be attributable to differences in growth rates since LOX cells double more rapidly (22-26 h) than MALME-3 cells (43-46 h). In principle, rapidlydividing cells should be more sensitive to inhibition of polyamine biosynthesis than slowly dividing cells, since pools wouldbe expected to be more rapidly depleted via daughter celldilution. Furthermore, as indicated in Table 1, uptake ofBESPM was similar in both cell lines, and there was no high-pressure liquid Chromatographie evidence for BESPM metabolism by either line.

The basis for the greater sensitivity of the MALME-3 cellswas further investigated by examining BESPM effects onpolyamine-related enzymes (Table 1). Total suppression of bothODC and AdoMetDC activities was achieved by 48 h inMALME-3 cells, whereas in the LOX cells, ODC activity wasfully suppressed and AdoMetDC reduced by about 80%. Whilethe decarboxylases were similarly affected in both cell lines,SSAT activity was not. By 48 h, BESPM-induced SSAT activitywas greater than 12,405 pmol/min/mg as compared with 120in LOX cells (Table 1). The induced values may somewhat

BESPM Growth InhibitionHuman Melanoma Cells

100

XMM

Zn

Cell Doubling UnitsFig. 2. Effects of 1 and 10 Â«IMBESPM (closed symbols) on the growth of

MALME-3 (O) and LOX (O) cells. Note that growth of MALME-3 cells is muchmore sensitive than that of LOX cells. Time is expressed in cell doubling unitsfor control cells, which was 22-24 h for LOX cells and 43-48 h for the MALME-3 cells. Data represent mean values taken from duplicate experiments in whichagreement was within 10% of the mean.

3716




underestimate the actual SSAT activity since the analogueswere not dialyzed away from the enzyme-containing extractbefore assaying. The kinetics of this increase are shown in Fig.3A. With both LOX and MALME-3 cells, SSAT rapidly roseto near-maximum activities and then plateaued at levels thatwere markedly different for the 2 cell lines. The magnitude ofthis difference suggests the possibility that SSAT could berelated to the greater growth sensitivity of MALME-3 cells.

At least a portion of the difference in SSAT response betweencell lines may be attributable to differences in the T,/2 of theSSAT protein. In LOX cells treated with BESPM, it was foundto be 1.6 h and greater than 12 h in the MALME-3 cells (Fig.3B). Given the extremely low levels of true SSAT activity inuntreated cells, it was not possible to reliably measure enzymehalf-life in the absence of BESPM in either cell line.

The rapid and dramatic increase in SSAT activity in theMALME-3 cells relative to LOX cells suggested that polyaminepools may be differentially depleted in the 2 cell lines as a resultof increased excretion of acetylated polyamines by MALME-3cells. SSAT has been shown to be involved in the interconversion of spermidine and spermine (25-27) and implicated inregulating polyamine export (28, 29). Table 2 compares thedecline of polyamine pools in cultures of BESPM-treated LOXand MALME-3 cells. Despite having a longer doubling time,MALME-3 cells were much more rapidly depleted of polyaminepools than LOX cells. Consistent with high SSAT levels, N-acetylspermidine pools were raised in MALME-3 cells byBESPM treatment while remaining below detectable levels inLOX cells. Finally, BESPM induced excretion of putrescine,spermidine and, particularly /V-acetylspermidine to a muchgreater extent in MALME-3 cells than LOX cells. By thin layerchromatography, the /V-acetylspermidine was found to be 90%/V'-acetylspermidine in BESPM-treated cell culture medium.Only trace amounts of /V-acetylspermine were detected (Table2).

When the same polyamine data for LOX and MALME-3cells are converted to pmol/106 cells and expressed as shown

in Fig. 4, it is apparent that BESPM treatment causes the poolsof MALME-3 cells to fall much more rapidly than predictedby dilution via cell division, but not those of LOX cells. Although polyamine depletion occurs more rapidly in MALME-3 cells, significant growth inhibition is not registered until after48 h (Table 1) due to the long doubling time of these cells (44-48 h). It is possibly also relevant that putrescine pools wereretained to a greater extent in MALME-3 cells, presumablybecause of back-conversion from spermidine via the action of

SSAT Effects by BESPMHainan Melinomi Cells

BSSAT T1/2 by BESPM

Human Melinomi Celli

DoublmÂ«Uniti Time <hrt>

Fig. 3. Comparison of effects of treatment with 10 /JM BESPM on accumulation of SSAT activity (A) and enzyme half-life (B) in MALME-3 (Â»)and LOX(â€¢)cells. Using SSAT-specific antiserum, it was found that authentic SSATroutinely accounted for approximately 27% of the assay-detected activity underbasal conditions in MALME-3 cells and greater than 96% after BENSPMtreatment. Similar results were seen with SSAT from LOX cells. Data representmean values taken from duplicate experiments in which agreement was within10% of the mean.

the induced SSAT activity together with polyamine oxidase.To further characterize the phenomenon of SSAT induction

in MALME-3 cells, the effects of two homologues of BESPM(Fig. 1) were compared with BESPM, since in previous studies(12,13) they were found to differentially induce SSAT in LI210cells. BENSPM and BESPM produced nearly identical effectson the biosynthetic enzyme activities, ODC and AdoMetDC,and on polyamine pools (Table 3). BEHSPM had a similareffect on the enzymes, but was less effective in depleting spermine pools. Of the 3 homologues, BENSPM produced the mostimpressive stimulation of SSAT activity, achieving levels in therange of 60,000 pmol/min/mg after only 48 h (Fig. 5B).BESPM increased SSAT to about 10,000 pmol/min/mg andBEHSPM, to 1200 pmol/min/mg. Interestingly, all 3 homologues caused SSAT activity to rise to a maximum after onecell doubling and remain relatively constant thereafter. In contrast to expectations, growth of MALME-3 cells was not differentially affected by the 3 homologues (Fig. 5A).

All 3 homologues accumulated to relatively the sameamounts in MALME-3 cells (Table 3), indicating that differences in uptake could not account for differential SSAT responses. Half-life determinations of the stimulated SSAT activities revealed that for all 3 homologues, the values were greater

Table I A comparison of BESPM effects on polyamine-related enzymes in MALME-3M and LOX human melanoma cell lines"

Cell line andtreatmentMALME-3Control,

48 hlOÃÃ.viBESPMControl,

96 hlOfiMBESPMLOXControl,

48 h10 MMBESPMControl.

96 h10 MMBESPMGrowth

(% ofcontrol)100

78100

24100

91100

55BESPM*

(pmol/106

cells)None

4,504None

5,576None

4,976None

4.944Dccarboxylase

activities(nmolCO2/h/mg)ODC1.30

NDC1.41

ND0.63

ND0.48

NDAdoMetDC1.72

ND1.63

ND0.94

0.170.71

NDSSAT

activity(pmol/min/mg)49

12.40538

8,72016

12029

180Â°Data represent mean values taken from duplicate experiments in which agreement was obtained within 10% of the mean.* No indication of BESPM metabolism by either cell line was observed by high-performance liquid chromatography.c ND, not detectable (i.e., 0.05 nmol CO2/h/mg for ODC or AdoMetDC).

3717



MELANOMA SENSITIVITY TO POLVAMINE ANALOGUES

than 12 h and could not be accurately determined by the useof cycloheximide (data not shown). The increase in SSATin MALME-3 cells treated with the most potent inducer,BENSPM, was almost totally blocked by concomitant exposureto cycloheximide and inhibited by approximately 50% withactinomycin D (Table 4), suggesting that the effect was dependent on new and/or continued protein synthesis and partiallydependent on new and/or continued mRNA synthesis. In LOXcells, cycloheximide also blocked enzyme induction, but actinomycin D did not, suggesting a differential dependence onRNA synthesis between the 2 cell lines (Table 4).

DISCUSSION

In the absence of other comparably different BESPM-me-diated polyamine effects between LOX and MALME-3 melanoma cell lines, it is tempting to at least partially relate the 80-fold greater accumulation in SSAT activity in MALME-3 cellsto their much greater sensitivity to growth inhibition by theanalogue. This possibility is further suggested by the studies ofCasero et al. (15), which showed that, as with the melanomacells, the differential sensitivity of human large and small celllung carcinoma cell lines also correlated with substantial differences in analogue-induced SSAT induction. SSAT catalyzesthe /V'-acetylation of the terminal amines of the aminopropyl

moieties of spermine and spermidine, which in turn can eitherbe excreted by the cell (28, 29) or back-converted to spermidineand putrescine, respectively, after oxidation by the cytosolicenzyme, polyamine oxidase (25-27, 30). In either case, the endresult could lead to an enhanced depletion of spermidine andspermine pools over that achieved by analogue-mediatedsuppression of ODC and AdoMetDC alone.

While the importance of individual polyamine pools to cellgrowth is undetermined for most cell types, recent experimentsby Kramer et al. (31) indicate that any of the 3 polyamines cansupport cell growth to some degree and that maximum growthinhibition is achieved by maximum depletion of all three. Thus,more rapid and extensive pool depletion represents one way inwhich SSAT induction could contribute directly to growthinhibition. This seems to be the case since, as illustrated in Fig.4, the nonacetylated polyamine pools of MALME-3 cells de

tox Melinomi CelliB

MALME-3 Melanoma Cells

It -

-TU-

0 kr < kr 12 kr 14 hi 41 kf

Treatment (10 nM BESPM)41 kr

Treu me n t (10 u M BESPM)

Fig. 4. Decline of polyamine pools in BESPM-trealed LOX (A) and MALME-3 (B) melanoma cells. The nonacetylated intracellular polyamine pool datapresented in Table 2 have been converted to pmol/106 cells and plotted cumula

tively for each treatment time. Note that the total cellular polyamine pools declinemore rapidly than predicted by pool dilution due to cell division ( ) TorMALME-3 cells but not LOX cells. The predicted line for MALME-3 cellsactually overestimates polyamine pool depletion by cell division since it is basedon the doubling time of control cells (43-48 h) and not that of treated cells, whichwould be longer. â€¢SPM; B. SPD; D, PUT.

cline during BESPM treatment at a rate significantly greaterthan can be accounted for by pool dilution by cell division. Bycontrast, the LOX cell pools decline at close to the rate predicted by cell division. The analysis of total polyamine pools incells and medium of BESPM-treated MALME-3 cultures reveals that a significant portion of the missing polyamines isapparent as A'-acetylspermidine in cells and as putrescine, spermidine, and especially A'-acetylspermidine in the culture me

dium. In addition, these cells retained a significant pool ofputrescine during treatment, which is probably indicative ofback-conversion from spermidine and ultimately, spermine

pools. Taken together, the data are in agreement with recentreports by Pegg et al. (32) and provide evidence for a mechanismof enhanced polyamine depletion and possibly growth inhibition in cell types in which SSAT is inducible to high levels.

The phenomenon of SSAT induction in MALME-3 cells wasfurther investigated using homologues of BESPM, which werepreviously shown to have profoundly different effects on SSAT

Table 2 Comparison of effects of BESPM on intracellular and extracellular polyamine pools in melanoma cell cultures

Cells/treatment (MMBESPM)MALME

3cells'Control

6hBESPM6hControl12hBESPM12hControl

24hBESPM24hControl

48hBESPM48hLOX

cells'Control

6hBESPM6hControl12hBESPM12hControl

24hBESPM24hControl

48hBESPM48hÂ°

Data represent meaninPUT1.20.71.90.51.90.42.9ND0.3N

D0.7ND0.6ND0.5NDvalues

based on diIntracellular"

(nmol/culture)SPD3.62.23.30.94.2ND5.9ND3.21.64.11.57.71.612.41.4jplicate

sampAcSPD0.41.10.31.30.10.70.10.1NDNDNDNDNDNDN

DNDles.

PUT, |SPM3.93.83.82.64.61.26.10.22.72.13.32.26.03.611.73.2jutrescine;SPD.Total9.17.89.35.310.82.315.00.36.23.78.13.714.35.224.64.6spermine;Extracellular,

medium"(nmol/culture)PUT0.60.20.10.1ND0.1ND1.31.01.0NDND1.0ND1.0NDAcSPD,

;V-acet;SPDND'1.80.10.8ND0.8ND0.6ND1.1ND1.51.02.11.02.1ylspermidine:AcSPD0.40.70.72.20.74.90.36.1NDNDNDNDNDNDND1.0;SPM, spSPM*NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDermine.Total1.01.70.93.10.75.80.36.71.02.1ND1.52.02.12.03.1* Only trace amounts of acetylated spermine were detected in MALME-3 cell culture medium.c Cell line doubling times in this experiment: MALME-3, 46 h; LOX, 20 h.d ND, not detectable at <0.1 nmol/culture.

3718




Table 3 A comparison ofBESPM homologue effects on polyamine metabolism in MALME-3M human melanoma cell lines"

TreatmentControl,

48h10KMBESPMlO^MBEHSPM10

KMBENSPMControl,

96h10^MBESPM10/iMBEHSPM10â€žM

BENSPMGrowth

(%control)100788674100242020Decarboxylase

activities(nmolCO2/h/mg)ODC1.30NDCNDND1.41NDNDNDAdoMetDC1.72ND0.40ND1.63ND0.13NDPolyamine

pools* (pmol/106cells)PUT2.3NDNDND5.1NDNDNDSPD6.3NDN

DND15.9NDNDNDSPM8.2ND2.1ND19.3ND0.4NDAnalogueNone15.316.513.9None21.419.319.6SSAT

activity(pmol/min/mg)4912,4051,56561,385388.7201,24063,460

" Data represent mean values taken from duplicate experiments in which agreement obtained within 10% of the mean.* PUT, putrescine; SPD, spermidine: SPM. spermine.c ND. not detectable (i.e., <0.1 nmol/culture or 0.05 nmol CO2/h/mg for ODC or AdoMetDC).

B

Analog Growth InhibitionMALME-3 Oil!

Analog IncreaseÂ«in SSATMALME-3 CellÂ«

Cell Doubiinj UnitÂ« Cell Doublini UnitÂ«

Fig. 5. Comparison of the relative effects of the homologues BESPM (*,broken line), BENSPM (T), and BEHSPM (A) on MALME-3 cell growth (A)and SSAT accumulation (B). Cells were seeded 24 h before treatment withanalogues at 10 //\i. Data for BESPM are the same as presented in Fig. 2, hencethe broken line.

induction in other less responsive cell types (12, 13, 16) whilehaving similar effects on the polyamine biosynthetic enzymes.Although these homologues produced significantly differenteffects from BESPM on SSAT accumulation in MALME-3cells, it was unexpected to observe that, in contrast to comparisons between LOX and MALME-3 cells, there was no correlation between SSAT accumulation and cell growth inhibition.In fact, all 3 analogues resulted in similar growth inhibition(Fig. 5). This finding is similar to observations made in LI210(13) and HT-29 cells (16). In the case of the MALME-3 cells,it must be noted that even the weakest effector of SSATaccumulation, BEHSPM, produced enzyme levels at least 10times greater than those produced by BESPM in LOX cells.Thus, differential sensitivity between cell lines may be relatedto the level of SSAT achieved while, between homologueswithin the MALME-3 cell line, this level may be in excess ofthat necessary to differentially affect growth.

At least some indication for the mechanistic basis for SSATaccumulation was obtained. Studies with inhibitors of proteinsynthesis and transcription revealed that both processes seemto be necessary for SSAT induction in MALME-3 cells (Table4). These findings are in agreement with the recent report byPegg et al. (32) and Casero et al. (33), showing that inductionof SSAT is due to an induction of enzyme protein related tothe combined effects of increased enzyme-specific mRNA accumulation and decreased enzyme protein degradation. Previously, we have shown that in LI210 cells in which enzymeaccumulation was comparable with that observed in LOX cells,

prolongation of enzyme half-life by BESPM appeared to be afactor (12). Further evidence for this involvement was alsoapparent in half-life comparisons between different melanomacell lines. In BESPM-treated LOX cells, the SSAT T,/2 was 1.6h and greater than 12 h in the treated MALME-3 cells. Sincethe analogue contains 2 terminal ethylaminopropyl groups thatbear structural resemblance to the acetamidopropyl moiety ofthe enzyme product, it might be expected to behave as anenzyme inhibitor. In fact, kinetic studies reported elsewhere(34) revealed that all 3 homologues were effective competitiveinhibitors of SSAT and that their potency as inhibitors correlated with the number of aminopropyl moieties present withintheir structures (and hence with the substrate requirement ofthe enzyme) and with the level of SSAT accumulation producedby each analogue (Table 5).

Although speculative at this time, it is possible that therapidly turning over SSAT protein is stabilized by competitivehomologue inhibition, thus allowing it to accumulate in cellsto levels at which an exchange equilibrium is reached betweenunbound and analogue-bound enzyme. Free enzyme would thusbe available to either react with the substrate as indicated bythe accumulation of A'-acetylspermidine in MALME-3 cells

(Table 2) or to be acted upon by degradative processes. Suchan equilibrium and ultimately, the level of SSAT accumulation,would be determined by the intracellular concentration of thehomologue and by its particular affinity for the enzyme. Thequalitative correlation between relative K, values and enzymeaccumulation (Table 5) is consistent with this possibility. As an

Table 4 Effect of actinomycin D and cycloheximide on SSAT accumulation inBENSPM-treated human melanoma cells"

MALME-3 LOX

Inhibitor* Control 10 /IM BENSPM Control BENSPM

None 17.2 Â±1.8 2183 Â±27Cycloheximide (0.18 HIM) <1.0 157.1 Â±14Actinomycin D (4 ^M) 33.2 Â±4.1 1040 Â±33

I8.6Â± 2.9 57.8 Â±2.419.8Â± 1.5 24.6 Â±4.824.1 Â±1.2 58.7 Â±3.3

" Data represent mean (Â±SEM)taken from duplicate experiments.* Cells were treated simultaneously with the inhibitor plus 10 JIM BENSPM

analogue for 6 h. In both cell types, inhibitor treatment reduced total protein andRNA synthesis by approximately 90%.

Table 5 Comparison ofSSA T inhibition and SSA T induction in MALME-3 Mcells by spermine analogues

AnalogueBEHSPM

BESPMBENSPMAminopropyl

units0

23SSAT

inhibition"(Ki,

UM)17.0

1.90.8SSAT

increase*

(10 ,iM, 48h)24-fold

200-fold1200-fold

' Apparent K, versus spermidine. Data taken from Libby et al. (34).' Based on total activity.

3719




alternative explanation for these observations, SSAT accumulation could be due to differential increases in enzyme synthesis.Although increased transcription and enzyme synthesis seemto occur, this possibly would require that transcription bemediated by the enzyme-analogue complex itself or by a proteinwith analogue binding properties similar to those of the enzymein order to account for the correlations seen in Table 5.

As proposed by Seiler (28) and Wallace (29), acetylation mayrepresent a cellular mechanism for regulating intracellularpolyamine levels and distribution. It has been demonstratedhere that analogue-induced increases in SSAT activity stronglyaffect cellular polyamine homeostasis (Fig. 4). Thus, the perturbations produced by polyamine homologues could be expected to contribute significantly to homeostatic imbalances,polyamine depletion, and ultimately to the cellular growthresponse. Depending on the prevalence of this unusual enzymeresponse among other human melanoma, large cell lung carcinoma or even unrelated tumor cell lines, the phenomenon couldhave importance as a determinant for tumor selectivity by theanalogues or as a target for approximately designed chemo-therapeutic strategies.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the skilled technical assistanceof Edwin Kelly and John Miller and the expert secretarial assistance ofJessie Crowe and Anne Culligan.

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1991;51:3715-3720. Cancer Res Carl W. Porter, Barbara Ganis, Paul R. Libby, et al. LinesPool Depletion, and Growth Inhibition in Human Melanoma Cell

-Acetyltransferase Activity, Polyamine1NSpermidine/Spermine Correlations between Polyamine Analogue-induced Increases in

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