Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP

Eur. J. Biochem. 21 7, 89-96 (1993) 0 FEES 1993

Correlation between the inhibition of cell growth by accumulated polyamines and the decrease of magnesium and ATP Yong HE', Keiko KASHIWAGIl, Jun-ichi FUKUCHI', Kiyoshi TERAO', Akira SHIRAHATA' and Kazuei TGARASHI I ' Faculty of Pharmaceutical Sciences, Chiba University, Japan ' Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan ' Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan

(Received April 30, 1993) ~ EJB 93 062716

The mechanism of the antiproliferation effect of spermidine and spermine was studied using a cell culture system of mouse FM3A cells. The addition of either 10 mM spermidine or 2 mM spermine to the growth medium containing 0.9 mM Mg2+ greatly inhibited cell growth (more than 90%). A decrease in the Mg2+ concentration to 50 pM in the growth medium, but without the polyamine addition, did not intluence ccll growth. Howcver, the concentrations of spermidine and spermine necessary for the inhibition of cell growth when cells were cultured in the presence of 50 pM Mg2+ were much smaller (2 mM spermidine and 0.15 mM spermine). Nevertheless, the amount of polyamines accumulating in cells which could cause the inhibition of cell growth was almost the same, regardless of the large difference in the added polyamine concentrations. At the early stage of polyamine accumulation, the inhibition of cell growth correlated with the decrease of Mg2+ content, but not with a decrease of the ATP content. The decrease in Mg" content correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. Thus, the inhibition of cell growth at the early stage of polyamine accumulation was thought to be due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines. The decrease in Mg" content was mainly caused by the inhibition of Mg2+ transport by polyamines. At the later stage of polyamine accumulation, a decrease in ATP content was also observed. This was followed by swelling of the mitochondria, which may be a symptom of the subsequent cell death.

Polyamines are essential for the maintenance of eukaryo- tic-cell proliferation and inhibitors of polyamine biosynthesis have been developed as antiproliferative reagents [l, 21. Similarly, bis(ethy1)polyamine analogues have recently been developed as antiproliferative reagents [3, 41. These reagents can not only negatively regulate the synthesis of ornithine decarboxylase (OrnDC) and S-adenosylmethionine decarboxylase (AdoMetDC) (51, but can also induce spermidinel spermine N'-acetyltransferase (SSAT) activity [6 , 71. Thus, bis(ethy1)polyamine analogues can deplete intracellular polyamines almost completely and they are thought to inhibit cell growth through this process. However, we found that N',W2- bis(ethy1)spermine (Et,spermine), the most active derivative in reducing polyamine contents [4], can substitute for the functions of spermine in several respects [8, 91. This obser- vation suggested the possibility that the accumulation of Et2- spermine is involved in the inhibition of cell growth in addition to polyamine deficiency [9].

Correspondence to K. Igarashi, Faculty of Pharmaceutical Sci- ences, Chiba University, 1-33 yayoi-cho, Inage-ku. Chiba, Japan, 263

Far: f 8 1 43 255 1574. Abbreviationr. Et, spermine, N',N'*-bis(ethyl)spermine; OrnDC,

ornithine decarboxylase; AdoMetDC, S-adenosylmethionine decarboxylase ; SSAT, spermidindspermine W-acetyltransferase.

Enzymes. Ornithine decarboxylase (EC 4.1.1.17) ; S-adenosylmethionine decarboxylase (EC 4.1.1 SO); spermidinehpermine W - acetyltransferase (EC 2.3.1 5 7 ) .

If this proposal is correct, the accumulated polyamines should also inhibit cell growth. The in vitro cytotoxicity of spermidine and spermine at the early period of polyamine accumulation is dependent on the presence of a serum amine oxidase, which is found in bovine serum [lo, 111. However, using bovine serum together with aminoguanidine, an inhibitor of Cu'+-dcpendent amine oxidasc 1121, or horse serum, in which the serum amine oxidase is not present [13], it was possible to show that spermidine or spermine itself is involved in thc inhibition of cell growth 114-171. Thc accumulated spermidine and spermine inhibited cell growth by decreasing the Mg2' and ATP contents. The decrease in Mg" content correlated well with the inhibition of protein synthesis, probably due to the inactivation of ribosomes through the replacement of Mg" on magnesium-binding sites by polyamines. The decrease in ATP content was followed by swelling of the mitochondria, which might have some role in cell death caused by polyamines.

MATERIALS AND METHODS

Cell culture and assays for DNA and protein synthesis

Established mouse mammary carcinoma FM3A cell lines were kindly supplied by Dr H. Matsuzaki (Saitama Univer- sity, Japan). The cells (1 X lo4 cells/ml) were cultured in ES growth medium (Nissui Pharmaceutical Co.) supplemented

90

with 50U/ml streptomycin, 100U/ml penicillin G and 2% heat-inactivated fetal calf serum at 37°C in an atmosphere of 5% CO,, according to the method of Ayusawa et al. [18]. When the effect of spermidine or spermine on cell growth was examined, either 1 mM aminoguanidine was added to the medium to inhibit amine oxidase in the serum, or 4% horse serum was used instead of 2% fetal calf serum. To prepare the medium containing 5.5 pM Mg”. fetal calf serum was dialyzed twice against 137 mM NaCI, 1.47 mM KH,PO,, 8.06 mM Na,HPO, ’ 7 HZO and 2.68 mM KCI, pH 7.5 (NaCUP,), and ES medium was prepared without add- ing Mg”. [ ’HIThyniidine or [’Hlleucine incorporation into whole cells was determined after a 2-h incubation of the cells in 2 nil ES medium with 37 kBq [’Hlthymidine (124 MBq/ mmol) or 101 kBq [-‘H]leucine (5.25 GBq/mmol). Radioac- ti\ity was measured by the method of Seyfried and Morris ~ 9 1 .

Enzyme assays

FM3A cells ( 5 X lo’ cells) were suspended in 0.8 ml 10 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 20% glycerol, 1 mM EDTA and 20 pM 6-amino-2-naphthyl-4-guanidino- benzoate dihydrochloride, a strong proteinase inhibitor [20]. The cells were frozen, thawed and homogenized with a Tef- lon homogenizer. The 12 000 X g supernatant was used for the enzyme assays. OrnDC, AdoMetDC and SSAT were as- sayed as described previously [ 2 1. 221 with some modifica- tions. To measure the enzyme activity accurately, the substrate concentration was increased and the concentrations used were 0.16 mM ornithine, 0.2 mM S-adenosylmethionine and 10 pM acetyl CoA. The incubation times for OrnDC, AdoMetDC and SSAT assays were 20, 20, and 5 min, respectively. The protein concentration was determined by the method of Lowry et al. [23].

Measurement of polyamines, MgZ+, ATP, ADP and AMP Polyamine contents were determined by HPLC as de-

scribed previously 1241 after extraction with trichloroacetic acid. Mg” was analyzed in the presence of strontium chloride (1 mg/ml) by means of atomic-absorption spectrometry. ATP was determined by the luciferase enzyme system [25]. The rate of light emission was linearly dependent on the square of the ATP concentration. For the measurement of the cellular ATP content, FM3A cells (4X 10” cells) were harvested and extracted with 0.2 ml 0.2 M HCIO,. The extracted ATP was measured after neutralization with 1 M KOH containing 5 0 m M K,HPO,. For the measurement of ATP, ADP and AMP, HPLC was performed using a DEAE- silica gel (TSK gel DEAE-2SW, Tosoh) column (250 mm X 4.6 mm). 80-pl aliquots of the above samples were loaded on the column and separated with a solvent composed of 75 mM sodium phosphate/acetonitrile (4: 1, by vol.) pH 7. The flow rate was 1 ml/min and the absorbance at 260 nm was measured using a Tosoh HPLC system. The retention times for AMP, ADP and ATP were 8.5, 16.3 and 29.3 min, respectively. The ATP content measured by this method was the same as that measured by the luciferase enzyme method.

Assays for spermine and Mgz+ transport Assays were performed with 5 pM [“Clspermine or

S O pM Mg” as substrate and included 5 X 10” FM3A cells

according to a previous study 1261. Mg”-deficient cells cultured in the presence of 5.5 pM Mg’+ were wed for the assay of Mg’+ transport. Mg2+ content was measured as described above. The amount of [’T]sperniidine radioactivity in the cells was measured in 10 ml Triton/toluene scintillant after sonication with 1 ml 1% SDS.

Flow-cytometry analysis FM3A cells (3 X lo7 cells) were fixed in 70% ethanol and

stained with 3,8-diamino-5-[3-(diethylmethylammonio)pro- pyll-6-phenylphenanthridinium diiodide monohydrate (0.05 mg/ml in 1.1% sodium citrate) according to the method of Crissman and Steinkamp [27l. Stained samples were analyzed with a 488-nm laser excitation using a Coulter Epics Elite instrument according to the manufacturer’s instructions. Each histogram was generated from the analysis of 1 X 10“ nucleated cells.

Electron microscopy

FM3A cells (2 X lo7 cells) were fixed in 4% glutaralde- hyde in 50 mM potassium phosphate, pH 7.0, for 12 h at 4”C, and were fixed again in 1 % OsO, for 2 h at 24°C. After dehydration with a graded series of ethanol solutions, the samples were embedded in Epon 81 2. Ultrathin sections were stained with uranyl acetate at the saturated concentration in ethanol, and subsequently with 30 mM lead citrate. The stained specimens were examined with a Hitachi H700H electron microscope.

Drugs

Aminoguanidine hydrochloride. an inhibitor of serum amine oxidase, was kindly supplied by Nippon Carbide Indu- stries. Spermidine trihydrochloride and spermine tetrahydro- chloride were purchased from Nacalai Tesquc.

RESULTS

Inhibition of cell growth by polyamines

As shown in Fig. 1, 10 mM spermidine or 2 mM spermine greatly inhibited cell growth (more than 90%) in the presence of the standard Mg’- concentration (0.9 mM), con- firming previous results [ 14, 151. The effective concentration of spermidine was five-times higher than the effective concentration of spermine. When Mg2 ’ in the medium was decreased to 50 pM, a level which did not influence cell growth, the concentrations of spermidine and spermine necessary to inhibit cell growth to a similar extent decreased to 1 .5 mM and 0.15 nlM, respectively. When the ME’ + concentration in the medium was 0.3 mM, the concentrations of spermidine and spermine necessary to inhibit cell growth by more than 90% were 3.0 mM and 0.5 mM, respectively. These results indicate that the inhibition of cell growth by polyamines was affected by Mg”.

The recovery of the polyamine inhibition of cell growth by Mg2+ was examined (Fig. 2). Cclls were cultured in the presence of SO pM Mg2- and either 1 .5 mM spermidine or 0.15 mM spermine. The addition of 1 mM Mg” on day 3 caused the recovery of cell growth, but when this addition was performed on day 5 , cell growth did not recover. The precence of polyamines eventually caused cell death on day 7. which was later than that observed with Et,spermine.

91

1x106

5x105

1x10~

4 5x10

0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5

Time ( Days ) Fig. 1. Polyamine toxicity for cell growth in the presence of either 0.9 mM, 0.3 mM or 50 pM Mg2+. (A) Incubation in the presence of 0.9 mM Mg2+. Control (0); 5 mM spermidine (A); 10 mM spermidine (A); 1 mM spermine (0); 2 mM spermine (M). (B) Incubation in the presence of 0.3 mM Mg’ ’ . Control (0); 2 mM spermidine (A); 3 mM spermidine (A); 0.3 mM spermine (0); 0.5 mM sperniine (W). (C) Incubation in the presence of 50 pM Mg”. Control (0); 1.5 mM spermidine (A); 2.5 mM spermidine (A); 0.15 mM spermine (0): 0.25 rnM spermine (M). Each value is the average of three determinations. The standard error was within 2 10% for each data point.

0 1 2 3 4 5 6 7

Time (Days )

Fig.2. Recovery of cell growth by the addition of Mg”. Cells were cultured in the presence of 50 pM Mg’+. Control (0); 1.5 mM spermidine (0-0); 1 mM Mg2+ was added to the medium containing 1.5 mM spermidine on day 3 (O----O) and on day 5 (0----O), respectively: 0.15 mM spermine (A-A); 1 mM Mg2- was added to the medium containing 0.15 mM spermine on day 3 (A----A) and on day 5 (A----A), respectively. Arrows indicate the addition of I mM Mg”. Each value is the average of three determinations. The standard error was within ? 10% for each data point.

Furthcrrnore, thc inhibition of cell growth by Et,spermine was not reversed even if Et,spermine was removed from the medium on day 2 [9]. However, cell growth was inhibited by polyamines from day 1 onwards, while Et,spermine inhibited cell growth significantly from day 2 after its addition.

Thc cell-cycle distribution was analyzed for the cells on day 3. The amounts of control cells in the G,, S and G, phases were 58.1, 19.8 and 22.1 %, respectively (Fig. 3A); the amounts or bpermine-treated cells in the G,, S and G, phases were 93.5, 4.6 and 1.9%, respectively (Fig. 3B).

Some cells may have been damaged during ethanol fixation, since cells (or cell debris) with a lower DNA content were observed (Fig. 3B). The results indicate that accumulated polyamines interrupted the cell cycle and arrested cell growth in the G, phase.

Polyamine contents and polyamine-metabolizing enzymes in FM3A cells treated with spermidine or spermine

The activities of polyamine-metabolizing enzymes were measured after dialysis of the ccll lysate to remove polyamines (Table 1). Both spermidine and spermine inhibited OrnDC activity completely and AdoMetDC activity significantly. SSAT activity was strongly enhanced by spermidine and spermine.

The polyamine contents were determined and are shown in Table 1. When cell growth was inhibited by polyamines in the presence of 0.9 mM or 50 pM Mg”, almost the same amounts of polyamines were accumulated in the cells. After the addition of spermidine, 35 -40 nmol spermidine/mg protein accumulated in thc cells, with a slight decrease in the amount of spermine at both Mg2+ concentrations. In the casc of spermine, 30-40 nmol spermine/mg protein accumulated in the cells, with a drastic decrease in the amount of spermidine. The accumulated amounts of polyamines in thc cellh were nearly the same as the amount of Etzspermine when cell growth was inhibited by these polyamines [9].

Correlation between growth inhibition and the decrease of Mgz+ and ATP contents in polyamine-treated FM3A cells

Since the inhibition of cell growth by polyamines was affected by Mg2+ (Fig. 1 ) and thc ATP content was decreased by Et,spermine [ 9 ] , the Mg” and ATP contents in cells cultured for 3 days were measured (Table 2). The contents of both Mg” and ATP in the cells were greatly decreased in polyamine-treated cells and the decrease of MgZ ’ and ATP correlated well with the inhibition of macromolecular synthesis, especially protein synthesis. The chargc contcnt. corrc- sponding to the decrease of Mg” in polyamine-treated cells,

92

4 5 } A G i S Gz

' 30 0

E, 20 25

n

z 1 1 5 0

10

5

-

n 0 40 80 120 160 200 240

Charnel (Relative DNA Content)

GI S Gz 1 1 I

0 40 80 120 160 200

Channel (Relative DNA Content)

Fig. 3. DNA-distribution analysis of control cells (A) and spermine-treated cells (B). Flow-cytometry analysis was performed as described in Materials and Methods. The cell-cycle phases are also indicated.

Table 1. Polyamine contents and polyamine-metabolizing enzyme activities in FM3A cells cultured in the presence and absence of polyamines. Cells were cultured for 3 days in the presence and absence of polyamines. The values are expressed as the mean t S. D. for three determinations. Control incubations have no added polyamines.

Pol yamine addition ME'- Content of Enzyme activity of concentration putrescine spermidine spermine OrnDC AdoMetDC SSAT

Control Spermidine (5 mM) Spermidine ( 10 mM) Spermine (1 mM) Spermine (2 mM) Control Sperrnidine (1.5 mM) Sperrnine (0.15 mM)

mM

0.9 0.9 0.9 0.9 0.9 0.05 0.05 0.05

nmol/mg protein

2.45 2 0.29 2.61 2 0.32 2.65 ? 0.41 (0.01 CO.01 2.09 ? 0.14 2.97 2 0.1 8 <0.01

11.5 5 1.0 25.4 2 1.8 37.1 t 2.7

1.59 i- 0.24 2.23 2 0.29

10.9 2 1.4 36.7 % 2.8

1.65 ? 0.41

pmoVminlmg protein

8.12 ? 0.73 146 i- 24 58.4 i- 1.31 80.8 ? 6.3 6.85 2 0.55 6.37 2 0.41 <0.2 10.1 i- 0.7 334 2 32

27.4 5 1.51 36.8 t 2.13 <0.2 16.5 2 1.3 392 i- 36 13.0 ? 1.27 133 -+ 18 50.4 2 3.7 96.8 2 8.5 9.45 2 0.88 <0.2 13.4 5 1.8 265 * 34

31.2 i- 2.9 <0.2 19.8 +- 1.8 324 2 23

Table 2. Mg*+, ATP, ADP and AMP contents and the synthesis of DNA and protein in FM3A cells cultured in the presence and absence of polyamines. Cells were cultured for 3 days in the presence and absence of polyamines. The values are expresed as the mean 2 S. D. for three determinations. n.d., not determined. Control incubations have no added polyamines.

Polyamine addition Mg' ' Content of Incorporation concentration Mg2+ ATP ADP AMP ['Hlthy- ['Hlleu-

midine cine

mM nmoVmg protein nmollpg DKA -

Control 0.9 126 i- 12 14.1 i- 0.9 n.d. n.d. 2720 2 214 859 t- 73 Spermidine (1 0 mM) 0.9 45.0 ? 3.9 5.77 2 0.48 n.d. n.d. 1294 i 112 322? 31 Spermine (2 mM) 0.9 57.2 ? 6.4 6.40 2 0.50 n.d. n.d. 1442 2 129 371 t 38 Control 0.05 135 -+ 11 13.8 2 0.9 2.35 2 0.15 1.01 2 0.12 2860 2 235 828 +- 91 Spermidine (1.5 mM) 0.05 40.3 2 3.8 8.33 i- 0.51 3.45 2 0.28 2.48 2 0.31 1388 t 124 292 5 33 Spermine (0.15 mM) 0.05 55.8 I 4.3 8.10 _f 0.74 3.58 t 0.33 2.83 ? 0.24 1586 i- 138 348 5 24

was nearly equal to the charge content corresponding to the increase of spermhe"- (Tables 1 and 2). In contrast, ADP and AMP contents were increased in accordance with the decrease of ATP in polyamine-treated cells (Table 2). Al- though the swelling of mitochondria was not clearly observed in the cells cultured for 3 days, the 5-day culture con- tained cells which distinctly showed swollen mitochondria (Fig. 4). In the case of Et2spermine-treated cells, the Mg2+ content was not decreased (data not shown). These results

imply that the decrease of Mg" is necessary for the binding of polyamines to macromolecules and their subsequent accumulation, because free polyamines, not Et,spermine, are ready to be metabolized by SSAT.

Characteristics of the inhibition of cell growth in Mgz+-deficient FM3A cells

To determine whether cell growth is inhibited by Mg" deficiency, cells wcre cultured in the presence of various

93

Fig.4. Electron micrographs of FM3A cells. The cells were cultured in the presence of SO pM Mg2‘ (A-C) or 5.5 pM Mg” (D) and were harvestcd on day 5 . (A) Control cell; (B) 0.15 mM sperrnine-treated cell; (C) 1.5 mM spermidine-treated cell; (D) Mg”-deficient cell. N, nucleus; the arrow heads and bar indicate mitochondria and I pni, respectively. Fourfold enlargements of the mitochondria are also shown in the insets.

Mg” concentrations. As shown in Fig. 5, cell growth was inhibited when the Mg” concentration added to the medium was less than 30 yM. Cell growth was inhibited by 90% in the presence or 5.5 pM Mg”. The addition of 1 mM Mg” to the medium on day 5 allowed for the recovery of cell growth, indicating that the inhibition is reversible. Under this condition (5.5 pM Mg”), the amount of polyamines in the cells was slightly decreased and the amount of Mg” was greatly decreased (Table 3). The decrease of Mg2+ content in the cells cultured in the presence of 10 1 M Mg” was nearly equal to the decrease observed in the polyamine-treated cells. However, the ATP content in the cells did not decrease sig-

nificantly. This was confirmed by electron microscopy, where no swelling of mitochondria was observed in the Mg”-deficient cells cultured for 5 days (Fig. 4D). Thc inhibition of cell growth in Mg’+-deficient cells correlated with the inhibition of macromolecular synthesis, especially protein synthesis (Table 3). These findings suggest that the cell growth of Mg2+-deficient cells is inhibited at the level of protein synthesis, due to the inactivation of ribosomes as reported previously [28]. Since it was impossible to decrease the Mgz+ concentration in the medium to less than 5.5 pM, we could not determine the level of dcficieney of Mg” which would cause cell death.

94

Table 3. Polyamine, Mg” and ATP contents and the synthesis of DNA and protein in FM3A cells cultured in the presence of various Mgz+ concentrations. Cells were cultured for 3 days in the presence of various concentrations of Mg*+. The values are expressed as the mean i S. D. for three determinations.

Mg’ ’ Content of Incorporation of concentration putrescine spermidine spermine Mg” ATP [’Hlthymidine [ ’Hlleucine

PM nmol/mg protein nmolipg DNA

5.5 0.18 2 0.04 3.08 i 0.41 7.92 i 0.81 33.9 2 1.8 10.8 ? 0.8 1232 t 115 312 i 35 10 0.20 i- 0.05 4.61 i 0.42 10.9 5 1.4 47.9 i 3.2 13.5 i 1.2 1638 2 131 402 i 34 30 1.85 2 0.21 8.51 t 0.08 11.0 i 1.7 121 i 15 13.2? 1.2 2716 2 218 792 i 87

0 1 2 3 4 5 6 7

Time ( Days )

Fig.5. The effect of MgZ+ on cell growth. The cells were cultured In the presence of variouh concentrations of Mg”. 0.9 mM Mg” (0). 50pM Mg’- (0); IOpM Mg’- (0); 5.5 pM Mg” (A-A); the cells were cultured in the presence of 5.5 pM Mg2+ until day 5. at which time 1 mM Mg” was added as indicated by the arrow (&---A). Each value is the average of three determinations. The standard error was within -C 10% for each data point.

Shift from reversible to irreversible inhibition for FM3A cells treated with polyamines

The contents of polyamines, Mg” and ATP, and protein synthesis in FM3A cells were measured by changing the incubation time with spennine (Fig. 6). The Mg2+ content decreased significantly in cells incubated for 2 h with spermine. After a 4-h incubation, the increase of spermine content and the inhibition of protein synthesis appeared to occur simulta- neously. The ATP content started to decrease in the cells incubated for 8 h with spermine. The swelling of mitochondria and the irreversible inhibition of cell growth were observed on day 5 and cell death occurred on day 7, as men- tioned abovc (Figs 2 and 5) . Although these phenomena occurred later in sperminc-treated cells than in Etzspermine- treated cells, the characteristics of cell death were similar except that a decrease in the Mg” content is necessary for accumulating spermine in the cells. The results suggest that the damage of mitochondria might have some role in the cell death caused by polyamines.

Inhibition of Mgz+ transport by spermine

Much higher concentrations of spermidine and sperinine were necessary to inhibit cell growth compared to Et,spermine and the effective polyamine concentrations decreased with the decrease of Mg” concentration in the medium. To clarify how spermidine or spermine could accumulate in the

r r I

0 24 48 72

Time ( h )

Fig.6. Changes of M e , spermine and ATP contents and protein synthesis of FM3A cells cultured in the presence of 50 pM Mgz+ and 0.15 mM spermine. Maximum values (100%) for Mg”. spermine and ATP contents and protein synthesis were 124, 1 1.8 and 13.9 nmol/mg protein and 814 nmol leucine incorporated/pg DNA, respectively, and the data are expressed relative to these values. Each value is the average of three determinations. The standard error was within ? 10% for each data point. Spermine (0); ATP (A); protein synthesis (0) ; Mg’ ’ (0).

0 0 10 20

Time ( min )

Fig.7. EtTect of spermine on Mg2+ transport. The cells were cultured in the presence of 5.5 pM Mg” for 3 days and used for the assay of Mg” transport. Mg” transport in the presence of 50 pM Mg” (0); Mg” transport in the presence of 50pM Mg” and 0.3 tnM spermine (0). Each value is the average of three determinations. The standard error was within 5 10% for each data point.

cells, the effects of spermine on Mg’- transport and the effects of Mg” on spermine transport were examined. As shown in Fig. 7, Mg” transport was clearly inhibited by spermine. However, spermine transport was not inhibited by

95

the addition of 1-10 mM Mg2+ (data not shown), which confirmed the previous results with bovine lymphocytes [26] . The present findings indicate that the inhibition of Mg2- transport by spermine is the major reason for the decrease of Mg” content, which is followed by an increase of spermine content in the cells.

DISCUSSION

We confirmed previous results [14- 171 that high concentrations of polyamines inhibit cell growth. Furthermore, when [”Clspermine was added to the medium, 85% and 7% of the radioactivity in the cells were recovered as spermine and acetylspermine, respectively, indicating that the accumulated spcrmine itself is toxic. This was also supported by the finding that a high concentration of putrescine (1 -4-diamino- butane), which is not a substrate for amine oxidase in serum, inhibited cell growth (data not shown). Recent work by Piercc and coworkcrs [14, 291 has suggested that polyamine oxidation in cells is involved in the subsequcnt cytotoxicity associated with the mechanism of programmed cell death. However, this hypothesis was refuted by a recent study by Bruton et al. [ 151. It has been reported by Guarino and Cohen [30] that a high concentration of putrescine was bactericidal against the cyanobacterium Anncytis nidulnizs. These authors proposed that putrescine directly inhibited protein synthesis, perhaps by competing with other cations for the binding sites on ribosomes, lcading to ribosome inactivation.

At the early stage of the inhibition of cell growth by polyamines, protein synthesis was inhibited in concert with the decrease of Mg2 I in the cells. Cell growth was also inhibited in Mg”-deficient cells. It is believcd that the major Mg” and polyamine binding sites in growing cells are ribosomes [31]. It has also been reported that a critical level of MgZ is required for the maintenance of ribosome function in both Escherichia coli [32, 331 and rat liver [28]. A decrease in the polypeptide-synthesizing activity of rat liver polysonies occurred when more than 40% of the Mg’ ’ origi- nally bound to ribosomes was replaced by spermidine [28]. Thus, the inhibition of cell growth by polyamines at the carly stage of inhibition was probably due to the inactivation of ribosomes as proposed by Guarino and Cohen [30]. Recently, Morris also proposed a similar idea, that the toxic errcct of polyamines is due to the replacement of Mg” at critical sites in the ribosomes by polyamines [34]. The functions of the polyamine-ATP complex may be different from the functions of the MgZ+-ATP complex, as suggested previously [35] . These different functions may also play some role in the inhibition of cell growth by polyamines.

At the later stage of the polyamine inhibition or cell growth, the ATP content in the cells also decreased and sub- sequenl swelling ol‘ the mitochondria was observed by electron microscopy. Irreversible inhibition of cell growth was apparent together with 1hc structural changes observed for the mitochondria. In a previous study [9], we proposed that the increase in free Ca2+ due to the damage of mitochondria may be involved in the cell death caused by Et,spermine, since there was a study 1361 which suggested that neu- ronal death in vitro was correlated with the increase in cyto- plasmic Ca”’. The increase in free Caz+ may also be the reason for the ccll death caused by spcrmidine or spermine accumulation,

Although the functions of polyamincs and Etzspermine are similar in terms of the stimulation of globin and OrnDC

synthesis, the stimulation of rat liver Ile-tRNA formation and Z-DNA structure formation, the recovery of cell growth for polyamine-deficient bovine lymphocytes, the inhibition of spermidine uptake by bovine lymphocytes and the antiulcer- ation effect [8,9], differences were observed in the inhibition of cell growth by polyamines and Et,spermine. These differences included the following : (a) spermidine and spermine accumulated in cells only when the Mg” content dccreased; polyamines may be metabolized by SSAT when they exist in a free form; (b) the inhibition of cell growth by polyamines was observed even on day 1 due to the decrease of Mg’i, while significant inhibition by Et,spermine accumulation was observed on day 2, since the polyamine contents decreased together with the accumulation of Et,spcrmine on day 1 ; (c) the damage to mitochondria was observed later in the polyamine-treated cells compared to the Et,spermine-treated cells, suggesting that Et,spermine may accumulate in mitochondria earlier than polyamines.

The experimental data showed that Mg” transport was inhibited by spermine, but spermine transport was not inhib- itcd by Mg”. When Mg2’ transport was inhibited by a 2-h incubation with spermine, a significant accumulation of spermine in the cells was not observed (Fig. 6). Thus, Mg” transport may be regulated by spermine outside of the cells. However, although it is not a very likely possibility, it still cannot be completely ruled out that the decrease of Mg’ content in the cells is due to the competition for intracellular binding sites with spermine. Proton magnetic resonance was used to try to determine whether Mg” and spermine could form a complcx. The results indicated that most of the spermine exists in a free form (data not shown).

In conclusion, the results indicate that the accumulation of both polyamines and Etzspermine causes mitochondria1 damage. This finding may be related to the process of cell death and, if this proves to be the case, this should be consid- ered in the design of polyamine analogues as antiproliferative agents.

We would like to express our thanks to Dr K. Samejima for his encouragement during the course of this study and to Drs S. Nakaike and K. Samata for their help with the flow-cytornetry analysis. Thanks are also due to Dr. H. Matsuzaki for his kind supply of mouse mammary carcinoma FM3A cell lines. This work was supported by a Grant-in-Aid for Scientific Rescarch from the Ministry of Education, Scicnce and Culture, Japan, and by Research Aid from the Yamada Science Foundation.

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