Correlation between the inhibition of cell growth by bis(ethyl)polyamine analogues and the decrease in the function of mitochondria

Eur. J. Biochem. 221, 391 -398 (1994) 0 FEBS 1994

Correlation between the inhibition of cell growth by bis(ethy1)polyamine analogues and the decrease in the function of mitochondria Yong HE', Toshikazu SUZUKI', Keiko KASHIWAGI', Kuniko KUSAMA-EGUCHI', Akira SHIRAHATA3 and Kazuei IGARASHI' ' Faculty of Pharmaceutical Sciences, Chiba University, Japan ' College of Pharmaceutical Sciences, Nihon University, Funabashi, Japan

Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan

(Received December 16, 1993) - EJB 93 1870/1

The antiproliferating effect of nine kinds of bis(ethy1)polyamine analogues [three kinds each of bis(ethyl)triamine, bis(ethy1)tetraamine and bis(ethy1)pentaaminel was compared using FM3 A cells. The inhibitory effect was in the order BE4444 > BE3443 > BE4334ZBE444 > BE343 > BE333 > BE44 > BE34 > BE33. Our results indicate that not only polyamine deficiency but also the accumulation of polyamine analogues is involved in the inhibition of cell growth. Accumulation of bis(ethy1)polyamine analogues caused the inhibition of protein synthesis and the decrease in the ATP content. The protein synthetic system in mitochondria was more strongly inhibited by bis(ethy1)polyamine analogues than that in the cytoplasm. Under conditions such that cytoplasmic protein synthesis was inhibited by 50% by bis(ethy1)polyamine analogues, mitochondrial protein synthesis was almost completely inhibited. Mitochondria1 Ile-tRNA formation was inhibited by bis(ethy1)polyamine analogues at the concentrations that cytoplasmic Ile-tRNA formation was stimulated. This may be one of the reasons for the selective inhibition of mitochondrial protein synthesis. This inhibition was followed by the decrease in ATP content, swelling of mitochondria and depletion of mitochondrial DNA. These results suggest that the early event of metabolic change caused by bis(ethy1)polyamine analogues in cells is the inhibition of protein synthesis, especially of mitochondrial protein synthesis.

Since the polyamines putrescine, spermidine and spermine are essential for the maintenance of eukaryotic cell pro- liferation [ 11, inhibitors of polyamine biosynthesis to deplete cellular polyamines have been developed as antiproliferative reagents [2, 31. Bis(ethy1)polyamine analogues have also been developed as antiproliferative reagents [4, 51. These reagents can not only negatively regulate the synthesis of omithine decarboxylase (OmDC) and S-adenosylmethionine decarboxylase (AdoMetDC) [6], but can also induce sper- midinekpermine A"-acetyltransferase (SSAT) activity [7, 81. Thus, the analogues can deplete intracellular polyamines almost completely, and they are thought to inhibit cell growth through this process. We found that A", W'-bis(ethyl)spermine (BE343) can substitute for the functions of spermine in various aspects and accumulate in cells at a concentration fivefold that of spermine in control cells [9, lo], and suggested that not only polyamine deficiency but also the accumulation of BE343 may be involved in the inhibition of cell growth [lo]. Recently, it has been reported that the inhibition of cell growth by BE343 correlated with the intracellular ac-

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

Fax: +81 43 290 2900. Abbreviations. OmDC, ornithine decarboxylase; AdoMetDC,

S-adenosylmethionine decarboxylase; SSAT, spermidinehpermine W-acetyltransferase.

Enzymes. Ornithine decarboxylase (EC 4.1.1.17) ; S-adenosylmethionine decarboxylase (EC 4.1.1.50) ; spermidinehpermine W-acetyltransferase (EC 2.3.1 S7).

cumulation of the analogue [ll]. We also found that the accumulated spermidine and spermine inhibited cell growth through the decrease in Mg2+ and ATP contents [12]. The decrease in Mg2+ content correlated well with the degree of inhibition of protein synthesis, due to the inactivation of ribosomes through the replacement of Mg" on magnesium- binding sites by polyamines.

In this study, we investigated the antiproliferating effect of nine kinds of bis(ethy1)polyamine analogues systematically. Among those, three which had until now received little attention, bis(ethy1)pentaamines (BE4334, BE3443 and BE4444), were included, and abbreviations and structures for these bis(ethy1)polyamine analogues are shown in Fig. 1. We found that accumulation of bis(ethy1)polyamine analogues caused the inhibition of protein synthesis, especially mitochondrial protein synthesis, then the decrease in ATP content.

MATERIALS AND METHODS

Cell culture and assays for 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 medium (Nissui Pharmaceutical Co., Ltd.), supplemented with 50U/ml streptomycin, lOOU/ml penicillin G and 2% heat-inactivated fetal calf serum at 37 "C in an atmosphere of 5% COz, according to the method of Ayusawa et al. [13]. When the effect of bis(ethy1)polyamine analogues on cell

392

BE 33

BE 34

BE 44

BE 333

BE 343

BE 444

BE 4334

BE 3443

BE 4444

N l , N 7-bis(ethyl)norspermidine CzH5N H(CHz)zN H(CHz)3N HCzH5

N ,N a-bis(ethy1)sperrnidine CzHsN H(CHz)sN H(CHz)4N HCzHs

N 1 ,N 9-bis(ethyl)homosperrnidine C z H d H(CHz)4N H(CHz)dN HCzH5

N 1 ,N ll-bis(ethyl)norspermine CzH5N H(CHz)3N H(CHn)sN H(CHz)3N HC2H5

N ,N l2-bis(ethyI)spermine C z H d H(CHz)3N H(CHz)4N H(CHz)3N HC2H5

N N 14-bis(ethyl)hornosperrnine CzH5N H(CHz)4N H(CHn)dN H(CHz)4N HC2H5

l117-bis(ethylamino)-5,9, 13-triazaheptadecane C2H5N H(CH+N H(CHz)3N H(CHz)3N H(CHn)d HCzH5

1 , I 7-bis(ethylarnino)-4,9,14-triazaheptadecane C 2 H d H(CHz)3N H(CHn)4N H(CHz)4N H(CHn)3N HCzH5

1,19-bis(ethylarnino)-5,10,15-triazanonadecane CZH5N H(CHzJ4N H(CHz)4N H(CHz)4N H(CHz)4N HCzH5

Fig. 1. Abbreviations and structures of bis(ethy1)polyamine analogues.

growth was examined, 1 mM aminoguanidine, an inhibitor of amine oxidase in serum [14], was added to the medium. [3H]Leucine incorporation into whole cells was determined after 2 h incubation of the cells in 2 ml medium with 101 kBq [3H]leucine (5.25GBq/mmol). Radioactivity was measured by the method of Seyfried and Morris [15].

Enzyme assays

FM3A cells (5x10’) were suspended in 0.8 ml buffer A containing 10 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 20% glycerol, 1 mM EDTA and 20 pM 6-amino-2-naphthyl- 4-guanidinobenzoate dihydrochloride, a strong proteinase inhibitor [16]. They were frozen, thawed, then homogenized with a Teflon homogenizer. The homogenate was centrifuged for 10min at 12000Xg. The supernatant was dialyzed against buffer A and used for the enzyme assays. Assays of OrnDC, AdoMetDC and SSAT were performed as described previously [17, 181 with some modifications [12]. Assay for spermidine uptake was performed with 5 pM [I4C]spermidine as substrate and 5 X lo6 FM3 A cells according to the previous publication [19]. Protein was determined by the method of Lowry et al. [20].

Measurement of polyamines, bis(ethy1)polyamine analogues, Mg2+ and ATP

FM3A cells (6x107 were harvested and extracted with 0.3 m10.2 M HC10,. The supernatant thus obtained was used for the following assays. Polyamines (putrescine, spermidine and spermine) were measured as described previously [21]. Bis(ethy1)polyamine analogues were dansylated according to the previous publication [22], and dansylpolyamines were then analyzed by a Toyo Soda HPLC system on which a TSK gel ODS 80TM column (4.6 mmXlOO mm), heated at 35°C was mounted. The eluents used for bis(ethyl)triamine, bis-

(ethy1)tetraamine and bis(ethy1)pentaamine analysis were 80, 85 and 90% methanol, respectively. The flow rate of the eluent was 1 ml/min, and fluorescence was measured at an excitation wavelength of 360nm and an emission wavelength of 510 nm. The retention times for the dansyl derivatives of BE33, BE34 and BE44 were 27, 30 and 38min, respectively. The retention times for BE333, BE343 and BE444 derivatives were 23, 27 and 32 min, and those for BE3443, BE4334 and BE4444 derivatives were 17, 19 and 22 min, respectively. Magnesium ions were analyzed in the presence of strontium chloride (1 mg/ml) by means of atomic absorption spectrometry. ATP was measured by the luciferase enzyme system [23] after neutralization with 1 M KOH containing 50 mM K,HP04.

Protein synthesis in a rabbit reticulocyte cell-free system Rabbit reticulocyte lysate and its nuclease-treated lysate

were prepared as described by Pelham and Jackson [24]. The nuclease-treated lysate was passed through a Sephadex G-25 column twice in order to remove polyamines, as described previously [25], and the concentration of spermidine remain- ing in the lysate was 10 pM. Protein synthesis was performed as described previously [26] using 0.015 A,,, unit of globin mRNA as mRNA. Incubation was carried out at 33°C for 20 min. Under these conditions, the rate of protein synthesis was linear up to 30 min after a short lag.

Analysis of total and mitochondrial protein synthesis FM3 A cells (5 X lo6) cultured as described above were

incubated for 2 h with 3.7 MBq [35S]methionine in the presence and absence of emetine (0.2 mg/ml), which inhibits cytoplasmic protein synthesis specifically [27, 281. The cells cultured in the absence of emetine were harvested, and total protein synthesis was measured by counting the radioactivity insoluble in hot trichloroacetic acid. Fluorography of total protein, separated by SDS/polyacrylamide gel electrophoresis, was performed using 20 pg protein. The mitochondria1 fraction was obtained from the cells cultured in the presence of emetine by homogenization with a buffer containing 0.25 M sucrose, 1 mM EGTA and 10 mM Hepes/KOH, pH 7.4, followed by differential centrifugation [28]. Mito- chondrial protein synthesis was measured by counting the hot trichloroacetic acid insoluble radioactivity in the mitochondrial fraction. The mitochondrial proteins (25 pg) were separated by SDS/urea/polyacrylamide gel electrophoresis. Radioactivity on dried gel was quantified by Fujix Imaging Analyzer Fuji BAS 2000 (Fuji Photo Film Co. Ltd.).

Assays for aminoacylation Cytoplasmic Ile-tRNA formation was assayed as de-

scribed previously [29] using liver tRNA and aminoacyl- tRNA synthetase complex as enzyme source. The ionic conditions of the reaction mixture were 50mM Tris/HCl, pH 7.5, 100 mM KCl and 2.5 mM Mg”. The digitonin- treated mitochondria were prepared according to the method of Eberly et al. [30]. Mitochondria1 tRNA and the 1OOOOOXg supernatant fraction were prepared by the method of Kuma- zawa et al. [31]. The lOOOOOXg supernatant of mitochondria (1 86 mg protein) was applied to a column of DEAE-cellulose (1.5 cmX24 cm), previously equilibrated with buffer B (10 mM Tris/HCl, pH 7.8, 2 mM magnesium acetate, 6 mM 2-mercaptoethanol and 10% glycerol) containing 50 mM

393

0.5mM 1 xlO6

1x105

E iX104

0,

- - . in al - -

1 XlO6 c 0 .- c

E C 1x105

1 ~ 1 0 4

1 X I 06

1 x i 05

1x104

a) 0 C 0

- - a, 0

Control

0.5mM -- 0.5mM

1 mM

BE 33 1 BE34 1 BE44 Control

BE 333 I BE343 I BE444 Control Control Control

Control BE 4334 I BE4444 Control

0.25rnM

Time (Days)

Fig.2. Effects of bis(ethy1)polyamine analogues on cell growth. Each value is the average of three determinations. Standard error was within L 10% for each point.

KCI. The column was washed with the same buffer and eluted with buffer B containing 250 mM KC1. This fraction was used as enzyme source. Mitochondria1 Ile-tRNA formation was assayed as described previously [32]. The ionic con- dition of the reaction mixture was 50 mh4 Tris/HCI, pH 7.5, 100 mM KCl and 5 mM Mg”.

Southern-blot analysis of mitochondrial DNA Plasmid PAM1 is a pACYC177 derivative containing the

entire mitochondria1 genome from mouse LA9 cells [33] and was generously provided by Dr D. A. Clayton of Stanford University. The PAM1 was digested with Hue11 and HindIII, and the mixture of 6.9 kbp and 6.5 kbp fragments was used for the DNA probe. The DNA (10 pg) prepared from FM3 A cells according to the method of Maniatis et al. [34] was digested with Hue11 and HindIII, fractionated by gel electrophoresis on 0.7% agarose, transferred to a Gene Screen Plus membrane (Du Pont-New England Nuclear) and hybridized with the 72P-labeled DNA probe described above (2X lo9 c p d p g DNA). Radioactivity was quantified by Fujix Imaging Analyzer Fuji BAS 2000 (Fuji Photo Film Co. Ltd.).

Synthesis of bis(ethy1)polyamines BE343 and BE4444 were synthesized as described [7,

351. Others were synthesized by removal of the protecting groups from benzylated and p-toluenesulfonated bis(ethy1)- polyamines prepared by N-alkylation of benzylamine, benzylated diamine or benzylated triamine with N-ethyl-N- (3-bromopropyl)-p-toluenesulfonamide (1) or N-ethyl-N- (4-bromobutyl)-p-toluenesulfonamide (2) according to the method for the synthesis of spermidine, spermine [36] or pentaamine [37]. The structures were confirmed by elemental

analysis within 0.3% of the calculated values for C, H and N. BE33 trihydrochloride from benzylamine with (l), crystals from watedethanol, melting point > 300°C, Anal. (C,,,H,,N, 3HC1) ; BE34 trihydrochloride from benzylamine with (1) and (2), crystals from watedethanol, melting point > 300°C, Anal. (C,,H,,N, 3HCl) ; BE44 trihydrochloride from benzylamine with (2), crystals from watedethanol, melting point > 300°C, Anal. (C,,H,,N, 3HCl) ; BE333 tetrahydrochloride from N,N’-dibenzyl-l,3-propanediamine with (1 ), crystals from watedethanol, melting point > 300°C, Anal. (C,,H,,N, 4HC1) ; BE444 tetrahydrochloride from N,N’-dibenzylputrescine with ( 2 ) , crystals from watedetha- no], melting point > 3OO0C, Anal. (C16H3,N4 4HC1); BE3443 pentahydrochloride from N’,NS,Ng-tribenzylhomospermidine with (l) , melting point > 300°C, Anal. (C,,H,,N, 5HC1); BE4334 pentahydrochloride from N1,N4,W-tribenzylnorsper- midine with (2), melting point > 300°C, Anal. (C,,H4,N, 5HC1).

RESULTS Antiproliferating effect of bis(ethy1)polyamine analogues

Although the antiproliferating effect of bis(ethy1)tetraamines (BE333, BE343 and BE444) has been studied extens- ively [38-401, those of bis(ethy1)triamines (BE33, BE34 and BE44) and of bis(ethy1)pentaamines (BE4334, BE3443 and BE4444) have not. Thus, the antiproliferating effects of the above nine kinds of bis(ethy1)polyamine analogues were compared systematically using mouse FM3A cells (Fig. 2). Although cell growth was inhibited significantly by low concentrations (10-30 pM) of bis(ethy1)tetraamines as reported previously [5, 8, lo], the degree of the inhibition of cell growth by bis(ethy1)polyamine analogues was not dose-de- pendent. For strong inhibition (more than 90%), relatively

394

Table 1. Change of polyamines and bis(ethy1)polyamine analogues and polyamine-metabolizing enzymes in FM3A cells. Cells were cultured in the presence and absence of bis(ethy1)polyamine analogues. At the designated times, cells were harvested and the amount of polyamines and their analogues and the activities of polyamine metabolizing enzymes were measured. Each value is the average of three determinations. Standard error was within -t 10% for each point.

Culture Time Content of Enzyme activity of

F'utrescine Spermidine Spermine Analogues OrnDC AdoMetDC SSAT

h nmoVmg protein pmol min-' mg protein-'

- - - - 11.3 17.5 8.69 12 10.3 14.3 8.75 24 8.25 10.3 8.37 48 7.25 9.45 8.00 - 134 18.5 81.9 72 4.50 9.63 7.53 159 13.0 74.4

BE34 6 3.14 9.34 7.87 19.6 12.9 13.2 788 12 2.83 3.31 4.51 24.8 7.9 7.4 2940 24 2.47 2.21 3.69 44.2 5.1 4.6 3870

(1

48 1.17 < 0.1 2.28 46.7 3.2 2.6 4380 72 < 0.1 < 0.1 2.31 49.7 <1 < 0.1 5250

BE343 6 3.55 9.72 8.05 19.0 9.6 8.0 538 (0.25 mM) 12 3.45 3.30 4.75 21.2 5.5 4.2 1690

24 1.32 1.48 4.15 37.7 < 1 2.1 2790 48 0.89 < 0.1 1.57 44.3 < 1 0.4 3880 72 < 0.1 < 0.1 <0.1 36.5 < 1 < 0.1 4550

BE3443 6 3.25 8.15 7.40 20.8 3.9 3.4 277 (0.25 mM) 12 3.52 2.36 4.52 26.1 < 1 1 .o 916

24 2.04 < 0.1 3.78 34.3 <1 0.4 2300 48 < 0.1 < 0.1 0.78 36.9 < 1 < 0.1 3070 72 < 0.1 < 0.1 <0.1 34.7 <1 < 0.1 3490

- - - Control 6

-

- - - -

~

high concentrations of bis(ethy1)polyamine analogues were necessary: they were 1-2 mM for bis(ethyl)triamines, and 0.25 -0.5 mM for bis(ethy1)tetraamines and bis(ethy1)pentaamines. The inhibitory effect was in the order BE4444 > BE3443 > BE4334 2 BE444 > BE343 > BE333 > BE44 > BE34 > BE33. The results imply that the presence of diaminobutane moiety in the bis(ethy1)polyamine analogues, especially in the backbone of the structure, causes the stronger inhibition of cell growth. The inhibition was not restored by provision of polyamines, which confirmed the previous results [ 101.

Mechanism of the polyamine-deficiency of cells caused by bis(ethy1)polyamine analogues

Polyamine contents and metabolizing enzymes in FM3 A cells treated with bis(ethy1)polyamine analogues were measured (Table l). When cells were treated with BE343 or BE3443 for 72 h, no significant amounts of polyamines remained in cells. When cells were treated with BE34, small amounts of spermine remained in cells. BE34 accumulated in cells at a concentration 6.6-fold that of spermine in control cells, while BE343 or BE3443 accumulated at a 4.8-fold or 4.6-fold concentration. Similar results were obtained among bis(ethy1)triamines (BE33, BE34 and BE44), bis(ethy1)tetraamines (BE333, BE343 and BE444) or among bis(ethy1)pentaamines (BE4334, BE3443 and BE4444 ; data not shown).

The decrease in polyamine contents started from putrescine, followed by the decrease in spermidine and spermine. Thus, the decrease in polyamine contents by the treatment of cells with bis(ethy1)polyamine analogues was generally parallel with the decrease in OrnDC and AdoMetDC activities. The induction of SSAT was in the order BE34 > BE343

> BE3443, which correlated inversely with the degree of inhibition of cell growth. Furthermore, although the addition of bis(ethy1)polyamine analogues to the assay mixture did not influence the activities of OrnDC and AdoMetDC, it inhibited SSAT activity strongly, which confirmed the previous results [8, 391. As a typical example, the effect of BE3443 on the activities of polyamine metabolizing enzymes is shown in Fig. 3. The results suggest that most fractions of induced SSAT exist in bis(ethy1)polyamine-analogue-treated cells as an inactive form. It was reported that the induction of SSAT rather than the inhibition of OrnDC and AdoMetDC correlated the polyamine deficiency of cells and their growth inhibition by BE343 [39, 411. However, our results suggest that the inhibition of OrnDC and AdoMetDC is strongly involved in the polyamine deficiency. Decrease of polyamines at the early stage of cells treated with bis(ethy1)polyamine analogues may be due to the excretion of polyamines through the replacement of polyamines on polyamine-binding sites by the analogues.

Polyamine transport was inhibited by bis(ethy1)polyamine analogues, and the degree of inhibition by the analogues generally paralleled that of cell growth (data not shown). This suggests that the inhibition of polyamine transport may also be involved in the polyamine-deficiency of cells, since significant amounts of polyamines exist in the medium.

Decrease in ATP content and inhibition of protein synthesis by the accumulated*bis(ethyl)polyamine analogues

We previously reported the correlation between the inhibition of cell growth by BE343, the amount of BE343 accu-

395

A AdoMetDC - OrnDC I

\--- , SSA: ~

0 0.1 0.2 0.3 0.4 0.5

BE3443 (rnM)

Fig. 3. Effect of BE3443 on the activities of polyamine-metabolizing enzymes. Activities of OrnDC (0), AdoMetDC (A) and SSAT (0) were measured as described in Materials and Methods in the presence of the various concentrations of BE3443 shown in the fig- ure. Maximum values (100%) for OmDC, AdoMetDC and SSAT were 125, 19.8 and 83.4 pmol . min-' . mg protein-', respectively, and the data are expressed relative to these values. Each value is the average of three determinations. Standard error was within 2 10% for each point.

mulated and the decrease in ATP content [lo]. The degree of inhibition of cell growth by nine kinds of bis(ethy1)polyamine analogues was nearly parallel with the decrease in ATP content (data not shown), confirming the previous results. We also reported that the accumulated spermine or spermidine inhibited protein synthesis due to the inactivation of ribosomes through the replacement of Mg2+ on magnesium-binding sites by polyamines [12]. In case of bis(ethy1)polyamine analogues, a decrease in magnesium content in the treated cells was not observed (data not shown). However, the inhibition of protein synthesis by BE34, BE343 and BE3443 was observed during the early period by treatment of cells with the analogues, followed by the decrease in ATP content (Fig. 4).

Inhibition of protein synthesis by the accumulated polyamines and bis(ethy1)polyamine analogues was confirmed in a cell-free protein synthetic system (Fig. 5). The concentration necessary for the inhibition of protein synthesis was in the order BE33 > BE34 > BE44 > spermidine > BE333 > BE343 > BE444 > spermine > BE4334 > BE3443 > BE4444, which correlated inversely with the degree of inhibition of cell growth by bis(ethy1)polyamine analogues. It was also noted that the degree of stimulation of protein synthesis at low concentrations of bis(ethy1)polyamine analogues was lower than that of spermidine and spermine at low concentrations.

Selective inhibition of mitochondrial protein synthesis In order to clarify the relationship between the inhibition

of protein synthesis and the decrease in ATP content, the degree of inhibition of total and mitochondrial protein synthesis by bis(ethy1)polyamine analogues was compared. When mitochondrial protein synthesis was measured, emetine was added together with [35S]methionine into the medium to inhibit cytoplasmic protein synthesis specifically

0 24 48 Time ( h )

Fig. 4. Comparison of decrease in ATP content and inhibition of protein synthesis in BE34-treated, BE343-treated and BE3443- treated cells. ATP content (- -) and [3H]leucine incorporation into whole cells (protein synthesis) (-) were measured as described in Materials and Methods. A, 1 mM BE34; 0, 0.25 mM BE343; 0, 0.25 mM BE3443. Maximum values (100%) for ATP content and protein synthesis were 13.7 nmoVmg protein and 897 nmol leucine incorporatedpg DNA, respectively, and the data are expressed relative to the value. Each value is the average of three determinations. The standard error was within ? 10% for each point.

[27, 281, and the amount of [35S]methionine incorporated into mitochondrial protein was measured. As shown in Fig. 6A, the degree of inhibition of mitochondrial protein synthesis by BE343 and BE3443 was much greater than that of cytoplasmic protein synthesis. This was confirmed by fluorography of total and mitochondrial protein (Fig. 6B). The synthesis of almost all kinds of cytoplasmic proteins was inhibited to a comparable degree (about 50%) by BE343. Mito- chondrial protein synthesis was inhibited thoroughly by BE343. Essentially the same results were obtained with BE3443 (data not shown).

The effect of BE343 and BE3443 on cytoplasmic and mitochondrial Ile-tRNA formation was then examined, since most mitochondrial tRNAs have an imperfect cloverleaf structure and are thus less stable compared to the structure of cytoplasmic tRNAs [42]. As shown in Fig. 7, mitochondrial Ile-tRNA formation was stimulated by spermine, but not significantly by BE343 and BE3443. The addition of high concentrations (2-3 mM) of BE343 and BE3443 inhibited mitochondrial Ile-tRNA formation. On the contrary, cytoplasmic Ile-tRNA formation was stimulated to a comparable degree by spermine, BE343 and BE3443. Only high concentrations (greater than 4mM) of BE3443 inhibited Ile- tRNA formation. These results suggest that the inhibition of mitochondrial aminoacylation by bis(ethy1)polyamine analogues may be one of the reasons for the selective inhibition of mitochondria1 protein synthesis.

It has been reported that BE343 causes selective depletion of mitochondrial DNA [43]. Depletion of mitochondrial DNA occurred 48 h after the addition of BE3443 (data not shown), which was at a later period compared with the previous results [43]. This may be due to the different cells used in the experiments, since we obtained the same results with BE343. The amount of nuclear DNA did not change significantly 48 h after the addition of BE3443. These results indicate that bis(ethy1)polyamine analogues first inhibit mitochondrial protein synthesis, which is then followed by the

396

A SPD (o), BE33 (0) BE34 (A), BE44 (A)

B BE333 (o), BE343 (0) c SPM (o), BE3443 (0) BE444 (A) 1 BE4334 (A), BE4444 (A)

0 1 2 0 0.1 0.2 0 0.06 0.12

Polyamine or bis(ethy1)polyamine (mM)

Fig. 5. Effect of polyamines and bis(ethy1)polyamine analogues on globin synthesis in a rabbit reticulocyte cell-free system. Globin synthesis was performed under standard conditions except that polyamine or bis(ethy1)polyamine analogue was added to the reaction mixture. Each value is the average of three determinations. Standard error was within 5 10% for each point.

A

0 12 24 36 48 Time (h)

Fig. 6. Effect of BE343 and BE3443 on total and mitochondrial protein synthesis. (A) ["SIMethionine incorporated into protein was measured. A and A represent total and mitochondrial protein synthesis, respectively, in 0.2 mM BE343-treated cells; 0 and 0 represent total and mitochondrial protein synthesis, respectively, in 0.2 mM BE3443-treated cells. Maximum values (100%) for total and mitochondrial protein synthesis were 1.47X10' and 7.77 x lo5 cpm [35S]methionine incorporatedmg protein, respectively, and the data are expressed relative to the value. Each value is the average of three determinations. Standard error was within ? 10% for each point. (B) Fluorography. Samples were prepared from cells cultured for 48 h. Lane 1, 20 kg total protein from control cells; lane 2, 20 pg total protein from cells treated with 0.2 mM BE343 for 48 h; lane 3, 25 pg mitochondrial protein from control cells; lane 4, 25 pg mitochondrial protein from cells treated with 0.2 mM BE343 for 48 h. Numbers on the left represent molecular masses in kDa.

decrease in ATP content, swelling of mitochondria and fi- nally depletion of mitochondrial DNA. The sequential events are in good accordance with the finding that most polyamines exist as a polyamineRNA complex in cells [44].

DISCUSSION The effect of nine kinds of bis(ethy1)polyamine analogues

on cell growth was studied to determine whether there is a

I A B

In 1

0 1 I 1 1 I I I I I

0 1 2 3 4 0 1 2 3 4 Spermine or bis(ethy1)polyamine (mM)

Fig.7. Effect of spermine, BE343 and BE3443 on cytoplasmic (A) and mitochondrial (B) Ile-tRNA formation. Ile-tRNA formation was performed under standard conditions except that spermine (O), BE343 (A) or BE3443 (0) was added to the reaction mixture. * indicates that the reaction mixture becomes muddy during incubation. Each value is the average of three determinations. Standard error was within ? 10% for each point.

better antitumor drug reagent than BE333 [45], and also to clarify the mechanism of the inhibition of cell growth by bis(ethy1)polyamine analogues. Especially, it is of interest to know the antiproliferative effect of bis(ethyl)pentaamines, which have not been studied thus far. The results indicate that the mode of action of the nine kinds of bis(ethy1)polyamine analogues is the same; both polyamine deficiency and accumulation of the analogues are involved in the inhibition of cell growth. However, the most effective concentration for inhibiting cell growth was the lowest in bis(ethy1)pentaamines. It has been reported that BE333 is the most effective against human melanoma xenografts among bis(ethy1)tetraamines since the drug has the least adverse reactions [45]. It is important to know why bis(ethyl)tetraamines, which contain diaminobutane moiety in the backbone of the structure have strong adverse reactions because these drugs inhibit cell growth to a greater extent than the drugs which contain the diaminopropane moiety in the backbone of the structure.

397

BE4334 showed the weakest effect among three bis(ethy1)- pentaamines. However, it is worthwhile to study the antitumor activity of BE4334, together with BE3333, since these drugs seem to less adverse reactions.

Although it became clear that accumulation of bis(ethy1)- polyamine analogues is important for the inhibition of cell growth by the analogues [lo-12, 461, it is not clear whether polyamine deficiency is involved in the growth inhibition which is not restored by provision of polyamines. It was reported that eIF-5 A containing hypusine, derivative of spermidine, was essential for cell viability in yeast [47], and that a potent inhibitor of deoxyhypusine synthase, which is involved in the first step of hypusine synthesis on the precursor protein of eIF-5A, inhibited cell growth of cultured Chinese hamster ovary cells [48]. These results raise the possibility that the effect of bis(ethy1)polyamine analogues might be due to depletion of hypusine brought about by the deficiency of spermidine in the cells. However, recent study with 5’-{ [(Z)- 4-amino-2-butenyl]methylamino} - 5’-deoxyadenosine, an irreversible inhibitor of AdoMetDC [49], shows that inhibition of cell growth correlated with an accumulation of precursor protein of eIF-5A is restored by the provision of polyamines [50]. Effect of N-(n-butyl)-l,3-diaminopropane, an inhibitor of spermine synthase, reported by Pegg and Coward [51] suggested that depletion of spermine caused growth inhibition in several cell lines, but the growth inhibition was observed only in a long-term culture in the presence of the inhibitor and was cytostasis. It is likely that both mecha- nisms, accumulation of analogues and deficiency of polyamines, are involved in the growth inhibition with bis(ethy1)- polyamine analogues. However, the cellcidal effect by bis- (ethy1)polyamine analogues observed in the present study cannot be explained by the deficiency of polyamines.

The accumulated bis(ethy1)polyamine analogues caused the inhibition of protein synthesis, especially of mitochondrial protein synthesis. Thus, proteins involved in the ATP production were markedly decreased, followed by the decrease of ATP content and swelling of mitochondria. Fi- nally, deletion of mitochondrial DNA was observed. We recently reported that the accumulated polyamines (spermidine and spermine) also caused the inhibition of protein synthesis [12]. However, mitochondria1 damage appeared at a later stage of cell culture compared to the damage caused by BE343. Since mitochondrial Ile-tRNA formation was much more strongly influenced by accumulated BE343 and BE3443 than spermine, the difference in the inhibition of mitochondrial protein synthesis by spermine and bis(ethy1)- polyamine analogues probably reflects this damage. Experi- ments are now in progress to clarify whether or not the degree of inhibition of mitochondrial ribosomal activity by bis(ethy1)polyamine analogues is the same as that of cytoplasmic ribosomal activity.

The results taken together indicate that the interaction between RNA and polyamines (or polyamine analogues) is important for their functions in cells.

We would like to express our thanks to Dr K. Samejima for his encouragement during the course of this study and to Dr S. Ohta for his advice on the analysis of mitochondrial protein synthesis. Thanks are also due to Dr H. Matsuzaki and D. A. Clayton for their kind supply of mouse mammary carcinoma FM3A cell lines and plasmid pAMl, respectively. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

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Correlation between the inhibition of cell growth by bis(ethyl)polyamine analogues and the decrease in the function of mitochondria