Spermidine/spermine N1-acetyltransferase transient overexpression restores sensitivity of resistant human ovarian cancer cells to N1,N12-bis(ethyl)spermine and to cisplatin

Gaetano Marverti *, Maria Giuseppina Monti, Anthony E. Pegg 1, Diane E. McCloskey 1, Saverio Bettuzzi 2, Alessio Ligabue, Andrea Caporali 2, Domenico D'Arca and Maria Stella Moruzzi

Dipartimento di Scienze Biomediche, Sezione di Chimica Biologica, Università di Modena e Reggio Emilia, Via Campi 287, I-41100 Modena, Italy, 1 Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA and 2 Dipartimento di Medicina Sperimentale, Sezione di Biochimica, Università di Parma, Via Volturno 39, I-43100 Parma, Italy

* To whom correspondence should be addressed Email: marverti.gaetano{at}unimore.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The limited induction of spermidine/spermine N1- acetyltransferase (SSAT) activity has been implicated as an important determinant of the reduced response to the spermine analogue N1,N12-bis(ethyl)spermine (BESpm) by the cisplatin or cis-diamminedichloroplatinum(II) (cDDP)- resistant human ovarian carcinoma cell line (C13*). We checked whether or not under conditions of SSAT overexpression, enzyme induction and cell sensitivity to both, BESpm and cDDP, were restored to levels comparable with those of more responsive cDDP-sensitive 2008 cells. We transiently transfected the SSAT repressed C13* cells with two expression vectors driving human SSAT overexpression by diverse promoters. We then analysed their responses in the absence and in the presence of BESpm. SSAT activity was promptly, but briefly, expressed by transfection with both pOP/SSAT and pCMV-SSAT plasmids. However, only in the presence of BESpm, did SSAT activity reach the highest levels of induction for longer duration, with different time-courses for the two vectors, that paralleled the effect on cell growth. Under these conditions, growth sensitivity to BESpm of the less-responsive C13* cells was 25% reverted to cell growth inhibition displayed by 2008 cells. More interestingly, the sensitivity to cDDP cytotoxicity also increased in parallel to SSAT overexpression. BESpm induction of pCMV-SSAT-transfected cells caused a further 20–30% reduction of cell survival induced by cDDP, almost recovering the sensitivity of 2008 cells. The enhanced effectiveness of cDDP was also confirmed by the comet assay, showing an increase in the number and length of tails of damaged DNA. These findings confirm that SSAT overexpression inhibits cell growth and enhances growth sensitivity to BESpm in C13* cells, showing for the first time that restoring high inducibility of SSAT activity subverts the reduced sensitivity to cDDP of SSAT-deficient cells, making them almost indistinguishable from the responsive parental 2008 cells.

Abbreviations: BESpm, N1,N12-bis(ethyl)spermine; CHENSpm, N'-ethyl-N''-[(cycloheptyl)methyl]-4,8-diazaundecane; CHO, Chinese hamster ovary; CPENSpm, N'-ethyl-N''-[(cyclopropyl)methyl]-4,8-diazaundecane; cDDP, cisplatin or cis-diamminedichloroplatinum(II); CMV, cytomegalovirus; RSV, Rous sarcoma virus; Spd, spermidine; Spm, spermine; SSAT, spermidine/spermine N1-acetyltransferase


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cisplatin or cis-diamminedichloroplatinum(II) (cDDP) is one of the most widely used chemotherapeutic agents. However, human tumours exhibit a wide spectrum of response to cDDP leading to resistance. This compels clinicians to a dose escalation that, although necessary to overcome even a 2-fold increase in resistance, usually results in unacceptable toxicity to normal tissues (1). The occurrence of the cDDP resistance is mainly based on the onset of intracellular modifications that arise following even few sessions of exposure to the compound, thus limiting its usefulness (2,3). These events have also been correlated to the development of a broad cross-resistance to other unrelated chemotherapeutic compounds in ovarian cancer (4). In this regard, we have demonstrated previously that the cDDP-resistant human ovarian cancer C13* cells were also cross-resistant to the spermine analogue N1,N12-bis(ethyl)spermine (BESpm), when compared with their parental cDDP-sensitive 2008 cells (5). In particular, we showed that the lower sensitivity to BESpm of C13* cells was partly associated with a deficient expression of spermidine/spermine N1-acetyltransferase (SSAT) owing to differential regulation at transcriptional and post-transcriptional levels (6). More recently, we attributed to a labile repressive system the limitation of SSAT induction using inducing agents to levels as high as in sensitive cells (7). Therefore, we concluded that the impaired expression of the SSAT gene in the cDDP-resistant cells could be somehow linked to the appearance of the acquired resistance phenotype.

SSAT is the rate-limiting enzyme in polyamine catabolic pathway. It acetylates spermine (Spm) and spermidine (Spd) that are then excreted from cell or back-converted to Spd and to putrescine, respectively, through the action of FAD-dependent polyamine oxidase (8). In this way, SSAT is essential for maintaining tightly regulated intracellular concentrations of the naturally occurring polyamines. Recently, a novel mammalian FAD-dependent enzyme distinct from polyamine oxidase, designated spermine oxidase on the basis of substrate specificity, has been characterized previously (9). The identification of this enzyme capable of directly oxidizing Spm to Spd has important implications not only for understanding polyamine homeostasis, but also, since it is an analogue-inducible enzyme, for determining the sensitivity of various human tumours to specific polyamine analogues (10). In fact, since high polyamine levels have been found in many different tumours (11) and suggested to be probably required to maintain the transformed phenotype (12), a novel strategy recently developed took advantage of Spm analogues with the aim of interfering with polyamine metabolism. In particular, bis(ethyl)derivatives have proved to be most effective, and some of them have been undergoing evaluation in phase I and phase II clinical trials (13). These compounds have been shown to act at different intracellular compartments, and one of the most interesting effects is the superinduction of SSAT. Depending on cell type, the induction of SSAT by Spm analogue has been often correlated with cell sensitivity and cell growth inhibition (14,15). However, this correlation is not a general response of all these compounds. SSAT can be highly induced by 1,12-dimethylSpm, without cytotoxicity (16). On the contrary, CHENSpm (N'-ethyl-N''-[(cycloheptyl)methyl]-4,8-diazaundecane), an unsymmetrically substituted Spm analogue, causes only 3-fold increase in SSAT activity in NCI H157 cells, but it induces cytotoxicity at levels comparable with CPENSpm (N'-ethyl-N''-[(cyclopropyl)methyl]-4,8-diazaundecane), a several 100-fold inducer of SSAT activity (17,18). The different induction of SSAT by CHENSpm and CPENSpm may be included in the different mechanisms by which these analogues induce apoptosis in many cell lines (19). However, in some cell types such as HL-60 cells, the significant induction of SSAT by CPENSpm was not sufficient for the initiation of apoptosis, suggesting a cytoprotective, rather than cytotoxic, function of SSAT activity when accompanied with little toxicity of the inducing agent (12). Analogue superinduction of SSAT has also been exploited for research purposes to better study the enzyme expression, because the very short life of SSAT enzyme and its very low basal activity make it difficult to study SSAT expression without inducers. However, an alternative approach to study SSAT gene regulation without inducing agents is to use transfection techniques to cause SSAT overexpression and then investigate possible changes in cell function and growth. In this regard, SSAT has been overexpressed in different cell lines such as MCF-7 breast cancer cells and in LNCaP prostate carcinoma cells by transfection of human SSAT cDNA, obtaining polyamine depletion, cell growth inhibition and enhanced growth sensitivity to the analogues (20,21). Chinese hamster ovary (CHO) cells, selected for their remarkable resistance to the polyamine analogues, BESpm and N1, N11-bis(ethyl)norspermine, showed altered SSAT activity and/or regulation owing to a point mutation in their SSAT gene. The sensitivity to the drugs was again restored through the expression of wild-type SSAT in these resistant cell clones. These analogue-resistant CHO cells were transfected with a construct where SSAT cDNA was under the control of the cytomegalovirus (CMV) promoter, simultaneously restoring both SSAT activity and analogue sensitivity (22). Accordingly, we transiently transfected the SSAT-deficient C13* cells with the same plasmid and with another expression vector bearing SSAT cDNA under the control of the strong universal Rous sarcoma virus (RSV) promoter (23), both driving human SSAT overexpression. Then, the cell response in the absence and the presence of BESpm was studied to check whether or not induction and sensitivity to BESpm was restored at levels comparable with those of responsive parental 2008 cells.

Moreover, it has been reported that, depending on cell type and protocol used, the binding of polyamines and their analogues to DNA affects the action of DNA alkylating agents such as cDDP, often increasing the effectiveness of this anticancer drug. Combinations of cDDP and polyamine analogues have shown synergistic interactions against neoplastic cells of different types both in vitro and in vivo (24,25). However, the therapeutic potential of these combinations might be limited at present by the lack of data in normal cells, since its selectivity has not yet been determined.

We show here that SSAT overexpression enhances growth sensitivity to cDDP of the SSAT-deficient C13* cells, restoring, at least in part, the effectiveness showed against the sensitive 2008 cells.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Drugs and chemicals
BESpm was kindly supplied by the Hoechst Marion Roussel (Cincinnati, OH). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO), except where otherwise indicated.

Cell lines
The 2008 cell line, established from a patient with serous cystadenocarcinoma of the ovary and the cDDP-resistant C13* subline, generated as described previously (3), were grown as monolayers in RPMI 1640 medium (BioWhittaker Europe, Verviers, Belgium) containing 10% heat-inactivated fetal bovine serum (BioWhittaker Europe, Verviers, Belgium) and 50 µg/ml gentamycin sulphate. Cultures were equilibrated with humidified 5% CO2 in air at 37°C. All studies were performed in Mycoplasma negative cells, as routinely determined with the Mycotest detection kit (Euroclone, Switzerland).

Constructs
The full-length cDNA fragment coding for human SSAT, as described previously (23), was cloned in pOP-SVI-MCS to originate the pOP/SSAT expression vector, a construct that drives SSAT overexpression under the control of the strong universal RSV promoter (26). Instead, the pCMV-SSAT vector drives SSAT expression under the control of the CMV promoter (27).

Cell transfection procedures
High-quality, endotoxin-free plasmid preparations were obtained routinely using the Jetstar Plasmid Maxi Kit (Genomed, GmbH, Bad Oeynhausen, Germany). Vectors were transfected into cells by the liposome-mediated gene transfer method. A total of 200 000 cells were plated in 2 ml of growth media in cell culture dishes (35 x 10 mm2), and grown until cells were ~60% confluent. Purified SSAT-bearing plasmids, 3 µg, or empty vector (as a control) and liposome reagent, 10 µl (DOSPER; Roche, Manheim, Germany), were added to each dish. The plasmid mixtures were allowed to form complexes at room temperature for 15 min in polystyrene tubes. After formation of the complexes, 1 ml of serum-free medium per plate was added to the tube containing the liposome–DNA complexes. Cells were rinsed once with serum-free medium, then the diluted complex solution was mixed gently, overlaid onto the rinsed cells, and then incubated for an initial 6 h at 37°C. Then 1 ml of complete medium was added to the plates. After 24 h incubation, the medium was replaced with fresh culture medium. Transfection efficiency was assessed by transfecting cells with a CMV-EGFP mammalian expression vector, and the percentage of positively transfected cells which were expressing enhanced green fluorescent protein under the control of the CMV promoter were detected by confocal laser spectral microscopy. Experiments were performed by adding the same amount of each plasmid to be tested, on an equimolar basis, to each cell culture for the time indicated. Controls were prepared by adding, under the same experimental conditions, the appropriate amount of the corresponding empty vector alone to the cell cultures. Dosper reagent was chosen among other transfection reagents, because it was found to be more efficient under these experimental conditions. In fact, 70–80% of cells appeared to be positive when observed by confocal laser spectral microscopy (data not shown), and very low cytotoxicity was evident.

Assay of SSAT activity
SSAT activity was measured essentially as described previously (28). The cells were harvested, washed twice in phosphate-buffered saline, and suspended in a buffer containing 10 mM Tris(hydroxymethyl)aminomethane (pH 7.2) and 1 mM dithiothreitol. This suspension was frozen and thawed twice, then cytosol aliquots were incubated in 100 mM Tris(hydroxymethyl)aminomethane (pH 8.0), 3 mM Spd and 0.5 nmol 1-[14C]acetyl coenzyme A in a final volume of 50 µl for 10 min at 30°C. The reaction was stopped by adding 10 µl of 1 M NH2OH HCl and boiling in water for 3 min. The resulting samples were spotted onto P-81 phosphocellulose discs and scintillation counted. The amount of cytosol added to the final reaction mixture was adjusted to maintain the enzyme/substrate concentrations within the linear range. Enzyme activity is expressed as picomoles of [14C]acetylspermidine formed/min/mg protein. Previous works (29) have shown that SSAT assay also measures other Spd-acetylating enzymes and that, particularly in untreated cells of different cell lines, the authentic SSAT accounts for ~27% of the assay-detectable activity versus 96% or more in BESpm-treated cells.

Protein determination
Protein content in the various assays was determined by the method of Lowry et al. (30).

Cell growth assay
Cell growth was determined by a modification of the crystal violet dye assay (31). On selected days, after removal of the tissue culture medium, the cell monolayer was fixed with methanol prior to staining with 0.25% crystal violet solution in 80% absolute ethanol for at least 30 min. After washing several times with distilled water to remove the dye excess, the cells were allowed to dry. The incorporated dye was solubilized in acidic isopropanol (1 N HCl: 2-propanol, 1:10). After appropriate dilution, dye was determined spectrophotometrically at 540 nm. The extracted dye was proportional to cell number. Percentage of cytotoxicity was calculated by comparing the absorbance of exposed to non-exposed (control) cultures.

Determination of intracellular polyamine content
The cells were allowed to adhere overnight and then were transfected and were either treated or not treated with BESpm. At scheduled times, cells were harvested, pelleted, washed with phosphate-buffered saline and extracted with 0.25 M HClO4 for the determination of intracellular polyamine content, essentially according to the method described by Seiler et al. (32), after dansylation. The dansyl derivatives were then extracted with CHCl3 and separated by high-performance liquid chromatography. The acid-insoluble cell pellet was resuspended in 0.3 M NaOH and aliquots were used for protein determination.

Western analysis
Cell extracts were resolved by SDS–PAGE using a 12% gel. Electrotransfer to nitrocellulose membrane was followed by staining with a polyclonal anti-SSAT antibody [prepared as described previously (8)] and detection using the Lumi-Light Plus detection solution (Roche).

Alkaline single cell gel electrophoresis (comet assay)
To determine the extent of DNA damage in cells, alkaline single gel electrophoresis, also called the ‘comet assay’, was performed essentially according to Johnson and Loo (33). Following treatment, aliquots of cells were harvested, washed and suspended in 1% low-melting point agarose dissolved in phosphate-buffered saline (without Ca2+ and Mg2+). Then 80–100 µl of this mixture was pipetted onto a cold microscope slide that had been pre-coated with 1% normal melting point agarose. Without delay, a glass cover slip was placed on top of the slide, and the agarose/cell mixture was allowed to completely congeal in a cold room for 10 min. After removing the cover slip, the gel slides were submerged with cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100 and 10% dimethyl sulphoxide were added fresh) in plastic dishes, and kept at 4°C for at least 1 h. Slides were then placed in alkaline electrophoresis buffer (300 mM NaOH and 1 mM EDTA, pH 13.5) and left at 4°C for 50 min to allow unwinding of the DNA. Slides were then transferred onto a horizontal gel electrophoresis box with fresh alkaline electrophoresis buffer, which just covered the slides. Electrophoresis was performed at 20 V for 25 min and the resulting current was ~300 mA. Following electrophoresis, the slides were carefully immersed in cold neutralizing buffer (0.4 M Tris–HCl, pH 7.5), which was changed three times after 5 min. Subsequently, the gels were stained with 60 µl of 2 µg/ml ethidium bromide for 15 min, and washed in deionized water for 10 min to eliminate excess dye in the dark. Gels were then covered with a cover slip, stored at 4°C overnight and analysed the following day. On each slide, the nuclei of 100 randomly selected cells were examined using an Axioscope 40 epifluorescence microscope (Zeiss, Germany) equipped with an excitation filter of 515–560 nm for the detection of DNA migration pattern. Individual tail lengths, measured from the edge of the extrapolated head region, were quantified at a 400-fold magnification by image analysis software (Axiovision 3.1 from Zeiss). The median of DNA migration lengths of 100 analysed cells was calculated and expressed as median DNA migration in µm. Additionally, from each sample the proportion of damaged cells, being determined by the number of cells with tail, was calculated and expressed as percentage of non-damaged control cells.

Statistical analyses
All values report the means ± SEM, unless otherwise indicated. Statistical significance was determined by Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to study the correlation between SSAT overexpression and inhibition of cell growth, we transiently transfected C13* cells with two different expression vectors carrying human SSAT cDNA: pOP/SSAT, that drives SSAT overexpression under the control of the strong universal RSV promoter, and pCMV-SSAT, which takes advantage of the CMV promoter. Since transient transfection allows an episomal gene expression that usually does not exceed 48–72 h, the effect of SSAT induction in transfected C13* cells was studied up to 72 h.

As shown in Figure 1A, SSAT activity induced in pCMV-SSAT C13* transfected cells peaked at 24 h, and then returned to the level of mock controls (Figure 1B) at 48 and 72 h. Instead, SSAT activity expressed by pOP-SSAT plasmid (Figure 1C) reached the maximum level at 24 h and then fell to the level of control plasmid only at 72 h, confirming that episomal SSAT expression lasts no longer than 24–48 h after transient transfection with pCMV-SSAT and pOP-SSAT, respectively. On the contrary, in the presence of BESpm the induction of total SSAT activity remained elevated for a longer time, up to 72 h in both cases; however, the time course of induction was vector-dependent. Transfection with pCMV-SSAT induced SSAT activity up to 200 pmol/min/mg already at 12 h. This induction persisted and reached its maximum level of 305 ± 12 pmol/min/mg at 72 h. The difference between BESpm-treated versus BESpm-untreated cells was 2.5-fold (P < 0.05) at 24 h and reached 12-fold (P < 0.01) at 72 h. Interestingly, BESpm stimulation of SSAT activity of pCMV-SSAT-transfected C13* cells was always significantly higher than induction in the same cells in the presence of control vector. In fact, by subtracting the values of induction by BESpm on genomic SSAT, the catalytic activity of plasmid-derived SSAT was ≥200 pmol/min/mg. A comparison of time course of SSAT induction by BESpm in pOP-SSAT-transfected C13* cells showed an effect that was very similar to that seen in 2008 cells, but the amount of induction was always lower in relation to pCMV-SSAT-transfected cells; although at 72 h, the analogue induction of SSAT activity was very similar for both vectors. It is interesting to note that the highest levels of SSAT induction in C13* cells for the shortest time was obtained with pCMV-SSAT plasmid.



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Fig. 1. Time-dependent induction of SSAT activity in C13* cells transiently transfected with pCMV-SSAT (A), with pOP-SSAT (C) compared with 2008 cells not transfected and C13* cells transfected with the empty vector (B). To evaluate SSAT induction in the presence of the analogue 12 h after transfection, cells were treated with 10 µM BESpm for the indicated times, harvested and then processed for SSAT enzyme activity determination. Circles, pOP-SVI-MCS; triangles, pCMV-SSAT; inverted triangles, pOP-SSAT; diamonds and dashed lines, 2008 cells and 10 µM BESpm. In each panel, the presence of BESpm is indicated by the closed symbols. Results represent the means of three separate experiments performed in duplicate. Error bars, SEM; where not visible, error bars did not exceed symbol size.

 
The effect of these experimental conditions on Spm content is shown in Table I. It is evident that analogue exposure progressively lowered Spm content of C13* cells with time. However, while in mock controls the Spm content was still ~70% of non-treated cells, in pOP-SSAT and pCMV-SSAT-transfected cells BESpm reduced Spm content by a further 30 and 40%, respectively. It is noteworthy that transfection with both SSAT plasmids resulted in a total Spm content that approached or was even lower than that determined in 2008 cells. Putrescine and Spd contents were also reduced following analogue administration to the transfected cells. However, the observed reduction of these polycations in cells containing SSAT plasmids compared with mock control cells was not as high as that obtained for Spm pool (data not shown).


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Table I. Effect on intracellular spermine content of transfection with pCMV-SVI-MCS (empty plasmid) and with SSAT-bearing plasmids (pCMV-SSAT and pOP-SSAT) in C13* cells and in 2008 cells not transfected, in the absence and in the presence of 10 µM BESpm

 
Previously, a large number of studies have shown that the cytotoxicity of different polyamine analogues is closely related with their ability to induce SSAT in a given cell type (14,15). Therefore, we have determined whether cell growth of transfected C13* and 2008 cells is similarly affected by BESpm exposure under our experimental conditions.

The correlation between SSAT activity and cell growth inhibition at 72 h after SSAT overexpression by transfection with pCMV-SSAT or pOP-SSAT and BESpm induction is shown in Figure 2. Under these conditions, SSAT activity induction by vector alone was already back to near the control levels (Figure 1A). As a consequence, cell growth was inhibited by only 20%. Nevertheless, this effect is comparable with that produced by BESpm administration alone in mock control cells, where SSAT induction was approximately three times higher. This could be a consequence of the steep and early increase of SSAT expression from the pCMV-SSAT plasmid at 24 h. However, the SSAT superinduction due to the combination of the early expression by pCMV-SSAT and BESpm stimulation showed the strongest inhibitory effect on C13* cell growth. In fact, cell proliferation was inhibited 6-fold (P < 0.01) and ~4-fold (P < 0.05) by this treatment, when compared, respectively, to mock control cells transfected with the empty plasmid (pOP-MCS) in the absence or in the presence of the analogue. Under these conditions, growth sensitivity to BESpm of the less-responsive C13* cells was brought to the level displayed by 2008 responsive cells. Similar results were obtained at 48 h, although cell growth inhibition was 10–20% lower than at 72 h (data not shown). The data here presented are in agreement with previous studies from McCloskey and Pegg. These studies showed that the derivative of CHO line C55.7, C55.7Res is 10-fold resistant to BESpm, because of a point mutation in the C55.7Res SSAT cDNA; and therefore, has reduced capability to respond with SSAT induction, and the restoration of sensitivity to BESpm can be successfully achieved by transfection with wtSSAT cDNA (22).



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Fig. 2. Correlation between levels of induction of SSAT activity (A) and cell growth inhibition (B) after 72 h treatment with 10 µM BESpm. C13* cells transfected with pCMV-SSAT, with pOP-SSAT or with the empty vector pOP-SVI-MCS and 2008 cells not transfected were treated with 10 µM BESpm for 72 h prior to determination of enzyme activity and cell growth inhibition as described in the Materials and methods section. Black bar, pOP-SVI-MCS plasmid; grey bar, pOP-SVI-MCS and 10 µM BESpm; dark grey bar, pCMV-SSAT plasmid; light grey bar, pCMV-SSAT and 10 µM BESpm; grey bar with coarse pattern, pOP-SSAT plasmid; dark grey bar with coarse pattern, pOP-SSAT + 10 µM BESpm; open bar, 2008 cells and 10 µM BESpm. Results are expressed as means of at least three determinations conducted with duplicate plates; error bars, SEM. *P < 0.05; **P < 0.01 by Student's t-test versus empty vector samples.

 
To further confirm that restoring sensitivity to BESpm in SSAT overexpressing C13* cells was actually because of SSAT induction and not because of a non-specific effect of the vector used, we also performed a cell viability assay on cells transfected with pOP-SSAT, which showed a different timing profile of SSAT induction. In this case, cell growth inhibition was comparable with the effect by pCMV-SSAT. However, the increase in SSAT activity driven by this plasmid plus BESpm did not increase at earlier times, and remained at least 50 pmol/min/mg lower than that observed with pCMV-SSAT even at 72 h. This difference may account for the ~15% diminished cell cytotoxicity that we have measured under these experimental conditions, as compared with the pCMV-SSAT plasmid plus the analogue. Also pOP-SSAT effect on cell growth observed at 48 h was 10–20% lower than that at 72 h, even if the difference on cell growth inhibition in the presence and in the absence of BESpm was lower than that observed in the case of pCMV-SSAT (data not shown), according with the timing of SSAT induction depicted in Figure 1. As reported in the Materials and methods section, the chosen ratio between Dosper and DNA was capable of combining a transfection efficiency of 70–80% with a low-cytotoxicity. However, it has been reported that depending on cell type, exposure time and the amount of reagents used per well, the transfection conditions affect cell viability (34), thereby probably explaining the cell growth inhibition by 9 ± 1% observed even with empty vector.

Since the effect of SSAT induction on Spm content and cell growth in pOP-SSAT-transfected C13* cells in the presence or absence of BESpm was always lower than in the case of the transfection with pCMV-SSAT, we decided to continue our experiments with cells transfected only with the latter plasmid for 72 h. At this time SSAT protein expression, as shown by the western analysis reported in Figure 3, was the highest achievable in our system. SSAT protein was undetectable in untreated cells and barely detectable in cells transfected in the absence of the analogue or in analogue-treated mock-transfected control cells (data not shown). BESpm exposure greatly enhanced SSAT protein amount in pCMV-SSAT-transfected cells, reaching levels even higher than those of BESpm-treated 2008 cells.



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Fig. 3. Western blot analysis of SSAT protein in pCMV-SSAT-transfected C13* cells and in 2008 cells. C13* cells after transfection (lanes 1–4) and 2008 cells not transfected (lanes 5–8) were incubated in the absence (lanes 1, 3, 5 and 7) or in the presence of 10 µM BESpm (lanes 2, 4, 6 and 8) for 48 h (lanes 1, 2, 5 and 6) and for 72 h (lanes 3, 4, 7 and 8), then western analysis was carried out using a polyclonal antibody as described in the Materials and methods section. Cell extracts containing 60 µg of protein were loaded onto each lane.

 
Since our manoeuvres restored sensitivity to BESpm in C13* cells, we decided to check whether or not C13* cell sensitivity to cDDP was also affected by SSAT overexpression and BESpm induction. Figure 4 shows that cDDP effectiveness is actually correlated with the degree of SSAT induction as shown in Figure 1. Figure 4A shows that cDDP cytotoxicity in mock-transfected C13* control cells (open circles) was increased ~15–20% by BESpm pre-treatment (closed circles), although the slope of the respective lines is quite parallel. Analogue pre-treatment sensitized resistant cells to cDDP more than transfection with pCMV-SSAT vector (Figure 4B, open triangles). It is noteworthy that SSAT overexpression due to combination of BESpm administration plus pCMV-SSAT transfection (Figure 4B, closed triangles) increased the sensitivity of C13* cells to cDDP by a further 30–40%, as is also evident from the greater slope of the line, restoring cell growth inhibition to levels comparable to those showed by sensitive 2008 cells.



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Fig. 4. Comparison of cDDP cytotoxicity in C13* cells transfected in different conditions. Twelve hours after transfection, C13* cells transiently transfected with pCMV-SSAT (B) or with the empty vector and 2008 cells not transfected (A), were treated with 10 µM BESpm (closed symbols) for 48 h and then co-treated with the indicated concentrations of cDDP for another 72 h. Cell monolayers in 24-well plates were fixed and stained with crystal violet solution in ethanol. After solubilization of the dye in acidic isopropanol, the optical density at 540 nm of the treated samples was compared with controls. Triangles, pCMV-SSAT; circles, pOP-SVI-MCS; diamonds and dashed lines, 2008 cells and 10 µM BESpm. In each panel, the presence of BESpm is indicated by the closed symbols. Results represent the means of three separate experiments conducted with duplicate plates. Error bars, SEM; where not visible, error bars did not exceed symbol size.

 
It has been shown that Spm analogue pre-treatment enhanced cDDP cytotoxicity by increasing DNA crosslinks (35). Thus, in order to check whether or not the enhanced cDDP cytotoxicity that we have observed could be ascribed to increased DNA damage, we performed single cell gel electrophoresis or ‘comet assay’ on C13* cells with highest SSAT overexpression and induction of cytotoxicity. The comet assay is a widely used method to analyse the genotoxic effects of chemical agents, since it enables DNA strand breaks to be detected with high sensitivity at the single cell level (36). In Figure 5, the damaging effects of cDDP on DNA of C13* cells transiently transfected with pCMV-SSAT are shown in the absence or in the presence of BESpm induction (Figure 5B). These effects are compared with those produced by the Pt compound on DNA of sensitive untransfected cells that have been treated with BESpm (Figure 5A). Figure 5B shows that in 10% of the cells transfected with pCMV-SSAT, and in 37% of the cells transfected and then treated with BESpm a light DNA damage occurred as showed by a comet tail length, respectively, of ~8 and 15 µm. This finding is consistent with the observation by the same technique that Spm depletion by itself affects DNA structure and synthesis, causing DNA strand breaks (37). However, cDDP exposure inflicted a higher DNA damage that further increased during SSAT overexpression. Experimental conditions such as SSAT transfection plus BESpm induction caused a 1.5- and 1.8-fold lengthening of DNA tails, accounting for the increased cDDP sensitivity. The enhancements of comet tail length brought about by these conditions are significant when compared with cDDP-treated cells (P < 0.05), and with cells exposed to the Spm analogue alone (P < 0.01). For comparison, Figure 5A shows that the DNA damage by cDDP exposure in the presence of the analogue in sensitive cells induces the formation of DNA tails that are almost as long as those of resistant cells treated with cDDP under SSAT overexpression conditions.



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Fig. 5. Comet assay analysis of DNA-damaging effects of cDDP in 2008 cells not transfected (A) and in C13* cells transiently transfected with pCMV-SSAT (B), in the absence or in the presence of BESpm, under the same conditions reported in Figure 4. The median of DNA migration lengths of 100 analysed cells was calculated and expressed as median DNA migration in µm. Additionally, from each sample the proportion of damaged cells, being determined by the number of cells with tail, was calculated, and expressed as percentage of non-damaged control cells. (A) Black bar indicates control 2008 cells; light grey and grey bar indicate cDDP 8 and 16 µM, respectively, in the presence of BESpm. (B) Black bars, C13* cells transfected with pCMV-SSAT; open bars, plus 10 µM BESpm; light grey and grey bar, cDDP 8 and 16 µM, respectively; light and grey bars with coarse pattern, same cDDP treatment in the presence of BESpm. The results represent the means of three experiments conducted with duplicate plates. Errors bars, SEM.

 
The DNA tails produced by the different experimental conditions described above are shown in Figure 6. Nuclei of the C13* and 2008 control cells (Figure 6A and G) show a compact area of fluorescence, and no DNA tails were detected. Analogue-treated C13* cells (Figure 6B) show a reduced DNA migration. In contrast, cells treated with cDDP alone (Figure 6C and D) display an increased electrophoretic mobility of the DNA fragments, accounting for the presence of strand breaks within nuclear DNA. These DNA lesions, which significantly impaired cell viability, are further enhanced by cDDP under SSAT overexpressing conditions, as evidenced by elongated comet-like tails (Figure 6E and F). Under these conditions, lengths of DNA tails became comparable with these of 2008 cells treated with cDDP plus analogue (Figure 6H and I).



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Fig. 6. The ethidium bromide-stained nuclei of 2008 and C13* cells prepared for the SCGE assay. (A) The nuclei of C13* cells transiently transfected with pCMV-SSAT are shown. (B) The nuclei of the same cells transfected and treated with 10 µM BESpm are shown. The other panels show different tail lengths of the typical comet-like pattern of the transfected C13* cells treated with 8 and 16 µM cDDP in the absence (C and D) or in the presence of BESpm induction (E and F). (H and I) By comparison, the tail length of the nuclei of 2008 cells treated with 8 and 16 µM cDDP plus BESpm is shown. Nuclei of control 2008 cells are shown in (G). The tail length measured from the edge of the extrapolated head region to the end of the tail is proportional to the applied damaging conditions.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A large number of studies have shown a correlation between the cytotoxic effects of Spm analogues N1,N11-bis(ethyl)norspermine and BESpm and their ability to induce SSAT activity (15,38). We have recently reported cross-resistance to BESpm in C13* cells, a human ovarian carcinoma cell line selected for resistance to cDDP. In this cell line we observed a decreased capability to induce SSAT activity in response to BESpm, with a consequent lower depletion of polyamine content than in sensitive 2008 cells (5). This is surprising since 2008 and C13* cells have no differences in their basal SSAT activity (39), in agreement with recent reports showing that SSAT gene is not among the genes altered following the development of the resistance phenotype, as revealed by DNA microarray analysis (40). As a consequence, cDDP-sensitive and cDDP-resistant cell lines have similar polyamine content, indicating that polyamine metabolism is not involved in the development of cDDP resistance. However, the response to more potent inducers such as BESpm is modulated by cDDP resistance, which hinders SSAT induction by means of one or more labile repressor proteins (7).

The correlation that we observed between the deficient expression of SSAT gene and the reduced sensitivity to SSAT inducing agents displayed by the cDDP-resistant C13* cells compared with their sensitive counterparts, prompted us to perform transfection experiments, that restore the constitutively reduced SSAT expression and thus led to restored sensitivity to BESpm and, in particular, to cDDP.

In these experiments, the greater and more prolonged increase of SSAT activity induced by the analogue is probably the result of widely known effects of BESpm on SSAT transcription and mRNA stabilization, which in this case involve both genomic and episomal SSAT. In our experiments, induction of SSAT activity driven by the two vectors seems to play the major role. This is particularly evident with the pCMV-SSAT plasmid. The swift increase in SSAT activity induced by BESpm in pCMV-SSAT-transfected cells, when compared with cells transfected with pOP-SSAT, could probably be ascribed to the greater strength of the CMV promoter compared with both endogenous SSAT and RSV promoter. In addition, the well-known post-transcriptional effects exerted by the analogue, which include enhanced mRNA translation and stabilization of SSAT protein by protection against rapid proteasomal degradation (41), may account for the increased and long-lasting induction of SSAT activity.

Recent attempts to increase SSAT activity, which in turn caused a decrease in polyamines content and inhibited cell growth, have been carried out successfully by stable transfection of SSAT cDNA in MCF-7 cells (20) and in SSAT-deficient CHO cells (22). Consistent with these studies, we show here that induction of SSAT activity by transfection paralleled enhanced sensitivity to the analogue, overcoming the cross-resistance still exhibited by control cells. The direct correlation between SSAT induction and a significant reduction of Spm content and cytotoxicity is demonstrated not only by the time course of exposure to the analogue with each plasmid, but also by the different results obtained between the two vectors. pOP-SSAT caused a weaker expression of SSAT than pCMV-SSAT under BESpm induction, and consequently it more weakly supported analogue action in inhibiting C13* cell growth. In consequence, it is noteworthy that the combination of pCMV-SSAT with the analogue inhibited cell growth of resistant cells with a more pronounced synergism than with pOP-SSAT.

Our results provide definitive evidence that, in resistant C13* cells, the re-establishment of BESpm sensitivity requires increased expression of SSAT, which we have recently shown as being depressed by labile repressor protein(s) in these cells (7).

Furthermore, our results demonstrate for the first time that SSAT overexpression not only brings C13* cell sensitivity to BESpm back to a level comparable to parental 2008 sensitive cells, but also restores sensitivity to cDDP. Depending on cell type and experimental protocol used, some authors have shown that other polyamine analogues synergized with cDDP against neoplastic cell lines (24,25). Very recently, it has been reported that this positive effect was the result of SSAT gene overexpression (42), since Pt drugs, oxaliplatin in particular, were shown to upregulate SSAT gene (43). However, we did not notice any induction of SSAT activity by cDDP alone, nor any appreciable effect on polyamine content in resistant and even in sensitive cells (data not shown), probably owing to different experimental conditions, such as drug treatment schedule and since cDDP appeared to be a much weaker inducer of SSAT activity than oxaliplatin.

Our group has also already reported that BESpm and cDDP acted synergistically to inhibit cell growth of 2008 cells and even of C13* cells in spite of documented cross-resistance, indicating that the multiple-site mechanism involved in analogue action could also modulate the action of cDDP (5). In the present study, we found evidence that, among these multiple mechanisms, SSAT overexpression is the most important factor in the enhanced cDDP cytotoxicity, owing to the high correlation between the elevated SSAT activity, causing Spm depletion and cDDP efficacy which was comparable to that exerted on sensitive cells.

It has been shown that the polyamine-depleted state increased cell sensitivity to monofunctional alkylating agents and that, under physiological conditions, Spm protected DNA from the induction of double-strand breaks (44), while its deficiency led to enhanced sensitivity to antiproliferative drugs (45). Consistent with these findings, we have already reported that in the resistant cells, BESpm induced Spm depletion, that facilitates DNA damage by cDDP, was lower than in sensitive cells. The impaired SSAT expression by repressor proteins in resistant cells might represent a mechanism developed during resistance selection to cDDP with the aim to protect cells against excessive polyamine depletion protecting DNA from attack by this alkylating agent. Conversely, sensitive cells, without such mechanism, have a more responsive SSAT gene. SSAT overexpression induced in resistant cells would make it possible to counteract this mechanism and thus to reduce Spm pool, facilitating the DNA-damaging action of cDDP.

Regarding the direct interaction with DNA, it has been reported that bis(ethyl)derivatives of polyamines are much weaker DNA aggregators than parent polyamines, and thus are good inhibitors of cell growth (46). Therefore, the analogue concentration itself may explain why DNA migration and tails were detected by comet assay even in samples untreated with cDDP, a condition that caused cell growth inhibition (Figures 5 and 6). The capability of reducing DNA aggregation exerted by polyamine analogues, along with Spm depletion (47), would affect the structure and/or organization of linker DNA, leading to relaxation of chromatin structure which seems to be preferred by DNA-binding anticancer agents such as cDDP with regard to the condensed status (48). Accordingly, polyamine depletion caused by SSAT induction and analogue pre-treatment probably renders chromatin more prone to nucleophilic attack by cDDP in resistant C13* cells, favouring cDDP–DNA adducts formation, increasing cytotoxicity and partially reducing cDDP resistance which is often associated with decreased cDDP–DNA adduct formation (49).

In summary, these findings demonstrate that SSAT overexpression, leading to Spm depletion, inhibits cell growth and re-establishes cell sensitivity to BESpm. Even more interestingly, cDDP sensitivity was also restored to levels very similar to those of sensitive parental cells. Taken all together, our data and those reported from other groups suggest the possibility of a therapeutic strategy based on drug combinations that, using selective inducers of SSAT, would be more efficient in causing cytotoxicity and, hopefully, overcoming the limitations due to cross-resistance.


    Acknowledgments
 
This work was supported by a grant from MURST 60%, and from Associazione Angela Serra per la Ricerca sul Cancro, Azienda Ospedaliera Policlinico di Modena, Modena, Italy. This work has been also partially supported by PRINN (Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale) 2004, MIUR, Italy and Associazione Assistenza Tumori Alto Adige–Südtiroler Krebshilfe, Bolzano, Italy.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received January 17, 2005; revised May 3, 2005; accepted May 13, 2005.





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