Department of Biochemistry and Immunology, St.Georges Hospital Medical School, University of London, London SW17 ORE, UK
1 To whom correspondence should be addressed at: Department of Biochemistry and Immunology, St.Georges Hospital Medical School, Cranmer Terrace, London SW17 ORE, UK. e-mail: g.whitley{at}sghms.ac.uk
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Abstract |
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Key words: apoptosis/extravillous trophoblast/hydrogen peroxide/nitric oxide/polyamines
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Introduction |
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The polyamines, spermine, spermidine and putrescine, are ubiquitous regulators of cell proliferation and differentiation (Sooranna and Das, 1995; Facchiano et al., 2001
; Maccarrone et al., 2001
). Putrescine is synthesized from arginine via ornithine and can be converted to spermidine and eventually to spermine through the sequential addition of propylamine groups. Their exact function within cells is still uncertain but they appear to exert a wide range of effects including ion channel control, protein synthesis and cell cycle regulation (Facchiano et al., 2001
). In addition, high levels of polyamine synthesis have been demonstrated in cells stimulated to grow and divide, and also in neoplastic tissue (Pegg, 1988
; Celano et al., 1989
). It has also been suggested that polyamines may be important in placental growth and for a successful pregnancy (Sooranna and Das, 1995
). Elevated levels of polyamines have been reported during pregnancy, with the highest levels detected during the first trimester (Sooranna and Das, 1995
). The enzymes involved in the oxidation of polyamines have also been detected in maternal serum, the placenta and the decidua during pregnancy (Illei and Morgan, 1979
; 1980).
In addition to their role in regulating many essential cellular processes, polyamines have also been implicated in the regulation of cell survival (Poulin et al., 1995; Stefanelli et al., 1998
; 2000). In some cell types, such as smooth muscle cells (Facchiano et al., 2001
), polyamines have been shown to induce apoptosis, while in others, such as thymocytes (Brune et al., 1991
), they play a protective role.
The aim of this study was to determine the effect of polyamines on extravillous trophoblasts. We demonstrate that polyamines rapidly induce apoptosis in the extravillous trophoblast cell line SGHPL-4. We also show that the induction of apoptosis is mediated through the oxidation of polyamines by amine oxidases and the production of hydrogen peroxide. Nitric oxide (NO) is known to react with hydrogen peroxide and to share a common biosynthetic origin with the polyamines. It has also been shown to inhibit apoptosis in many cell types and we have demonstrated that it inhibits apoptosis in extravillous trophoblasts (Dash et al., 2003). We therefore investigated the role of NO in the regulation of polyamine-induced apoptosis.
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Materials and methods |
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Cell lines
SGHPL-4 cells are derived from primary extravillous trophoblasts transfected with the early region of SV40. Characterization of these cells has confirmed an extravillous trophoblast-like phenotype including expression of cytokeratin-7, BC-1, HLA Class I, CD9, hPL and hCG (Choy and Manyonda, 1998; Cartwright et al., 1999
; Shiverick et al., 2001
). SGHPL-4 cells were cultured using standard tissue culture techniques in Hams F10 media supplemented with glutamine (2 mmol/l), penicillin (100 units/ml), streptomycin (0.1 mg/ml) and 10% (v/v) fetal calf serum. Jurkat cells were used as a positive control in some of the apoptosis assays as it is well established that apoptosis is induced in these cells following exposure to 10 µmol/l camptothecin for 4 h.
DNA fragmentation (Comet) assay
Cells were treated with 10 µmol/l spermine for 5 h, pelleted and resuspended in PBS. Frosted microscope slides were covered with normal melting point (NMP) agarose. Approximately 104 cells were mixed with 65 µl low melting point agarose and placed on top of the layer of NMP agarose. A further layer of NMP agarose (75 µl) was then placed on top of the cell suspension. The slides were then placed in cold lysis solution [2.5 mol/l NaCl, 100 mmol/l EDTA, 10 mmol/l Tris, adjusted to pH 10 with 1% (v/v) Triton X-100 added fresh] for 1 h at 4°C. Slides were then placed in a horizontal electrophoresis tank and covered with electrophoresis buffer (1 mmol/l EDTA, 300 mmol/l NaOH) for 30 min before being electrophoresed at 25 V for 20 min. The slides were neutralized using 0.4 mol/l Tris (pH 7.5) and DNA was visualized by fluorescent microscopy following the addition of ethidium bromide.
Time-lapse digital image microscopy
Apoptosis was observed over time using an Olympus Ix70 inverted fluorescence microscope equipped with a Hamamatsu C474295 digital camera. The microscope and stage were enclosed within a heated and humidified chamber (37°C) in the presence of 5% CO2 in air. Images and time-lapse sequences were analysed using Image Pro Plus software from Media Cybernetics. In each treatment 40 cells per field of view were randomly chosen, with three fields of view examined and repeated in triplicate. These cells were observed over time and were scored according to whether they became apoptotic. Apoptotic cells were also scored according to the time at which clear apoptotic morphology was first observed. Apoptotic morphology was considered as cytoplasmic and nuclear shrinkage and a change to a phase-bright appearance, often with the formation of membrane blebs/blisters.
Caspase activity assays
Cells (5x106) were pelleted, resuspended in 500 µl lysis buffer [10 mmol/l Hepes, 2 mmol/l EDTA, 0.1% (v/v) NP40, 5 mmol/l dithiothreitol (DTT), 1 mmol/l phenyl-methyl-sulphonyl-fluoride (PMSF), 10 µg/ml pepstatin A, 20 µg/ml leupeptin and 10 µg/ml aprotinin] and incubated on ice for 15 min. The lysates were centrifuged at 10 000 g for 2 min, the supernatant removed and protein content quantified using the Bradford assay. In a microtitre plate reader 178 µl of reaction buffer [100 mmol/l Hepes, 20% (v/v) glycerol, 0.5 mmol/l EDTA and 5 mmol/l DTT] was mixed with up to 20 µl of cell lysate (containing 100200 µg total protein) and 2 µl of a colorimetric caspase substrate. Volumes were adjusted to 200 µl where necessary and plates incubated at 37°C. Absorbance was measured at 405 nm every 30 min to demonstrate enzyme activity. The value for caspase activity was determined after 2 h and is represented as a percentage of the caspase activity in untreated cells (modified from Kim et al., 1998).
Greiss reaction
NO was measured indirectly by the Greiss reaction to detect nitrite in the media. The reaction mixture was made by adding 1 ml of a sulphanilamide solution [2% (w/v) sulphanilamide, 5% (v/v) phosphoric acid] to 1 ml of a 0.2 % (w/v) solution of napthylethylenediamine. The reaction mixture (25 µl) was then added to the sample (75 µl) and the absorbance read at 540 nm. A standard curve was generated using serial dilutions of a stock solution of sodium nitrite (0200 µmol/l).
Western blot analysis
Following SDS-PAGE, proteins were transferred onto Hybond P membrane (Amersham, UK). Membranes were blocked for 1 h in 5% (w/v) non-fat milk. The primary antibody incubation was performed for 1 h at a dilution of 1:1000 for both PARP and caspase-3. The secondary antibodies were used in both cases at a dilution of 1:1000. Detection was performed using Electrochemiluminescence (ECL) Plus (Amersham, UK) according to the manufacturers instructions. Western blots shown are representative of at least three separate experiments.
Transient transfections
Transient transfections of SGHPL-4 cells were performed in 6-well plates in the presence of 10% FCS using poly-L-ornithine (15 000 Mw) mixed with DNA at a ratio of 0.9:1 (wt:wt). The fusion protein (iNOS-EGFP) plasmid used was produced by Purdie et al. (2002). Cells were incubated with 100 µmol/l chloroquine in tissue culture media for 30 min prior to the addition of the poly-L-ornithine/DNA mixture at a final concentration of 5 µg DNA per well. The cells were then incubated for 5 h, washed with PBS and fresh media added. Analysis of the cells was carried out 24 h post-transfection.
Statistics
The time taken to induce apoptosis in 50% of cells, the EC50 value, was determined by non-linear regression using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Triplicate EC50 values were then analysed for their statistical significance using Students t-test and error values expressed as standard errors.
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Results |
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Spermine-induced apoptosis is independent of caspase-2, -3, -6, -8 and -9 activity but is inhibited by the broad spectrum caspase inhibitor Z-VAD-fmk
The involvement of caspases in spermine-mediated cell death was investigated by measuring the level of caspase activity following exposure to 10 µmol/l spermine for 5 h. Cell lysates were tested for caspase-2, -3, -6, -8 and -9 activity. Caspase activity was expressed as a percentage of untreated SGHPL-4 cells, while a combination treatment of TNF and actinomycin D was used as a positive control (Figure 4A). Spermine-treated cells showed no increased activity in any of the caspases tested compared with untreated cells. By contrast, cells treated with TNF
and actinomycin D showed increased activity in all the caspases, particularly caspases -3 and -6. Caspase activity was also assayed at different time-points (1, 2, 4, 6, 7 and 8 h exposure to 10 µmol/l spermine), but no activity was detected.
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Oxidation of spermine contributes to the induction of apoptosis
It has been reported that oxidation products of polyamines may account for their ability to induce apoptosis in other cell types. To determine whether this is the case for spermine-induced extravillous trophoblast apoptosis, SGHPL-4 cells were pre-incubated with 3 mmol/l pargyline, an inhibitor of amine oxidases (Figure 5). Treatment with pargyline was found to significantly (P < 0.001) inhibit the onset of apoptosis induced by spermine (the EC50 value increased from 5.5 to 14.7 ± 0.8 h), and also to reduce the number of cells that are apoptotic over the time-course of the experiment. This result indicates that the induction of apoptosis by spermine was, at least in part, a consequence of its oxidation.
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Discussion |
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Polyamines induce apoptosis in many cell types, including endothelial cells and smooth muscle cells (Facchiano et al., 2001) but are also known to protect from apoptosis in other cell types such as thymocytes (Brune et al., 1991
). In extravillous trophoblasts we found that polyamines rapidly induced apoptosis, in the case of spermine at doses of 10 µmol/l and upwards. The onset of apoptosis occurred only a few hours after exposure to spermine and typically led to 100% apoptosis within 45 h. Such rapid induction of apoptosis in the entire cell population is unusual and may indicate that these cells are particularly sensitive to the induction of apoptosis by spermine. Spermidine was also an effective inducer of apoptosis, but to a lesser extent than spermine, while putrescine had little effect.
It is unclear how polyamines such as spermine are able to induce apoptosis, although there is much speculation in the literature. It has been proposed that polyamines can directly activate caspases (Stefanelli et al., 1998; 1999), promote cytochrome C release from the mitochondria (Stefanelli et al., 2000
), activate transglutaminase activity (Facchiano et al., 2001
) or produce toxic metabolites such as aldehydes (Szabo et al., 1994
) or hydrogen peroxide (Maccarrone et al., 2001
). We examined characteristic biochemical markers of apoptosis such as PARP cleavage and caspase-3 processing in order to determine how spermine was able to induce apoptosis in extravillous trophoblasts. It was found that spermine treatment resulted in morphological changes and DNA fragmentation consistent with apoptosis, but showed no signs of either PARP cleavage or processing of caspase-3. A more detailed examination of caspase activity demonstrated no increased activity of caspases-2, -3, -6, -8 or -9 at any time-point following treatment with spermine. Despite this, treatment of cells with the broad-spectrum caspase inhibitor Z-VAD-fmk was able to significantly inhibit cell death. This suggests that some caspase activity was involved in the cell death, but that it proceeds independently of the major caspase activation pathways. This was an unexpected finding as, to our knowledge, polyamines have not previously been shown to induce apoptosis in this way in other cell types.
This unusual mechanism of apoptosis induction was further investigated by examining the potential role of the metabolic products of polyamines. There is evidence in some cell types that the induction of cell death by polyamines is due to their oxidation products rather than through direct effects (Szabo et al., 1994; Maccarrone et al., 2001
). Polyamines are typically oxidized by a class of enzymes known as amine oxidases which include specific polyamine oxidases such as spermine oxidase. It has been shown that amine oxidases are present in the serum, amniotic fluid and retroplacental serum during pregnancy, and can be produced by the decidua at high local concentrations (Illei and Morgan, 1979
; 1980).
The products of polyamine oxidation are hydrogen peroxide, ammonia and the relevant aldehyde, all of which have been reported to induce cellular stress and apoptosis. Pargyline, an inhibitor of amine oxidases, and catalase, an enzyme involved in the metabolism of hydrogen peroxide, were able to significantly increase the survival times of extravillous trophoblast cells exposed to spermine, suggesting that at least some of its apoptosis-inducing effect is due to its oxidation and the formation of hydrogen peroxide. The inability of putrescine to induce apoptosis may reflect the fact that putrescine is oxidized by separate enzymes to spermine and spermidine (Morgan, 1987; Sessa and Perin, 1994
). It is likely that oxidation occurs outside the cells in the culture medium, as it has been shown that amine oxidases are present in serum and we have observed that apoptosis does not occur in the absence of serum (unpublished data). This hypothesis is supported by the effect of catalase, which is too large a molecule to readily penetrate the plasma membrane.
Hydrogen peroxide is a well-known reactive oxygen species capable of initiating lipid peroxidation, cellular stress and apoptosis. There is some evidence that, in certain circumstances, hydrogen peroxide is able to inhibit caspase activity (Hampton et al., 1998). It is possible that, in extravillous trophoblasts, hydrogen peroxide produced from polyamines suppresses caspase activity but promotes cellular stress, ultimately leading to the activation of specific apoptotic pathways that are independent of the major caspases. The addition of hydrogen peroxide directly to the tissue culture media was found to induce cell death in SGHPL-4 cells at doses comparable to that which can be theoretically produced from the oxidation of 10 µmol/l spermine (data not shown). However, this cell death was morphologically distinct from that induced by spermine and occurred over a much longer period, suggesting that the production of hydrogen peroxide is necessary, but not sufficient, for the induction of apoptosis by spermine.
We and others have previously shown that the signalling molecule NO is involved in pregnancy through its action as a vasodilator (Williams et al., 1997) and as an important regulator of trophoblast functions such as implantation, differentiation, motility, invasion and apoptosis (Lyall et al., 1998
; Cartwright et al., 1999
; 2002; Gagioti et al., 2000
). NO is derived from L-arginine in a reaction catalysed by NOS, of which three isoforms exist. Cells of the placenta express at least two of these isoforms, the calcium-calmodulin dependent isoform, eNOS, and the calcium independent isoform, iNOS, that is expressed following growth factor stimulation (Zarlingo et al., 1997
; Martin and Conrad, 2000
; Yoshiki et al., 2000
; Hambartsoumian et al., 2001
).
The effect of NO on polyamine-induced extravillous trophoblast apoptosis was investigated using NO donors and iNOS over-expression. Exogenous NO from the NO donors DPTA-NONOate and PAPA-NONOate both inhibited spermine-induced apoptosis. DPTA-NONOate (half-life of decomposition to release NO of 3 h) delayed the onset of apoptosis, while the highest dose of PAPA-NONOate (100 µmol/l, half-life of 15 min) completely prevented apoptosis in 90% of trophoblast cells treated with spermine. NO produced endogenously by cells transfected to over-express iNOS also significantly inhibited apoptosis, with effects comparable with those of DPTA-NONOate. We have previously demonstrated that SGHPL-4 cells express eNOS and iNOS (Cartwright et al., 1999; 2002). Inhibition of this basal NO production with the general NOS inhibitor L-NAME had no effect on the sensitivity of cells to spermine-induced apoptosis. This was unexpected, as we have previously demonstrated that basal NO production in extravillous trophoblasts plays an important role in protecting these cells from apoptotic stimuli such as TNF
and actinomycin D. This result suggests that the relatively low concentration of NO produced basally by these cells is insufficient to offer any significant protection from the apoptotic effects of hydrogen peroxide. It is possible that NO levels must be stimulated to rise above a threshold to have any protective effect from spermine, and that this occurs when iNOS is over-expressed in cells or when NO donors are used. This may also occur physiologically when iNOS expression is stimulated by growth factors or cytokines, when eNOS activity is enhanced following phosphorylation or due to increased NO production by cells in the local environment.
NO can react with hydrogen peroxide to form a variety of reaction products and can protect from reactive oxygen species toxicity (Wink et al., 1993). We have implicated hydrogen peroxide production as a constituent of spermine-induced apoptosis and it is therefore possible that rapid production of NO is needed to counter the effects of hydrogen peroxide. There are a number of ways in which NO may act to prevent the effects of hydrogen peroxide. It may react with it in the media and convert it to other, shorter lived species such as peroxynitrite or the hydroxyl radical (Filep et al., 1997
). These are more reactive species than hydrogen peroxide which, if produced in the media, may react harmlessly with serum proteins before they are able to diffuse into cells and cause damage. Another possibility is that hydrogen peroxide toxicity is mediated through lipid peroxidation and subsequent cellular stress, as it has been suggested that NO may be able to inhibit lipid peroxidation by interfering with the propagation stage of the peroxidation chain reaction (Hogg et al., 1993
; Kelley et al., 1999
). Since both amine oxidases and polyamines are present in the placenta the synthesis of hydrogen peroxide is possible. Under these circumstances NO may play an important role in regulating the exposure of the placenta to potentially damaging reactive oxygen species.
As well as its potential role in limiting the damage caused by hydrogen peroxide and other reactive oxygen species, it is known that NO can exert its anti-apoptotic effects through the direct suppression of caspase activity following nitrosylation of the active site cysteine residue (Mannick et al., 1999). We have previously demonstrated that NO can suppress caspase activity in SGHPL-4 cells following induction of apoptosis with TNF
and actinomycin D, and that even basal NO production is sufficient for the complete nitrosylation of caspase-3 (Dash et al., 2003
). Although no activity of caspases-2, -3, -6, -8 or -9 was detectable in cells treated with spermine it remains possible that as yet unidentified caspases may be inhibited by NO through nitrosylation.
Further work will seek to determine the mechanisms through which NO protects extravillous trophoblasts from polyamine- and hydrogen peroxide-induced apoptosis. Understanding the balance between the factors which can regulate oxidative stress within the placental environment may have profound implications for trophoblast function in early pregnancy.
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Acknowledgement |
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References |
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Submitted on November 13, 2002; accepted on January 20, 2003.