* Dept. of Aquatic Biosciences, NFH, University of Tromsø, N-9037 Tromsø, Norway, and Laboratoire Arago, CNRS-UMR 7628 UPMC, F-66650 Banyuls-sur-Mer, France
Received March 5, 2004; accepted May 28, 2004
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ABSTRACT |
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Key Words: 2-trans-4-trans-decadienal; polyunsaturated aldehyde; sea urchin; cell cycle.
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INTRODUCTION |
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The ,ß,
,
-unsaturated aldehydes have cytotoxic effect on several cell types, e.g., cell division is blocked in sea urchin embryos (Miralto et al., 1999
) and cell proliferation is inhibited in Streptococcus pneumoniae (Bisignano et al., 2001
) and Caco2 human colon adenocarcinoma cell lines (Miralto et al., 1999
). In a study using ascidian oocytes, 2-trans-4-trans-decadienal and 2-trans-4-cis-7-cis-decatrienal were found to reversibly inhibit the fertilization current which is generated in the oocyte after contact with a spermatozoon (Tosti et al., 2003
) thus inhibiting fertilization of the eggs. Apoptosis is a morphologically and biochemically distinct cell death program that is initiated under a variety of physiological and pathological conditions (Arends and Wyllie, 1991
), and DNA fragmentation is one of the hallmarks of apoptosis (Zhang and Xu, 2000
). Copepod and sea urchin embryos treated with 2-trans-4-trans-decadienal test positive in the terminal-deoxynucleotidyl-transferase-mediated dTPU nick-end labelling (TUNEL) assay and displays DNA laddering (Romano et al., 2003
), indicating that DNA fragmentation and thus apoptosis have been induced.
The ability of the PUAs to inhibit cell divisions in sea urchin embryos depends on the presence of the Michael element, thus the ,
-double bond or the stereochemistry of this bond is not essential for the biological activity of the compound (Adolph et al., 2003
). The polarity of the aldehyde is to a certain degree important, as shorter and more oxidized aldehydes have lower biological activity (Adolph et al., 2003
), probably due to differences in bioavailability of the PUAs. On the other hand, the chemical reactivity of the PUAs are influenced by the substitution on the ß-carbon. Any electron-releasing substituent that lessens the partial positive charge on this carbon atom will decrease the electrophilic reactivity of the
,ß-double bond, and vice versa (Witz, 1989
).
This study investigated the cellular effects of the ,ß,
,
-unsaturated aldehyde 2-trans-4-trans-decadienal (Fig. 1B) on mitotic cell divisions in order to obtain more knowledge of the underlying mechanism of cytotoxicity. Sea urchin embryos were used for this study because the mechanisms of cell cycle regulation are well characterized in this model (reviewed by Sluder et al., 1999
). Extracts from marine algae producing polyunsaturated aldehydes are known to affect mitotic processes like microtubular organization in sea urchin embryos (Buttino et al., 1999
), but to our knowledge nobody has tested the cellular effects of the pure toxin. Furthermore, alteration in the activity of the mitosis promoting factor (MPF) by PUAs has never been evaluated, thus we examined the effect of 2-trans-4-trans-decadienal on cell cycle time-course, DNA replication, mitotic spindle formation, and cyclin B/Cdk1 kinase activity.
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MATERIALS AND METHODS |
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Sea urchins. Sea urchins (Sphaerechinus granularis) were collected in the Mediterranean Sea near Banyuls-sur-Mer (France) and kept in running seawater until use. Spawning was induced by shaking the sea urchins. Eggs were filtered three times with a 100 µm mesh size sieve, and washed three times with filtered (0.22 µm membrane filter) seawater (FSW), and stored at 19°C until use. The sperm was kept undiluted on ice until use. All later manipulations and incubations of the eggs and embryos were done at 19°C unless otherwise specified.
Cytotoxicity test. The washed eggs were diluted to appropriate concentration (1000 eggs/ml) and sperm was diluted 1:100 with FSW. The egg suspension was fertilized with 1 µl of diluted sperm per ml egg suspension, and elevation of the fertilization membrane was controlled 1 min after fertilization. When a minimum of 95% of the eggs were fertilized, 50 µl of the egg suspension were transferred to 96-well plates containing different concentrations of 2-trans-4-trans-decadienal (Acros Organics, Geel, Belgium) in 50 µl FSW. Each well contained maximum 1% methanol, and blank controls containing the same amount of methanol as the dilutions of the extracts were included in every experiment. The number of dividing eggs was recorded 120 min after fertilization using an inverted microscope.
Incorporation and detection of BrdU. For incorporation and detection of 5-bromo-2-deoxyuridine (BrdU) (Amersham Biosciences Europe GmbH, Freiburg, Germany), eggs were fertilized in presence of 0.3 mg/ml BrdU and 1 mM of 3-amino-1,2,4-triazole (ATA), the latter to avoid hardening of the fertilization membrane (Schowman and Foerder, 1979). After 1 min, the presence of fertilization membranes was confirmed in a microscope. As soon as the cells had sedimented, the supernatant was removed and 0.1 mg/ml pronase in FSW was added in order to remove the fertilization membrane. The eggs were allowed to sediment and the solution was exchanged with 1% bovine serum albumin (BSA) in FSW. When the eggs had sedimented they were washed twice with FSW. The eggs were diluted to 5% (v/v) density with FSW containing 0.3 mg/ml BrdU, dispatched in 1 ml in four well plates and incubated in 5 µg/ml 2-trans-4-trans-decadienal, 10 µg/ml aphidicolin (negative control) and FSW containing the same amount of methanol as the PUA (positive control). Samples from each treatment were fixed 60 and 120 min after fertilization. The eggs were fixed in 4 M HCl for 120 min, post-fixed in methanol for 30 min and washed twice in AT-buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4 and 0.05% Tween-20) before they were incubated in anti-BrdU mouse monoclonal antibody (Amersham Biosciences Europe GmbH) at 4°C overnight and subsequently rinsed in AT-buffer. Incorporated BrdU was stained in darkness with 0.1% fluorescein 5(6)-isothiocyanate (FITC) conjugated anti-mouse antibody in AT-buffer for 60 min, and the stained eggs were washed twice in AT-buffer before mounting on pre-washed glass-plates in Mowiol (Clariant, Frankfurt, Germany).
Staining of microtubules. For immunofluorescence staining of microtubules, eggs were fertilized and the fertilization membranes were removed as described above. The fertilized eggs were diluted to 5% concentration with FSW, transferred to four-well plates, and incubated in 2-trans-4-trans-decadienal. Controls were incubated with FSW containing the same amount of methanol as the PUA. Eggs were sampled 30, 60, and 180 min after fertilization and treated with a microtubule-preserving buffer for 80 min. The buffer consisted of 10 mM Na-EGTA, 0.55 mM MgCl2, 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.8), 25% glycerol, and 1% nonylphenylpolyethylene glycol (NP40). The eggs were fixed for 60 min in 75% methanol and 25% glycerol. After being washed twice in AT-buffer, the eggs were preincubated in 5% goat serum in AT-buffer for 60 min, and incubated in 0.2% anti-mouse -tubulin and 4,6-diamidino-2-phenyllindole (DAPI) in AT-buffer overnight at 4°C. The eggs were washed three times with AT-buffer and incubated in darkness with FITC conjugated anti-mouse antibody in AT-buffer for 60 min. After washing three times in AT-buffer, the eggs were mounted on pre-washed glass-plates in Mowiol.
Western blot and kinase assay of cyclin B-Cdk1. To allow accurate detection of cyclin B in Western blot and measurement of immunoprecipitated cyclin B/Cdk1 activity in triplicates, 50 ml of a 5% (v/v) egg suspension were fertilized as described above and 2-trans-4-trans-decadienal (5 µg/ml) was immediately added. Aliquots of egg suspension containing a cell volume of 5 µl were briefly centrifuged, and the pellet was suspended in 250 µl IPNP-buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 ml -naphtyl phosphate, 0.1% soybean trypsin inhibitor, 1 mM phenylmethylsulfonyl fluoride, 10 mM phenylphosphate, 0.1% Triton X-100, 50 mM NaF, 10 mM pyrophosphate, 0.1 mM orthovanadate, and 20 µM ZnCl2. Samples were harvested immediately and 15, 30, 60, 90, and 110 min after fertilization and frozen in liquid nitrogen. Frozen samples were sonicated for 30 s and centrifuged at 4°C and 10,000 x g for 15 min. The supernatant was used for kinase assay or immunodetection by Western blot.
For kinase assays, 2 µg affinity purified anti-S. granularis cyclin B antibodies and 5 µl Protein A-Sepharose beads were added to the supernatant. The mixture was incubated for 1 h at 4°C under continuous shaking and the beads were pelleted and washed 3x in a buffer containing 150 mM NaCl, 50 mM Tris-HCl pH 7.4 and 0.1% Tween 20, and twice in the same buffer without Tween. Then 10 µl of a phosphorylation mix (10 mM MgCl2, 100 µM ATP, 1 µg histone H1, 80 mM Hepes pH 7.4 and 100 µCi/ml -[32P]ATP (3000 Ci/mmol)) were added to the immobilized complexes and incubated at 20°C for 10 min. The reaction was stopped by adding 10 µl 4 x Laemmli buffer and boiling for 3 min, and the proteins were separated by 12.5% SDS-PAGE. The gel was dried and submitted to autoradiography.
For immunoblot analysis, proteins were resolved on 8% SDS-PAGE and transferred to PVDF membranes (Amersham Biosciences Europe GmbH). Membranes were saturated overnight in buffer S: 200 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 25 Mm Tris-HCl pH 7.5, 0.05% Tween 20 containing 5% stim milk and probed with affinity purified anti-S. granularis cyclin B antibodies (1 µg/ml) in buffer S supplemented with milk powder. After 2 h incubation the membranes were washed in buffer S, and the bound antibodies were detected with mouse anti-rabbit IgG conjugated to peroxydase and ECL kit (Amersham Biosciences Europe GmbH).
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RESULTS |
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Figure 2 shows the dose-dependent effect of 2-trans-4-trans-decadienal on cell divisions in S. granularis. In the control treatment 98% of the eggs were developing normally and the embryos were at the two-cell stage 120 min after fertilization. A weak inhibition of cell division could be observed in 0.8 µM 2-trans-4-trans-decadienal where 86% of the embryos developed normally. At 1.6 and 3.3 µM the inhibition was increasingly stronger (29 and 3% normally developing embryos, respectively), and at concentrations from 6.6 µM and higher the inhibition of cell divisions was complete. By using a nonlinear least square model estimation we found EC50 to be 1.3 µM, which corresponds to 0.2 µg/ml. It should be noted that this value was obtained when experiments were performed with 50 eggs in a total volume of 100 µl in 96-well plates. As we increased the density of the embryos in the sample plates, the amount of 2-trans-4-trans-decadienal required for a complete block of cell division (100% arrested eggs) increased, from 6 µM for 50 eggs to 15 µM for 500 eggs per 100 µl; with a 5% egg suspension the minimal required concentration was 32.9 µM both in 24-well plates and 50 ml beakers (data not shown). 2-trans-4-trans-Decadienal has a high potential for nonspecific interactions, and we suspect that the variation in EC50-values might be due to interactions between the PUA and nucleophilic targets or hydrophobic sites on fertilization envelope, the plasma membrane or the cytosol of the eggs. Hydrophobic interactions with different plastic materials could also have an additional effect.
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In the treatments with the highest concentrations of 2-trans-4-trans-decadienal (i.e., five times the minimal concentration required to inhibit all eggs from dividing), we observed substantial membrane blebbing. This modification of egg morphology appeared about 100 min after fertilization when cytokinesis of the first cell division occurred in control eggs. Trypan blue exclusion assays set up along the two first hours post fertilization demonstrated that the plasma membrane permeability was not significantly affected by the 2-trans-4-trans-decadienal treatment even at the highest concentration.
2-trans-4-trans-Decadienal Inhibits DNA-Replication
To investigate whether 2-trans-4-trans-decadienal affects DNA synthesis, we monitored DNA replication by incorporation of the nucleotide analogue BrdU followed by staining with fluorescent labelled anti-BrdU antibodies. BrdU was incorporated in the chromatin of positive control eggs incubated in FSW as visualized in eggs harvested 60 and 120 min after fertilization (Figs. 3A and 3C, respectively). It was verified in a parallel experiment that no BrdU was incorporated in the presence of aphidicolin (20 µg/ml), an inhibitor of DNA polymerase (Sheaff et al., 1991
) (data not shown). In eggs incubated in 2-trans-4-trans-decadienal no DNA-synthesis occured as no BrdU had been incorporated in the chromatin either at 60 or 120 min post fertilization (Figs. 3B and 3D, respectively).
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DISCUSSION |
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It was not possible to reinitiate the cell cycle by removing the PUA from the incubation medium. This irreversible effect argues in favor of covalent modifications of proteins or DNA by this PUA as was previously shown for other ,ß-unsaturated aldehydes (Ishii et al., 2003
; van Iersel et al., 1997
). However we cannot exclude that the PUA being rather hydrophobic it could accumulate in the cells, and could be difficult to remove by simply washing with FSW.
In our cytotoxicity assay, embryos incubated in relatively high concentrations of 2-trans-4-trans-decadienal (five times the minimal dose for complete arrest of all eggs) displayed an extensive membrane blebbing already detectable 100 min after fertilization. Although this morphological characteristic has been associated with sea urchin embryos undergoing apoptosis (Lockshin et al., 1998; Roccheri et al., 1997
; Voronina and Wessel, 2001
) and lends support to other reports that 2-trans-4-trans-decadienal and related PUAs induce apoptosis in sea urchin at similar concentrations as in our assays (Miralto et al., 1999
; Romano et al., 2003
), we cannot be certain that apoptosis was occuring in the nondividing embryos displaying membrane blebbing in this study.
In presence of the PUA DNA-replication was inhibited in fertilized eggs as indicated by the absence of BrdU incorporation (Fig. 3). Unsaturated aldehydes have been reported to directly inhibit DNA polymerase in rats (Wawra et al., 1986
), and to interact with DNA and form adducts both in vitro and in vivo (Marnett, 1994
). However, these results were obtained using the
,ß-unsaturated aldehyde 4-hydroxy-nonenal (HNE), which is a relatively stronger electrophile than 2-trans-4-trans-decadienal due to its ß-substituted hydroxyl group. Nevertheless it is possible that 2-trans-4-trans-decadienal blocks DNA replication through the same mechanism even though we cannot exclude that the effect is secondary to interactions with other cellular targets required for DNA replication.
Initiation of DNA replication is normally preceded by the migration and fusion of male and female pronuclei although DNA synthesis is not strictly dependent on this process (Schatten and Schatten, 1981; Sluder et al., 1995
). A few minutes after sperm entry in an egg, the sperm head swells and changes into a sperm pronucleus. A microtubule aster forms around the sperm centriole and the male and female pronuclei migrate towards each other propagated by the elongating astral rays (Okazaki, 1975
). Eggs incubated in 2-trans-4-trans-decadienal a few minutes after fertilization showed no sign of pronuclear migration (Fig. 4). As the migration process is dependent on elongation of microtubule astral fibers this result suggest that polymerization of microtubules has been affected.
In order to assay the ability to assemble tubulin into microtubules in the nondividing embryos, we visualized microtubules by immunofluorescence with anti--tubulin antibodies (Fig. 5). The staining revealed an almost total absence of tubulin polymerization, and neither interphasic microtubular arrays nor distinct mitotic spindles were formed in the presence of 2-trans-4-trans-decadienal. One striking exception was that a significant portion of the nondividing eggs had single thick microtubules that were a few µm long (Fig. 5F), indicating that some abnormal polymerization had occurred. Tubulin subunits are known to interact with a broad range of ligands (Bai et al., 1996
) and several aldehydes have been demonstrated to inhibit colchicine from binding to tubulin. Colchicine is an alkaloid known to prevent tubulin polymerization by binding to tubulin (Hastie, 1991
). Tubulin contain thiol-groups that are important for its function, and these thiol-groups are suspected to be targets for the electrophilic aldehydes (Gabriel et al., 1977
). The
,ß-saturated aldehyde HNE is known to interact with tubulin and the adduction occurs via Michael addition (Neely et al., 1999
), and this could also be the case for 2-trans-4-trans-decadienal.
Beside a direct action on microtubule polymerization, PUA can also alter the activity of the MPF that is known to regulate microtubule dynamic (Verde et al., 1992). MPF consists of two proteins: A cyclin dependent kinase (Cdk1), which is more or less constitutively expressed, and a cyclin B regulatory subunit, which oscillates throughout the cell cycle. The association of Cdk1 with its partner is driven by the cyclin B synthesis, and the cyclin B/Cdk1 complex is stabilized by phosphorylation of the Thr161 residue on the catalytic subunit. The activity of the complex is restrained during S and G2 phases by inhibitory phosphorylation of the Thr14 and Tyr15 residues in Cdk1. At mitosis entry these residues are dephosphorylated by the Cdc25 phosphatase (for reviews see Porter and Donoghue, 2003
; Smits and Medema, 2001
). We monitored the activity of the cyclin B/Cdk1 complex to see if the lack of mitotic events could result from alteration of this G2-M phase progression controller. We found that the cyclin B/Cdk1 kinase activity was completely inhibited in 2-trans-4-trans-decadienal treated eggs (Fig. 6A). This alteration was not due to an inhibition of cyclin B synthesis as the abundance of cyclin B reached similar levels in the treated and control eggs (Fig. 6B), even if its accumulation in the treated eggs was slower. Thus, either the PUA prevents the association of cyclin B with Cdk1 or the complex is maintained inactive by inhibition of Tyr15 dephosphorylation. This does not necessarily imply that the aldehyde directly interacts with cyclin B/Cdk1 complexes, an interaction with an upstream target leading to the regulation of the MPF is another valid explanation. However, the absence of cyclin B/Cdk1 activation cannot be a consequence of the inhibited DNA replication as we previously have shown that inhibition of DNA synthesis by aphidicolin does not prevent cyclin B/Cdk1 activation in sea urchin embryos (Geneviere-Garrigues et al., 1995
).
The lack of cyclin B/Cdk1 kinase activation might explain the absence of mitotic events and the cell cycle arrest. However, it does not explain the loss of pronuclear migration and the inhibition of DNA replication as these two processes have been demonstrated to be independent of Cdk1 as well as Cdk2 kinase activities in sea urchin early embryogenesis (Moreau et al., 1998). We thus conclude that 2-trans-4-trans-decadienal cytotoxicity relies on several cellular mechanisms. This PUA affects (1) the assembly of sperm aster and the accompanying pronuclear migration, (2) the DNA synthesis, and (3) the mitotic events likely through the inhibition of cyclin B/Cdk1 kinase activity. Thus we expect that 2-trans-4-trans-decadienal act on several intracellular pathways to lead to a cell cycle arrest and at higher concentration to apoptosis.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: +4777646020. E-mail: espenh{at}nfh.uit.no.
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