Station Biologique de Roscoff, Cycle Cellulaire et Développement, Unité Mer & Santé (FRE 2775), Centre National de la Recherche Scientifique (CNRS) and Université Pierre et Marie Curie (UPMC), BP 74, 29682 Roscoff, France
Received July 5, 2004; accepted September 9, 2004
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ABSTRACT |
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Key Words: CDK1-cyclin B; formulated glyphosate; cell cycle checkpoint; DNA synthesis.
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INTRODUCTION |
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In all eukaryotic cells, CDKs are initially activated by their association with cyclin subunits, by phosphorylation on a threonine residue located at position 161 (based on the cdc2 Schizosaccharomyces pombe sequence) and by dephosphorylation of two crucial residues Tyr 15 and Thr 14, located within the ATP-binding loop of the CDKs (reviewed in Nurse, 2002). The dephosphorylation of Thr 14 and Tyr 15, mediated by dual specificity phosphatases of the cdc25 family, is the rate-limiting step for CDK activation (Donzelli and Draetta, 2003
; Margolis and Kornbluth, 2004
; Wolfe and Gould, 2004
). Cyclin-dependent protein kinases function as key targets of the checkpoints response. Thus, upon DNA damage or replication errors, activation of the checkpoint pathways leads to cell cycle arrest in G1, S, or G2 (reviewed in Motoyama and Naka, 2004
). It has been demonstrated that G2 arrest is triggered by the maintenance of the inhibitory phosphorylations of CDK1, the CDK responsible for the G2/M transition (reviews in Norbury and Zhivotovsky, 2004
; Nurse, 2002
). The checkpoint operates for the most part through the regulation of cdc25 activity (Donzelli and Draetta, 2003
; Margolis and Kornbluth, 2004
; Wolfe and Gould, 2004
).
Among the most obvious models used to study cell cycle regulation, yeasts, mammalian cultured cells or oocytes and embryos from amphibians and invertebrates, sea urchin embryos won their lettres de noblesse by being at the origin of the discovery of cyclins (Evans et al., 1983). The molecular basis of cell cycle control is highly conserved from single unicellular eukaryotes to complex metazoans such as humans and Echinoderm gametes, and embryos constitute a recognized powerful model for the study of cell cycle regulation (Nurse, 2002
). Fertilization of ovulated sea urchin eggs arrested in a G1-like state of the cell cycle triggers entry into S phase followed by the G2 and M phases. Fertilized eggs then divide and subsequent cell cycles proceed as rapid rounds of highly synchronous S and M phases without gap (G1 or G2) phases. A number of reports suggested that cells from rapidly developing embryos (from fertilization to the mid-blastula transition) were devoid of mitotic checkpoints and apoptotic response (Clute and Masui, 1997
; Epel 2003
; Finkielstein et al., 2001
; Newport and Dasso, 1989
). Nevertheless, the presence and activation of checkpoint signaling mechanism have been thoroughly demonstrated during the first mitotic embryonic cell cycle (Greenwood et al., 2001
; Ikegami et al., 1978
; Kawahara et al., 2000
; Kim et al., 1999
; Lesser et al., 2003
; Meijer et al., 1991
; Sluder et al., 1995
; Stokes and Michael, 2003
; Voronina and Wessel, 2001
; You et al., 2002
).
Widely spread pesticides containing glyphosate as the active herbicide (Malik et al., 1989) are currently of no health or environmental concern (Williams et al., 2000
). However, formulated glyphosate was recently shown to provoke cell cycle dysfunction when applied to sea urchin embryos (Marc et al., 2002
, 2003
, 2004
). Formulated glyphosate affected CDK1/cyclin B activation, thus impeding entry into the M phase of the cell cycle (Marc et al., 2002
, 2003
, 2004
). The mechanism of action of the pesticide was shown to be independent of the formation of the CDK1/cyclin B complex (Marc et al., 2003
), which normally occurs for the G2/M transition as a result of cyclin B synthesis (Murray, 2004
; Nurse, 2002
). Therefore, formulated glyphosate may affect CDK1/cyclin B activation through inhibition of the dephosphorylations of Thr 14/Tyr 15. In this work, this hypothesis was tested and we present evidence that formulated glyphosate acts on the G2/M transition through mobilization of the DNA-response checkpoint as a consequence of interference with DNA replication during S phase.
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MATERIALS AND METHODS |
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Handling of eggs and embryos and cytological observations. The Sphaerechinus granularis sea urchins were collected in the Brest area (France), kept in seawater and used within 5 days. Spawning of gametes was induced by intracoelomic injection of 0.1 M acetylcholine. Eggs were collected in 0.22 µm Millipore-filtered seawater and rinsed twice by centrifugation at 2,000 rpm for 2 min. For DNA synthesis determination experiments, eggs were dejellied by swirling twenty seconds in 3.5 mM citric acid pH 5 and rinsed three times in filtered seawater prior to fertilization. For fertilization, eggs were suspended in Millipore-filtered seawater (5% suspension) containing 0.1% glycine. Diluted sperm was added to the eggs and withdrawn after fertilization envelope elevation. Experiments were only performed on batches exhibiting greater than 90% fertilization, and each experiment used gametes from a single female. Cultures were performed at 16°C with constant agitation. Thousands of embryos were incubated for each experimental determination. Pesticide solutions in Millipore-filtered seawater were adjusted to pH 7.5 and added to the embryo suspension 10 min after fertilization. Approximately 100 embryos were scored at short time intervals by phase contrast microscopy for cytokinesis. For some experiments, 0.2 ml aliquots of the embryo suspension at various times after fertilization were fixed for at least 2 h in 0.5 ml methanol/glycerol (3:1) in the presence of the DNA dye bisbenzimide (0.1 µg/mL) and were mounted in 50% glycerol. Chromatin state observation under fluorescence microscopy was used to determine metaphase stage timing.
Affinity purification of CDK1/cyclin B from embryos. The CDK1 protein was affinity purified at different times after fertilization using p13suc1-Sepharose beads (Dunphy et al., 1988). Every 10 min after fertilization, embryos from 2 ml samples of the 5% suspension were rapidly packed by centrifugation for 5 s at full speed in an Eppendorf centrifuge, immediately frozen in liquid nitrogen, and kept at 80°C until further processing. Embryos were homogenized through a 25-gauge needle in 400 µL ice-cold buffer (60 mM ß-glycerophosphate pH 7.2, 15 mM p-nitrophenyl phosphate, 25 mM 4-morpholinepropanesulfonic acid (MOPS), 15 mM ethylene glycol tetraacetic acid (EGTA), 15 mM MgCl2, 2 mM dithiothreitol (DTT), 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM disodium phenylphosphate, 10 µg/ml soybean trypsin inhibitor (SBTI), 100 µM benzamidine, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 0.1% Triton). The homogenates were clarified by centrifugation at 14,000 rpm for 10 min at 4°C. The CDK1/cyclin B complex was isolated by incubating the 14,000 rpm supernatant (400 µL) at 4°C for 45 min under constant rotation in the presence of 50 µL p13suc1-Sepharose beads (1/5 dilution) prepared using bacterially produced p13suc1 protein coupled to Sepharose beads as described elsewhere (Arion and Meijer, 1989
). After incubation, the beads were collected by centrifugation, washed twice in ice-cold bead buffer and once in 1 mL ice-cold 50 mM Tris-HCl pH 7.5. Proteins bound to the beads were eluted by boiling for 3 min in 35 µL electrophoresis sample buffer (Laemmli, 1970
).
Western blot analysis. After clearing by centrifugation, 15 µl of the affinity-purified proteins were resolved by one-dimensional electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel (Laemmli, 1970). Proteins were transferred to nitrocellulose (Towbin et al., 1979
), stained with Ponceau red and processed for Western blotting. The anti-cyclin B antibody and the anti-PSTAIR were used, respectively, at a dilution of 1:1000 and 1:3000 in 5% skimmed milk, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Tween 20. The anti p-Tyr was used at a dilution of 1:1000 in 5% bovine serum albumin (BSA), 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Tween 20. Membranes were then incubated with peroxidase-coupled secondary antibodies (Biorad): goat anti-mouse IgG (H + L)/HRP conjugate for PSTAIR detection (1:5000) and goat anti-rabbit IgG (H + L)/HRP conjugate for cyclin B detection (1:10 000). The antigenantibody complexes were revealed by the chemiluminescence system according to the manufacturer's instructions (Amersham Biosciences). When needed, membranes were stripped in dehybridization buffer (100 mM ß-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) for 40 min at 60°C under constant rocking and processed as above with a new antibody.
Phosphatase assays. The specific 3-O-methylfluorescein phosphate (OMFP) artificial substrate was used for the estimation of cdc25 activity (Gottlin et al., 1996). Pure human cdc25C was assayed at 50 m units (1 unit = nanomole of 3-O-methylfluorescein phosphate hydrolyzed per minute). Embryo extracts were prepared as described for the CDK1/cyclin B purification procedure, except that the homogenization buffer was 80 mM ß-glycerophosphate pH 7.3, 20 mM EGTA, 15 mM MgCl2, 1 mM DTT, 25 µg/mL aprotinin, 25 µg/mL leupeptin, 1 mM benzamidine, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), 10 µg/mL soybean trypsin inhibitor (SBTI), 10 µg/ml pepstatin A. Supernatants used for phosphatase assays were from metaphase-stage embryos as judged by the chromatin state.
Phosphatase assays were performed in 1 mL reactions at 30°C for at least 1 h. The mixture contained either 50 m units of commercial recombinant cdc25C or embryo extracts (100 µg protein) in substrate buffer (500 µM 3-O-methylfluorescein phosphate (OMFP), 100 mM Tris-HCl pH 8.2, 40 mM NaCl, 1 mM DTT, 10 mM glutathione, 20% glycerol; Gottlin et al., 1996). Absorbance was measured at 477 nm in a Pharmacia Biotech Ultrospec 2000 spectrophotometer. Activity of pure cdc25 C was 0.12 OD units per hour.
Determination of DNA synthesis in vivo. Ten minutes post-fertilization, methyl-3H-thymidine (10 µCi/ml final concentration), was added to the embryo suspension as described by Arion and Meijer (1989). When used, formulated glyphosate at the indicated concentration or aphidicolin (10 µg/mL final) were added 10 min post-fertilization. At different times thereafter, embryos from 1 mL samples of the suspension were rapidly packed by centrifugation for 5 s in an Eppendorf centrifuge, rinsed in 1 mL Millipore-filtered seawater, and suspended in 1 mL ice-cold 20% trichloroacetic acid (TCA). Aliquots were taken for the determination of total thymidine uptake. After precipitation for one night at 4°C, the pellets were collected by centrifugation, then washed twice with 20% TCA. The pellets were dissolved in 1 ml 0.125 M NaOH. Radioactivity was measured on duplicate 500 µL aliquots of the dissolved samples in the presence of Optiphase Supermix scintillation liquid in a 1450 Wallac MicroBeta-Counter (Wallac, EG&G Instruments). The protein content was measured on the remaining sample using the BCA protein assay kit.
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RESULTS |
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Together, these results demonstrate that the delay in cytokinesis due to the prevention of CDK1/cyclinB complex activation induced by formulated glyphosate is exerted through the inhibition of CDK1 dephosphorylation on tyrosine in vivo, which occurs as the result of an indirect effect of formulated glyphosate. Since the prevention of CDK1/cyclin B dephosphorylation is reminiscent of the DNA damage or unreplicated-DNA checkpoint of the cell cycle (review in Motoyama and Naka, 2004; Nurse, 2002
), the effect of formulated glyphosate on DNA synthesis was investigated.
Formulated Glyphosate Inhibits DNA Synthesis In Vivo
Incorporation of radiolabeled thymidine into DNA was measured as a function of time post-fertilization in control and formulated glyphosate-treated embryos. In control embryos, incorporation was first detected at 20 min post-fertilization, increased rapidly, reaching a plateau at 50 min (Fig. 2). Incorporation of thymidine into DNA was totally inhibited by the presence in the incubation medium of aphidicolin, a DNA polymerase 1 inhibitor, indicating that the thymidine incorporation measurement reflected the DNA synthesis activity of the embryos (Fig. 2). In a series of independent experiments, 10 µg/mL aphidicolin delayed cytokinesis by a mean of 105 min (S.D. = 30; n = 7). This result is in accordance with existence of mitotic checkpoints as already reported in Sphaerechinus granularis embryos (Meijer et al., 1991) and in other sea urchin species (Patel et al., 1997
). When the embryos were treated with a dose of formulated glyphosate that prevented entry into M phase, DNA synthesis was reproducibly inhibited by around 75% (Fig. 2). A comparison between the inhibitory effects of formulated glyphosate on DNA synthesis and on the first cell cycle demonstrated a strict correlation between the two events (Fig. 3).
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DISCUSSION |
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It is well established that the transition from the G2 to the M phase of the cell cycle requires activation of the mitotic kinase CDK1/cyclin B, and that activation of this complex is restrained by Wee1/Myt1-mediated phosphorylation of CDK1 (Murray, 2004; Nurse 2002
). Moreover, reversal of these phosphorylations (on Thr 14 and Tyr 15) is catalyzed by a dual-specific phosphatase of the cdc25 phosphatase family (Donzelli and Draetta, 2003
; Margolis and Kornbluth, 2004
; Wolfe and Gould, 2004
). Implication of cdc25 protein phosphatases in the activation of CDK1/cyclin B during the cell cycle is clearly established. On the other hand, the mechanism of cdc25 activation and regulation during the normal cell cycle is not fully understood. In mammals, cdc25 phosphatases are translated from three different genes leading to three proteins (cdc25 A, B, and C). The three cdc25 protein phosphatases share highly similar structural and mechanistic properties, and it is likely that their specific biological roles are determined by the timing of their activation, their subcellular localization and substrate recognition. Regulation of cdc25 activity has been reported to be mediated by both the abundance of the protein resulting from modulation of expression and degradation (for cdc25A), and the modulation of the activity of the enzyme through the phosphorylation of a number of alternative sites, which affects its localization (through 1433 binding) and possibly also its catalytic properties (Donzelli and Draetta, 2003
; Margolis and Kornbluth, 2004
; Wolfe and Gould, 2004
). Formulated glyphosate had no direct effect of purified cdc25 activity or on cdc25 extractable activity from the metaphase embryos judged as 3-O-methylfluorescein phosphate (OMFP) hydrolysis.
Formulated glyphosate may have maintained the Tyr 15 phosphorylation of CDK1/cyclin B through an indirect pathway reminiscent of the cell cycle checkpoint, which inhibits the activation of the CDK1/cyclin B kinase as a consequence of damaged or unreplicated DNA (review in Motoyama and Naka, 2004; Nurse, 2002
). An effect of formulated glyphosate at the level of DNA synthesis was investigated and demonstrated during the S phase of the cell cycle. DNA synthesis inhibition correlated well among experiments with the timing of the inhibition of cell cycle progression, strongly suggesting a link between the two effects on formulated glyphosate acting at S phase and resulting in an inhibition of G2/M progression. The fact that formulated glyphosate acts during the first hour of the first cell cycle (Marc et al., 2002
) is compatible with an initial effect of the pesticide at the level of S phase.
Altogether, the effect of formulated glyphosate presents two essential features of G2/M cell cycle checkpoint activation: sensing damaged or unreplicated DNA in S phase and preventing cell cycle progression at M phase through inhibition of CDK1/cyclin B activation (review in Motoyama and Naka, 2004; Nurse, 2002
). Obviously, we cannot formally exclude the possibility of multiple formulated glyphosate targets mimicking a checkpoint mechanism.
The adverse effect of formulated glyphosate toward the cell cycle is in the millimolar range concentration when applied for short time exposure to sea urchin embryos. At soil or water residual concentrations of glyphosate in the nanomolar range, there is no indication that glyphosate exerts any genotoxic effect (Williams et al., 2000). However, mobilization of the DNA response checkpoint may reflect interference of the product at the DNA level, potentially leading to genetic instability, which is recognized as one the main forces driving the onset and progression of carcinogenesis (Hoeijmakers, 2001
; Molinari, 2000
; Schneider and Kulesz-Martin, 2004
). Because formulated glyphosate is sprayed for herbicide usage at a recommended concentration of 40 mM, it would be of interest to analyze the consequence for humans of the inhalation of the micro-droplets in the vicinity of the pesticide usage.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Station Biologique, BP 74, 29682 Roscoff Cedex, France. Fax: 33 (0) 2 98 29 23 06. E-mail address: belle{at}sb-roscoff.fr.
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