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1 Department of Biomembrane and Biofunctional Chemistry, and 2 Department of Synthetic and Industrial Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan
3 College of Pharmacy, Chungbuk National University, Chongju 361-763, South Korea
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Abstract |
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Introduction |
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In addition to its extracellular functions, S1P is thought to act intracellularly as a second messenger for various growth factors and cytokines (Pyne & Pyne 2000; Spiegel & Milstien 2003). Intracellularly, S1P is formed by the phosphorylation of sphingosine (Sph), catalyzed by Sph kinase, the activity of which is transiently increased by certain stimuli (Pyne & Pyne 2000). Although its target molecule remains unidentified, accumulation of intracellular S1P is thought to induce various cellular responses including cell proliferation (Zhang et al. 1991; Van Brocklyn et al. 1998; Olivera et al. 1999, 2003), Ca2+ mobilization (Ghosh et al. 1990, 1994; Zhang et al. 1991), activation of phospholipase D (Desai et al. 1992; Van Brocklyn et al. 1998) and inhibition of apoptosis (Cuvillier et al. 1996; Edsall et al. 1997; Van Brocklyn et al. 1998).
Of the proposed intracellular functions of S1P, cell proliferation is the most extensively characterized. Initially, the addition of Sph was shown to induce proliferation of quiescent Swiss 3T3 fibroblasts (Zhang et al. 1990). A later study demonstrated that S1P converted from the added Sph, not the Sph itself, provided the mitogenic effect (Zhang et al. 1991). Additionally, treatment of quiescent Swiss 3T3 cells with platelet-derived growth factor (PDGF) and foetal calf serum (FCS) was found to increase the amount of intracellular S1P, and DNA synthesis induced by these stimuli was inhibited by DL-threo-dihydrosphingosine, a competitive inhibitor for Sph kinase (Olivera & Spiegel 1993). These results suggested the involvement of S1P as a second messenger in PDGF and FCS signaling. Subsequently, PDGF-induced S1P formation was found to initiate the activation of two cyclin-dependent kinases (p34cdc2 kinase and Cdk2 kinase) and of mitogen-activated protein kinase (Rani et al. 1997).
The extracellular actions of S1P also include signaling cell proliferation (An et al. 2000; Kimura et al. 2000; Kluk & Hla 2001; Tamama et al. 2001) and at least in some cell-types, intracellular S1P can be released to stimulate S1P receptors in an autocrine or paracrine fashion (Hobson et al. 2001; Olivera et al. 2003). This activity has been called ‘inside-out signaling.’ Therefore, it is still under some debate as to whether the intracellular role of S1P is indeed mediated intracellularly and not extracellularly through the S1P receptors (Hla et al. 1999; Pyne & Pyne 2000; Spiegel & Milstien 2002). However, several lines of evidence have supported a role for intracellular S1P in proliferation. Both the microinjection of S1P and its intracellular generation by the photolysis of incorporated caged S1P induced DNA synthesis in Swiss 3T3 fibroblasts (Qiao et al. 1998; Van Brocklyn et al. 1998). An increased intracellular S1P level in NIH3T3 fibroblasts, caused by the over-expression of Sph kinase type 1 (SPHK1), was sufficient to promote growth in low-serum media, expedite the G1/S transition and increase DNA synthesis without any detectable S1P secretion in the medium (Olivera et al. 1999). Moreover, the effect of SPHK1 over-expression on DNA synthesis was also observed in s1p2/s1p3 double knockout mouse embryonic fibroblasts that had been treated with pertussis toxin to remove any functional S1P receptors (Olivera et al. 2003).
S1P is metabolized by two pathways (Fig. 1). S1P phosphohydrolases (SPP1 and SPP2) dephosphorylate S1P to Sph, which is again used for sphingolipid synthesis (Mandala et al. 2000; Ogawa et al. 2003). Alternatively, S1P lyase, encoded by the SPL gene, converts S1P to hexadecenal and phosphoethanolamine (van Veldhoven & Mannaerts 1993; Zhou & Saba 1998). Hexadecenal is metabolized to hexadecanal, palmitate and palmitoyl-CoA, which are then used for precursors of sphingolipids and glycerophospholipids (van Veldhoven & Mannaerts 1993); the phosphoethanolamine is used for phosphatidylethanolamine synthesis via CDP-ethanolamine (van Veldhoven & Mannaerts 1993). Although in all of the above experiments studying intracellular S1P in cell proliferation, the amount of S1P did increase, the levels of the S1P metabolites would likely have been increased as well. Since both phosphoethanolamine and the ethanolamine generated from its dephosphorylation reportedly possess mitogenic properties (Kano-Sueoka et al. 1979; Kiss et al. 1997), one cannot exclude the possibility that the proposed actions of intracellular S1P are elicited by the S1P metabolites and not S1P itself.
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Results |
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To investigate the intracellular function of S1P in mitogenesis more precisely, we created several F9 mouse teratocarcinoma cell lines differing in the ability to regulate S1P levels, thereby allowing various levels of accumulation. Previously, we had generated F9-7 cells (SPL–/–), which lack the S1P-degrading enzyme S1P lyase (SPL) (Kihara et al. 2003; Ikeda et al. 2004) and F9-9 cells (SPL+/+/HA-SPHK1a), which stably overproduce the Sph kinase SPHK1a as a hemagglutinin (HA)-tagged protein (HA-SPHK1a) (Kihara et al. 2003). In the present study, we also established F9-12 cells (SPL–/–/HA-SPHK1a), which are F9-7 clones that stably express HA-SPHK1a.
We first examined the amounts of SPL and SPHK1 in each cell line by immunoblotting. The absence of the SPL protein was confirmed in the SPL–/– cells (F9-7 and F9-12) and compared to the SPL+/+ cells (F9 and F9-9) (Fig. 2A). F9 and F9-7 cells expressed only low amounts of SPHK1, under the limit of detection of our anti-SPHK1 antibodies. Additionally, an in vitro Sph kinase assay revealed that these cells possess low Sph kinase activity (
15.6 pmol/mg/min). On the other hand, in F9-9 and F9-12 cells HA-SPHK1a was detected in immunoblots, both with anti-HA and anti-SPHK1 antibodies (Fig. 2A). The amount of HA-SPHK1a was about 3-fold higher in F9-9 cells than in F9-12 cells. Similarly, an in vitro Sph kinase assay demonstrated that F9-9 and F9-12 cells contained approximately 370-fold and 130-fold higher Sph kinase activities than F9 cells.
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We also measured the amounts of Sph and dihydrosphingosine (dihydroSph), the precursors of S1P and dihydroS1P, respectively. Sph levels were slightly lower in F9-7 and F9-9 cells, but 3-fold higher in F9-12 cells, compared to F9 controls (Fig. 2C). The amounts of dihydroSph were slightly higher in F9-7 and F9-9 cells and much higher in F9-12 cells than in F9 cells (Fig. 2C). Again, the ratio of dihydroSph to Sph was equal or greater in F9-7, F9-9 and F9-12 cells than in F9 cells, in which the opposite was found (Fig. 2C).
Intracellular S1P levels show no correlation with the stimulation of DNA synthesis
We examined the ability of the cell lines described above to undergo DNA synthesis in low-serum conditions (0.5% FCS) by measuring [3H]thymidine incorporation. As previously observed for NIH3T3 or HEK 293 cells (Olivera et al. 1999), the overproduction of SPHK1 in F9 cells (F9-9) resulted in a marked induction of DNA synthesis (Fig. 3A). However, although the SPL-null cells (F9-7) accumulated S1P to a similar extent as the F9-9 cells, no increase in DNA synthesis was detected (Fig. 3A). Moreover, F9-12 cells, which accumulated the most S1P, also failed to stimulate DNA synthesis (Fig. 3A). Thus, the intracellular amounts of S1P did not correspond to the cell's ability to induce DNA synthesis. This phenomenon was not due to the usage of specific clones, since we obtained similar results using a transient expression system. Specifically, transient over-expression of SPHK1 in F9 cells resulted in an increase in DNA synthesis, whereas SPHK1 overproduction in F9-7 cells did not (data not shown). On the other hand, co-overproduction of SPL and SPHK1 in F9-7 cells induced DNA synthesis (data not shown), so re-introduction of SPL restored the ability to elicit the effects of SPHK1 over-expression.
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The overproduction of SPHK1 caused increased DNA synthesis only in the SPL+/+ background (F9-9 cells) and not in the SPL–/– (F9-12 cells), suggesting that enhanced sphingolipid metabolism, through the Sph kinase/S1P lyase pathway, rather than S1P might function in mitogenesis. To test this possibility, we transiently over-expressed SPL in F9-9 cells. The increase in DNA synthesis observed in the F9-9 cells was further enhanced by the overproduction of the SPL protein (Fig. 4A). SPL over-expression would be expected to cause a reduction in S1P levels, so this result clearly demonstrated that it is the increased S1P metabolism that has a mitogenic role and not S1P itself. On the other hand, overproduction of another S1P-degrading enzyme, S1P phosphohydrolase SPP1, resulted in a slight decrease in DNA synthesis (Fig. 4A). Competition from SPP1 with SPL in degrading S1P may cause this reduction. Similar results were obtained when SPL was transiently re-introduced into SPL–/– cells over-expressing SPHK1 (F9-12 cells). Overproduction of SPL but not that of SPP1 induced DNA synthesis (Fig. 4B).
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Our results demonstrated that not only S1P but also dihydroS1P was increased in F9-7, F9-9 and F9-12 cells (Fig. 2B). Since dihydroS1P has been reported to have different effects from S1P, at least on cell survival (Van Brocklyn et al. 1998; Rosenfeldt et al. 2001), we tried to investigate their effects separately. For this purpose, we treated cells with 5 nM Sph or 5 nM dihydroSph, which are converted intracellularly to S1P or dihydroS1P, respectively, then tested the DNA synthesis. As shown in Fig. 6, treatment with Sph stimulated proliferation in F9 cells, 1.3-fold over untreated cells. DihydroSph stimulated DNA synthesis to a similar extent. Although the stimulatory effects of Sph and dihydroSph were also observed in F9-9 cells, no such changes were detected in F9-7 and F9-12 cells. Re-introduction of HA-SPL into F9-7 cells (F9-8) restored the ability to respond to Sph and dihydroSph. Thus, the effects of Sph and dihydroSph on DNA synthesis were again completely dependent on the existence of SPL. These results demonstrated that the metabolism of either Sph or dihydroSph through the SPL pathway can exhibit mitogenic effects.
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Ceramide involvement in mitogenic activities of the F9 derivatives
Ceramide is also an important spingolipid metabolite that causes inhibition of DNA synthesis and induces G0/G1 cell cycle arrest (Hannun 1996; Mathias et al. 1998), so the observed mitogenic effects might possibly have been caused by a reduction in cellular ceramide. Therefore, we measured the ceramide levels in F9 cells and the derivative cell lines by diacylglycerol kinase (DGK) assay. As shown in Fig. 7, an apparent reduction in ceramide levels was observed in F9-9 cells (70% ceramide in F9 cells), whereas F9-7 and F9-12 cells contained slightly increased amounts of ceramide. A reduction in ceramide levels would be reasonable in F9-9 cells, since they over-express Sph kinase HA-SPHK1a, which competes with ceramide synthase for their common substrates Sph and dihydroSph. In contrast, in cells with the SPL–/– background (F9-7 and F9-12 cells), S1P and dihydroS1P are dephosphorylated by S1P phosphohydrolase, thereby regenerating Sph and dihydroSph, which are then used for ceramide synthesis. Thus, there is some correlation between the ceramide decreases and the mitogenic effect observed in F9-9 cells.
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Treatment with certain S1P metabolites induces DNA synthesis
We next investigated the possibility that certain metabolites of S1P (dihydroS1P) have mitogenic properties. We treated F9 cells with direct metabolites of dihydroS1P, specifically hexadecanal and phosphoethanolamine, the hexadecanal metabolite palmitate and ethanolamine (which is synthesized from phosphoethanolamine by phosphatase), each at a concentration of 5 nM. As shown in Fig. 8, palmitate and ethanolamine were as effective as Sph on DNA synthesis and hexadecanal and phosphoethanolamine also had stimulatory properties, although somewhat weak. Since S1P (dihydroS1P) metabolism generates fatty-aldehyde with phosphoethanolamine, we next treated F9 cells with hexadecanal or its metabolite palmitate, in combination with phosphoethanolamine or ethanolamine. Additive effects were observed in all cases. The most striking effects were drawn from the combination of hexadecanal and ethanolamine and that of palmitate and ethanolamine.
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Discussion |
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A key question is how metabolism of sphingolipids by Sph kinase and S1P lyase stimulates DNA synthesis. Several possibilities can be considered. First, since ceramide reportedly causes cell cycle arrest at the G0/G1 phase (Hannun 1996; Mathias et al. 1998), it is possible that a decrease in ceramide causes the mitogenic effects. Activation of the Sph kinase/S1P lyase pathway is thought to lead to decreases in intracellular ceramide levels through enzymatic competition. Sphingosine kinase competes with ceramide synthase for Sph and dihydroSph; S1P lyase competes for S1P and dihydroS1P with S1P phosphohydrolase (which regenerates Sph and dihydroSph). Indeed, F9-9 (SPL+/+/HA-SPHK1a) cells contained reduced amounts of ceramide compared to F9 cells (Fig. 7). However, ceramide levels did not always correspond to ability to increase DNA synthesis. The addition of Sph or dihydroSph to the culture medium also stimulated DNA synthesis in an SPL-dependent manner (Fig. 6), without a reduction in ceramide. In fact, ceramide levels significantly increased (Fig. 7), probably due to an increase in available substrates.
Second, it is possible that some metabolites of S1P (dihydroS1P), resulting from degradation by SPL, directly act as mitogens. Indeed, mitogenic effects of phosphoethanolamine and ethanolamine have been reported (Kano-Sueoka et al. 1979; Kiss et al. 1997). As shown in Fig. 8, treatment with hexadecanal, palmitate, phosphoethanolamine and ethanolamine, especially in combination, did stimulate DNA synthesis, although how each of these compounds was metabolized intracellularly and what portion was imported into the cells are unknown.
The third possibility is that a change in the overall lipid composition confers global effects to the cells, including the ability to grow in low serum conditions. Only the Sph kinase/S1P lyase pathway can convert sphingolipids to glycerophospholipids (Ikeda et al. 2005). Thus, stimulation of this pathway may cause an overall reduction in the sphingolipid content and an increase in the glycerophospholipid content. Indeed, when F9, F9-7, F9-9 and F9-12 cells were labeled with [3H]palmitic acid, a remarkable increase in phosphatidylethanolamine was observed only in F9-9 cells (data not shown).
Oncogenic functions for Sph kinase have been reported (Xia et al. 2000; Nava et al. 2002). Over-expression of SPHK1 resulted in a transformed phenotype in NIH3T3 cells (Xia et al. 2000) and promoted estrogen-dependent tumorigenesis of MCF-7 cells (Nava et al. 2002). Moreover, expression of SPHK1 was significantly elevated in a variety of tumors, compared with levels in normal tissue from the same patient (French et al. 2003). Although it has been considered that increased S1P stimulates the growth of cancer cells, our results imply an involvement of the Sph kinase/S1P lyase pathway. Here we demonstrated that activation of the Sph kinase/S1P lyase pathway stimulates mitogenesis at least in the cells tested (F9 and HeLa cells). However, further studies using other cancer cell lines are required to elucidate oncogenic functions of the Sph kinase/S1P lyase pathway.
Although the results presented here indicate that intracellular S1P has no role in the induction of DNA synthesis, we cannot exclude all of the functions of S1P as an intracellular signaling mediator. We have recently demonstrated that SPL-null F9 cells exhibit accelerated primitive endoderm differentiation (Kihara et al. 2003). Since F9-9 cells also display similar effects (Kihara et al. 2003), it is likely that S1P (dihydroS1P), not its metabolism, functions in the differentiation. Differentiation is accompanied by global changes in gene transcription and intracellular S1P might have roles in regulating some of this transcription. Interestingly, the S1P-accumulating cells (F9-7, F9-9 and F9-12 cells) contained higher amounts of dihydroSph and dihydroS1P compared to F9 cells (Fig. 2). DihydroSph is produced only in the de novo sphingolipid synthesis pathway. Therefore, it is possible that intracellular S1P affects the transcription of certain sphingolipid biosynthetic genes.
SPL-null cells (F9-7) and Sph kinase-overproducing cells (F9-9 and F9-12) are useful in discriminating whether a certain cellular response is drawn from S1P itself or from activation of the Sph kinase/S1P lyase pathway. Although both S1P metabolism and dihydroS1P metabolism, utilizing the SPL pathway, exhibit similar effects on DNA synthesis (Fig. 6), it has been reported that S1P and dihydroS1P differ in the ability to protect against apoptosis (Van Brocklyn et al. 1998; Rosenfeldt et al. 2001). Thus, S1P, but not dihydroS1P, might act as a signaling molecule in cell survival. Additional studies will be required to identify target molecules of S1P to elucidate its precise functions as an intracellular bioactive lipid molecule.
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Experimental procedures |
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Mouse F9 embryonal carcinoma cells and HeLa cells were grown in Dulbecco's modified Eagle's medium (DMEM) D6429 (Sigma) or D6046, respectively, containing 10% FCS supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin. F9 cells were grown in 0.1% gelatin-coated dishes. Transfections were performed using LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA, USA) or Lipofectamine PlusTM Reagent (Invitrogen).
Plasmids
The pCE-puro HA-SPL and pCE-puro HA-SPHK1a plasmids are derivatives of pCE-puro vector and encode N-terminally HA-tagged mouse SPL protein and N-terminally HA-tagged mouse SPHK1a protein, respectively (Kihara et al. 2003). The SPHK1a mRNA is one of the transcription variants of the SPHK1 gene (Kohama et al. 1998). The plasmid pCE-puro mSPP1-HA, which encodes C-terminally HA-tagged mouse SPP1 protein, was constructed by cloning the mSPP1-HA gene from pcDNA3 mSPP1-HA (Ogawa et al. 2003) into the pCE-puro vector.
Preparing stable transformants of F9 cells
F9-7 (SPL–/–), F9-8 (SPL–/–/HA-SPL) and F9-9 (SPL+/+/HA-SPHK1a) cells have been previously described (Kihara et al. 2003; Ikeda et al. 2004, 2005). To obtain F9-7 derivatives that stably express the mouse HA-SPHK1a gene, the pCE-puro HA-SPHK1a plasmid was transfected into F9-7 cells. Cells were subjected to puromycin selection at 0.5 µg/mL for 1 week. Of the clones obtained, the F9-12 cells expressed the highest levels of HA-SPHK1a and were used for further analyses.
Immunoblotting
Immunoblotting was performed as previously described (Kihara et al. 2003). Anti-SPL antibodies (1/1000 dilution) (Kihara et al. 2003), anti-SPHK1 antibodies (1/1000 dilution) (Fukuda et al. 2003), anti-HA 12CA5 antibody (0.2 µg/mL; Roche Diagnostics, Indianapolis, IN, USA) and anti-actin antibodies (0.6 µg/mL; Sigma-Aldrich) were used as primary antibodies. HRP-conjugated anti-mouse or anti-rabbit IgG F(ab’)2 fragment (both from Amersham Biosciences and diluted 1 : 7500) was used as the secondary antibody. Labeling was detected by the ECL detection method (Amersham Biosciences).
Thymidine incorporation assay
To measure DNA synthesis, we performed a [3H]thymidine incorporation assay. Cells were seeded in a 24-well plate at a density of 2 x 104 cells/well in DMEM containing 10% FCS and incubated for 12 h. After washing with DMEM containing 0.5% FCS, cells were suspended in the same media and incubated for 36 h at 37 °C. Cells were then treated with 0.25 µCi of [3H]thymidine (18.0 Ci/mmol; PerkinElmer Life Sciences, Ontario, Canada) for 4 h at 37 °C, washed with phosphate-buffered saline (PBS), then treated with 1 mL 10% trichloroacetic acid (TCA) for 30 min on ice. After washing with 5% TCA twice, labeled DNA was solubilized with 0.5 mL 0.5 N NaOH. The incorporated radioactivity was measured using a liquid scintillation counter (LSC-3600; Aloka, Tokyo, Japan).
Quantification of lipids
Intracellular amounts of Sph, dihydroSph, S1P and dihydroS1P were measured by HPLC as previously described (Min et al. 2002). To measure the cellular amounts of ceramide, a DGK assay was performed. Lipids were extracted according to the method of Bligh & Dyer (1959). Cells grown in a 6-well plate to 70% confluency were washed with PBS twice, suspended in buffer A (PBS, 1 mM dithiothreitol, 1 mM EDTA, 1 x protease inhibitor mixture (CompleteTM; Roche Diagnostics, Indianapolis, IN, USA) and 1 mM phenylmethylsulfonyl fluoride) then collected by scraping. After centrifugation, cells were suspended in 500 µL chloroform/methanol (2 : 1, v/v) and 133 µL H2O was added. Cells were then sonicated and centrifuged at 1000 g for 3 min at room temperature and the resulting supernatant was treated with 167 µL chloroform and with 167 µL H2O, each with vigorous mixing by a vortex. After phase separation by centrifugation (1000 g for 5 min at room temperature), the organic phase was collected and dried.
Using these lipids, a DGK assay was performed as previously described (Preiss et al. 1986). Briefly, lipids were suspended in 10 µL of 25 mM dioloyl-phosphatidylglycerol in 7.5% octyl-ß-D-glucopyranoside solution. Then, 87 µL reaction buffer was added (final concentration 50 mM imidazole, pH 6.6, 50 mM LiCl, 12.5 mM MgCl2, 1 mM ethyleneglycol bis(2-aminoethyl ether)tetraacetic acid, 0.2 mM diethylenetriaminepentaacetic acid, 2 mM dithiothreitol, 1.2 mM cold ATP). The reaction was initiated by mixing with 1 µL E. coli DGK (2.4 U/mL, Calbiochem (EMD Biosciences, La Jolla, CA, USA) and 2 µL [
-32P]ATP (2 Ci/mmol; 10 Ci/mmol; PerkinElmer). After an incubation at 30 °C for 1 h, lipids were extracted by adding 600 µL chloroform/methanol (1 : 1, v/v) and 265 µL 1 M KCl. After the phases were separated by centrifugation at 1000 g for 5 min at room temperature, the organic phase was collected. The obtained lipids were subjected to alkaline treatment (90 µL 1 M NaOH at 40 °C for 1 h) to hydrolyze the glycerophospholipids, then neutralized with 100 µL 1 M HCl and treated with 100 µL 1 M KCl. Phases were separated by centrifugation (1000 g for 5 min at room temperature) and the organic phase was again treated with 50 µL 1 M KCl. After phase separation, the organic phase was collected, dried and suspended in 20 µL chloroform/methanol (1 : 1, v/v). Lipids were resolved by thin layer chromatography on Silica Gel 60 high performance thin layer chromatography plates (Merck) with chloroform/acetone/methanol/acetic acid/water (10 : 4 : 3 : 2 : 1, v/v). Radioactivities associated with ceramide 1-phosphate were quantified using a Bio-Imaging Analyzer, BAS-2500 (Fuji Photo Film, Tokyo, Japan).
Chemical synthesis of hexadecanal
Hexadecanal was synthesized from 1-hexadecanal (Sigma-Aldrich) using the Dess-Martin oxidizing agent as previously described (Dess & Martin 1983; Meyer & Schreiber 1994).
Annexin V/PI bivariate flow cytometry
Annexin V-PI staining was performed using the MEBCYTO® Apoptosis Kit (Medical & Biological Laboratories, Nagoya, Japan). Cells were detached from a 6-well plate by incubating with a 0.25% Trypsin/0.02% EDTA solution, washed with PBS, then suspended in 85 µL binding buffer (10 mM HEPES-NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Cells were then treated with 10 µL fluorescein isothiocyanate-labeled Annexin V (50 µg/mL) and 5 µL PI (100 µg/mL). After a 15 min incubation at room temperature in the dark, cells were diluted with 0.4 mL binding buffer, followed by analysis using a FACSortTM cell sorter (Immunocytometry Systems, BD Biosciences, San Jose, CA, USA) and CELL QuestTM software.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yigarash{at}pharm.hokudai.ac.jp
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Received: 20 January 2005
Accepted: 16 March 2005
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