1 Institute of Cosmetic Sciences, Club Cosmetics Co. Ltd, 145-1 Ichibu-cho, Ikoma-shi, Nara 630-0222, Japan
2 Tezukayama Junior College, 3-1-3 Gakuen-minami, Nara 631-8585, Japan
3 Japan BCG Laboratory, 3-1-5 Matsuyama, Kiyose-shi, Tokyo 204-0022, Japan
4 Department of Host Defense, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan
Correspondence
Ikuyo Sakaguchi
Ikuyos{at}clubcosmetics.co.jp
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ABSTRACT |
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INTRODUCTION |
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Since the bacterial ceramides possess unique structures with branched, long-chain base and fatty acid (Nakayama, 2000), we have investigated their ability to induce apoptosis in HL-60 cells in vitro, when administered exogenously. N-2-Hydroxyisopentadecanoyl isoheptadecasphinganine was more than twice as active as mammalian ceramide in inducing typical features of apoptotic cell death. Furthermore, SPLs such as a novel ceramide phosphorylmannose (cerPM-1), ceramide phosphorylethanolamines (cerPE-1 and cerPE-2) and ceramide phosphorylinositols (cerPI-1 and cerPI-2), all of which are good chemotaxonomic markers unique to the genus Sphingobacterium, were found to have similar apoptotic activity. This is the first report describing that bacterial ceramides and sphingolipids induce programmed cell death in mammalian cells when administered exogenously.
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METHODS |
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The TLC purification was repeated until a single spot was obtained. Analysis and identification of bacterial ceramides and SPLs were performed essentially according to the previous report (Yano et al., 1983), by gas chromatography/mass spectrometry (GC/MS) of each constituent and fast atom bombardment-mass spectrometry (FAB/MS) of the intact molecule.
Cell culture.
Human myeloid leukaemia HL-60 cells (from the ATCC) were maintained in RPMI 1640 medium (Nissui) supplemented with 20 % (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine. Human promonocytic U-937 cells (ATCC) and human leukaemia Jurkat cells (Dainippon Pharmaceutical) were maintained in RPMI 1640 medium supplemented with 10 % (v/v) FBS and 2 mM L-glutamine. For apoptotic experiments, cells were resuspended in RPMI 1640 medium supplemented with 1 % (v/v) FBS and 2 mM L-glutamine.
Co-culture of HL-60 cells and bacteria or heat-inactivated bacteria.
S. spiritivorum was grown and prepared as above to yield a suspension of OD610 >0·6. Bacterial cells were collected by centrifugation and passed through a 0·45 µm disposable filter. This bacterial suspension (100 µl) was added to HL-60 cells (5x105 cells) and these were incubated for 1 h at 37 °C. HL-60 cells were washed once with phosphate-buffered saline (PBS) and further cultured in RPMI 1640 medium containing 1 % (v/v) FBS for 24 h at 37 °C in 5 % CO2. We then analysed the cell cycle by flow cytometry. Heat inactivation of S. spiritivorum was performed for 30 min at 65 °C. After cooling to room temperature, the heat-inactivated bacteria (100 µl) were added to HL-60 cells (5x105 cells) and cultured in RPMI 1640 medium containing 1 % (v/v) FBS for 24 h at 37 °C in 5 % CO2. We then analysed the cell cycle by flow cytometry.
Measurement of the cell cycle by flow cytometry.
Cell cycle analysis was done according to a standard protocol (Taylor, 1980). Briefly, aliquots of 2·5x106 cells were washed twice with PBS and fixed in 70 % ethanol for 5 min on ice. The cell pellets were washed twice with PBS, and resuspended in PBS containing RNase A (1 µg ml-1; Nippon Gene), and incubated for 30 min at 37 °C. The cells were stained with propidium iodide at 20 µg ml-1 for 5 min and analysed by flow cytometry. The percentage of apoptotic cells was calculated using the internal software system of the FACScan (Becton Dickinson).
Flow cytometric analysis of Fas antigen expression.
Cell-surface expression of Fas antigen was determined by flow cytometric analysis (Yonehara et al., 1989). HL-60 cells (5x105 cells ml-1) were seeded in 35 mm plastic dishes and treated with sphingolipids [500 nM dissolved in ethanol/dodecane (98 : 2, v/v)], in RPMI 1640 medium containing 1 % (v/v) FBS. At the indicated time points, the cells were harvested, washed once with PBS, and reacted on ice for 1 h with 0·1 ml PBS containing 1 % (v/v) FBS, 0·02 % (v/v) NaN3 and anti-Fas IgM antibody (20 µg ml-1; MBL, Nagoya, Japan). Cells were washed once with PBS, then for an additional 1 h with 0·1 ml PBS containing 0·02 % (v/v) NaN3 and 10 µg ml-1 FITC-conjugated goat anti-mouse IgM (Cappel). After washing once with PBS, cell-surface-bound FITC-anti-mouse IgM was analysed by flow cytometry. The percentage of cells expressing Fas antigen was calculated from fluorescence intensity using the internal software system of the FACScan.
Apoptotic morphology.
HL-60 cells (5x105 cells ml-1) were seeded in 35 mm plastic dishes and treated with sphingolipids [0500 nM dissolved in ethanol/dodecane (98 : 2, v/v)], in RPMI 1640 medium containing 1 % (v/v) FBS. At the indicated time points, the cells were harvested and fixed with 1 % (v/v) glutaraldehyde in PBS for 1 h. After washing once with PBS, the cells were stained with 0·2 mM Hoechst 33258 for 10 min in the dark. Chromatin condensation was examined by fluorescence microscopy. Experiments were performed at least in triplicate.
Analysis of DNA fragmentation.
DNA fragmentation was analysed by agarose gel electrophoresis (Gorczyca et al., 1993). After the appropriate period of incubation (04 h) of HL-60 cells (5x105 cells ml-1) with 500 nM sphingolipids, cells were harvested by centrifugation at 400 g for 5 min, washed with PBS, and incubated with digestion buffer [0·1 M NaCl, 10 mM Tris/HCl (pH 8·0), containing 25 mM EDTA and 0·5 % (w/v) SDS]. Proteinase K (0·2 mg ml-1; Takara) was then added and the incubation was continued at 50 °C overnight. The cellular lysates were centrifuged at 13 000 g for 20 min to separate the low-molecular-mass DNA from the chromatin. Fragmented DNA was extracted from the supernatant by phenol/chloroform/isoamyl alcohol (25 : 24 : 1, by vol.). The upper aqueous layer was placed in a fresh tube and 0·5 vol. 7·5 M ammonium acetate solution followed by 2 vols absolute alcohol (4 °C) were added to precipitate the DNA. After washing with 70 % (v/v) ethanol, to remove residual RNA, 0·1 % (w/v) SDS solution containing 0·625 mg ml-1 RNase A was added, and the sample was incubated for 4 h at 37 °C. After repeated extraction with phenol/chloroform/isoamyl alcohol and ethanol precipitation, the DNA was dissolved in TE buffer (10 mM Tris/HCl pH 8·0, 1 mM EDTA), loaded on 1·5 % (w/v) agarose gel with ethidium bromide and electrophoresed for 30 min at 100 V. Bands were visualized by UV illumination.
Estimation of activities of ICE (-like) and CPP32/Yama (-like) proteases (caspases) (Shimizu et al., 1996).
HL-60 cells (5x105 cells ml-1) were treated with ceramides or sphingophospholipids [500 nM dissolved in ethanol/dodecane (98 : 2, v/v)], in RPMI 1640 medium containing 1 % (v/v) FBS. After 0·5, 1, 2, 3 and 4 h, cells were washed three times with PBS, suspended in 50 mM Tris/HCl (pH 7·4), 1 mM EDTA, 1 mM EGTA and 0·2 % (v/v) Triton X-100 and incubated at 37 °C for 10 min. Cell lysates (supernatants) were prepared by centrifugation at 25 000 g for 3 min, and cleared lysates containing 40 µg protein were incubated with 50 µM enzyme substrate Ac-Try-Val-Ala-Asp-MCA (Peptide Institute, Osaka, Japan) and Ac-Asp-Glu-Val-Asp-MCA (Peptide Institute) at 37 °C for 1 h. Levels of released 7-amino-4-methylcoumarin (AMC) were measured with a spectrofluorometer (Hitachi F-4000) with excitation at 380 nm and emission at 460 nm. Excitation and emission slit widths were adjusted to 10 mm and 20 mm, respectively. One unit was defined as the amount of enzyme required to release 0·22 nmol AMC min-1 at 37 °C.
Inhibition of apoptosis by caspase inhibitors.
HL-60 cells (5x105 cells ml-1) were treated with 250 µM caspase-1 specific inhibitor (Ac-Tyr-Val-Ala-Asp-H, Peptide Institute) or 250 µM caspase-3 specific inhibitor (Ac-Asp-Glu-Val-Asp-H, Peptide Institute) for 2 h at 37 °C in 5 % CO2. HL-60 cells were then treated for 4 h with ceramide or SPLs [500 nM dissolved in ethanol/dodecane (98 : 2, v/v)], in RPMI 1640 medium containing 1 % (v/v) FBS. The cells were harvested and washed once with PBS. After this, they were fixed with 70 % (v/v) ethanol for 5 min on ice, stained with propidium iodide and assayed by flow cytometry, as described above.
Statistical analysis.
Data are expressed as mean values±SD from 39 experiments. Significant difference was evaluated by an unpaired Student's t-test.
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RESULTS |
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Morphological changes in HL-60 cells with exogenous sphingolipids
To observe specific apoptotic changes of nuclei in HL-60 cells, the cells were treated with 500 nM cer-A for 4 h, stained with the DNA-labelling fluorochrome Hoechst 33258, and examined by phase-contrast and fluorescence microscopy. As shown in Fig. 4, many cells showed fragmented and/or condensed nuclei. Membrane blebbing and condensation and a marked shrinkage of the cells were also observed, showing that a typical apoptosis event occurred.
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DISCUSSION |
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The existence of ceramide or sphingolipid as a major component in bacteria is unique and they may well play a role as a virulence factor. However, it is rather difficult to define whether bacterial ceramide and sphingolipids are a pathological or beneficial component. Kawasaki et al. (1994) reported that in Sphingomonas sp. sphingoglycolipids were a major membrane component, and that these molecules play an important role as a substitute for LPS. In S. spiritivorum, about 20 % of the crude lipid consists of sphingolipids, and LPS may be replaced completely or partially. In Gram-negative bacteria, LPS is contained in the outer-membrane bilayer, whilst in Sphingobacterium, sphingolipids are contained in the inner-membrane bilayer. Therefore, it was considered that the sphingolipid of Sphingobacterium could not contact host cells directly and did not play a role in induction of apoptosis. In this study, when HL-60 cells were exposed to live or heat-inactivated S. spiritivorum, apoptosis did not occur, whereas when HL-60 cells were exposed to live Escherichia coli, apoptosis did occur (data not shown). On the other hand, crude lipids of S. spiritivorum, bacterial ceramides and SPLs were found to induce apoptosis. In Gram-negative bacteria, LPS is regarded as the characteristic and essential component which confers potent apoptosis-inducing activity.
In mammalian systems, ceramide plays important roles in the intracellular signal transduction system (Hannun & Obeid, 1995). However, it is difficult to demonstrate the function of ceramide exogenously in vitro because eukaryote-derived ceramides are usually difficult to dissolve in water and do not permeate membranes freely. To conquer this problem, analytical studies on ceramide function have been carried out by sphingomyelinase stimulation or exogenous C2 (acetyl) ceramide administration in vitro (Okazaki et al., 1989
, 1990
; Schutze et al., 1992
). Synthetic C2 or C6 ceramide can induce apoptosis when administered exogenously and is used experimentally for intracellular second messenger analysis in HL-60 cells (Mansat et al., 1997
; Okazaki, 1999
; Okazaki & Domae, 1994
). Mammalian ceramides have a straight-chain fatty acid, while bacterial ceramides have a branched-chain fatty acid. In the context of molecular structure and water solubility, branched-chain fatty acids are more soluble than straight-chain fatty acids for molecules with the same number of carbon atoms. It is considered that since bacterial ceramide is more soluble and permeable than mammalian ceramide for HL-60 cell membranes, the bacterial ceramides may be more potent for apoptosis induction. When bacterial ceramide was added to HL-60 cells exogenously, the ceramide content of the cells was significantly increased (Karasavvas et al., 1996
), indicating that ceramide is taken into the cell. In the present study, we have shown that for two types of free bacterial ceramides, cer-B (which possesses a 2-hydroxy fatty acid) showed greater activity for induction of apoptosis than cer-A (which possesses a non-hydroxy fatty acid). However, almost equivalent activities were observed in the cases of SPLs derived from S. spiritivorum, indicating that the presence of a 2-hydroxy fatty acid in the ceramide moiety seems not to be crucial for inducing apoptosis in vitro. Moreover free fatty acid with an iso-C15 chain did not show any apoptosis-inducing activity, whilst iso-C17 sphinganine from the ceramide showed a weak but significant apoptosis-inducing activity after 2 h incubation in vitro. From these results, taken together with other reports, it was concluded that the minimum ceramide structure necessary for apoptosis induction in HL-60 cells is one possessing isoheptadecasphinganine and non-hydroxy- or 2-hydroxyisopentadecanoic acid.
Ceramide is produced by the action of sphingomyelinase and is thought to be involved in mediating effects of various cytokines (tumour necrosis factor-, interleukin-1
, interferon-
), neuronal growth factor, Fas ligand, and ionizing radiation (Saba et al., 1996
). Some reports have indicated that ceramide is a primary signalling molecule in Fas-induced cell death (Tepper et al., 1995
). To clarify the mechanism of exogenous ceramide-induced apoptosis in HL-60 cells, we examined the possible involvement of Fas surface antigen. Fas is already known to be a type 1 membrane protein and to be activated by binding of the Fas ligand or an antagonistic anti-Fas antibody in Fas-bearing cells (Ferrarini et al., 1999
; Schlegel et al., 1996
). Fas activation results in caspase activation, which is an apoptosis-inducing signal (Suzuki et al., 1999
). DNA endonuclease activation is stimulated as part of the apoptotic process and causes fragmentation of DNA (Mizushima et al., 1996
), condensation of nuclei and cytoplasm, convolution of plasma membranes and nuclear or cellular fragmentation. In our study, bacterial ceramides were found to induce Fas expression 24 h after stimulation with cer-A or cer-B. Activation of caspase-3, but not caspase-1, by bacterial ceramide administration was maximal at 1 h after stimulation and then gradually decreased. Also, marked DNA fragmentation was observed even at 1 h after stimulation. These results suggest that at least two (and possibly more) mechanisms are involved in apoptosis, both mediated by ceramide derived from S. spiritivorum: a Fas-independent mechanism at an early stage of stimulation and a Fas-dependent mechanism at a later stage. However, direct evidence for ceramide-induced binding of Fas-ligand to Fas remains to be demonstrated.
Bacterial SPLs induced Fas expression at 24 h after stimulation with cerPE-1, cerPE-2, cerPI-1, cerPI-2 or cerPM-1. Activation of caspase-3, but not caspase-1, by these five SPLs was maximal at 3 or 4 h after stimulation. However, marked DNA fragmentation was observed as early as 1 or 2 h after stimulation. These results suggest that a caspase-3-independent mechanism is involved in the SPL-induced apoptosis at an early stage of stimulation and a caspase-3-dependent mechanism at a later stage. The data also suggest that there is a difference in the response of HL-60 cells to bacterial ceramides and SPLs.
Some targets are known to be the downstream signal of ceramide. From this study, it is suggested that bacterial ceramides activated signalling molecules via caspase cleavage, and then induced apoptosis. However, we have not yet studied the details of the phosphorylationdephosphorylation system involved in signalling. Such analysis will allow clarification of the versatility of ceramide signalling in cells, with regard to chronological and spatial regulation and cross-talk with other lipid signalling. Further detailed biochemical or immunological studies are necessary to clarify the function of bacterial sphingolipids in the host animals.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Damjanovski, S., Amano, T., Li, Q., Ueda, S., Shi, Y. B. & Ishizuya-Oka, A. (2000). Role of ECM remodeling in thyroid hormone-dependent apoptosis during anuran metamorphosis. Ann N Y Acad Sci 926, 180191.
Decaudin, D., Geley, S., Hirsch, T., Castedo, M., Marchetti, P., Macho, A., Kofler, R. & Kroemer, G. (1997). Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 57, 6267.[Abstract]
Deng, G. & Podack, E. R. (1993). Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2. Proc Natl Acad Sci U S A 90, 21892193.[Abstract]
Eischen, C. M., Schilling, J. D., Lynch, D. H., Krammer, P. H. & Leibson, P. J. (1996). Fc receptor-induced expression of Fas ligand on activated NK cells facilitates cell-mediated cytotoxicity and subsequent autocrine NK cell apoptosis. J Immunol 156, 26932699.[Abstract]
Ferrarini, M., Imro, M. A., Sciorati, C., Heltai, S., Protti, M. P., Pellicciari, C., Rovere, P., Manfredi, A. A. & Rugarli, C. (1999). Blockade of the Fas-triggered intracellular signaling pathway in human melanomas is circumvented by cytotoxic lymphocytes. Int J Cancer 81, 573579.[CrossRef][Medline]
Folch, J., Lees, M. & Sloane Stanley, G. H. (1959). A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226, 497509.
Furuya, S., Ono, K. & Hirabayashi, Y. (1995). Sphingolipid biosynthesis is necessary for dendrite growth and survival of cerebellar Purkinje cells in culture. J Neurochem 65, 15511561.[Medline]
Goodman, Y. & Mattson, M. P. (1996). Ceramide protects hippocampal neurons against excitotoxic and oxidative insults, and amyloid beta-peptide toxicity. J Neurochem 66, 869872.[Medline]
Gorczyca, W., Gong, J., Ardelt, B., Traganos, F. & Darzynkiewicz, Z. (1993). The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res 53, 31863192.[Abstract]
Hannun, Y. A. & Obeid, L. M. (1995). Ceramide: an intracellular signal for apoptosis. Trends Biochem Sci 20, 7377.[CrossRef][Medline]
Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr & Bell, R. M. (1986). Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets. J Biol Chem 261, 1260412609.
Harel, R. & Futerman, A. H. (1993). Inhibition of sphingolipid synthesis affects axonal outgrowth in cultured hippocampal neurons. J Biol Chem 268, 1447614481.
Hartfield, P. J., Mayne, G. C. & Murray, A. W. (1997). Ceramide induces apoptosis in PC12 cells. FEBS Lett 401, 148152.[CrossRef][Medline]
Ishizuya-Oka, A. (1996). Apoptosis of larval cells during amphibian metamorphosis. Microsc Res Tech 34, 228235.[CrossRef][Medline]
Ito, A. & Horigome, K. (1995). Ceramide prevents neuronal programmed cell death induced by nerve growth factor deprivation. J Neurochem 65, 463466.[Medline]
Iwata, M., Hotta, H., Higuchi, N., Nishiuchi, N., Fujiwara, N., Arakawa, T., Yano, I. & Kobayashi, K. (2002). Induction of thymic apoptosis by shigatoxin from Escherichia coli O157 : H7 in vivo and in vitro. Osaka City Med J 47, 1122.
Karasavvas, N., Erukulla, R. K., Bittman, R., Lockshin, R. & Zakeri, Z. (1996). Stereospecific induction of apoptosis in U937 cells by N-octanoyl-sphingosine stereoisomers and N-octyl-sphingosine. The ceramide amide group is not required for apoptosis. Eur J Biochem 236, 729737.[Abstract]
Kawasaki, S., Moriguchi, R., Sekiya, K., Nakai, T., Ono, E., Kume, K. & Kawahara, K. (1994). The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis. J Bacteriol 176, 284290.[Abstract]
Kondo, T., Matsuda, T., Tashima, M., Umehara, H., Domae, N., Yokoyama, K., Uchiyama, T. & Okazaki, T. (2000a). Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis. J Biol Chem 275, 88728879.
Kondo, T., Matsuda, T., Kitano, T. & 7 other authors (2000b). Role of c-jun expression increased by heat shock- and ceramide-activated caspase-3 in HL-60 cell apoptosis. Possible involvement of ceramide in heat shock-induced apoptosis. J Biol Chem 275, 76687676.
Lakics, V. & Vogel, S. N. (1998). Lipopolysaccharide and ceramide use divergent signaling pathways to induce cell death in murine macrophages. J Immunol 161, 24902500.
Lavie, Y., Cao, H., Volner, A., Lucci, A., Han, T. Y., Geffen, V., Giuliano, A. E. & Cabot, M. C. (1997). Agents that reverse multidrug resistance, tamoxifen, verapamil, and cyclosporin A, block glycosphingolipid metabolism by inhibiting ceramide glycosylation in human cancer cells. J Biol Chem 272, 16821687.
Linardic, C. M. & Hannun, Y. A. (1994). Identification of a distinct pool of sphingomyelin involved in the sphingomyelin cycle. J Biol Chem 269, 2353023537.
Liu, P. & Anderson, R. G. (1995). Compartmentalized production of ceramide at the cell surface. J Biol Chem 270, 2717927185.
Mansat, V., Laurent, G., Levade, T., Bettaieb, A. & Jaffrezou, J. P. (1997). The protein kinase C activators phorbol esters and phosphatidylserine inhibit neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin. Cancer Res 57, 53005304.[Abstract]
McConkey, D. J., Nicotera, P. & Orrenius, S. (1994). Signalling and chromatin fragmentation in thymocyte apoptosis. Immunol Rev 142, 343363.[Medline]
Mizushima, N., Koike, R., Kohsaka, H., Kushi, Y., Handa, S., Yagita, H. & Miyasaka, N. (1996). Ceramide induces apoptosis via CPP32 activation. FEBS Lett 395, 267271.[CrossRef][Medline]
Nakamura, S., Kozutsumi, Y., Sun, Y., Miyake, Y., Fujita, T. & Kawasaki, T. (1996). Dual roles of sphingolipids in signaling of the escape from and onset of apoptosis in a mouse cytotoxic T-cell line, CTLL-2. J Biol Chem 271, 12551257.
Nakashima, K., Ohtsuka, A. & Hayashi, K. (1998). Comparison of the effects of thyroxine and triiodothyronine on protein turnover and apoptosis in primary chick muscle cell cultures. Biochem Biophys Res Commun 251, 442448.[CrossRef][Medline]
Nakayama, M. (2000). Structure and apoptosis-inducing activity of sphingolipids isolated from Sphingobacterium spiritivorum. Seikatsu Eisei 42, 135148.
Okazaki, T. (1999). Implications of "ceramide-regulating biostat system" from the discovery of "sphingomyelin cycle" [in Japanese]. Tanpakushitsu Kakusan Koso 44, 10521058.[Medline]
Okazaki, T. & Domae, N. (1994). Physiological activity of sphingosine. J Clin Exp Med 171, 913916.
Okazaki, T., Bell, R. M. & Hannun, Y. A. (1989). Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation. J Biol Chem 264, 1907619080.
Okazaki, T., Bielawska, A., Bell, R. M. & Hannun, Y. A. (1990). Role of ceramide as a lipid mediator of 1 alpha, 25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 265, 1582315831.
Okazaki, T., Bielawska, A., Domae, N., Bell, R. M. & Hannun, Y. A. (1994). Characteristics and partial purification of a novel cytosolic, magnesium-independent, neutral sphingomyelinase activated in the early signal transduction of 1 alpha, 25-dihydroxyvitamin D3-induced HL-60 cell differentiation. J Biol Chem 269, 40704077.
Ozeki, Y., Kaneda, K., Fujiwara, N., Morimoto, M., Oka, S. & Yano, I. (1997). In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6'-dimycolate). Infect Immun 65, 17931799.[Abstract]
Parton, R. G. (1994). Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae. J Histochem Cytochem 42, 155166.
Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R. & Anderson, R. G. (1992). Caveolin, a protein component of caveolae membrane coats. Cell 68, 673682.[Medline]
Saba, J. D., Obeid, L. M. & Hannun, Y. A. (1996). Ceramide: an intracellular mediator of apoptosis and growth suppression. Philos Trans R Soc Lond B Biol Sci 351, 233240; discussion 240231.
Saito, T. & Kurasaki, M. (2000). Apoptosis and endocrine disrupters. Biomed Res 21, 353359.
Sawai, H., Okazaki, T., Yamamoto, H. & other authors (1995). Requirement of AP-1 for ceramide-induced apoptosis in human leukemia HL-60 cells. J Biol Chem 270, 2732627331.
Schlegel, J., Peters, I., Orrenius, S., Miller, D. K., Thornberry, N. A., Yamin, T. T. & Nicholson, D. W. (1996). CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem 271, 18411844.
Schnitzer, J. E., Oh, P. & McIntosh, D. P. (1996). Role of GTP hydrolysis in fission of caveole directly from plasma membranes. Science 274, 239242.
Schutze, S., Potthoff, K., Machleidt, T., Berkovic, D., Wiegmann, K. & Kronke, M. (1992). TNF activates NF-kappa B by phosphatidylcholine-specific phospholipase C-induced "acidic" sphingomyelin breakdown. Cell 71, 765776.[Medline]
Schwarz, A. & Futerman, A. H. (1997). Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth. J Neurosci 17, 29292938.
Shimizu, S., Eguchi, Y., Kamiike, W., Matsuda, H. & Tsujimoto, Y. (1996). Bcl-2 expression prevents activation of the ICE protease cascade. Oncogene 12, 22512257.[Medline]
Suzuki, A., Tsutomi, Y., Miura, M. & Akahane, K. (1999). Caspase 3 inactivation to suppress Fas-mediated apoptosis: identification of binding domain with p21 and ILP and inactivation machinery by p21. Oncogene 18, 12391244.[CrossRef][Medline]
Taylor, I. W. (1980). A rapid single step staining technique for DNA analysis by flow microfluorimetry. J Histochem Cytochem 28, 10211024.[Abstract]
Tepper, C. G., Jayadev, S., Liu, B., Bielawska, A., Wolff, R., Yonehara, S., Hannun, Y. A. & Seldin, M. F. (1995). Role for ceramide as an endogenous mediator of Fas-induced cytotoxicity. Proc Natl Acad Sci U S A 92, 84438447.[Abstract]
Tessitore, L., Valente, G., Bonelli, G., Costelli, P. & Baccino, F. M. (1989). Regulation of cell turnover in the livers of tumour-bearing rats: occurrence of apoptosis. Int J Cancer 44, 697700.[Medline]
Verheij, M., Bose, R., Lin, X. H. & 10 other authors (1996). Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis. Nature 380, 7579.[CrossRef][Medline]
Wang, B., Ohyama, H., Haginoya, K., Odaka, T., Itsukaichi, H., Yukawa, O., Yamada, T. & Hayata, I. (2000). Adaptive response in embryogenesis. III. Relationship to radiation-induced apoptosis and Trp53 gene status. Radiat Res 154, 277282.[Medline]
Wiegmann, K., Schutze, S., Machleidt, T., Witte, D. & Kronke, M. (1994). Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78, 10051015.[Medline]
Yang, Z., Costanzo, M., Golde, D. W. & Kolesnick, R. N. (1993). Tumor necrosis factor activation of the sphingomyelin pathway signals nuclear factor kappa B translocation in intact HL-60 cells. J Biol Chem 268, 2052020523.
Yano, I., Tomiyasu, I. & Yabuuchi, E. (1982). Long chain base comparison of strains of three species of Sphingobacterium gen. nov. FEMS Microbiol Lett 15, 303307.
Yano, I., Imaizumi, S., Tomiyasu, I. & Yabuuchi, E. (1983). Separation and analysis of free ceramides containing 2-hydroxy fatty acids in Sphingobacterium species. FEMS Microbiol Lett 20, 449453.[CrossRef]
Yonehara, S., Ishii, A. & Yonehara, M. (1989). A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J Exp Med 169, 17471756.[Abstract]
Zhang, Y. H., Takahashi, K., Jiang, G. Z., Kawai, M., Fukada, M. & Yokochi, T. (1993). In vivo induction of apoptosis (programmed cell death) in mouse thymus by administration of lipopolysaccharide. Infect Immun 61, 50445048.[Abstract]
Received 1 August 2002;
revised 28 March 2003;
accepted 28 April 2003.