Blood Cells, Molecules, and Diseases 33 (2004) 68 – 76 www.elsevier.com/locate/ybcmd
Ceramides and glycosphingolipids in maturation process: leukemic cells as an experimental model Gabriela Smolen´ska-Sym, a,* Justyna Spychalska, a Ewa Zdebska, a Jolanta Woz´niak, b ZdzislJawa Traczyk, c Ewa Pszenna, c StanislJaw Maj, d Witold Danikiewicz, e Tomasz Bien´kowski, e and Jerzy Kos´cielak a b
a Department of Biochemistry, Institute of Hematology and Blood Transfusion, Warsaw, Poland Department of Pathophysiology, Institute of Hematology and Blood Transfusion, Warsaw, Poland c Internal Diseases Clinic, Institute of Hematology and Blood Transfusion, Warsaw, Poland d Hematology Clinic, Institute of Hematology and Blood Transfusion, Warsaw, Poland e Institute of Organic Chemistry, Polish Academy of Science, Warsaw, Poland
Submitted 9 April 2004 (Communicated by M.A. Lichtman, M.D., 11 April 2004) Available online 13 May 2004
Abstract Leukemic cells were used as experimental material to demonstrate changes in the content of GSLs during the development and maturation of neutrophils. The most abundant cellular GSL is LacCer. An elevation in the LacCer level occurs twice during the maturation process: initially, on formation of azurophil granules, and subsequently, (a more significant rise) on formation of specific granules. The formation of the latter is accompanied by an increase in the level of GalGalCer. During the maturation of myeloblasts, there is a simultaneous growth in the content of LacCer and GM3 as well as that of their common precursors, that is, free ceramides. Like other tumor cells, GM3 rich myeloblasts in the peripheral blood from patients with AML are characterized by shedding of gangliosides. The quantitative Cer/GlcCer ratio in these cells seems to be advantageous for the efficacy of chemotherapy in the induction of apoptosis. Myelo- and metamyelocytes achieve the highest level of GSLs. Their entry into the full maturity stage is accompanied by a decrease in the level of GSLs. Patterns of GSLs expression change greatly during development and maturation. However, with respect to the composition and content of GSLs, there are no significant differences between normal and leukemic mature neutrophils. At each stage of the development and maturation of myelogenous leukemic cells, as well as in normal mature neutrophils, there occurs the synthesis of the same molecular species both free ceramides and ceramide portions of LacCer, precursor of more complex GSLs. D 2004 Elsevier Inc. All rights reserved. Keywords: Glycosphingolipids; Ceramides; Ganglioside shedding; Human neutrophil maturation; CML; AML
Introduction
Abbreviations: Cer, ceramide; GalCer, galactosylceramide; GalGalCer, galabiosylceramide, Gala1-4GalCer; Gb3Cer, globotriaosylceramide, Gala1-4Galh1-4GlcCer; Gb4Cer, globotetraosylceramide, globoside, GalNAch1-3Gala1-4Galh1-4GlcCer; GlcCer, glucosylceramide; GM3, NeuAca2-3Galh1-4GlcCer; LacCer, lactosyceramide, Galh1-4GlcCer; Lc3Cer, lactotriaosylceramide, GlcNAch1-3Galh1-4GlcCer; nLc4Cer, neolactotetraosylceramide, Galh1-4GlcNAch1-3Galh1-4GlcCer; SPG, sialoparagloboside, NeuAc-nLc4Cer. * Corresponding author. Department of Biochemistry, Institute of Hematology and Blood Transfusion, Chocimska 5, 00-957 Warsaw, Poland. Fax: +48-22-848-06-37. E-mail address:
[email protected] (G. Smolen´ska-Sym). 1079-9796/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2004.04.002
Glycosphingolipid (GLS) clusters exist on virtually all mammalian cell plasma membranes. They are amphipathic molecules consisting of a ceramide lipid moiety, inserted in the outer leaflet of the membrane, linked to different externally oriented oligosaccharide structures. A subclass of GLSs that contain sialic acid residues is known as gangliosides. GLSs have been implicated in many fundamental cellular processes including: growth, differentiation, development, signal transduction, cellular interactions, oncogenic transformation [1 –4]. They are believed to be integral components of detergent-resistant plasma membrane microdomains known as rafts or caveolae (specific form of rafts) that are rich in
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sphingolipids and cholesterol, assemble receptors, signaling molecules, and proteins [5 –7]. Microdomains involved in glycosylation-dependent cell adhesion or recognition coupled with signal transduction were termed glycosynapses [8]. A number of studies suggested that changes in sphingolipid contents alter raft lipid and protein constituents [9]. Neutrophils have an exceptionally higher GSLs content than the remaining blood cell types [10]. Detergent-insoluble microdomains are present not only in the plasma membranes but also in the granule membranes of neutrophils, and contain the integral membrane protein, stomatin [11]. Iwabuchi and Nagaoka [12] provided evidence, that lactosylceramide (LacCer) forms GSL signaling domains coupled with Src family kinase Lyn and that LacCer-mediated Lyn activation causes the activation of phosphatidylinositol-3 kinase, p38 mitogen-activated protein kinase, and protein kinase C, leading to NADPH oxidase activation. GSLs of neutrophils were assessed in the 1980s [13] with the main focus on determination of their chemical structure. Researchers also used leukemic cells lines such as, for example, HL-60, which were studied most frequently [14]. Particular reports described either neutral GSL fraction or only gangliosides; each time, different methods of quantitative analysis were employed, hence, it was impossible to compare the results yielded from different or the same material. Using leukemic cells as an experimental model helped study the maturation process in vivo and GSLs proved to be markers of successive maturation stages. It is worth noting that the in vivo studies have an advantage over the most frequently conducted experiments on cell lines. Extrapolating from tissue culture experiments to intact animals, particularly to humans, always is hazardous (carries a significant risk). Likewise, our experiments allowed us to prove whether malignant transformation exerts any effect on molecular species of GSLs present in the cells, and GSL profile which the cell can reach at the full maturity stage. Moreover, we would like to focus on two issues: shedding of GM3 from leukemic cells to the environment and change in their phenotype as well as the Cer/GlcCer ratio in leukemic cells on initiation of chemotherapy and its results.
Materials and methods Patients Blood samples were collected from 12 healthy donors, 14 patients with AML (M-1, n = 7; M-2, n = 4; M-3, n = 3) and 4 patients with CML to obtain appropriate blood cells. Apart from that, blood samples were collected from 12 healthy donors and 8 patients with AML (M-1 and M-2) to obtain plasma. None of the patients had been previously treated with cytostatic agents. All blood samples were collected with the informed consent of involved individuals in accordance with the standards of the local ethical committee. To
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secure a sufficient amount of material for assessment, the patients were selected for high counts of leukemic cells. The disease subtype was classified according to the French– American –British criteria, and immunophenotypes of leukemic cells. Isolation of normal and leukemic cells Normal white blood cells were isolated from a buffy coat of healthy donors. Blood of leukemic patients was fractionated directly omitting the buffy coat isolation. Fractions of particular cell (normal or leukemic) types were isolated in self-generated gradient of Percoll using the method of Pertoft et al. [15]. In the case of normal white cells, the method allowed separating a lower band containing pure mature neutrophils (d = 1.099 g/cm3), and an upper band that was a mixture of lymphocytes and monocytes (d = 1.063 g/cm3). Leukemic myeloblasts and promyelocytes, which banded in the gradient of Percoll presented above together with lymphocytes and monocytes, were analyzed as such without further purification, provided that the leukemic cells corresponded to at least 95% of total cells, according to examination by flow cytometry. In CML cases, apart from the band d = 1.099 g/cm3, there was an additional band (d = 1.080 g/ cm3) that was a mixture of myelo-and metamyelocytes. Fraction purity was evaluated for the presence of characteristic cell-surface and intracellular antigens by FACS analysis (CD34, HLA-DR, CD71, CD45/CD14, CD13/CD33, CD15/ CD117, CD16/CD66 and MPO/LF) and by light and electron microscopy, which confirmed their morphological features. Isolation of cell-free and platelet-free plasma Plasma was prepared directly by centrifuging whole anticoagulated blood at 2500 g for 15 min. Five ml of supernatant was collected and centrifuged again under the same conditions. Isolation and determination of free ceramides and GSLs from cells Cer and GSLs from the isolated cells were extracted and purified, and their amount was determined by the methods described previously [16]. Briefly, the lyophilized material (equivalent to 2 – 20 108 viable cells) was sequentially extracted with chloroform/methanol/water (1:1:0.2, v/v), chloroform/methanol (2:1, v/v), chloroform/methanol (1:2, v/v) and propanol/hexane/water (55:25:20, v/v). The extracts were combined and desalted using octadecyl Bakerbond spe columns. The total lipids in the dried extracts were acetylated and chromatographed on Florisil columns (Bakerbond spe) [17]. Fractions containing sphingolipids were deacylated, and subsequently separated into individual compounds on silica gel 60 HPTLC plates (Merck) with the solvent system chloroform/methanol/water (65:35:8, v/v). Cer and GSLs were visualized with
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primulin spray and spots containing Cer, monoglycosyl-, diosyl-, triaosyl-, tertaosylceramides, and gangliosides were scraped from the plates. Spots corresponding to Cer were assessed for the sphingosine content, whereas spots with GSLs were subjected to qualitative and quantitative analysis of monosaccharides.
Palmer 74900 series syringe pump. Mass spectra were acquired over a range of mass-to-charge ratio 150– 2000 with data accumulation of 9 s per a single spectrum. Twenty to 40 individual spectra were acquired for each sample and averaged to improve the signal-to-noise ratio. Statistical analysis
Isolation and determination of GSLs from plasma Lyophilized plasma specimens were suspended in 1 ml of water, sonicated, and subsequently extracted with a mixture of chloroform/methanol (2:1 and 1:1 v/v respectively). The resulting lipid extract was purified on octadecyl Bakerbond spe columns and silica gel Bakerbond spe columns. Fractions containing GSLs were fractionated using HPTLC (as in the system above). Spots corresponding to GM3 were scraped from the plates and analyzed for the sialic acid content. Determination of carbohydrates and sphingosine Spots corresponding to individual GSLs were hydrolyzed with 2 M trifluoroacetic acid (TFA) for 4 h at 100jC to release neutral sugars and hexosamines. To determine sialic acid, the spots containing GM3 were hydrolyzed with 0.2 M TFA for 1 h at 80jC. Hydrolysates were analyzed for carbohydrates by High pH Anion Exchange Chromatography (HPAEC, Dionex Series 4500i system) [18]. Monosaccharides analyses by HPAEC permits simultaneous quantitative assessment GalCer and GlcCer in monoglycosylceramides, GalGalCer and LacCer in diosylceramides and globo, lacto and neolacto series in the remaining spots. The amount of GSL was expressed in pmol/106 cells. Hydrolysis of free ceramides was carried out with 1 N hydrochloric acid in aqueous methanol (methanol/water, 82:18, v/v) for 18 h at 70jC. Sphingosine in hydrolysates was determined by a fluorometric method [19].
The unpaired two-tailed Student’s t test was used for statistical analysis. Values of P < 0.05 were considered to indicate statistically significant differences between data sets.
Results Stages of neutrophil development and maturation Leukemic cells isolated from peripheral blood of AML and CML patients represent different maturation steps. Myeloblasts obtained from AML type M-1 with minimum maturation (with or without granules) and from type M-2 with maturation (with granules) represent development stages I and II respectively. Stage III corresponds to the promyelocytes (AML M-3). More mature cells present in CML include myelo-/metamyelocytes (stage IV), and band plus segmented cells (fully mature neutrophils, stage V). The stage V cells were compared with normal neutrophils (Vn) at the same stage of maturity obtained from healthy donors. Stages I – III show a gradual synthesis of azurophil granules, and cell divisions are still present. At stage IV, specific granules are formed, and the cell divisions cease. Stage V involves formation of gelatinase granules and secretory vesicles as well as an alteration in the shape of the nucleus. The above findings are confirmed by light, electron microscopy, and also flow cytometric analyses.
Mass spectrometry Mixtures of free ceramides and GSLs isolated from normal and leukemic cells were used for analysis by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). ESI mass spectra were obtained with a Mariner (PerSeptive Biosystems) ESI-TOF mass spectrometer equipped with a standard ion spray source. Electrospray ionization was carried out in the negative mode with spray tip voltage of 3.7 kV. The nozzle potential was set to 120, 170, or 190 V depending on a sample. The nozzle temperature was 140jC. The flow rates of nebulizing gas and curtain gas (nitrogen in both cases) were 0.4 l/min and 2 l/min, respectively. Solutions for MS analysis were prepared by dissolving 10 nmol of dry samples in 100 Al of chloroform (analytical grade) and diluted with methanol (HPLC grade) to a desired methanol – chloroform (v/v) concentration 4:1. Samples of solutions were directly infused at a flow rate 5 Al/min using a Cole-
Fig. 1. Altered GSLs and LacCer content in the successive stages of the neutrophil development and maturation.
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Total GSLs and LacCer The level of GSLs undergoes dynamic changes in successive stages of development and maturation (Fig. 1). Myeloblasts with no maturation and without granules (AML type M-0) contain only 17 pmol/106 cells of the total GSLs (data not presented). A slight elevation (3.7-fold) in stage II, as compared with stage I, precedes an over 10fold increase in the level of GSLs in stage IV. Full maturation is accompanied by a reduction in the level of GSLs (statistically significant: P < 0.001). Simultaneous to the changes in the total content of GSLs is those in the LacCer amount. LacCer is the most abundant glycosphingolipid in the cell. At the first three stages of maturation, the LacCer content does not exceed 50% of the total GSLs; however, at the two last stages its level amounts to 65%. The increase in the LacCer amount in stages I and II is due to the formation of azurophil granules. In stage III, the LacCer level does not increase and is even lower (but not statistically significant) than that at stage II. This may be associated with a completed process of primary granule formation, and incessant cell divisions at this stage of development. The formation of specific granules (stage IV) is accompanied by a dramatic rise in the LacCer level (because the specific granules in the cell are twice abundant as azurophilic granules). Reaching full maturity is associated with a statistically significant diminished level of LacCer (by 32%). With respect to the total content of GSLs and LacCer, mature CML neutrophils (stage V) are not distinct from normal mature neutrophils (Vn). Gangliosides At the initial maturation stage, gangliosides account for 34.4% of the total GSLs. In myeloblasts (stage I), GM3 is the most abundant ganglioside; the contents of GM3 and
Fig. 3. GM3 content in individual plasma samples of donors and AML patients with myeloblasts CD34+ and myeloblasts CD34ÿ.
LacCer are almost equimolar. The cell maturation is accompanied by a reduction in the GM3 level, and a gradual increase in the SPG amount (Fig. 2). In normal mature neutrophils, neutral GSLs are predominant and gangliosides (SPG) represent merely 3.2% of the total GSLs. Such an abundant content of GM3 in myeloblasts attracted our attention; hence our decision to investigate if, like in other tumor cells, shedding of cell surface gangliosides may be characteristic of myeloblasts. Therefore, we studied the content of GM3 in plasma from eight patients diagnosed as AML M-1 and M-2 cases, and from 12 control healthy donors (Fig. 3). In six patients, the GM3 level showed a statistically higher significance ( P < 0.05) than that in the controls; in the two remaining patients, the values were similar to those in the control subjects. The immunophenotype characteristics of myeloblasts revealed the presence of CD34+ cells in the six patients with the increased plasma GM3 levels, and phenotype CD34ÿ cells in the other two cases. Gala, neolacto, and globo series GSLs
Fig. 2. Altered GM3 and SPG content in the successive stages of the neutrophil development and maturation.
Irrespective of the cell maturity stage, GalCer is present in trace amounts (Fig. 4). The content of GalGalCer, however, increases rapidly, on formation of specific granules (stage IV), and subsequently, it shows a slight decrease (stage V). The gala series GSLs in mature neutrophils contain about 20% of the total GSLs. There is no statistically significant difference in the GalGalCer content between mature CML (stage V) and normal neutrophils. The amounts of Gb4Cer (globo series) and nLc4Cer (neolacto series) and their biosynthetic precursors Gb3Cer and Lc3Cer (Fig. 5) do not represent more than 13% at stages I and II, and they further decrease to approximately 5% at stages IV and V (vs. the total GSLs). It is worth emphasizing that in stages IV and V, the amounts of Gb4Cer and nLc4Cer are small whereas normal mature neutrophils contain only nLc4Cer.
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Fig. 4. Altered gala series GSLs content in the successive stages of the neutrophils development and maturation.
Fig. 6. Altered free ceramides and GlcCer content in the successive stages of the neutrophils development and maturation.
Free ceramides and GlcCer
patients (Fig. 6). Leukemic cells from all of the subjects showed sensitivity to chemotherapeutic agents. On average, 5 days (4 –7 days) were required to reduce the white blood cells count to less than 5% of the initial value.
In the course of neutrophil maturation, the level of free ceramides and GlcCer remains almost unaltered (Fig. 6): stage II is the only exception. However, the reported increase in Cer and GlcCer is not statistically significant. The quantitative Cer/GlcCer ratio is approximately 2 at stages I – II and at successive maturation stages it falls below 1. Daunorubicin and arabinofuranosylcytosine, that is, agents, which elevate cellular ceramide concentration and produce apoptosis, were administered to the patients with ALM M-1 and M-2. Before the treatment, Cer had prevailed over GlcCer in myeloblasts from peripheral blood of the
ESI-TOF mass spectra of free ceramides and glycan-bound ceramides Negative ion ESI-TOF mass spectra of free ceramides, mono- and diglycosyloceramides are shown in Fig. 7. Molecular masses of ions (m/z) always correspond to chloride adducts (M+Clÿ). Ions with m/z 572, 682, and 684 were assigned to free ceramides containing C16:0 (dominant), C24:1 and C24:0 fatty acids and d18:1 sphingosine, respectively. Chloride adduct of ceramide (at m/z 572) had been previously identified in Jurkat leukemic cells undergoing apoptosis induced by ionizing radiation or anti-Fas antibody [20]. Their mono-and diglycosylated derivatives were observed at m/z 734, 844, 846 and 896, 1006, 1008, respectively. ESI-TOF mass spectra indicate that the same three molecular species of free ceramides are present in all the types of the assessed cells and they are used in LacCer biosynthesis, that is, the precursor of more complex GSLs. Metelmann et al. [21] reported the presence of ceramide components of the same structure as above in gangliosides from mature neutrophils.
Discussion
Fig. 5. Altered neolacto and globo series GSLs content in the successive stages of the neutrophils development and maturation.
Leukemia arises following malignant transformation and subsequent clonal expression of a single hematopoietic progenitor, which is unable to complete its maturation program [22]. A severely blocked maturation is character-
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Fig. 7. Representative negative ion ESI-TOF mass spectra of free ceramides and their mono-and diglycosylated derivatives isolated from neutrophils in the successive stages of development and maturation.
istic of AML. The FAB classification distinguishes morphological variants of AML. Less or more mature myeloblasts and promyelocytes are consistent with subtypes of AML M-1, M-2, and M-3. Maturation of leukemic blast cells approximates the normal process in CML. Neutrophils at all stages of development are present in the blood of CML patients, and they generally show normal appearance. The prevalence of myelo-/metamyelocytes and band/segmented cells is observed.
In view of the data presented, it seems that leukemic cells may represent a relevant cellular model to assess successive stages in the maturation of myeloblasts. Therefore, leukemic cells were chosen as an experimental model in our study. We defined five stages of the neutrophil development and maturation. Each stage is characterized by a definite GSLs profile. We used uniform techniques both with respect to characterization and separation of normal and leukemic cells, and methods of GSL analysis. On the other hand,
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using ESI MS, it was possible to deal with the complexity of the mixture of individual free ceramides and GSLs as intact molecules and to obtain precise information about each molecular species. GSLs, with signal transducers and other membrane proteins, form functional units termed rafts, caveolae or glycosynapses, may play a key role in defining a phenotypic change of cells during the development and maturation process [1]. GSLs present in such microdomains have been used as their ‘‘markers’’, and a cell-type specific glycosphingolipid pattern in the membrane, essentially reflects the composition of glycosphingolipid-enriched microdomains. LacCer, the predominant GSL in neutrophils, is bound mainly with granule membranes (only 10% is present in plasma membranes) [23,24] and may be considered to be marker of both granule synthesis and degranulation processes. Iwabuchi and Nagaoka [12] showed that in neutrophils LacCer forms enriched GSL signaling microdomains in granule membranes as well as in plasma membranes where it is involved in the superoxide generation. This confirms previously reported data on the role of LacCer in the processes of neutrophil activation [25 –27]. At the full maturity stage of neutrophils, we noted a decrease in the LacCer amounts. Therefore, it could be postulated that once neutrophils have entered the blood stream, they assume their biological functions, and in response to low concentrations of inflammatory mediators (e.g., priming by TNFa, LPS, PAF, LTB4), granule displacement, fusion with the plasma membrane, and degranulation may occur. Borregaard et al. [28] indicate that stimulus-response coupling leading to granule exocytosis is intact in neutrophils from patients with myeloproliferative disorders (including these with CML). Recently, activated circulating neutrophils have been reported to produce microparticles (ectosomes) that a play role in inflammation and cell signaling [29]. Myeloblasts of phenotype CD 34+ from AML M-1 and M-2 patients are characterized by shedding of GM3 into plasma. In the case of leukemic cells, only one report has been published which describes increased level of gangliosides in plasma of children with T-ALL [30]. Shedding of cell surface gangliosides is a prominent component of tumor gangliosides metabolism, which is characteristic of numerous types of tumor cells [31]. The recent studies suggest that membrane vesicles shedding originate in plasma membrane domains enriched in gangliosides and caveolin-1 [32]. On the other hand, changes in the expression of gangliosides might contribute to the onset of a resistant phenotype in tumor cells. Prinetti et al. [33] showed that GM3 synthase mRNA level and GM3 synthase activity were remarkably higher in resistant cells. The role of gangliosides in multidrug-resistant (MDR) cell lines has been also reported. Plo et al. [34] show that gangliosides enhanced MDR1 P-glycoprotein (P-gp) function in immature acute myeloid leukemia KG1a cells. The study suggests that gangliosides are important P-gp regu-
lators perhaps through their capacity to modulate P-gp phosphorylation. It is also known that caveolin-1 is upregulated in a variety of drug-resistant cells which express a high level of P-gp [35,36]. Co-immunoprecipitation of P-gp and caveolin suggest that there is a physical interaction between them. Co-localization of P-gp and caveolin-1 was found in caveolae-enriched microdomains from brain capillaries, indicating that this association also occurs in vivo [37]. Moreover, MDR cells have a far higher activity of GlcCer synthase and content of GlcCer [38 –40]. However, GlcCer was proved to be a major glycosphingolipid constituent of caveolar membranes [41 – 44]. Summarizing, MDR is usually mediated by overexpression of P-gp, and acquisition of MDR is accompanied by upregulation of lipids and proteins that constitute lipid rafts and caveolar membranes, notably GlcCer and caveolin. An additional link seems to exist between the upregulation of P-gp and caveolin-1 within caveolae and enhancement of the P-gp function by gangliosides. In conclusion, several interesting relationships presented above seem to suggest that the shedding of GM3 from myeloblasts CD 34+ (which are more frequently characterized by a higher expression P-gp [45]) might result in a loss of MDR cellular properties. The shedding might lead to loss of caveolae with GM3, GlcCer, and P-gp from cells. It is known that immunosuppressive activity is a general biological property of gangliosides [46]. GM3 in the environment inhibits cell motility, produces an inhibitory effect on epidermal growth factor receptor (EGF-R) and proliferation [47]. Therefore, shed GM3 to the microenvironment would allow tumor cells to escape host immune destruction and the loss of P-gp and GlcCer with caveolae might increase sensitivity of cells to chemotherapeutic agents. Increased maturity stage of neutrophils is accompanied by the loss of GM3. This may be due to an increased activity of ganglioside sialidase, the enzyme converting GM3 to LacCer and sialic acid. It was proved that in neuroblastoma cells plasma membrane ganglioside sialidase cofractionates with the lipid raft markers [48]. Recently, Kakugawa et al. [49] reported an up-regulation of plasma membrane-associated sialidase in human colon cancer, and its involvement in suppression of apoptosis. Resulting LacCer is claimed to inhibit apoptosis, mainly through an increased Bcl 2 and decreased caspase expression. The results indicate that a high expression of sialidase in cancer cells provides to protection against programmed cell death, probably modulating gangliosides. Desialylated GSL core structures may affect some, yet-unknown process to enhance tumor growth, invasiveness, and metastasis [2]. In a similar way, GM3 may be lost in the final stages of neutrophil maturation. At that particular time, as we showed earlier [50], the expression of LacCer/CDw17 on the cell surface increases. However, myeloblasts (with a high GM3 content) do not exhibit the presence of this antigen on the cell surface but only inside the cell, which is associated with the formation of azurophilic granules.
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Many papers have shown that GlcCer promotes cell proliferation and growth, whereas Cer induces apoptosis and/or growth inhibition [51 – 53]. The balance between the pro-and anti-apoptotic growth controllers seems to be more critical than the absolute concentration of any single sphingolipid. Poorly differentiated myeloblasts in patients with AML M-1 and M-2 are characterized by a 2-fold higher content of Cer than GlcCer. The clinical assessment of these patients indicated that a relatively high rate of myeloblasts reduction in peripheral blood occurred in the first line therapy with daunorubicin/arabinofuranosylcytosine combination, that is, agents which elevate cellular ceramide concentration and produce apoptosis. Gala series GSLs are characteristic of neutrophils [13]. GalCer (e.g., sulfatide precursor) is found in trace amounts in all the stages of maturation. However, relatively much GalGalCer are present in the final maturation stages of neutrophils. So far, it has not been evident if the compound, similarly to LacCer, is bound with granule and plasma membranes. In their study, Kniep and Skubitz [24] were not able to establish it due to the then quantitative method used in determining GSLs. Moreover, there are no data on its probable role in the formation of rafts. Apart from that, a question arises whether GSLs, such as nLc4Cer and SPG (found in small quantities in granule and plasma membrane), participate in the formation of rafts having specific functions. GSL profiles of mature neutrophils, normal and leukemic from CML patients do not differ significantly. Free ceramides and ceramide components of GSLs have the same structures regardless of the maturity stage and cell origin (normal and leukemic). Patterns of GSLs expression change greatly during development and maturation but molecular species of GSLs are not affected by hematopoietic disorders. Still, it might be interesting and essential to define the GSLs distribution in normal myeloid cells in the bone morrow in different stages of maturity and to compare the results with the data reported in the present paper.
References [1] S. Hakomori, Structure, organization and function of glycosphingolipids in membrane, Curr. Opin. Hematol. 10 (2003) 16 – 24. [2] S. Hakomori, Glycosylation defining cancer malignancy: new wine in an old bottle, Proc. Natl. Acad. Sci. 99 (2002) 10231 – 10233. [3] S. Hakomori, Cell adhesion/recognition and signal transduction through glycosphingolipid microdomain, Glycoconjugate J. 17 (2000) 143 – 151. [4] T. Yamashita, R. Wada, T. Sasaki, C. Deng, U. Bierfreund, K. Sandhoff, R.L. Proia, A vital role for glycosphingolipid synthesis during development and differentiation, Proc. Natl. Acad. Sci. 96 (1999) 9142 – 9147. [5] D.A. Brown, E. London, Structure and function of sphingolipidand cholesterol-rich membrane rafts, J. Biol. Chem. 275 (2000) 17221 – 17224. [6] K. Kasahara, Y. Sanai, Functional roles of glycosphingolipids in signal transduction via lipid rafts, Glycoconjugate J. 17 (2000) 153 – 162. [7] K. Simons, D. Toomre, Lipids rafts and signal transduction, Nat. Rev., Mol. Cell Biol. 1 (2000) 31 – 39.
75
[8] S. Hakomori, The glycosynapse, Proc. Natl. Acad. Sci. 99 (2002) 225 – 232. [9] M. Nagafuku, K. Kabayama, O. Daisuke, A. Kato, S. Tani-ichi, Y. Shimada, Y. Ohno-Iwashita, S. Yamasaki, T. Saito, K. Iwabuchi, T. Hamaoka, J. Inokuchi, A. Kosugi, Reduction of glycosphingolipid levels in lipid rafts affects the expression state and function of glycosylphosphatidylinositol-anchored proteins, but does not impair signal transduction via the T cell receptor, J. Biol. Chem. 278 (2003) 51920 – 51927. [10] K. Kiguchi, C.B. Henning-Chubb, E. Huberman, Glycosphingolipid patterns of peripheral blood lymphocytes, monocytes and granulocytes are cell specific, J. Biochem. 107 (1990) 8 – 14. [11] E. Feuk-Lagerstedt, M. Samuelsson, W. Mosgoeller, C. Movitz, A. Rosqvist, J. Bergstrom, T. Larsson, M. Steiner, R. Prohaska, A. Karlsson, The presence of stomatin in detergent-insoluble domains of neutrophil granule membranes, J. Leukocyte Biol. 72 (2002) 970 – 977. [12] K. Iwabuchi, I. Nagaoka, Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils, Blood 100 (2002) 1454 – 1464. [13] B.A. Macher, W.M.F. Lee, M.A. Westrick, Glycosphingolipids of normal and leukemic human leukocytes, Mol. Cell. Biochem. 47 (1982) 81 – 95. [14] H. Nojri, T. Takaku, T. Tetsuka, K. Motoyoshi, Y. Miura, M. Saito, Characteristic expression of glycosphingolipid profiles in the biopotential cell differentiation of human promyelocytic leukemia cell line HL-60, Blood 64 (1984) 534 – 541. [15] H. Pertoft, M. Hirtenstein, L. Kogedal, Separation of human mononuclear cells (MNC) and polymorphonuclear (PMNC) in cell populations, in: E. Reid (Ed.), Methodological Surveys, (B) Biochemistry, vol. 9, 1979, pp. 67 – 80. [16] G. Smolen˜ska-Sym, E. Zdebska, E. GoJaszewska, J. Woz´niak, T. Dur´zyn´ski, S. Maj, U. Mokras, J. Kos´cielak, Contents of total and protein-bound carbohydrates are low in leukemic leukocytes from patients with acute myelogenous leukemia, Acta Biochim. Pol. 45 (1998) 361 – 371. [17] T. Saito, S. Hakomori, Quantitative isolation of total glycosphingolipids from animal cells, J. Lipid Res. 12 (1971) 257 – 259. [18] E. Zdebska, J. Kos´cielak, A single-sample method for determination of carbohydrate and protein contents glycoprotein bands separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Anal. Biochem. 275 (1999) 171 – 179. [19] M. Naoi, Y.C. Lee, S. Roseman, Rapid and sensitive determination of sphingosine bases and sphingolipids with fluorescamine, Anal. Biochem. 58 (1974) 571 – 577. [20] R.L. Thomas Jr., C.M. Matsko, M.T. Lotze, A.A. Amoscato, Mass spectrometric identification of increased C16 ceramide levels during apoptosis, J. Biol. Chem. 43 (1999) 30580 – 30588. [21] W. Metelmann, J. Muthing, J. Peter-Katalinic, Nano-electrospray ionization quadrupole time-of-flight tandem mass spectrometric analysis of a ganglioside mixture from human granulocytes, Rapid Commun. Mass Spectrom. 14 (2000) 543 – 550. [22] M.A. Lichtman, The stem cell in the pathogenesis and treatment of myelogenous leukemia: a perspective, Leukemia 15 (2001) 1489 – 1494. [23] F.W. Symington, W.A. Murray, S.I. Bearman, S. Hakomori, Intracellular localization of lactosylceramide, the major human neutrophil glycosphingolipid, J. Biol. Chem. 23 (1987) 11356 – 11363. [24] B. Kniep, K.M. Skubitz, Subcellular localization of glycosphingolipids in human neutrophils, J. Leukocyte Biol. 63 (1998) 83 – 88. [25] F.W. Symington, CDw17: a neutrophil glycolipid antigen regulated by activation, J. Immunol. 142 (1989) 2784 – 2790. [26] F. Lund-Johansen, J. Olweus, V. Horejsi, K.M. Skubitz, J.S. Thompson, R. Vilella, F.W. Symington, Activation of human phagocytes through carbohydrate antigens (CD15, sialyl-CD15, CDw17, and CDw65), J. Immunol. 148 (1992) 3221 – 3229. [27] T. Arai, A.K. Bhunia, S. Chatterjee, G.B. Bulkley, Lactosylceramide
76
[28]
[29]
[30]
[31] [32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
G. Smolen´ska-Sym et al. / Blood Cells, Molecules, and Diseases 33 (2004) 68–76 stimulates human neutrophils to upregulate Mac-1, adhere to endothelium, and generate reactive oxygen metabolites in vitro, Circ. Res. 82 (1998) 540 – 547. N. Borregaard, L. Kjeldsen, H. Sengelov, Mobilization of granules in neutrophils from patients with myeloproliferative disorders, Eur. J. Haematol. 50 (1993) 189 – 199. O. Gasser, C. Hess, S. Miot, C. Deon, J.-C. Sanchez, J.A. Schifferli, Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils, Exp. Cell Res. 285 (2002) 243 – 257. W.D. Merritt, V. Der-Minassian, G.H. Reaman, Increased GD3 ganglioside in plasma of children with T-cell acute lymphoblastic leukemia, Leukemia 8 (1994) 816 – 822. Y. Kong, R. Li, S. Ladisch, Natural forms of shed tumor gangliosides, Biochim. Biophys. Acta 1394 (1998) 43 – 56. V. Dolo, R. Li, M. Dillinger, S. Flati, J. Manela, B. Taylor, A. Pavan, S. Ladisch, Enrichment and localization of ganglioside GD3 and caveolin-1 shed tumor cell membrane vesicles, Biochim. Biophys. Acta 1486 (2000) 265 – 274. A. Prinetti, L. Basso, V. Appierto, M.G. Villani, M. Valesecchi, N. Loberto, S. Prioni, V. Chigorno, E. Cavadini, F. Formelli, S. Sonnino, Altered sphingolipid metabolism N-(4-hydroxyphenyl)-retinamide-resistant A2780 human ovarian carcinoma cells, J. Biol. Chem. 278 (2003) 5574 – 5583. I. Plo, G. Lehne, K.J. Beckstrom, N. Maestre, A. Bettaieb, G. Laurent, D. Lautier, Influence of ceramide metabolism on P-glycoprotein function in immature acute myeloid leukemia KG1a cells, Mol. Pharmacol. 62 (2002) 304 – 312. Y. Lavie, G. Fiucci, M. Liscovitch, Up-regulation of caveolae and caveolar constituents in multidrug-resistant cancer cells, J. Biol. Chem. 273 (1998) 32380 – 32383. Y. Lavie, G. Fiucci, M. Czarny, M. Liscovitch, Changes in membrane microdomains and caveolae constituents in multidrug-resistant cancer cells, Lipids 34 (1999) 557 – 563 (suppl.). M. Demeule, J. Jodoin, D. Gingras, R. Beliveau, P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries, FEBS Lett. 466 (2000) 219 – 224. Y. Lavie, H. Cao, S.L. Bursten, A.E. Giuliano, M.C. Cabot, Accumulation of glucosylceramides in multidrug-resistant cancer cells, J. Biol. Chem. 271 (1996) 19530 – 19536. Y.Y. Liu, T.Y. Han, A.E. Giuliano, M.C. Cabot, Expression of glucosylceramide synthase, converting ceramide to glucosylceramide, confers adriamycin resistance in human breast cancer cells, J. Biol. Chem. 274 (1999) 1140 – 1146.
[40] Y.Y. Liu, T.Y. Han, A.E. Giuliano, M.C. Cabot, Ceramide glycosylation potentiates cellular multidrug resistance, FASEB J. 15 (2001) 719 – 730. [41] K. Iwabuchi, K. Handa, S. Hakomori, Separation of ‘‘glycosphingolipid signaling domain’’ from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling, J. Biol. Chem. 273 (1998) 33766 – 33773. [42] M. Liscovitch, Y. Lavie, Multidrud resistance: a role for cholesterol efflux pathways?Trends Biochem. Sci. 25 (2000) 530 – 534. [43] Y. Lavie, M. Liscovitch, Changes in lipid and protein constituents of rafts and caveolae in multidrug resistant cancer cells and their functional consequences, Glycoconjugate J. 17 (2000) 253 – 259. [44] Y. Lavie, G. Fiucci, M. Liscovitch, Upregulation of caveolin in multidrug resistant cancer cells: functional implications, Adv. Drug Delivery Rev. 49 (2001) 317 – 323. [45] D.M. Van der Kolk, E.G.E. de Vries, L. Noordhoek, E. van den Berg, M.A. van der Pol, M. Muller, E. Vellenga, Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia, Leukemia 15 (2001) 1544 – 1553. [46] R. McKallip, R. Li, S. Ladisch, Tumor gangliosides inhibit the tumorspecific immune response, J. Immunol. 163 (1999) 3718 – 3726. [47] X. Wang, P. Sun, M. O’Gorman, T. Tai, A.S. Paller, Epidermal growth factor receptor glycosylation for ganglioside GM3 binding and GM3mediated suppression of activation, Glycobiology 11 (2001) 515 – 522. [48] D. Kalka, C. Reitzenstein, J. Kopitz, M. Cantz, The plasma membrane ganglioside sialidase cofractionates with markers of lipid rafts, Biochem. Biophys. Res. Commun. 283 (2001) 989 – 993. [49] Y. Kakugawa, T. Wada, K. Yamaguchi, H. Yamanami, K. Ouchi, I. Sato, T. Miyagi, Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression, Proc. Natl. Acad. Sci. 99 (2002) 10718 – 10723. [50] J. Spychalska, G. Smolen˜ska-Sym, E. Zdebska, J. Woz´niak, J. Kos´cielak, Quantitative analysis of LacCer/CDw17 in human myelogenous leukaemic cells, Cell. Mol. Biol. Lett. 8 (2003) 911 – 917. [51] N.S. Radin, Killing cancer cells by poly-drug elevation of ceramide levels. A hypothesis whose time is come? Eur. J. Biochem. 268 (2001) 193 – 204. [52] P.P. Ruvolo, Ceramide regulates cellular homeostasis via diverse stress signaling pathways, Leukemia 15 (2001) 1153 – 1160. [53] H. Sietsma, R.J. Veldman, J.W. Kok, The involvement of sphingolipids in multidrug resistance, J. Membr. Biol. 181 (2001) 153.
