Quantitative analysis of phospholipids and gangliosides in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes in tumor-bearing animals
Arsen Zakaryan,
Konstantin Karageuzyan,
Laura Hovsepyan,
Leon Karabashyan and
Gayane Zakaryan
Institute of Molecular Biology of Armenian NAS, 7, Hasratyan St., Yerevan 375014, Republic of Armenia
Correspondence to:
A. Zakaryan
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Abstract
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The phospholipid and ganglioside composition in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes of intact rats and rats with sarcoma 45 were studied. The lymphocytes and their progenitors were isolated by Ficoll-Paque density centrifugation. The phospholipids and gangliosides were separated by thin-layer chromatography following standard chloroform:methanol extraction from the cells. Alterations in the lipid spectrum (both phospholipids and gangliosides) were shown to take place during lymphocyte differentiation. The rate of ganglioside sialylation diminished, which was expressed as an increase in mono- and di-, and a decrease in tri- and tetrasialoganglioside levels. Tumor-induced alterations in lymphocyte lipid composition involve all stages of lymphocyte differentiation. These shifts are believed to be connected with a disturbance of the antineoplastic function of lymphocytes and, consequently, the immune response of the tumor-bearing organism.
Keywords: cancer, ganglioside, lymphocyte, phospholipid
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Introduction
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Cell membrane lipids are known to be involved in cell differentiation (17). Phospholipids and gangliosides of the cell membrane take part in the processes of lymphocyte maturation from bone marrow progenitors to mature functional cells of the peripheral immune system (813). It was demonstrated earlier (14) that changes in fatty acid composition of membrane phospholipids alter their properties and some cell functions, including immune cell reactions. In several types of cancers different alterations in the phospholipid pattern were observed (15,16). Modifications in the phospholipid spectrum were also described in non-cancer cells of organisms affected by cancer (17,18).
Gangliosides produced by tumor cells were shown to be shed into the pericellular space (19,20) and to bind host immune cells (21), inhibiting the cellular immune response (22). Thus, cell membrane lipid composition seems to be involved in malignant transformation.
It is interesting to follow changes in phospholipids and gangliosides (in lymphocytes related to the ganglioseries) of immune cells during their maturation and the alterations of these lipids under the influence of solid tumors developing in the organism.
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Methods
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Rats
Sixteen animals (120150 g weight) were used. They were separated into two groups. The first group (eight rats) of intact animals was used as a control. Rats of the second group were grafted with sarcoma 45 (the source of rat sarcoma 45 tumor cells was nursery `Stolbovaya', Moscow, Russia) by s.c. injection of 1x106 tumor cells suspended in 0.2 ml saline into the right side of the thorax. The grafting success rate was shown to be ~95%. Animals that had well-expressed tumors after 14 days were used.
Isolation of lymphocytes and their progenitors
Bone marrow cells were obtained by washing of femurs with cold HBSS solution. Thymus tissue was homogenized in the same solution. The bone marrow lymphocyte progenitors and thymocytes were isolated by Ficoll-Paque (
= 1.07) density centrifugation (1500 r.p.m., 40 min). The blood was diluted by HBSS (1:1). Mature lymphocytes were isolated by Ficoll-Paque (
= 1.87) density centrifugation (1500 r.p.m., 40 min) (23). The cells obtained were washed 2 times with HBSS. Macrophages were removed by the differential adhesion technique (23). The homogeneity of the isolated fractions of lymphocyte progenitors, thymocytes and mature lymphocytes as analyzed microscopically by their morphology was 9095%.
Lipid extraction and analysis
Lipids were extracted from cells with a chloroform:methanol mixture (2:1, v/v) using the standard procedure of Folch et al. (24). The sediment was separated by centrifugation (3000 r.p.m., 10 min) and re-extracted twice. The supernatant was mixed with cold water and maintained at 4°C overnight. The lower chloroform phase was used to fractionate the phospholipids by thin-layer chromatography on glass plates HPTLC (Merck, Darmstadt, Germany) in a solvent system of chloroform:methanol:ammonium (65:35:5). The phospholipid bands on thin-layer plates were visualized with iodine vapor. Phospholipid identification was performed using appropriate high purity reagents (Sigma, St Louis, MO) as markers. The yellowbrown patches of phospholipids were outlined, scraped and collected into test tubes. Mineralization of lipid phosphorus was carried out in a mixture of sulfuric and nitric acids to transform organic phosphorus into inorganic phosphorous. The quantity of phospholipids was determined in a color reaction with ammonium molybdate and vitamin C (25), and expressed as µg/mg of protein measured by the Lowry method (26).
The upper water/methanol phase (35% of the total volume) containing gangliosides was dialyzed against water (4°C, 72 h). The ganglioside determination was performed by thin-layer chromatography on glass plates HPTLC (Merck) in a solvent system of chloroform:methanol:ammonium:water (60:35:2:6). The ganglioside fractions on thin-layer plates were visualized with iodine vapor. Identification of gangliosides was carried out using appropriate markers (Sigma). Ganglioside content was determined with the periodate/resorcine method (25) and expressed as µg/mg of protein.
Statistical analysis was performed with the MannWhitney distribution free test.
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Results
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Phospholipid composition of lymphocytes at different stages of maturation from intact animals
Results of the analysis of phospholipids in bone marrow progenitors, thymocytes and mature lymphocytes derived from intact rats are shown in Table 1
. Modifications of cell phospholipid composition during lymphocyte differentiation were observed. The comparison of phospholipids in bone marrow cells and thymocytes revealed a decrease in lysophosphatidylcholine (LPC), sphingomyelin (SPM) and cardiolipin (CL), and an increase in the level of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). In mature lymphocytes, LPC and SPM levels were lower, and phosphatidylinositol (PI), PE and phosphatidylserine (PS) concentrations were higher, than in bone marrow progenitors.
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Table 1. Contents of phospholipids (% of the total) in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes in intact animals (both thymocytes and mature lymphocytes were compared with bone marrow progenitors)
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Ganglioside composition of lymphocytes at different stages of maturation from intact animals
The gangliosides of investigated cells (Table 2
) from intact rats were separated into five fractions: mono-, two fractions of di- (that have different locations of neuraminic acid residues, denoted below as fractions 1 and 2), and tri- and tetrasialogangliosides. In thymic lymphocytes, the fraction of disialogangliosides was more abundant, and mono- and tetrasialoganglioside levels were lower, than in bone marrow progenitors. In mature lymphocytes, the level of all gangliosides except tetrasialogangliosides was the same as in bone marrow cells. Thus, alterations in phospholipids and gangliosides during cell maturation seem to be more expressed in thymocytes than in mature lymphocytes.
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Table 2. Contents of gangliosides (% of the total) in bone marrow progenitors of lymphocytes, thymocytes and mature lymphocytes in intact animals (both thymocytes and mature lymphocytes were compared with bone marrow progenitors)
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Phospholipid composition of lymphocytes at different stages of maturation from tumor-bearing rats
In cells derived from rats with sarcoma 45 the phospholipid levels (Fig. 1
) at any stage of lymphocyte maturation showed a drop in PI and PC, and an increase in LPC, in comparison with normal cells. The increase in SPM was observed in thymocytes and mature lymphocytes. PS was elevated in immature cells, but decreased in mature ones. PE diminished only in mature cells and CL increased only in thymocytes.

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Fig. 1. The contents of phospholipids in tumor-bearing rats (percent of the same in intact animal cells taken as 100%). (a) Bone marrow progenitors of lymphocytes, (b) thymocytes and (c) mature lymphocytes. Phospholipid fractions: 1, PI; 2, LPC; 3, SPM; 4, PC; 5, phosphatidylserine; 6, PE; 7, CL. Phospholipid fractions of cells from normal and tumor-bearing animals were compared.
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Ganglioside composition of lymphocytes at different stages of maturation from tumor-bearing rats
Ganglioside determination in animals with tumors (Fig. 2
) showed an increase in mono- and disialogangliosides, and a decrease in tri- and tetrasialogangliosides, in comparison with healthy rats.

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Fig. 2. The contents of gangliosides in tumor-bearing rats (percent of the same in intact animal cells taken as100%). (a) Bone marrow progenitors of lymphocytes, (b) thymocytes and (c) mature lymphocytes. Ganglioside fractions: 1, monosialoganglioside; 2, disialoganglioside-1; 3, disialoganglioside-2; 4, trisialoganglioside; 5, tetrasialoganglioside. Ganglioside fractions of cells from normal and tumor-bearing animals were compared.
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Discussion
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The results presented demonstrate that phospholipids and gangliosides are actively involved into the processes of lymphocyte differentiation, and that the lipid pattern is susceptible to tumors developing in the organism.
One of the most noticeable alterations in grafted rats is a drop in the PI level at all stages of lymphocyte maturation. It is known that PI is involved in intracellular signal transduction as a secondary messenger (27,28) and that this phospholipid may interact as a ligand with immune cell receptors (29). Thus, it may be proposed that the described decrease in levels reflects modifications of these functions as a sequence of the influence of developing solid tumors.
Another expressed shift is the decrease in PC and increase in LPC levels. This suggests the activation of phospholipase A2-mediated hydrolysis of PC with the formation of LPC and arachidonic acid (30,31) in lymphocytes derived from an organism with solid tumors. The loss of both PI and PC may be related to their hydrolysis by phospholipase C. In this process PI is believed to be a preferable substrate for which the hydrolysis products are the known second messengers phosphatidic acid and diacylglycerol (32). Furthermore, PS and PE phospholipid fractions are known to be decarboxylated and methylated, forming PC (33), which, in turn, can be transformed into LPC. This is thought to be a possible cause of the described increase in LPC and drop in PC levels at all stages of lymphocyte differentiation, and significant diminution of the PS and PE content in mature lymphocytes in tumor-bearing animals.
As to gangliosides, a reduction in their sialylation level in lymphocytes from organisms with solid tumors was observed. These lipids were demonstrated to play an active role in immune reaction formation, cell growth and other cell function regulation (13,34,35). Earlier (36) we observed that the ganglioside sialylation level is negatively correlated with the rate of proliferation of cultured chick embryo cells. It may be proposed that the diminution in ganglioside sialylation observed in the present work reflects the decrease in lymphocyte proliferative potential due to the influence of solid tumors developing in the organism.
Our results are a good basis to propose that gangliosides may play an important role in lymphocyte and tumor cell interaction. As the ganglioside sialylation level is believed to be connected with cell functional activity, the described alteration of this character seems to concern the modification of the immune reaction occurring in tumor-bearing animals.
Taken together our data suggest alterations of phospholipids and gangliosides occurring during lymphocyte maturation. The dynamics of these shifts is modified under the influence of tumors developing in the organism.
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Acknowledgments
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The authors are grateful to Dr G. Gasparyan for critical review of this manuscript.
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Abbreviations
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CL cardiolipin |
LPC lysophosphatidylcholine |
PC phosphatidylcholine |
PE phosphatidylethanolamine |
PI phosphatidylinositol |
PS phosphatidylserine |
SPM sphingomyelin |
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Notes
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Transmitting editor: G. Klein
Received 16 November 2001,
accepted 6 June 2001.
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