Brought to you by Portland Press Ltd.
Published on behalf of the International Federation for Cell Biology
Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2005) 29, 920–928 (Printed in Great Britain)
In vivo expression and characteristics of novel α-d-mannose-rich glycoprotein markers of apoptotic cells
Rostyslav Bilyya, Yuriy Kita, Ulf Hellmanb, Volodymyr Tryndyakc, Vitaliy Kaminskyya and Rostyslav Stoikaa*
aInstitute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine
bLudwig Institute for Cancer Research, Husargatan Street 3, Uppsala, SE-751 24, Sweden
cInstitute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine, Vasyl'kivska Street 45, Kyiv 03022, Ukraine


Abstract

We recently established that an increased expression of α-d-mannose (Man)- and β-d-galactose-rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89–95]. It was independent of cell line or apoptosis-inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-d-mannose- and beta-d-galactose-containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829–838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of α-Man-rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin-affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34kDa, and high MW of &007E;600 and 800kDa) whose expressions were increased during apoptosis. Triton X-114 treatment of cell membrane samples showed that the apoptotic cell-specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI-TOF MS analysis as the microtubule-actin cross-linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G-protein β-subunit like (Acc# BAA06185.1) and dystonin isoform β.


Keywords: Apoptosis, Glycoproteins, Plasma membrane, Lectins.

*Corresponding author. Tel./fax: +380 322 720087.


1 Introduction

Programmed cell death is accompanied by the expression of different biochemical markers in cytoplasm (activation of caspases and the appearance of cytochrome c (Chang and Yang, 2000; Fujimura et al., 1998; Perez-Pinzon et al., 1999; Cohen, 1997)), mitochondria (expression of pro- and anti-apoptotic proteins of Bcl-2 family (Reed, 1994)), nucleus (DNA fragmentation (Wyllie, 1980)), and plasma membrane (externalization of phosphatidyl serine (Fadok et al., 1992)). Bilyy and Stoika (2003) and other investigators (Heyder et al., 2003) demonstrated an increased expression of α-d-Man- and β-d-Gal-rich plasma membrane glycoproteins (GPs) in apoptotic cells. It was suggested that such GPs can be novel plasma membrane markers of the apoptotic cells. Their expressions were demonstrated in cell lines of various tissue origins, and associated with various inducers of apoptosis. We have demonstrated that these GPs can also be used for isolation of apoptotic cells from a mixed cell population (Bilyy et al., 2004).

Evidently apoptosis is accompanied by distinct changes in the level of plasma membrane GPs (Batisse et al., 2004; Chionna et al., 2003; Azuma et al., 2000; Chorna et al., 2004), however, specific functions of those GPs are poorly studied, and the in vivo expression of those GPs in the apoptotic cells has not been demonstrated. In the present study, we investigated an expression of α-d-Man-rich GPs during apoptosis in order to determine: (a) whether an increased expression of α-d-Man-rich GPs is intrinsic for cells in vivo; (b) which specific GPs possess an increased expression after apoptosis induction in vivo, and whether they are similar to the GPs found after apoptosis induction in vitro and (c) which compartment of plasma membrane is characteristic for the location of GPs whose expressions are elevated during apoptosis.

2 Materials and methods

2.1 Cells and animals

Murine leukemia cells of L1210 line were obtained from the Cell Culture Collection of the Institute of Experimental Pathology, Oncology and Radiobiology, National Academy of Sciences of Ukraine (Kiev, Ukraine). L1210 cells were maintained in DME medium (Sigma Chemical Co., USA) supplemented with 10% heat-inactivated fetal calf serum (Sigma) and gentamycin (50μg/ml, Sigma). The cells were cultured in a humidified atmosphere at 37°C and 5% CO2. Alternatively, F1 hybrids of DBA2×Black mouse lines (Mus musculus) were used for inoculation of L1210 cells. After inoculation these cells were allowed to grow intraperitoneally for 5–7 days. Apoptosis of L1210 cells in culture was induced by doxorubicin (2.0μg/ml, Ebewe, Austria) (Somerville and Cory, 2000); apoptosis of L1210 cells inoculated in mice was induced by doxorubicin in maximal survival dose of 2mg/kg (Chekhun et al., 2003). After 24h mice were anaesthetized and killed by cervical dislocation and L1210 cells were isolated from the ascitic fluid by centrifugation. Murine splenocytes were obtained as described (Krishnan et al., 2003), apoptosis of mouse splenocytes was induced by intraperitoneal injection of dexamethasone phosphate (LvivDialik, Lviv, Ukraine), 40mg/kg of mouse weight (Krishnan et al., 2003). After 24h mice were killed by cervical dislocation, and the spleen was removed and placed in phosphate-buffered saline (PBS). Splenocytes were isolated and suspended in PBS. The cells were pelleted by centrifugation and frozen at −80°C. Food and water for mice were available ad libitum before and after the treatments. The experimental animals were treated in accordance with the guidelines detailed in the NIH Guide for Care and Use of Laboratory Animals (US Public Health Service). Cell viability was controlled by trypan blue (0.1% w/v solution) dye exclusion test, and cells were counted in a hemocytometric chamber under light microscope.

2.2 Lectinocytochemistry

HRP-labeled PSL lectin and PSL–agarose (4.5mg/ml of resin with PSL lectin conjugated to resin through (CH2)9-spacer) were kindly gifted by Dr. V. Antonyuk (Lectinotest Laboratory, Lviv, Ukraine). αMMan (Sigma Chemical Co., USA) was used as sugar competitor of PSL (Fowlkes et al., 1980). Lectinocytochemical analysis was conducted as described previously by Herrington and McGee (1992) with some modifications. Cell smears were fixed in acetone:methanol:formalin mixture (19:19:2) for 90s at room temperature, and then air dried. Smears were washed twice with Tris-saline buffer pH 7.5 (TSB) for 2min, and incubated with HRP-labeled lectins (50μg/ml) at 4°C overnight. If needed, an appropriate sugar (0.1M) was added to the incubation mixture for blocking of lectin binding. Smears were washed twice with TSB for 10min and incubated with 0.5mg/ml 3,3′-diaminobenzidine (DAB, Sigma) and 4μl/ml H2O2 in TSB for 5min. NiCl2 solution was added to this incubation mixture (final concentration 1mg/ml) to improve cell contrasting. Smears were washed in distilled water, air dried and photographed. If needed, an additional Giemsa–Romanowsky staining was performed as described (Wittekind, 1983; Perea-Sasiain, 2003). Alternatively, a combined fluorescent microscopy using vital staining with 14μg/ml of PSL–FITC conjugate, 1μg/ml DAPI (Sigma Chemical Co., USA) or 1μg/ml PI was performed for 30min at 37°C. Cells were examined under MikMed-2-K12 microscope (LOMO, Russian Federation) using appropriate excitation/emission wavelengths. Densitometry of smears was conducted using images under the microscope (Zeiss, Jena) equipped with video capturing device. Densitometric analysis was performed using ImageJ (Wayne Rasband, National Institutes of Health, USA) program support and the UTHSCSA ImageTool program (University of Texas Health Science Center in San Antonio, Texas).

2.3 Isolation of plasma membrane GPs

Plasma membrane fractions were isolated as described by Bilyy and Stoika (2003). Cell pellet (250–1000μl) was thawed and suspended in hypotonic buffer (10mM Tris–HCl, pH 7.5, 1.5mM MgCl2, 1mM phenylmethylsulphonylfluoride, PMSF), and then homogenized in a Potter homogenizer. An appropriate volume of 2M sucrose was added immediately to the homogenate to achieve 0.25M final concentration, and the suspension was centrifuged for 15min at 2000×g for pelleting nuclei and intact cells. The pellet was homogenized once more in the hypotonic buffer. The supernatants of two homogenizations were combined and centrifuged for 90min at 25,000×g, and the sediment of plasma membrane fractions was collected. All operations were performed at 4°C. Approximately 6.8mg of protein (detected by Bio-Rad protein assay) was isolated from 1ml of cell pellet. Equal amounts of proteins, isolated from the intact and apoptotic cells, were used for further analysis.

Affinity chromatography was carried out by dissolving the obtained sediment of plasma membrane fraction in TSB containing 1% of Triton X-100 at 4°C for 1h, and incubating the obtained solution with PSL–agarose (4.5mg lectin/ml of resin) at room temperature for 2h. Protein (27.2mg) was applied for 1ml of PSL–agarose, then the affinity sorbent was washed three times and the GPs were eluted with equal volume of 1M mannose. Proteins were precipitated 30min by acetone at 4°C, and an appropriate quantity of Laemmli buffer was added to the protein samples.

Determination of protein location in plasma membrane was carried out using isolated plasma membrane fractions. Isolated membranes were dissolved in TSB buffer containing 2% Triton X-114, as described (Bordier, 1981). They were incubated for 1h on wet ice and then centrifuged at 2000×g for 5min. The solution (100μl) was then loaded over 100μl of 6% sucrose and heated to 37°C until the upper phase lost its opacity. Samples were centrifuged at 1500×g for 10min. Upper (peripheral proteins) and lower phases (integral proteins) were separated by centrifugation and denaturing buffer (65mM Tris–HCl, pH 6.8, 4% 2-mercaptoethanol, and 10% glycerol) was added for further analysis by PAGE electrophoresis.

The electrophoresis was carried out in 7.5–17.5% gradient PAGE by using Laemmli buffer system (Laemmli, 1970). The proteins were stained with Coomassie G-250 or with AgNO3 according to Shevchenko et al. (1996). For lectinoblot analysis membrane proteins of L1210 cells or splenocytes were electrophoretically transferred onto the nitrocellulose sheets (Millipore, HA type, 0.45μm), as described (Towbin et al., 1992). The membrane was treated with 0.1% BSA in TSB solution for 30min to block non-specific binding. The membrane was incubated overnight with HRP-labeled PSL lectin (1:400 dilution of 5mg/ml stock solution) under gentle shaking at 4°C. Glycoproteins were revealed on the blots using peroxidase-DAB/H2O2 detection system (Lutsik and Kusen, 1987).

2.4 MALDI-TOF MS

After electrophoresis, the appropriate bands from the PAGE were excised and treated for in-gel digestion according to Hellman (2000). After proteolysis and extraction of the generated peptides, the mixture was analyzed by peptide mass fingerprinting (PMF) using a Bruker Ultraflex TOF/TOF (Bruker Daltonics, Bremen, Germany) following procedures recommended by the manufacturer.

2.5 Comet assay

Cells were collected and lysed as described by Gulston et al. (2002). Slides were prepared and subjected to electrophoresis at 0.6V/cm for 15min. After drying for 30min at 37°C and fixing for 10min in solution containing 15% w/v trichloroacetic acid, 5% ZnSO4 and 5% glycerol, slides were re-hydrated for 10min in the distilled water. Silver staining was performed as described by Nadin et al. (2001), with some modifications, using a solution of 0.1% ammonium nitrate, 0.1% silver nitrate, 0.25% tungstosilicic acid, 0.2% formaldehyde and 3.2% sodium bicarbonate. After staining slides were washed in 1% acetic acid, distilled water, and then air dried.

DNA damage was evaluated by visual score on arbitrary scale of 0–4 (ranging from 0 to no DNA damage to 4 – extensive DNA damage) according to Heaton et al. (2002). A minimum of 500 cells was analyzed in each sample.

2.6 DNA-fragmentation assay

Fragmentation of DNA assay of intact and apoptotic L1210 cells and murine splenocytes was studied using 5×106 cells, as described by Gong et al. (1994).

2.7 Statistical analysis

Experiments were performed in triplicate and repeated three times. Significance of the difference in a typical experiment was assessed by Student's t-test. The level of significance was set at 0.05. Statistical interpretation of the densitometric data was done with Microcal Origin (Microcal Software, Inc., Northampton, MA, USA).

3 Results

3.1 Cytochemical studies of apoptotic cells

The expression of Man-rich glycoconjugates in intact and apoptotic murine of L1210 leukemia cells and in murine splenocytes was compared by means of the lectinocytochemistry using PSL lectin. It was found that the apoptosis of L1210 cells was accompanied by 1.43-fold increase (p<0.01) in PSL staining compared with the intact cells. The apoptosis of murine splenocytes led to a similar (1.44-fold) increase (p<0.01) in the intensity of staining of the apoptotic cells (Fig. 1). αMMan – sugar inhibitor of PSL lectin, almost totally eliminated the difference in lectin binding between the intact and the apoptotic cells.


Fig. 1

Densitometry (A,B) and lectinocytochemical study (C–F) of murine leukemia L1210 cells (A, C, D) and murine splenocytes (B, E, F). Staining with HRP–PSL lectin. C, E – Intact cells and D, F – apoptotic cells.


The apoptosis of L1210 leukemia cells and splenocytes was confirmed by comet (Fig. 2A) and DNA-fragmentation assays (Fig. 2B). An increased expression of Man-rich GPs on the apoptotic cells was confirmed by combined fluorescent microscopy using vital staining of cells with propidium iodide (PI) – for staining the apoptotic cells which lost their ability to pump out the dye; DAPI – for staining of cells with damaged plasma membrane and condensed (apoptotic) nucleus (Wyllie et al., 1998); and PSL–FITC conjugate – for staining of cells with increased levels of Man-rich GPs. The data for apoptotic L1210 cells (apoptosis induced by low concentration of doxorubicin, 0.5μg/ml, 20h) are presented in Fig. 3.


Fig. 2

Comet (A) and DNA fragmentation (B) assays of murine leukemia L1210 cells (1–2) and murine splenocytes (3–4) and comet class distribution of the studied cells. 1 – Intact L1210 cells, 2 – L1210 cells treated with doxorubicin (2mg/ml), 3 – intact splenocytes, and 4 – splenocytes treated with dexamethasone (40mg/kg).


Fig. 3

Combined fluorescent microscopy (vital staining) of apoptotic L1210 cells. Apoptosis was induced by relatively low concentration of doxorubicin, 0.5μg/ml for 20h. A – Light microscopy; B – staining with PSL–FITC, 14μg/ml; C – DAPI staining, 1μg/ml; insertion – combined light microscopy and DAPI staining; and D – staining with PI. The cell that possess a markedly increased levels of Man-rich GPs (revealed by PSL–FITC staining) is also characterized by a loss of membrane integrity (is permeable to PI and DAPI), and condensation of nucleus (revealed by DAPI staining) and, thus, can be considered to be an apoptotic one.



The smears of intact and apoptotic splenocytes stained with PSL–HRP were additionally stained by Giemsa–Romanovsky dye. Such double staining revealed heterogeneity in PSL staining of different cell types present in the splenocyte population: lymphocytes (85% of the population) were most intensively stained by PSL, while the monocytes (11%) which were also found in the population, were less intensively stained by labeled lectin. The granulocytes were detected in insignificant quantity (<4%), and the red blood cells were weakly stained and ignored during cell densitometry. Thus, the main impact to PSL staining of splenocyte population was contributed by the lymphocytes (Fig. 1). Intact murine splenocytes were characterized by increased levels of basal staining with PSL, which was almost double that for the unstained cells, while the intact L1210 bound PSL at a level similar to the unstained cells. This might be explained by different basal glycoprotein patterns of the two studied cell systems, since the possibility of spontaneous apoptosis in studied populations was excluded by cell staining with trypan blue and/or PI.

3.2 PSL-affinity chromatography for isolation of Man-rich GPs

Isolation of the plasma membrane fraction of the intact and apoptotic murine L1210 cells inoculated into the abdominal cavities of mice with subsequent PSL-affinity chromatography and 4.0–17.5% gradient PAGE electrophoresis revealed that plasma membrane possess several GP binding targets for the PSL (Fig. 4). The Man-rich GPs with MW 34kDa and with high MW GP A, as well as a set of GPs with MW range of 75–98kDa, were detected in the apoptotic cells, but not in the intact cells. The densitometric profile of the corresponding electrophoretic bands confirmed an increased expression of those Man-rich GPs (Fig. 4II). Separation of isolated plasma membranes of intact and dexamethasone-treated (apoptotic) murine splenocytes into the peripheral and integral proteins was achieved by using Triton X-114 solubility assay (Bordier, 1981). It revealed a markedly increased expression of integral high MW protein B in the apoptotic cells compared to the intact ones (Fig. 5I). The apoptotic cells also expressed the peripheral membrane high MW proteins C and D, which were not expressed in the intact cells. A subsequent PSL-lectinoblot analysis (Fig. 5II) confirmed that those GPs were rich in the Man-residues, also demonstrating an increased expression of the low MW (&007E;15kDa) integral GP in the apoptotic cells. The specificity of PSL-lectinoblot was controlled by using 100mM αMMan which resulted in a complete blocking of the labeled lectin binding (Fig. 5III). Localization of GPs in plasma membrane was achieved using Triton X-114 assay with subsequent lectinoblot. The results of our study suggest that the high MW GP B belongs to integral membrane proteins, while the GP with MW 34kDa, and GPs C and D were peripheral membrane proteins.


Fig. 4

I – Gradient (7.5–16%) SDS-PAGE electrophoresis of proteins (silver staining) isolated from L1210 cells by PSL-affinity chromatography. I – Intact cells; 2 – apoptotic (treated with doxorubicin) cells. II – Densitometry profile of gel (I). A – Man-rich GP with increased expression at apoptosis.


Fig. 5

I – Electrophoretic study of glycoproteins isolated from plasma membrane of intact and apoptotic cells in gradient PAGE (4.0–17.5%). Membrane solubilization in 2% Triton X-114 was used to discriminate between the integral and peripheral membrane glycoproteins. II – PSL-lectinoblot of the correspondent gel. III – PSL-lectinoblot of the correspondent gel in the presence of 100mM of αMMan (sugar competitor of PSL binding): 1 – integral proteins of intact cells; 2 – integral proteins of apoptotic cells (treated with dexamethasone); 3 – peripheral proteins of intact cells; and 4 – peripheral proteins of apoptotic cells.



MALDI-TOF MS analysis of high-molecular weight proteins A and B isolated from the apoptotic murine L1210 cells and the apoptotic murine splenocytes, correspondingly, revealed their similarity with the microtubule-actin cross-linking factor 1 (MW 608kDa). A 34-kDa protein characteristic for the apoptotic L1210 cells was found to be close to G-protein beta-subunit-like protein (Acc# BAA06185.1) involved in cell signal transduction. Finally, peripheral Man-rich GP C isolated from plasma membranes of murine splenocytes was shown to be close to dystonin isoform β (MW 833kDa) and dynein heavy chain (MW 530kDa), while the peripheral Man-rich GP D was shown to be close to dystonin isoform β (MW 833kDa).

Thus, our results prove that the in vivo induction of apoptosis in mice is accompanied by an elevated expression of certain Man-rich GPs similar to those observed in the in vitro cell culture systems. In both cases, the GPs which were involved in the rearrangement of cytoskeleton and in signal transduction exhibited changed expression.

4 Discussion

Recently we demonstrated that apoptotic cells can bind α-d-Man- and β-d-Gal-specific lectins much better than intact cells (Bilyy et al., 2004), suggesting that the apoptotic cells possess changed expression of specific plasma membrane glycoconjugates. Among the Man-specific lectins used in that study, HRP-labeled Pisum sativum lectin (PSL) provided the most intensive staining. PSL was also used to compare lectin-binding profiles of the apoptotic and intact cells obtained from two in vivo experimental models: (1) inoculated transformed murine leukemia cells of L1210 line, and (2) normal murine splenocytes. The results of the present study demonstrated that in in vivo animal cell systems apoptosis was accompanied by a significant increase in the expression of Man-rich GPs, and that changes are comparable with changes in the expression of similar GPs shown in in vitro cellular systems (Bilyy and Stoika, 2003). Earlier we found that the expression of Man-rich specific GPs during apoptosis was increased in mouse and human leukemia cells, transformed mouse fibroblasts, human lung and breast carcinoma cells (Bilyy and Stoika, 2003; Bilyy et al., 2004; Chorna et al., 2004), human lymphocytes of patients with autoimmune diseases (unpublished data), and under the action of different inducers of apoptosis: chemical – anticancer drugs (etoposide, methotrexate, cisplatin, and doxorubicin), or steroid hormones (dexamethasone), physical – hyperthermia or X-radiation (Bilyy and Stoika, 2003; Bilyy et al., 2004; Chorna et al., 2004), and biological – anti-CD95 antibodies or galectin-3 (unpublished data). Our approach is clearly useful for detection and study of the apoptotic cells. Moreover, we were able to isolate the apoptotic L1210 cells from mixed population by means of the PSL-affinity chromatography (Bilyy et al., 2004).

The analysis of L1210 cells grown in culture, shown to have a molecular weight of 32 and 49kDa, proved that the lectin-binding GP targets are predominantly located in the plasma membrane fraction (Bilyy and Stoika, 2003). Here we studied plasma membrane fraction isolated from the intact and apoptotic cells growing in mice organism. Due to various properties of the cellular composition of L1210 cells and splenocytes growing in vivo, the approach used for the isolation and identification of Man-rich GPs of these cells was different. PSL-affinity chromatography and SDS-PAGE electrophoresis were applied when the homogenous population of transformed murine leukemia L1210 cells was studied; separation of plasma membrane proteins for integral and peripheral cells was used when the heterogeneous population of normal murine splenocytes was under consideration.

In both in vivo systems studied the apoptosis was characterized by the elevated expression of several Man-rich plasma membrane GPs. The electrophoretic patterns of those GPs, however, were different in the transformed murine leukemia L1210 cells and murine splenocytes. For example, the peripheral GPs C and D (identified by mass spectrometry as GPs similar to dystonin isoform β) were observed in the apoptotic splenocytes, but were absent in the apoptotic L1210 cells inoculated into the abdominal cavity of mice. At the same time, an increased expression of MW 34kDa GP and of high MW GPs (A and B) was observed in both the apoptotic murine L1210 cells and the apoptotic murine splenocytes. Apoptosis was accompanied by two types of changes in GP expression: (1) the appearance of GPs was not observed in the intact cells (GP A in Fig. 4II), and (2) an increase in the level of GPs that were present in the intact cells (30kDa GP in Fig. 4II). The absence of GP B on the nitrocellulose membrane (Fig. 5II) might be explained by low efficiency of blotting high MW proteins (>300kDa) using our gradient (4.0–17.5%) PAGE electrophoresis system.

The apoptosis-dependent appearance of specific Man-rich membrane GPs can hardly be explained by the activation/deactivation processes involving the corresponding genes coding for those GPs. Changes in GP presentation in the plasma membrane, especially via the modification of pre-existing GP by desialation, can be an alternative explanation of the observed phenomena. This hypothesis (previously mentioned by Bilyy et al., 2004) is in agreement with the results of Azuma et al. (2000) and Kakugawa et al. (2002). Those researchers showed that an increase in Man-containing GPs during apoptosis was caused by the activation of endogenous sialidases which led to desialation of membrane GPs and the exposure of their Man-residues. Recently, we found that an increase in Man-residues on the surface of the apoptotic cells was accompanied by a simultaneous decrease in the sialic acid residues on the surface of the cells (the expression of sialic acids was estimated by the indirect method of cell agglutination in the presence of specific lectin) (unpublished data). Another group (Batisse et al., 2004) also showed a decrease in the level of sialic acid residues in plasma membrane of the apoptotic cells.

The MALDI-TOF MS of high MW GP isolated from L1210 cells (A) and from murine splenocytes (B) revealed their similarity to the microtubule-actin cross-linking factor 1 (MW 608kDa), suggesting a similarity between the apoptosis-dependent increase in the level of Man-rich GP(s) in the studied in vivo systems of both normal and transformed cells. MS data also highlight a discrepancy between the high MW of the GPs detected by the MS analysis and the much lower MW determined on the basis of protein migration rate during SDS-PAGE electrophoresis. There was no such discrepancy regarding the low MW range GP of 34kDa. We speculate that this discrepancy was due to the presence of the glycoconjugate part in the high MW GPs altering the electrophoretic behaviour of the studied GPs leading to the detection of multiple isoforms of GPs with different MW. It is known that a family of microtubule-actin cross-linking factors includes proteins with different molecular masses subject to alternative splicing, having possible MW range of 302–834kDa (Leung et al., 2001).

The MALDI-TOF MS data suggested specific functional roles for the apoptosis-dependent plasma membrane GPs in cytoskeleton rearrangements and signal transduction, implying that membrane GPs are involved in different signaling pathways activated during programmed cell death. It is known that the apoptosis (the programmed cell death of type I) is accompanied by dramatic rearrangements of cellular cytoskeleton, such as depolymerization or cleavage of actin, cytokeratins, lamins and other proteins participating in the cytoskeleton formation. One of the identified proteins, namely microtubule-actin cross-linking factor, possesses an actin-binding domain and the plakin domain at the NH2-terminal (Okumura et al., 2002), and can associate with both actin microfilaments and microtubules (Sun et al., 2001; Leung et al., 1999). Recent data (Kanzawa et al., 2005) also demonstrated that autophagy (the programmed cell death of type II) depended on the microtubule-associated protein light chain 3 (LC3), considered to be a specific marker of the autophagy (Kabeya et al., 2000; Asanuma et al., 2003). Microtubules and cytoplasmic dynein, a microtubule-dependent motor, are required for nuclei movement along the filamentous fungi Aspergillus nidulans hyphae. Heat-sensitive mutations in the nudA gene, which encodes dynein heavy chain, and in the nudF gene, which encodes G-protein beta-subunit-like protein, blocked nuclei migration (Willins et al., 1995). These data could be another example of specific interrelations between the dynein heavy chain and the G-protein beta-subunit-like protein, which was shown to increase its expression during apoptosis. Besides, it was demonstrated that missense point mutations in the cytoplasmic dynein heavy chain gene resulted in a progressive motor neuron degeneration in heterozygous mice (Hafezparast et al., 2003). However, 1,25-dihydroxyvitamin D3-induced cell death was accompanied with down-regulated expression of transcripts for the microtubule motor dynein heavy chain/MAP 1C (Baudet et al., 1998). It may be suggested that the cytoskeleton-dependent processes during apoptosis are cell specific.

Thus, our data give evidence that specific changes in the expression of plasma membrane GPs take place during apoptosis not only in vitro but also in vivo. Although the molecular mechanisms of those changes are still poorly understood, their potential role in the processes of cytoskeleton rearrangement and signal transduction in the target apoptotic cells is evident.

5 Conclusions

An increased expression of Man-rich GPs induced during apoptosis in two in vivo systems of normal and transformed cells was demonstrated. Those GPs possess a wide MW range (34 and high MW of 608 and 833kDa) and different localization (integral and peripheral) in plasma membrane. The results of the MALDI-TOF MS analysis suggest that apoptosis-dependent GPs may be involved in signal transduction and/or cytoskeleton rearrangements in the apoptotic cells.

Acknowledgments

This work was partly supported by the West-Ukrainian BioMedical Research Center (WUBMRC) grant awarded to R. Bilyy. The authors thank Dr. V. Antonyuk for supplying PSL lectin and PSL-bound affinity sorbent, and Dr. H.J. Gabius for galectin 3. Valuable ideas of Dr. H.J. Gabius during the article planning and comments of Dr. M. Lutsyk during the study performance were also highly appreciated.

References

Asanuma K, Tanida, I, Shirato, I, Ueno, T, Takahara, H, Nishitani, T. MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis. FASEB J 2003:17:1165-7
Medline   1st Citation  

Azuma Y, Taniguchi, A, Matsumoto, K. Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase. Glycoconj J 2000:17:301-6
Crossref   Medline   1st Citation   2nd  

Batisse C, Marquet, J, Greffard, A, Fleury-Feith, J, Jaurand, MC, Pilatte, Y. Lectin-based three-color flow cytometric approach for studying cell surface glycosylation changes that occur during apoptosis. Cytometry 2004:A62:81-8
1st Citation   2nd  

Baudet C, Perret, E, Delpech, B, Kaghad, M, Brachet, P, Wion, D. Differentially expressed genes in C6.9 glioma cells during vitamin D-induced cell death program. Cell Death Differ 1998:5:116-25
Crossref   Medline   1st Citation  

Bilyy RO, Antonyuk, VO, Stoika, RS. Cytochemical study of role of alpha-d-mannose- and beta-d-galactose-containing glycoproteins in apoptosis. J Mol Histol 2004:35:829-38
Crossref   Medline   1st Citation   2nd   3rd   4th   5th   6th  

Bilyy RO, Stoika, RS. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 2003:56A:89-95
Crossref   1st Citation   2nd   3rd   4th   5th   6th  

Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 1981:256:1604-7
Medline   1st Citation   2nd  

Chang HY, Yang, X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 2000:64:821-46
Crossref   Medline   1st Citation  

Chekhun VF, Tryndiak, VP, Todor, IM, Mykhailenko, VM, Kondrychyn, IH, Dovbeshko, HI. Phospholipid and cholesterol levels in the tumor cell plasma membranes with different sensitivity to doxorubicin. Ukr Biokhim Zh 2003:75:120-5
Medline   1st Citation  

Chionna A, Dwikat, M, Panzarini, E, Tenuzzo, B, Carla, EC, Verri, T. Cell shape and plasma membrane alterations after static magnetic fields exposure. Eur J Histochem 2003:47:299-308
Medline   1st Citation  

Chorna I, Bilyy, R, Datsyuk, L, Stoika, R. Comparative study of human breast carcinoma MCF-7 cells differing in their resistance to doxorubicin: effect of ionizing radiation on apoptosis and TGF-beta production. Exp Oncol 2004:26:111-7
Medline   1st Citation   2nd   3rd  

Cohen GM. Caspases: the executioners of apoptosis. Biochem J 1997:326:1-16
Medline   1st Citation  

Fadok VA, Voelker, DR, Campbell, PA, Cohen, JJ, Bratton, DL, Henson, PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992:148:2207-16
Medline   1st Citation  

Fowlkes BJ, Waxdal, MJ, Sharrow, SO, Thomas, CA, Asofsky, R, Mathieson, BJ. Differential binding of fluorescein-labeled lectins to mouse thymocytes: subsets revealed by flow microfluorometry. J Immunol 1980:125:623-30
Medline   1st Citation  

Fujimura M, Morita-Fujimura, Y, Murakami, K, Kawase, M, Chan, PH. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1998:18:1239-47
Medline   1st Citation  

Gong J, Traganos, F, Darzynkiewicz, Z. A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. Anal Biochem 1994:218:314-9
Crossref   Medline   1st Citation  

Gulston M, Fulford, J, Jenner, T, de, LC, O'Neill, P. Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19), hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites. Nucleic Acids Res 2002:30:3464-72
Crossref   Medline   1st Citation  

Hafezparast M, Klocke, R, Ruhrberg, C, Marquardt, A, Hmad-Annuar, A, Bowen, S. Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 2003:300:808-12
Crossref   Medline   1st Citation  

Heaton PR, Ransley, R, Charlton, CJ, Mann, SJ, Stevenson, J, Smith, BH. Application of single-cell gel electrophoresis (comet) assay for assessing levels of DNA damage in canine and feline leukocytes. J Nutr 2002:132:1598S-603S
Medline   1st Citation  

Hellman U. Sample preparation by SDS/PAGE and in-gel digestion. Proteomics in functional genomics. Protein structure analysis 2000:43-54
1st Citation  

Herrington CS, McGee, JO`D. Diagnostic molecular pathology. 1992:
1st Citation  

Heyder P, Gaipl, US, Beyer, TD, Voll, RE, Kern, PM, Stach, C. Early detection of apoptosis by staining of acid-treated apoptotic cells with FITC-labeled lectin from Narcissus pseudonarcissus. Cytometry 2003:55A:86-93
Crossref   1st Citation  

Kabeya Y, Mizushima, N, Ueno, T, Yamamoto, A, Kirisako, T, Noda, T. TLC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000:19:5720-8
Crossref   Medline   1st Citation  

Kakugawa Y, Wada, T, Yamaguchi, K, Yamanami, H, Ouchi, K, Sato, I. Up-regulation of plasma membrane-associated ganglioside sialidase (Neu3) in human colon cancer and its involvement in apoptosis suppression. Proc Natl Acad Sci U S A 2002:99:10718-23
Crossref   Medline   1st Citation  

Kanzawa T, Zhang, L, Xiao, L, Germano, IM, Kondo, Y, Kondo, S. Arsenic trioxide induces autophagic cell death in malignant glioma cells by upregulation of mitochondrial cell death protein BNIP3. Oncogene 2005:24:980-91
Crossref   Medline   1st Citation  

Krishnan N, Thellin, O, Buckley, DJ, Horseman, ND, Buckley, AR. Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo. Endocrinology 2003:144:2102-10
Crossref   Medline   1st Citation   2nd  

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970:227:680-5
Crossref   Medline   1st Citation  

Leung CL, Sun, D, Zheng, M, Knowles, DR, Liem, RK. Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J Cell Biol 1999:147:1275-86
Crossref   Medline   1st Citation  

Leung CL, Zheng, M, Prater, SM, Liem, RK. The BPAG1 locus: alternative splicing produces multiple isoforms with distinct cytoskeletal linker domains, including predominant isoforms in neurons and muscles. J Cell Biol 2001:154:691-7
Crossref   Medline   1st Citation  

Lutsik MD, Kusen, SI. Study of membrane glycoproteins of human erythrocytes using lectins. Ukr Biokhim Zh 1987:59:3-9
Medline   1st Citation  

Nadin SB, Vargas-Roig, LM, Ciocca, DR. A silver staining method for single-cell gel assay. J Histochem Cytochem 2001:49:1183-6
Medline   1st Citation  

Okumura M, Yamakawa, H, Ohara, O, Owaribe, K. Novel alternative splicings of BPAG1 (bullous pemphigoid antigen 1) including the domain structure closely related to MACF (microtubule actin cross-linking factor). J Biol Chem 2002:277:6682-7
Crossref   Medline   1st Citation  

Perea-Sasiain J. Giemsa stain's 100th year. Biomedica 2003:23:5-18
Medline   1st Citation  

Perez-Pinzon MA, Xu, GP, Born, J, Lorenzo, J, Busto, R, Rosenthal, M. Cytochrome C is released from mitochondria into the cytosol after cerebral anoxia or ischemia. J Cereb Blood Flow Metab 1999:19:39-43
Medline   1st Citation  

Reed JC. Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994:124:1-6
Crossref   Medline   1st Citation  

Shevchenko A, Wilm, M, Vorm, O, Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 1996:68:850-8
Crossref   Medline   1st Citation  

Somerville L, Cory, JG. Enhanced roscovitine-induced apoptosis is mediated by a caspase-3-like activity in deoxyadenosine-resistant mouse leukemia L1210 cells. Anticancer Res 2000:20:3347-55
Medline   1st Citation  

Sun D, Leung, CL, Liem, RK. Characterization of the microtubule binding domain of microtubule actin crosslinking factor (MACF): identification of a novel group of microtubule associated proteins. J Cell Sci 2001:114:161-72
Medline   1st Citation  

Towbin H, Staehelin, T, Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Biotechnology 1992:24:145-9
Medline   1st Citation  

Willins DA, Xiang, X, Morris, NR. An alpha tubulin mutation suppresses nuclear migration mutations in Aspergillus nidulans. Genetics 1995:141:1287-98
Medline   1st Citation  

Wittekind DH. On the nature of Romanowsky–Giemsa staining and its significance for cytochemistry and histochemistry: an overall view. Histochem J 1983:15:1029-47
Crossref   Medline   1st Citation  

Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980:284:555-6
Crossref   Medline   1st Citation  

Wyllie A, Donahue, V, Fischer, B, Hill, D, Keesey, J, Manzow, S. Apoptosis and cell proliferation. 1998:
1st Citation  


Received 14 June 2005/2 July 2005; accepted 10 August 2005

doi:10.1016/j.cellbi.2005.08.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)