Interaction of soybean agglutinin with leukemic T-cells and its use for their in vitro separation from normal lymphocytes by lectin-affinity chromatography

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<ul><li><p> ! "#$ % &amp; &amp; "'% ##() *</p><p>Received 22 August 2002; revised 4 October 2002; accepted 30 October 2002</p><p>ABSTRACT: A procedure for separation of leukemic T-cells from normal lymphocytes, using lectin-affinity column chromatogra-phy, is described. CNBr-activated Sepharose 6MB was used as a non-mobile phase. The gel was covalently coupled with soybeanagglutinin (SBA), then served as an affinity probe for fractionation of mixture of normal lymphocytes and leukemic cells. Leukemiccell lines, derived from acute lymphoblastic leukemia (Jurkat, MOLT-4, RPMI-8402), were tested. The elution of normallymphocytes was carried out by PBS(). The leukemic T-cells, interacting with SBA, were removed by N-acetyl-D-galactosamine orlow-concentration acetic acid. The type and viability of the separated cell fractions were analyzed by flow cytometry and fluorescentmicroscopy, using adequate fluorescent antibodies. The interaction of leukemic T-cells with free SBA, as well as with SBA-conjugated Sepharose beads, was examined fluorimetrically and visualized by fluorescent microscopy, using FITC-SBA as a marker.The rate of cell elution on SBA-affinity column decreased in order: normal leukemic T-cells. Both normal lymphocytes andleukemic T-cells were removed in a mixture from SBA-free Sepharose 6MB by PBS() and were not fractionated discretely. Theleukemic T-cells specifically interacted with SBA as well as with SBA-affinity adsorbent. In contrast, the normal lymphocytes did notinteract with free SBA as well as with SBA-conjugated Sepharose beads in the concentrations applied. The method potentiallycombines a discrete cell fractionation with manifestation of a specific target cytotoxicity of SBA against leukemic T-cells, withoutany influence on normal lymphocytes. Copyright 2003 John Wiley &amp; Sons, Ltd.</p><p>KEYWORDS: acute lymphoblastic leukemia; soybean agglutinin; lectin-affinity chromatography; fraction of cells</p><p>! "#$ !</p><p>Leukemia is an increasingly recognized health problem.At present, the research demands are directed to adevelopment of a new strategy for disease control,attending to the requirements of autologous transplanta-tion. Many anticancer drugs are also applied duringchemotherapy followed by bone marrow implantation,until leukemic cells are not observed in the blood.However, these drugs induce severe side-effects inpatients (Santos, 1988, 1990; Petros and Evans, 1990;Hozumi, 1994; Sweetenham, 1995; Reed, 2000; Solary etal., 2000). Sometimes a complete disease remission isinduced, but relapse is usual. The efforts of clinicians and</p><p>researchers are directed to reducing the risk of relapse byselective in vitro removal and utilization of tumor cellsfrom autologous graft (Santos, 1988, 1990; Marmont,1998). In this context, the separation of leukemic cellsfrom normal ones, based on their different affinity andsusceptibility to leukemia-cytotoxic drugs or respectiveantibodies, can form the basis for development of a newgeneration of methods for effective control and therapyof leukemia.</p><p>In the last 20 years some lectins (plant-derivedproteins) are approved as promising anti-leukemia agentswith their unique biological activities as cytoagglutina-tion, mitogenic activity and cytotoxicity, manifestingwith high target-selectivity for leukemic cells, withoutany significant influence on normal ones (Gabius et al.,1988; Mody et al., 1995; Gabius, 1997; Moriwaki et al.,2000; Sallay et al., 2000). The prerequisite step of theseactivities seems to be initiated by binding of lectins to thecell surface carbohydrate chains of tumor cells (Gabius etal., 1988; Mody et al., 1995; Gabius, 1997, 2001; Chayand Pienta, 2000). These lectin characteristics are broadlyuseful for demonstration of differences in the composi-tion of cell surface and intracellular glycoproteins andglycolipids in tissues at various stages of differentiation,in malignancy and in functional subsets of cells, as well</p><p>BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 17: 239249 (2003)Published online in Wiley InterScience ( 10.1002/bmc.218</p><p>*Correspondence to: R. Bakalova, Natural SubstanceComposedMaterials Group, Institute for Structural and Engineering Materials,Independent Administrative Institution, National Institute of AdvancedIndustrial Science and Technology, AIST-Kyushu, 807-1 Shuku, Tosu,Saga-ken 841-0052, Japan.E-mail: r.bakalova-zheleva@aist.go.jpAbbreviations used: ALL, acute lymphoblastic leukemia; FITC,fluorescent isothiocyanate; NAGal, N-Acetyl-D-galactosamine;PBS(), phosphate buffered saline (Ca2 and Mg2 free); PE,phycoerythrin; SBA, soybean biflorus agglutinin.Contract/grant sponsor: JSPS Invitation Fellowship Program forResearch in Japan; contract/grant number: L01505.</p><p>Copyright 2003 John Wiley &amp; Sons, Ltd.</p><p>ORIGINAL RESEARCH</p></li><li><p>as for isolation, identification and characterization oftumor cell surface receptors (Gabius et al., 1988; Gabius,1989, 1997, 2001; Mody et al., 1995; Chay and Pienta,2000). The lectins proved to be applicable for diagnosticpurposes, especially for the differential diagnosis ofanaplastic tumors (Gabius, 1989; Gabius et al., 1986,1988; Mody et al., 1995; Roth et al., 1996).</p><p>In leukemic cells a reduced molecular weight andchanges in the saccharide composition of surface orintracellular glycoproteins were found to be the maindistinguishing marks (Vaickus et al., 1991; Neame et al.,1994; Misra et al., 2000). Antibodies, produced againstlectin affinity-isolated glycoproteins, allowed the demon-stration of structural relationship of glycoproteins withidentical or diverse lectin binding pattern (Forsberg andMacher, 1987; Fischer et al., 1988; Lee et al., 1990).Therefore, the lectins with their specific affinities forsimple and complex sugars on tumor cell surface canrecognize fine differences between leukemic and normalcells. This characteristic may be a potentially useful toolfor separation of leukemic leukocytes from normal oneswith high potential for selective in vitro removal of tumorcells from the autologous graft. A separation wasreported of osteoclasts, erythrocytes and immunogenictumor cells based on their lectin affinity (Killion andKollmorgen, 1976; Pereira and Kabat, 1979; Itokazu etal., 1991). However, the use of lectin-affinity chroma-tography for separation of leukemic T- and B-cells fromnormal lymphocytes is only beginning to be exploited.</p><p>In our previous paper we already described aseparation of leukemic T-cells from normal lymphocytes,using Sepharose 6MB, conjugated with dolichos biflorusagglutinin (DBA) (Ohba et al., 2002). However, based onthe fact that the fine differences in quaternary structure ofthe lectins relate directly to the difference in theircarbohydrate specificity and the strength of binding withtumor cell surface receptors (Bouckaert et al., 1999), weexamined and compared the degree of lectin-cell bindingfor several lectins (DBA, SBA and WGA, wheat germagglutinin, and its isolectins) and its influence on theutilization of leukemic T-cells from normal ones, usingdifferent lectin-affinity adsorbents. It was established thatthe degree of lectin-cell binding increased in the orderDBA SBAWGA, however WGA and its threeisolectins interacted not only with leukemic cells, butalso with normal lymphocytes (unpublished data). Thismade WGA unsuitable for adsorbent saturation andseparation procedure. SBA was found to have a higherdegree of binding with leukemic cells, as well as a betterexpressed cytotoxic effect against leukemic cells, thanDBA. Thus, we gave preference to SBA as the bestcandidate for affinity probe among another lectins used,because of the potential to combine a discrete cellfractionation with specific cytotoxic effect of SBA on theleukemic T-cells, without any influence on the viabilityof normal lymphocytes.</p><p>In the present study, using SBA-conjugated Sepharose6MB as an affinity adsorbent, we separated discretelynormal lymphocytes from ALL-derived leukemic T-cells(Jurkat, MOLT-4, and RPMI-8402) and characterizedtheir type and viability after passing through the lectin-affinity column.</p><p>%&amp;'%(%!)*</p><p>' The human leukemic T-cell lines (Jurkat,MOLT-4, RPMI-8402; Hayashibara Biochemical Laboratories,Inc., Okayama, Japan) were cultured in RPMI-1640 mediumsupplemented with 10% heat-inactivated fetal bovine serum (FBS),100 g/mL streptomycin, and 100 U/mL penicillin in a humidifiedatmosphere at 37C with 5% CO2. The cell lines were a generousgift of Dr J. Minowada (Hayashibara Biochemical LaboratoriesInc., Okayama, Japan). Normal lymphocytes were purified fromheparinized peripheral blood obtained from normal adults (aged3840 years) by Lymphosepar I. The cells used for assay were in alogarithmic phase. They were sedimented by centrifugation (1000rpm, 10 min) and washed three times by PBS() before experi-ments.</p><p>* The followingbuffers were used to prepare CNBr-activated Sepharose 6MB forcolumn chromatography: coupling buffer (0.1 M NaHCO3/0.5 MNaCl, pH = 8.5), washing buffer (0.1 M CH3COONa/0.5 MCH3COOH, pH = 4.5), and blocking buffer (0.1 M NaHCO3/0.2 M glycine, pH = 8.5). The gel was washed consecutively withthe buffers as described in gel certificate, and then added to SBA(2.5 mg SBA/mL, dissolved in a coupling buffer). The mixture wasincubated 24 h at 4C for conjugation of SBA to the gel particles.The coupling procedure was repeated three times (the total SBAconcentration added to the Sepharose 6MB was 15 mg SBA/g gel).The non-binding SBA was measured in supernatant spectro-photometrically at = 280 nm. The concentration of SBAconjugated to the Sepharose beads was calculated using acalibration coefficient and was found to be more than 95%. Thefree non-SBA-saturated active sites of adsorbent were blocked bywashing several times with blocking buffer at room temperature(RT).</p><p>Cells (normal lymphocytes, Jurkat, MOLT-4, RPMI-8402) werewashed by PBS(), re-suspended in the same buffer to a con-centration of 2 106 cells/mL, mixed 1:1 (v:v, normal:tumor) andthe cell suspension (1 mL) was added to SBA-conjugated CNBr-activated Sepharose 6MB (2 mL) for 15 min at RT. The cells,interacting with gel beads, were removed by competitive inhi-bition, using stepwise elution with aliquots of PBS(), containingsaccharide (00.4 M N-acetyl-D-galactosamine, in the case ofJurkat or RPMI-8402), or low concentrated acetic acid (12.5%, inthe case of MOLT-4). A flow rate was 1.2 mL/min and the elutioncontinued until the eluate was cell-free. The same chromatographicprocedure was carried out on SBA-free Sepharose 6MB. The cellfractions were collected by ATTO Minicollector (SJ-1410).</p><p>+ The cell suspensions were detectedspectrophotometrically at 600 nm before and after separation onSBA-affinity or SBA-free columns. The adjacent cell fractions,</p><p>Copyright 2003 John Wiley &amp; Sons, Ltd. Biomed. Chromatogr. 17: 239249 (2003)</p><p>240 ORIGINAL RESEARCH R. Bakalova and H. Ohba</p></li><li><p>showing a high OD at 600 nm (OD600nm 0.050), were combined.The cells were sedimented by centrifugation (1800 rpm for 10 min)and re-suspended in PBS() to concentrate the cell fraction and toprepare it for flow cytometric and microscopic analyses. Thenumber of cells in each fraction was determined microscopically.</p><p>, The separated cell fractions wereincubated with specific fluorescent antibodiesFITC-CD90 forleukemic T-cells and PE-CD44 for normal lymphocytes. Fivemicroliters of each antibody were added to 0.5 mL of each cellfraction, and incubated for 30 min at RT before flow cytometricassay. As controls, 5 L PE-CD44 or FITC-CD90 antibodies wereadded respectively to normal or tumor cell suspensions (0.5 mL)and the samples were treated in the same manner. Suspensions ofnormal lymphocytes, leukemic T-cells and their mixture withoutantibodies were used as negative controls to determine thespontaneous cell fluorescence.</p><p>The viability and the type of cells were analyzed by flowcytometer Beckman Coulter-Epics XL. The flow cytometer wasoperated in accordance with the manufacturers recommendationsafter fine adjustments for optimization. The forward- and side-scatter parameters were adjusted to accommodate the inclusion ofboth leukemic T-cells and normal lymphocytes within theacquisition data. No cells excluded from the analysis, and 10,000cells were counted. Data were collected and analyzed by using XLSystem II software.</p><p>The results were presented as a dot plot of FITC-fluorescenceand PE-fluorescence with quadrant markers drawn to distinguishFITC- and PE-labeled cells. Quadrants A in Fig. 2 contain viablenormal lymphocytes, quadrants B contain viable leukemic T-cells,quadrants C contain all viable cells, and quadrants D contain alldead cells.</p><p>The percentage lysis of cells was calculated before (sponta-neous lysis) and after their passing through the column from thefollowing equations:</p><p> for leukemic T-cells: percentage lysis = [1 quadrant Bevents/(quadrant C quadrant D)] 100;</p><p> for normal lymphocytes: percentage lysis = [1 quadrant Aevents/(quadrant C quadrant D)] 100.</p><p>To estimate the effect of SBA-affinity column on the viability ofnormal and leukemic leukocytes, we applied different cell lines tothe column separately and incubated for 15 min at RT or 24 h at4C. To estimate the effect of incubation on the spontaneous lysis,the respective cell lines were incubated in PBS() for 15 min at RTor 24 h at 4C.</p><p>, - </p><p> Identification of the type of separatedcell fractions was examined also by fluorescent microscopy, usingFITC-CD90 (green light), and PE-CD44 (red light) antibodies. Tenmicroliters of each antibody were added to 0.5 mL of separated andconcentrated cell fractions, and were incubated for 30 min at RT.The cell fractions were washed twice by PBS() for elimination offree fluorescent antibodies and were analyzed by fluorescentmicroscopy, detecting the fluorescence of cellantibody conju-gates. As controls, 10 L of PE-CD44 and 10 L of FITC-CD90were added to normal or leukemic cells, respectively (0.5 mL,</p><p>2 106 cells/mL) and the samples were treated and analyzed at thesame conditions.</p><p>, .- +) FITC-SBA (0.3 M)was added to Jurkat, MOLT-4, RPMI-8402 or normal cells(2 106 cells/mL) and the mixtures were incubated 15 min at RT.The cells were sedimented by centrifugation at 1800 rpm for10 min, washed twice with PBS(), re-suspended in PBS() andFITCSBAcell conjugates were analyzed by fluorescent micro-scopy. In parallel, the degree of lectincell interaction wasestimated spectrofluorimetrically at ex = 485 nm, and em =538 nm, using BioRad Fluoromark (JASCO Co., Japan).</p><p>, - +)/ + 0( Jurkat, MOLT-4,RPMI-8402 or normal cells (1 mL, 2 106 cells/mL) wereincubated with FITC-SBA (0.3 M) for 15 min at RT. The cellswere sedimented by centrifugation at 1800 rpm for 10 min, washedtwice with PBS() and added to SBA-conjugated CNBr-activatedSepharose 6MB for 15 min at RT. The gel was filtrated by PBS()and then FITCSBAcell conjugates, bound to the lectin-saturatedSepharose beads, were analyzed by fluorescent microscopy. Thecontrol experiment consisted of gel particles incubate...</p></li></ul>


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