interaction of soybean agglutinin with leukemic t-cells and its use for their in vitro separation...
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Received 22 August 2002; revised 4 October 2002; accepted 30 October 2002
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 & Sons, Ltd.
KEYWORDS: acute lymphoblastic leukemia; soybean agglutinin; lectin-affinity chromatography; fraction of cells
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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
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.
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
BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 17: 239249 (2003)Published online in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/bmc.218
*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: email@example.comAbbreviations 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.
Copyright 2003 John Wiley & Sons, Ltd.
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).
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.
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.
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.
' 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.
* 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).
Cells (normal lymphocytes, Jurkat, MOLT-4, RPMI-8402) we