interaction of soybean agglutinin with human leukemia-lymphoma lines at various stages of...

Leukemia Research Vol. II, No. 7, pp. 589-595, 1987. 0145-2126/87 $3.00 + .00 Printed in Great Britain. © 1987 Pergamon Journals Ltd.

INTERACTION OF SOYBEAN AGGLUTININ WITH H U M A N L E U K E M I A - L Y M P H O M A LINES AT VARIOUS

STAGES OF DIFFERENTIATION

HANNAH BEN-BASSAT,* SARAH WEKSLER-ZANGEN,* ZIPORAH SHLOMAI* and MIRON PROKOCIMER'I"

*Laboratory of Experimental Surgery, Hadassah University Hospital, Jerusalem and tHematology Division, Beilinson Medical Center, Petah-Tikva, Israel

(Received 15 September 1986. Revision accepted 13 January 1987)

Abstract--Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for reactivity with soybean agglutinin (SBA). The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate conjugated SBA (F-SBA) by ultraviolet microscopy, and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between SBA labelling-- distribution and the stage of lymphoid cell differentiation. The SBA labelling on the membrane of null lines was undetectable by U.V. microscopy and flow cytometry. A gradual increase in SBA labelling correlating with the stage of differentiation was observed on cell lines of both B and T origin. However the maximal fluorescence intensity of the T lines was lower than the B lines. The redistribution pattern of SBA on the membrane of T lines was rings and mild patches, whereas that on the B lines was moderate to large patches. The reactivity of the lymphoid lines with SBA was not affected by growth conditions. The binding of SBA to normal lymphoblastoid lines was generally low and the fluorescence intensity weak. The reactivity of these lines with SBA was not associated with their origin or "age". It is suggested that the differences in the reactivity of SBA with human hematopoietic lines at various stages of maturation may be of value in future understanding the differences in structure and function of the surface membrane between normal and malignant cells, and the relation to normal and abnormal cell differentiation.

Key words: Soybean agglutinin, human leukemia-lymphoma lines, differentiation.

I N T R O D U C T I O N

HUMAN leukemia-lymphoma cell lines are genetically homogeneous with remarkable fixed expression of a certain marker profile representing a specific point dur- ing hematopoietic cell differentiation. It has been suggested that the information obtained from these stable monoclonal lines, may be utilized for a model of normal hematopoietic differentiation, postulating that the marker profile exhibited by the individual leukemia- lymphoma lines reflects normal hematopoietic cell clones arrested at a particular stage of differentiation [11.

Abbreviations: F-SBA, fluorescein-isothiocyanate-conjugated soybean agglutinin; FACS, fluorescent activated cell sorter; PBS, phosphate buffered saline; EBV, Epstein-Barr virus; CF, cationized ferritin; Ra-B95-8, Ramos EBV negative line converted in vitro to EBV-genome carrying line with EBV from the B95-8 cell line; Bj-B95-8, in vitro converted B jab cells; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ML, malignant lymphoma; ConA, concanavalin A; WGA, wheatgerm agglutinin.

Correspondence to: Dr. H. Ben-Bassat, Laboratory of Experimental Surgery, Hadassah University Hospital, POB 12000, Jerusalem, Israel il-91120, Israel.

Lectins have been used extensively as probes of mammalian cell surfaces. Studies with these probes have indicated changes in the expression of carbohydrate- containing molecules which accompany cellular differentiation and malignant transformation [2]. The application of concanavalin A for determination of differences in the structure and function of the surface membrane between normal and transformed cells is well established [3, 4]. Earlier studies with soybean agglutinin have shown that SBA agglutinates human, mouse and rat transformed fibroblast cell lines, but not normal cells [5]. Presently SBA is successfully applied in the fractionation of cells for bone-marrow transplantation in humans across the histocompatibility barrier [6, 7].

The purpose of the present study was: (1) to deter- mine the binding and redistribution pattern of SBA on human leukemia-lymphoma cell lines, reflecting hematopoietic clones at various stages of differentiation and (2) to evaluate possible association between the stage of maturation arrest and the reactivity with SBA. The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate-conjugated SBA (F-SBA) by ultraviolet microscopy and with a fluorescent activated cell sorter (FACS).

589

590 H. BENIBASSAT et al.

TABLE 1. LYMPHOID CELL LINES

Line Origin References

MATERIALS AND METHODS

Km-3 Non B non T ALL [1] Reh Non B non T ALL [1] Nalm-16 Non B non T ALL [1] HD-Mar T-Lymphoma [8] HPB-ALL T-ALL [1] CCRF-CEM T-ALL [1] Amsalem T-ALL [9] Be-13 T-ALL [10] ALL-Sil T-ALL * Peer T-ALL [11] CCRF-HSB2 T-ALL [1] Nalm-6 Pre B-ALL [1] Raji Burkitt's lymphoma [12] Daudi Burkitt's lymphoma [13] Ramos Burkitt's lymphoma [14] Ra-B95-8 Burkitt's lymphoma [15] DG-75 Burkitt's lymphoma [16] B jab Burkitt 's lymphoma [17] B j-B95-8 Burkitt 's lymphoma [18] U-266 Myeloma [19] ARH-77 Myeloma [20]

* H. Ben-Bassat, unpublished.

Cell fines

A list of the cell lines and their origin is presented in Table 1. The lines were propagated in RPMI 1640 medium, supplemented with 20% fetal bovine serum and antibiotics: penicillin 100 U/ml and streptomycin 100 mg/ml (Gibco). The lines were maintained at 37°C in a humidified incubator containing 5% CO2. Logarithmically growing cell cultures were used in the experiments.

Interaction with fluorescent soybean agglutinin (F-SBA)

Fiuorescein-isothiocynate-conjugated soybean agglutinin (F-SBA) was prepared by Vector Laboratories, Inc. (Calif., U.S.A.) at a ratio of 5.2 fluorescein to protein. For the experiments 0.1 ml cell suspension (4-6 x 106ceils/ml) were incu- bated with 0.1 ml F-SBA (1 mg/ml) for 30 min at 37°C, and then washed twice with phosphate-buffered saline (PBS). The binding capacity (percent of cells that bound F-SBA), the fluorescence intensity (scored on a scale from - to + + + +), and the distribution pattern into patches and caps were determined on a drop of living cells with a Zeiss Ultraviolet microscope. Four hundred cells were counted for each point and only single cells and very small clumps (2-5 cells) were counted

t l r . . . . . . . 1 [ , Lymphoid t Stem cetl ~, precursor I I I n u l l i _ . . . . . . . j

TdT la Ph'

Reh Km-3

Be-f3 ALL- Sil

CCRF-CEM Amsalem HPB-ALL CCRF-HSB2 HD-Mor Peer

C-ALL T-Ag T-Ag TdT ADA TDT ADA T-Ag E EAC E EAC E

T-Blast I 1-- [ T-BLast II I - -J t -Maturecet t ]

Pre-B I - -J B-Blast [

C-ALL Ia-Ag C-ALL Ia-Ag TdT Cy-Ig Sm-Ig EBV Ph' Eo EAC

B-BLast II ]~JB-Mature cel t l - - I Plasma cell I

Ia-Ag Sm-lg la-Ag la-Ag Sm-lg EBV EA EAC Sm-lg

Nalm 6 Daudi Bjab- B95-8 Raji B jab Ramos Ra -895-8 DG -75

FIG. 1. Scheme for human lymphoid cell differentiation. Under each compartment is marked: the relevant positive markers, the examined cell lines belonging to that compartment. Distri- bution is based on a combination of positive expression of markers. Cell lines are assigned to the different compartments according to marker combination. E = sheep erythrocyte rosettes; EA = rosettes formed by bovine erythrocyte-IgG antibody complex; EAC = rosettes formed by bovine erythrocyte-IgM antibody complement complex; Sm-Ig and Cy- Ig = surface membrane and cytoplasmic immunoglobulins, respectively; T-Ag = T cell antigen; Ia-Ag = Ia-like P28, 30 glycoprotein antigen; c-ALL = common All-associated antigen; TdT = terminal deoxunuclotidyl transferase; Pht = Philadelphia chromosome; EBV = Epstein-Barr virus;

ADA = adenosine deaminase.

U-266 ARH-77

Soybean agglutinin receptors on human hematopoietic lines

TABLE 2. INTERACTION OF LYMPHOID CELL LINES WITH SOYBEAN AGGLUTININ

591

F-SBA Line Differentiation stage Binding (%) Fluorescence intensity Redistribution pattern

Km-3 Non B non T <0.1 0 - - Reh Non B non T <0.1 0 - - Nalm-16 Non B non T <0.1 0 - - HD-Mar T-Blast 1 35 _+ rings + caps HPB-ALL T-Blast 1 10 -+ rings + caps CCRF-CEM T-Blast 1 72 _+ rings + caps Amsalem T-Blast 2 29 + rings + caps Be-13 T-Blast 2 50 + rings + caps ALL-Sil T-Blast 2 68 + rings + caps Peer T-Blast 2 20 + rings + caps CCRF-HSB2 T-Blast 2 82 + rings + caps

Nalm 6 Pre-B <0.1 0 patches Raji B-Blast 1 23 + patches Daudi B-Blast 1 65 + + + patches Ramos B-Blast 1 82 + + + patches Ra-B95-8 B-Blast 1 93 + + + patches DG-75 B-Blast 1 90 + + + patches B jab B-Blast 2 100 + + + + patches B j-B95-8 B-Blast 2 100 + + + + patches U-266 B-plasma 4 - very small patches ARH-77 B-plasma 23 --- very small patches

for the percentage of labelled cells. In parallel experiments binding and fluorescence intensity were determined by flow cytometry with a 440 FACS Becton Dickinson. The fluorescence analysis by U.V. microscope and flow cytometry were carried out immediately after binding of the lectin. To prevent changes in capping and patching 1% formaldehyde (in PBS) was added to the cells.

The binding of SBA to the surface membrane was specific, for it was completely inhibited by 0.2 M o-galactose [5].

R E S U L T S

Binding and redistribution of SBA receptors on the surface of human leukemia-lymphoma lines

The binding and redistribution pattern of SBA on the surface of human leukemia-lymphoma lines was examined. The cell lines were of null (non B non T), B or T origin; representing various stages of differentiation (Fig. 1). They were assigned to the compartments of the differentiation scheme according to a combination of positive expression of markers [1]. The results indicate that SBA binding to the surface membrane of null cell lines was undetectable by U.V. microscope or by flow cytometry (Table 2, Fig. 2A). In the B- and T-cell lines there was a gradual increase in the SBA-binding, correlating with the stages of the differentiation-maturation pathway (Figs 2B, C). This pattern was manifested by the increase in the percent of cells that bound SBA and by the fluorescence intensity as determined on a scale from - to + + + + . However, the maximal fluorescence intensity of the T lines was lower and did not exceed + compared to that of the B lines (Fig. 2D). A representative experiment is summarized in Table 2 and Fig. 2: thus HD-Mar and

HPB-ALL lines which represent an early T stage (blast 1) exhibited a lower binding capacity for SBA and a lower fluorescence intensity than the Peer or CCRF- HSB2 lines which represent T-blast II stage (Fig. 2B). A similar pattern was also exhibited by the B cell lines. Nalm 6 cells (Pre B) practically did not bind SBA; Raji and Daudi cells (B-blast I) had a moderate binding capacity for SBA (23 and 65%, respectively) and fluorescence intensity of (+ and + + +) which increased in the Bjab and Bj-B95-8 lines (B-blast II): The binding capacity of these lines for SBA was 100% and the fluorescence intensity very high (Fig. 2C). However the myeloma lines which represent well differentiated B cells had low binding capacity for SBA (4 and 23%) and the fluorescence intensity of the cells that bound the lectin was also very low (+).

The B and T lines differed also in the redistribution pattern of the SBA membrane receptors. The redistribution pattern of SBA on the membrane of T lines was rings and small caps, whereas that of the B lines was moderate to large patches.

SBA binding and redistribution pattern on lymphoblastoid cell lines

Human non-neoplastic lymphoblastoid cell lines were established from peripheral blood B lymphocytes spon- taneously [21]. The cordblood lymphocytic line was established by infecting the lymphocytes with EBV from the B95-8 line [21]. The group included newly established lines less than 10 passages in cultures, and "old" lines that have been passaged for many months. The lines were analysed for reactivity with F-SBA. As shown in Table 3 most of the lymphoblastoid cell lines reacted weakly with F-SBA: The percent of cells that bound SBA was between 10 and 50 for the various lines exam-

592 H. BEN-BASSAT et al.

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0

> I-- ,< . J Ul ee

A- nul l lines

I

~1 t Reh I ,' '~ Km-3

C - B lines

Raji ; ~ ( ARH -77

li I ' ~ ' ' ~ r " c ~ a ~ r ' 1 ~ . . ~ _ Ramos

B" T lines

,r 't HD- Mar

/ tl r "

D ~ ,~ null (Nalm 16)

' ,~ T (Amsalem) I ,~1 '~ / / myeloma(ARH-77)

,~, . ~, / n~---~B (B j -B95-8 )

FLUORESCENCE INTENSITY (log)

FIG. 2. FACS analysis of the reactivity of cell lines with F-SBA (100 mg/ml). Fluorescence intensity histogram of: (A) null cell lines indicating low reactivity scored as ( - ) in U.V. microscope. (B) T lines at blast I (HD-Mar) and blast II (Peer) stages, ind':cating increased reactivity with differentiation. HD- Mar is scored as (-+) and Peer as (+) in U.V. microscope. (C) B lines at blast I (Raji, Ramos), II (B j-B95-8) stages and fully differentiated myeloma line (ARH-77). Raji is scored as (+) in U.V. microscope, Ramos as ( + + + ) , Bj-B95-8 as ( + + + + ) and ARH-77 as (-+). (D) A comparative analysis of null (Nalml6), T (Amsalem), B (Bj-B95-8) and myeloma (ARH- 77) lines, demonstrating the weakest reactivity of null lines, intermediate of T lines, the strongest of B lines; and the

decreased reactivity of the myeloma lines.

ined and the f luorescence intensity very weak ( + ) to modera te (+ +) . Only two lymphoblastoid lines showed a modera te- increased f luorescence intensity. The redistribution pat tern was of small patches.

Cell replication and binding o f SBA The next exper iments were ca rded out in order to

de termine the effect of cell replication on the interact ion of lymphoid lines with F-SBA. A non B non T line

TABLE 3. INTERACTION OF NORMAL LYMPHOBLASTOID CELL LINES WITH F-SBA

Period of cultivation Line Origin (short/long)

F-SBA Fluorescence

Binding % intensity Redistribution patter

CBL-1C CBL-EBV Short* 8 - LBL-1 Healthy donor Short* 10 +- LBL-3 Healthy donor Short* 10 -+ LBL-K AML Short* 15 - LBL-SMal Hairy cell leukemia Short* 50 + + LBL-Oss ALL Short* 23 + + Im-Yos Infectious mononucleosis Long 10 + LBL-Sas All Long 8 +- LBL-Shev Hairy cell leukemia Long 20 + LBL-Ma ML Long 6 +

Small patches Small patches Small patches Small patches

Moderate patches Moderate patches

small patches Small patches Small patches Small patches

* Newly established lymphoblastoid lines, less than 10 passages in culture.

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Soybean agglutinin receptors on human hematopoietic lines

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~ . . . ~ . . . ~ - - - ~ K m-~ 8 o 4 6 8

o a r s A rER S U B C U L T U R E

~ R a - B 9 5 - e

FIG. 3. Cell number and F-SBA binding capacity of representative null (Km-3), T (Peer) and B (Ra-B95-8) lines. Cells were seeded at a concentration of 2 × 105 cells/ml and cell number (A) and the percent of cells that bind F-SBA (B) determined at 1-6 days after subculture. (D) Kin-3 (0) Peer

(4) Ra-B95-8.

593

(Km-3), a T line (Peer), and a B line (Ra-B95-8) were seeded at 2 × l0 s cells/ml and the reactivity with the lecting tested at 1-6 days after subculture. The results indicate that the interaction of the lines with F-SBA was not affected by cell replication. The degree of binding and fluorescence intensity of F-SBA of the null, B- and T-cell lines did not change significantly throughout the growth-curve experiment (Fig. 3),

D I S C U S S I O N

Our results on the interaction of SBA with human lymphoid cell lines show that different lines vary in the distribution of membrane receptors for SBA. The leukemia-lymphoma cell lines included in this study were chosen to reflect hematopoietic clones arrested at different stages of the differentiation-maturation pattern [1]. The results indicate that there is a correlation between the SBA binding and redistribution and the stage of lymphoid cell differentiation. The SBA labelling on the surface membrane of null lines was undetectable by U.V. microscope and flow cytometry. In the B and T lines there was a gradual increase in the SBA labelling correlating with the stage of differentiation. However, the maximal fluorescence intensity of the T lines was lower than that of the B lines. The B and T lines differed also in the redistribution pattern of the SBA receptors. The redistribution pattern of SBA- receptor complexes on the membrane of T lines was rings and small patches, whereas that on the B lines was moderate to large patches.

In a detailed study on lymphocyte cell surface glycoproteins which bind to soybean and peanut lectins the

leucocyte-common antigens (L-CAs) were suggested as the candidate for the interaction with soybean lecting [22]. It was also shown that the L-CAs of thymocyte and B and T lymphocytes constitute a set of large glycoproteins which share antigenic determinants but differ in their apparent molecular weights. It is proposed that the L-GAs consist of identical polypeptides which are glycosylated differently in different cell types, and that these changes in the expression of carbohydrates accompany cellular differentiation [22].

In a previous study using cationized ferritin (CF), an electron dense label of anionic sites, a similar correlation was observed between the stage of lymphoid differentiation and the redistribution pattern of this membrane probe [23]. Viable-unfixed null lines showed a low CF labelling density with few and small CF patches. The CF labelling density, the size and number of CF molecules increased gradually and correlated with the stage of differentiation on cell lines of both B and T origin [23]. It has also been shown that CF induced a time-dependent redistribution of surface anionic sites in the malignant cell lines but not in the normal ones [24].

Human normal and malignant lines of hematopoietic origin have been studied extensively by multiple marker analysis with techniques derived from different disci- plines to provide information for an exact classification which is necessary for prognosis and selection of proper treatment [1]. Using lectins as surface probes it was shown that Con A [25], W G A [26] and SBA [5] bind specifically to the surface membrane of normal and malignant cells, and that malignant cells can be agglutin- ated by these lectins. Previous studies with Con A have shown that human B-lymphoma lines exhibited a

594 H. BEN-BASSAT et al.

reduced redistribution of Con A receptors compared to T-lymphoid lines. Among the B-lymphoma lines the EBV-carrying lines showed a lower redistribution pattern and higher agglutinability than the EBV-negative lines [27].

It is noteworthy that the reactivity of the lines with SBA was not affected by growth conditions. The reactivity of the null B and T lines with SBA did not change throughout the growth curve experiments. In earlier experiments with Con A it was demonstrated that fibroblast transformed lines had to undergo at least one cell generation before they gained agglutinability [3, 25]. However the number of Con A molecules bound per cell was similar and not affected by days in culture or cell replication [3].

The interaction of SBA with the human lymphoblastoid lines is of interest, although its significance is presently not understood. The binding of SBA to the lymphoblastoid lines was generally low and the fluorescence intensity weak to moderate. The reactivity was not associated with the period of cultivation of the line or its origin. In previous experiments on the time- dependent CF-induced redistribution of surface receptors it was shown that lymphoblastoid lines of malignant origin differed in the labelling pattern of CF compared to lymphoblastoid lines of normal origin [24]. A some- what similar observation was made using yet another fluorescence probe (1,6-diphenyl-l ,3,5 hexatriane) embedded in the membrane lipid core of intact cells. It was shown that lymphoblastoid lines of malignant origin had a more fluid lipid layer in their surface membrane than lymphoblastoid lines of normal origin, but less than malignant lines [28].

In a recent study we have shown that the changes in the surface membrane of human lymphoma lines that result in decreased reactivity with SBA are associated with decreased tumorigenicity in v i vo [29]. It was demonstrated that SBA can be used for identification and selection of cell subpopulations within a human malignant cell line, by itself of clonal origin, which differ in their tumorigenicity. Previous studies with mammalian malignant lines have also demonstrated that selection of lectin-resistant mutants can alter the meta- static phenotype of tumor cells (detailed review [30]).

Thus among the lectins shown to date to be useful for cell separation and identification, the application of SBA, although limited, is of great importance since it can be used for fractionation of cells for bone marrow transplantation in humans across histocompatibility bar- riers, as well as for identification and selection of subpopulations within malignant cell lines which differ in their tumorigenicity in v ivo.

The present study may be of value in further understanding the differences in structure and function of the surface membrane between normal and malignant cells and the relation to normal and abnormal cell differentiation; particularly with the lymphoid cells.

Acknowledgements--The authors thank Dr H. Giloh and Dr Y. Oshri of the Interdepartmental Equipment Unit for excellent FACS analysis. This study was supported by a grant

from the Israel Cancer Association (H.B.B.) and the MJF Foundation.

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5. Sela B. A., Lis H., Sharon N. & Sachs L. (1970) Different locations of carbohydrate-containing sites in the surface membrane of normal and transformed mammalian cells. J. Membrane Biol. 3, 267.

6. Reisner Y., Kapor N., Kirkpatrick D., Pollack M. S., Cunningham-Rundes S., Dupont B., Hodes M. S., Good R. A. and O'Reilly R. J. (1983) Transplantation for severe combined immunodeficiency in HLA A, B, D, DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 61,341.

7. Sharon N. (1980) Cell surface receptors for lectins: markers of murine and human lymphocyte subpopulations. In Pro- gress in Immunology. IV (Fongereau M. & Dausset J., Eds), Vol. 3, p. 254. Academic Press, London and N.Y.

8. Ben-Bassat H., Rosenbaum-Mitrani S., Gamliel H., Naparstek E., Leizerowitz R., Korkesh A., Sagi M., Voss R., Cohn G. & Polliack A. (1980) Establishment in continuous culture of a T-lymphoid cell line (HD-Mar) from a patient with Hodgkin's lymphoma. Int. J. Cancer 25, 583.

9. Ben-Bassat H., Rabinowitz R., Polliack A., Hadar R. & Schlesinger M. (1985) Establishment of the Amsalem T cell line from a patient with acute lymphoblastic leukemia. Expression of E-receptor-associated antigens in cells incompatible of forming E-rosettes. Int. J. Cancer 35, 27.

10. Galili N., Galili U., Ravid Z., Schlesinger M. & Goldblum N. (1981) Induction of differentiation with phorbol ester in a human T-cell line (Be-13) expressing both pro- thymocyte and thymocyte characteristics. Hum. Lymph. Diff. 1, 123.

11. Ravid Z., Goldblum N., Zaizov R., Schlesinger M., Kertes T., Minowada J., Verbi W. & Greaves M. (1980) Estab- lishment and characterization of a new leukemic T-cell line (Peer) with an unusual phenotype. Int. J. Cancer 25, 705.

12. Pulvertaft F. J. V. (1965) A study of malignant tumors in Nigeria by short term tissue cell culture. J. clin. Path. 18, 261.

13. Klein G., Pesson C., Nadkarni J. L., Nadkarni J. J., Klein

Soybean agglutinin receptors

E., Henle G., Henle W. & Clifford P. (1968) Relation between Epstein-Barr viral and cell membrane immunofluorescence of Burkitt tumor cells--l. Dependence of cell membrane immunofluorescence on presence of EB virus. J. exp. Med. 128, 1011.

14. Klein G., Giovanella B., Westman A., Stehlin J. S. & Munford D. (1975) An EBV-genome-negative cell line established from an American Burkitt lymphoma, receptor characteristics, EBV infectibility and permanent coversion into EBV-positive sublines by in vitro infection. Inter- virology 5, 319.

15. Steinitz M. & Klein G. (1976) EBV induced changes in the saturation sensitivity and serum dependence of established EBV-negative lymphoma lines in vitro. Virology 70, 570.

16. Ben-Bassat H., Goidblum N., Mitrani S., Goldblum T., Yoffey J. M., Cohen M., Bentwich Z., Ramot B., Klein E. & Klein G. (1977) Establishment in continuous culture of a new type of lymphocyte from a "Burkitt-like" malignant lymphoma (Line DG-75). Int. J. Cancer 19, 27.

17. Menezes J., Leibold W., Klein G. & Clements G. (1975) Establishment of an EBV-negative lymphoblastoid B cell line (Bjab) from an exceptional EBV-genome negative African Burkitt's lymphoma. Biomedicine 22, 276.

18. Steinitz M. & Klein G. (1975) Comparison between growth characteristics of an EBV-genome negative lymphoma line and its EBV-converted subline in vitro. Proc. natn. Acad. Sci. U.S.A. 72, 8515.

19. Nilsson K., Bennich H., Johansson S. G. O. & Ponten J. (1970) Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgD) cell lines from an IgE myeloma patient. Clin. exp. Immun. 7, 477.

20. Burk K. H., Drewinko B., TrujiUo J. M. & Abeam M. J. (1978) Establishment of a plasma cell line in vitro. Cancer Res. 38, 2508.

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on human hematopoietic lines 595

Goldblum N. (1975) Concanavalin A receptors and other cell surface and antigenic characteristics of continuous lymphoblastoid cell lines derived from patients with Hodg- kin's Disease and other malignant lymphomas. Prog. med. Virol. 21, 177.

22. Brown W. R. A. & Williams A. F. (1982) Lymphocyte cell surface glycoproteins which bind to soybean and peanut lectins. Immunology 46, 713.

23. Marikovsky Y., Shlomai Z., Asher O., Lotan R. & Ben- Bassat H. (1986) Distribution and modulation of surface changes of cells from human leukemia-lymphoma lines at various stages of differentiation. Cancer 58, 2218.

24. Marikovsky Y., Ben-Bassat H., Leibovich S. J., Cividalli L., Fishier M. & Danon D. (1979) Surface charge characteristics of cells from malignant lines and normal lines of the hematopoietic system. J. natn. Cancer Inst. 62, 2857.

25. Inbar M., Ben-Bassat H. & Sachs L. (1972) Membrane changes associated with malignancy. Nature, New Biol. 236, 3.

26. Burger M. M. (1969) A difference in the architecture of the surface membrane of normal and virally transformed cells. Proc. natn. Acad. Sci. U.S.A. 62, 994.

27. Yefenof E., Klein G., Ben-Bassat H. & Lundin L. (1977) Differences in the Con-A-induced redistribution and agglutination patterns of EBV genome-free and EBV- carrying human lymphoma lines. Exp. Cell Res. 108, 185.

28. Inbar M. & Ben-Bassat H. (1976) Fluidity difference in the surface membrane lipid core of human lymphoblastoid and lymphoma cell lines. Int. J. Cancer 10, 293.

29. Ben-Bassat H., Weksler-Zangen S. & Shouval D. Dif- ferences in the tumorigenicity of human lymphoma (Daudi) subpopulations: Correlations with reactivity to soybean agglutinin, submitted.

30. Briles E. B. (1982) Lectin-resistant cell surface variants of eukaryotic cells. Int. Rev. Cytol. 75, 101.

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