Int. J. Cancer: 23, 464-473 (1979)

CELL SURFACE GLYCOPROTEIN CHANGES IN EPSTEIN-BARR VIRUS-POSITIVE AND -NEGATIVE HUMAN HEMATOPOIETIC CELL LINES Wim P. VAN BEEK Kenneth NILSSON 8 , George KLEIN and Peter EMMELOT Division of Cell Biology, Antoni van Leeuwenhoek Huis, The Netherlands Cancer Institute, Amsterdam, The Netherlands; The Wallenberg Laboratory, University of Uppsala, Uppsala; and Institute of Tumor Biology, Karolinska Institute, Stockholm, Swederz

It has previously been shown that differential fucose labelling of many normal and homologous t u m o r cells, followed by proteolytic release and degradation, yields glycopeptides which upon gel filtration shown an increase i n fast-eluting glycopeptides for the t u m o r cells. This technique has now been applied t o cell-surface glycoproteins of different human hernatopoietic cell lines. These lines included Epstein-Barr virus (EBV)-carrying lymphoblastoid cell lines of presumed non-neoplastic origin, and malignant EBV-genome-positive Burkitt lymphoma and EBV-negative non-Burkitt lymphoma, leukemia and myeloma lines. A s compared with normal peripheral lymphocytes, both the lymphoblastoid type of cell lines and the different types of lines of proven malignant ancestry contained the fast-eluting glycopeptides on their cell surface with very few exceptions. It i s therefore concluded that ( I ) malignant conversion of human lymphoid cells in vivo i s commonly, but not obligatorily, associated with a specific change i n the composition of the fucosyl glycopeptides, and (2) EBV infection of B lymphocytes does n o t lead only t o the well-documented immortalization in v i m but also, as a rule, t o the same type of alteration i n fucosyl glycopeptides as was demonstrated for the neoplastic cell lines. It proved possible t o distinguish several categories of hematopoietic cell lines due t o the effect that pretreatment of the glycopeptides with neuraminidase o r mild acid exerted on their subsequent chromatographic behavior.

Transformation in vitro by Epstein-Barr virus (EBV) cannot be equated with the malignant transformation of B cells that occurs in vivo and leads to the formation of aBurkitt lymphoma (Klein, 1975; Nilsson and Ponten, 1975). EBV exposure of B cells in vitro leads to the acquisition of one '' malignant. " feature-a potential for infinite growth in vitro-but not to the development of the characteristic chromoson~alabnormalities found in Burkitt lymphoma biopsy cells and derived cell lines (BLs) (Zech et al., 1976), nor to morphological and surface marker characteristics as found for BL cells (Nilsson and Ponten, 1975; Nilsson, 1978). The glycoprotein composition of the cell surface of lymphoblastoid cells (LB), labelled by the galactose oxidase-tritiated borohydride method and analyzed by polyacrylamide slab gel electrophoresis appeared to resemble that of normal B lyrnphoblasts (Nilsson e f a / . , 1977n). Recently, two of us with other co-workers (Nilsson et al., 19776) showed " new " cultures of LBs to be non-malignant according to the criteria of anchorageindependent growth and tumor-forming capacity

in nude mice, while " old " sxondary changed LBs app:ared to be tumorigenic in many cases studied. In the same paper, however, the validity of the methods used for discrimination between tumorigenic and normal hematopnietic cell lines were questioned. In contradiction, experiments of others based on hetcroor auto-transplaniation expyriments suggested the 1.B cells to be malignant (Christofines. 1969; A d a m et al.. 1970, 1973; lmamura et a/., 1970). This discrepancy, however, can be ascribed to the differences between " new " and " old " LB lines (Nilsson et al., 19776). For this reason we decided to investigate " old " and " new " LB lines for a specific alteration in cell-surface glycopeptides, frequently associated with malignant growth (for a review see Warren et al., 1978). The specific alteration rzsides in sialic-acidcontaining fucosyl glycopeptides from neoplastic cells which elute partly ahead of the normal control material in gel filtration columns, indicating a shift in size distribution towards higher molecular weights. I n most cases, neuraminidase (NANasz) digestion of the glycopeptides prior to chromatography causes the differences to disappear. Apparently, the sialic acid concentration of these malignant-cell-derived glycopeptides is increased. This glycopeptide alteration has been described for a great variety of transformed and tumorigenic cells in vifro (Bucket al.. 1970; Van Beek et al., 1973; Click, 1974a; Warren et al., 1974; Grimes et al., 1977) and in tumors grown in experimental animals (Click et ai., 1974b; Smets et al., 1975, 1977; Warren et a/,, 1975; Van Beek et al., 1977~).Similar changes have been observed for various cases of " early " and " latz '' stage human leukemia and lymphoma and in human glioma and osteosarcoina (Van Beek et al., 1975, 19776; Emmelot et al., 1977; Van Beek ef al., 1978a, b). Incidental negative observations in transformed fibroblasts (Ceccarini et al., 1975) can be explained as cases of transformed non-tumorigenic cells (Smets et al., 1976), hence emphasizing the common association of altered surface glycopeptides with the malignant condition. Control experiments revealed that this glycoprotein change is not related to growthperse (Van Beek etal., 1975, 19776), late embryonic expression (Van Beek et al., 1977a) or hyperplastic mode of growth (Smets et a/., 1977). Received: November 1, 1978.

GLYCOPROT'EIN OF EBV-GENOME-CARRYING

465

CELLS

TABLE I ANALYSIS O F CELL-SURFACE GLYCOPEPTIDES O F AFRICAN BURKITT LYMPHOMA LINES COMPARED WITH GROWTH CAPACITY OF THESE CELLS I N SEMI-SOLID AGAROSE AND NUDE MICE. Glycopeptide analysis Cell line

Reference

U-6262, new line U-47703, new line BJAB, new line GC-BJAB, new line BJABIB95-8, new line BJABlHRlK, new line Ramos, new line RamoslB95-8, new line Ramos/HRlK, new line U-984, new line U-52002, new line P,HRlK, old line Raji, old line Jijoye, old line Rael, old line Daudi, old line

Nilsson and Ponten (1975) Nilsson and PontCn (1975) Klein et al. (1974) Clements et al. (1 975) Fresen et al (I 976) Fresen et a1 (I 976) Klein et al. (1975) Klein et al. (1975) Fresen et a / . (1976) Nilsson and PontCn (1975) Nilsson and Ponten (1975) Hinuma and Grace (1967) Pulvertaft ( I 965) Klein et al. (1972) Klein et al. (1972) Klein et al. (1968)

EBV genome

Not treated

+ + + + + + + + + + + + + + + +

+ I

+ + + + + + + + + + + + + -

-

NANase treated

Acid treated

Growth capacity according to Nilsson e l a/. (19776) in: Nude mice Agarose

t

-.

L

__

. . .

-t

+ + + +

+ + + + + +

+ + -t +

-+-

1 ( + ) = elution profile enriched in more rapidly eluting fucose labelled glycopeptides compared ( - ) = elution profile coinciding with the control. Abbreviation used: NANase = neurarninidase.

t

-t

with normal lymphocyte control material:

neuraminidase o r mild acid treatment (Van Beek et a / . , 1977a) was performed in order to possibly

In this study we have investigated 16 Burkitt lymphoma lines consisting of two EBV genomenegative lines and 14 EBV genome-carrying lymphoblastic cell lines. In addition, established cell lines of six human leukemias, two myelomas and five non-Burkitt lymphomas were included ; these lines were all EBV-genome-negative. Degradation of the glycopeptides prior t o chromatography using

differeniiate the various categories of cell lines. The present results indicate that both old and new LB cells have a n increase in early eluting glycopeptides indistinguishable from those of Burkitt lymphoma cells. In most cases, neuraminidase digestion and mild acid treatment of glycopeptides

TABLE I I ANALYSIS O F CELL-SURFACE GLYCOPEPTIDES O F LYMPHOBLASTOID CELL LINES WITH GROWTH CAPACITY O F THESE CELLS I N SEMI-SOLID AGAROSE AND N U D E MICE Glycopeptide analysis Cell line

Reference

U-43909, new line U-974, new line U-718, new line U-704, new line Tg,new line HI, new line TSl1, new line TS-26, new line U-1163, new line U-296, old line U-61, old line U-255, old line Maku, old line PGLC-56B, old line PGLC-33H, old line U-1188, new line

Bechet et a / . (1974) Nilsson, unpubl. Glimelius et al. (1975) Glimelius et a / . (1975) Klein, unpubl. Klein, unpubl. Splinter, unpubl. Splinter, unpubl. Nilsson, unpubl. Nilsson (1971~) Ponten (1967) Nilsson (1971a, 6 ) Yata and Kelin (1969) Glade et al. (1968) Glade et al. (1968) Nilsson, unpubl.

~~~

EBV genome

Not treated

NANasetreated

+ +

t

-t

+ +'

-t-

Acidtreated

Growth capacity according to Nilsson ' a in: Nude mice Agarose

~~~

See Table 1. - See text.

+ ++ +

-t -t t

-t

++ +-

t -t

++

+ ++ ++

-t

+ + + +

t-

+

-

-

-

-

-t -t

+ +

-t

-

+-

+-

+

__

.t

-

+

466

VAN BEEK ET AL.

before analysis made it possible to distinguish Burkitt lymphoma cells from non-Burkitt lymphonia or leukemia cells. In addition, EBV-genome-positive Burkitt lymphoma cells could be distinguished from EBV-negative Burkitt lymphoma cells.

8[

B.D.

A

H

MATERIAL A N D METHODS

Cell and lahrlling procedures Hematopoietic cell lines were investigated including EBV-genome-carrying African Burkitt lymphoma and lyniphoblastoid cell lines in early passages (according t o the classification proposed by Nilsson and Ponten, 1975). The LB lines were all polyclonal (Bechet ef al., 1974), normal diploid lines (Zech e t a / . , 1976) and displayed functional, morphological and other phenotypic characteristics clearly distinguishing them from the BL lines (Nilsson and Pontkn. 1975;

'1

A

B.D.

H

B

B.D.

C 6 4

fraction number FIGURES 1-9 - (a)Pronase-digested glycopeptides; (b) following neuraminidase treatment of n ; (c) following mild acid (HCI) hydrolysis of a . BD = blue dextran2,000. FIGURE I - Elution profiles of surface glycopeptides from BJAB Burkitt lymphoma cells and control peripheral blood lymphocytes. The BJAB cells were labelled with 3H-fucose ( 0 ) and the control lymphocytes with 14Cfucose (0).

2 020

30

40

50

60

70

fraction number FIGURE 2 - Elution profiles of surface glycopeptides from BJAB/B95-8 Burkitt lymphoma cells ( 0 ) and control peripheral blood lymphocytes (0).

GLYCOPROTEIN OF EBV-GENOME-CARRYING CELLS

467

Van Oers et al. (1977) and the percentage of B cells estimated according to Zeijlemaker et al., (1976). 'Ol

Gel filtration analysis The procedures for the isolation of fucose-labelled cell-surface glycopeptides and of chromatographic analysis were essentially identical to those used previously (Van Beek et a/., 1973, 1975). Briefly, glycoproteins from normal lymphocytes labelled with 14[C]fucose and those from cells to be investigated, labelled with [3H]fucose, were released with

a

A

6

fraction number FIGURE 3 - Elution profiles of surface glycopeptides from Daudi Burkitt lymphoma cells ( 0 ) and control peripheral blood lymphocytes (0).

4 2

Nilsson ei al., 1974, 1977a: Glimelius ei al., 1975; Fragaeus et a/., 1975; Huber et a/., 1976). The BL lines are monoclonal (Fialkow et al., 1970; BBchet et a/., 1974) aneuploid (Zech et a/., 1976) and are representative for the neoplastic prototype of the explanted uniclonal tumor. Long-term cultivation of LBs ( > I year continuous cultivation) will result in secondary chromosomal alterations (Zech et nl., 1976) and changes of other phenotypic characteristics (Nilsson et nl., 1974; Nilsson and Ponten., 1975; Glimelius et a / . , 1975; Nilsson, 1978). For this reason both recently established ( t 6 month continuous cultivation) and old lines were included in the present study. The aneuploid BL lines similarly undergo chromosomal changes during prolonged culture (Zech, personal communication). For this reason, both old and new BL lines were investigated. A subdivision of the group of EBV-genome-negative lines (leukemia, lymphoma and myeloma) into new and old lines was not possible since new lines were not available. The origin of the cell lines and selected references are presented in Tables 1-111. All lines were grown as suspension cultures in RPMI 1640 medium supplemented with 10% fefal calf serum and antibiotics (penicillin 100 IU/ml, streptomycin 50 pg/ml). The cells were metabolically labelled by incubation for 24 h at 37" C in the presence of L-[3H]fucose (1-5 pCi/nil; generally labelled; 4.8 Ci/mmole; New England Nuclear, Boston, Mass.) Control lymphocytes were obtained from the venous blood of healthy donors by centrifugation of diluted samples on a Ficoll-Hypaque mixture (Boyum, 1968). Cells from the interphase were washed and suspended in growth medium in the presence of L-( 1 -14C)-fucose (60 mCi/mmole; The Radiochemical Centre, Amershanl, Bucks., England). T-cell-depleted lYn1phocytes concentrates were prepared according to

0 8 6

5

*-

30

'

#

2

0 8 6 4 2

0

50 60 70 fraction number FIGUR4 - Elution profiles of surface glycopeptides from U-43909 lymphoblastoid cells ( 0 ) and control peripheral blood lymphocytes (0).

20

30

40

VAN BEEK ET AL.

treatment before analysis. Both procedures aim at releasing sialic acid from the glycopeptides, as described earlier (Van Beek et al., 1977~).

B.D.

RESULTS

H

A,fricarr Burkitt lymphoma lines A

fraction number FIGURE 5 - Elution profiles of surface glycopeptides from U-296 lymphoblastoid cells ( 0 ) and control peripheral blood lymphocytes (0).

trypsin from the surface of intact cells. Cell-free [ 14C] and [3H]fucose-containing glycoproteins were combined and further digested by Pronase. The glycopeptide mixture was analysed by gel filtration. The [3H] and [14C]radioactivity of chromatographic fractions was differentially counted in a liquid scintillation counter and the elution profiles were constructed by plotting [3H] and [14C]radioactivities as percentages of total radioactivity eluting from the column. GlycopYptide degrudation procedures Glycopeptide mixtures of the control cells and the cells under investigation were subjected to digestion by neuraminidase (NANase) nad to mild acid

As summarized in Table I, all 16 BL lines studied, as well as six other BL lines not included in this report, contained the fast-eluting glycopeptides as compared with the peripheral blood lymphocytes used as controls (as illustrated for one of these lines in Fig. I n , fraction 31-39). No specific differences in the elution profiles were observed between EBVnegative (Fig. la) and EBV-positive (Fig. 2a) RL lines, except for the quantity of the fast-eluting material. Old BLs produced almost identical elution profiles (Fig. 3) as compared to the new BL lines (Fig. la). The glycopeptides of all BL lines invesligated were found to be relatively insensitive towards neuraminidase digestion as compared with the normal control glycopeptides (Figs. Ib, 26). Moreover, these glycopeptides were also insensitive towards mild acid treatment (Fig. 2c), excepl for the two EBV-negative BL lines studied (as illustrated in Fig. I r ) and only one out of 10 EBVpositive BL lines, which also showed a coinciding profile with the control after mild acid treatment (Table I, Raji). Lytnphoblastoid cell lines

The results of analysis of the glycopeptides from 15 LB lines, including three infectious mononucleosis-derived lines (PGLC-33H, PGLC-56B and U-l188), compared with those of normal peripheral blood lymphocytes, are summarized in Table 11. All lines investigated contained the fast-eluting glycopeptides (as illustrated in Fig 4a), exczpt for one LB derived from a patient with infectious mononucleosis (Tdhle 11, PGLC-56B line), whereas in one other line (TSII) a minor but reproducible

A

B

-.I

20

30

40

50

60

80

fraction numher

20

30

40

50

60

70

fraction number FIGURE 6 -Elution profiles of surface glycopeptides from (a) T-cell-depleted B cells stimulated to grow by pokeweed mitogen ( 0 )and resting T-cell-depleted B cells (0): (b) T lymphocytes (70%) (0)and B lymphocytes (56%) (0).

469

GLYCOPROTEIN OF EBV-GENOME-CARRYING CELLS TABLE I11 ANALYSIS OF CELL-SURFACE GLYCOPEPTIDES O F LYMPHOMA AND LEUKEMIA CELL LINES COMPARED WITH GROWTH CAPACITY O F THESE CELLS IN SEMI-SOLID AGAROSE AND NUDE MICE Glycopeptide analysis Cell line and type

U-937, HL u-1133, LL U-698M, LL U-715M, LL DG-75, LL JM, ALL Molt 4,ALL Molt 4 Kaly, ALL CCRF-CEM, ALL HSB-2, ALL K562, CML RPMI8226, myeloma U266B1, myeloma CML

Reference

EBV genome

Not treated

Sundstrom and Nilsson (1976) Fresh biopsy Nilsson and Sundstrom (1974) Nilsson and Sundstrom (1974) Ben-Bassat et at. (1977) Schwenk and Schneider (1975) Minowada et nl. (1972) Klein, unpubl. Foley el at. (1965) Adams et at. (1968) Lozzio and Lozzio (1975) Matsuoka et nl. (1967) Nilsson et al. (1970)

NANasetreated

Acidtreated

Growth capacity according to Nilsson a'' (1977b) in: Nude mice Agarose

+ +

+ +

+

+

+

+

+

+ + + +

-

.-

-

+ +

See Table I - * Abbreviations used: HL = histiocytic lymphoma; LL = lymphocytic lymphoma; ALL =acute lymphoblastic leukemia; == chronic myeloid leukemia; BL African Burkitt lymphoma; LB :- lymphoblastoid cell line. - See text. ~

A 8[

B.D.

H

'r

A

B.D.

6

4

fraction number

fraction number

FIGURE 7 - Elution profiles of surface glycopeptides from U-937 histiocytic lymphoma cells ( 0 ) and control peripheral blood lymphocytes (0).

FIGURE 8 - Elution profiles of surface glycopeptides from JM acute lymphoblastic leukemia cells ( 0 ) and control peripheral blood lymphocytes (0).

470

VAN BEEK ET AL.

Only the DG 75 line contained minor but detectable amounts of these fast-eluting glycopeptides. Neuraminidase treatment of glycopeptides from cells of three lines studied rendered the profiles coinciding with those of control lymphocytes (Fig. 76), a property which distinguishes non-Brukitt lymphoma glycopeptides from those of BLs and LBs.

B.D.

U

Leukemia and myeloma lines The results on the fast-eluting glycopeptides of six leukemia and two myeloma lines are summarized in Table 111. All leukemia lines except the CCRFCEM contained the fast-eluting material (as illustrated in Fig. 8a). Both myeloma lines did not contain the fast-eluting material as illustrated in Figure 9. Neuraminidase treatment of the glycopeptides before analysis made the profiles coincident (as illustrated in Fig, 86) except for the K562, a CML line, which was barely susczptible to both neuraminidase and mild acid treatment (cf. Van Beek et al., 1975, 1978a).

4 1

30

40

50

60

70

fraction number

FIGURE 9 - Elution profiles of surface glycopeptides from U-266 myeloma cells ( 0 ) and control peripheral blood lymphocytes (0).

amount was observed. The glycopeptide elution profiles of the LBs-whether old or new (compare Fig. 5 and 4 a j w e r e strikingly similar to those of the BL lines (compare Fig. 3 and 4a). In addition, the glycopeptides of the different LBs appeared to be relatively insensitive towards both neuraminidase digestion and mild acid treatment as was also the case with those of the EBV-containing BL lines (Table 11). Recent studies of Nilsson et al. (1977a) on the glycoproteins of the surface of LB cells, labelled by means of the galactose oxidase-tritium borohydride method and analysed by polyacrylaniide slab gel electrophoresis, revealed the resemblance of the LB glycoproteins with those of normal B lyniphocytes, stimulated to grow by pokeweed mitogen (PWM). For this reason, we investigated PWMstimulated B lymphocytes as an additional control fol LB cells. We stimulated a T-cell-depleted lymphocyte concentrate (40-72 % B lymphocytes) with PWM for 7 days. May-Grunwald-Giemsa stained smears revealed a majority of lymphoblasts, while radioautography with r3H]thymidine revealed 30-41 % thymidine-incorporating cells. As illustrated in Figure 6u, PWM-stimulated B lymphoblasts did not contain fast-eluting glycopeptides on their surface at all. Figure 66 shows the coinciding patterns of predominantly B-cell- (56%) and T-cell- (70%) derived glycopeptides. Nun-Burkitt lymphoma lines The glycopeptides of all five non-Burkitt lymphoma lines studied as summarized in Table 111 (first five entries), contained the fast-eluting glycopeptides as compared with those from peripheral control blood lymphocytes; one case is illustrated in Figure 7a.

TABLE I V

RELATIONSHIP BETWEEN THREE DIFFERENT ASSAYS FOR DETECTING TUMORIGENICITY, i.e. THE GLYCOPEPTIDE ANALYSIS CRITERION, GROWTH CAPACITY I N NUDE MICE AND COLONY FORMATION I N SEMI-SOLID AGAROSE Growth capacity Glycopeptide according to Nilsson P I ol. (19776) in:

Cell type

BL, new lines BL, old lines LB, new lines LB, old lines LL, HL, old lines ALL, CML, old lines

Myeloma, old lines

17/17 515 919 516 515 516 012

For abbreviations used see Table 111.

Nude mice

Agarose

416 414 013 214 214 214 012

215 212 013 213 I 14 3J4 212

See Table 1.

DISCUSSION

Fucose-containing glycopeptides of all malignant cells studied in this respect up to now specifically differ from those of their nonmalignant homologues in that part of the malignant-cell-derived glycopeptides elute in front of the control glycopeptides in gel filtration columns. This phenomenon has been observed in a great variety of cell systems both in vitro and in vivo, including also human derived cell lines (see opening paragraphs). This criterion for malignancy based on gl ycopeptide analysis has already proved its validity in correctly detecting or excluding malignancy in cases of human hematopoietic disorders where a clinical diagnosis could not yet be established (Van Beek et al., 1975, 1977, 1978~).The biological meaning of this phenomenon

GLYCOPROTEIN OF EBV-GENOME-CARRYING

in malignant growth is rather poorly understood. However, the consistent finding of this class of glycopeptides in tumorigenic cells is impressive and deserves attention. Glycopeptide changes in tumor-derived cell lines As summarized in Table IV, the correlation between the occurrence of the fast-eluting material and the neoplastic state in hematopoietic cell lines (BL, LL, HL, ALL, CML and myeloma lines) is high as compared with two other criteria for “ malignancy”, viz tumor formation in nude mice and colony-forming ability in semi-solid agarose (Nilsson et al., 19776). From the last two criteria, only tumor formation in nude mice agreed well with the presence of fast-eluting glycopeptides. The cell lines scored negative on account of the glycopeptide profile appeared to coincide with the negative growth capacity of these lines in nude mice (see Table 111). The finding of three cell lines (CCRFCEM, RPMI-8226 and U-266) of proven malignant origin without glycopeptide changes, but also nontumorigenic after inoculation in nude mice, may suggest that neoplastic transformation could occur in human hematopoietic cells without the elaboration of fast-eluting glycopeptides. Alternatively, the chromosomal alterations after prolonged culture, as demonstrated in these lines (Huang et al., 1969; Nilsson, 19716), may have been followed by changes in the surface glycopeptides towards the composition typical for non-neoplastic cells. Also, the results on fresh biopsies from patients with leukemic disorders are suggestive of an association between the presence of fast-eluting glycopeptides and hematopoietic neoplasia. Up to now, 49 out of 51 leukemias have been shown to contain fast-eluting glycopeptides on their cell surface, the exceptions being a case of aleukemic leukemia and a case of acute myelomonocytic leukemia that went into spontaneous remission (Van Beek et al., 1975, 1977, 1978a, and unpublished observations). All BL lines were recognized as malignant by the glycopeptide analysis criterion. EBV-genomenegative and -positive BL lines produced nearly identical profiles, including the faster-eluting glycopeptides. However, comparison of the quantity of fast-eluting peak material of the EBV-genomepositive BJABjB95-8 line (Fig. 2a) with that of the corresponding EBV-genome-negative parent BJAB line (Fig. la) suggests an additional increase in the fast-eluting peak material after EBV infection. This increase appears not to be a general one, because it was not established in the other BJAB- or Ramosderived lines. The neuraminidase treatment allowed non-Burkitt lymphoma and leukemia lines (except CML lines, being neuraminidase and acid-resistant) to be distinguished from BLs and LBs. Besides the absence of detectable amounts of EBV-determined nuclear antigen (EBNA), the BJAB and Ramos Burkitt lymphoma lines exhibited specific differences in their glycopeptide structure as compared with those of the EBNA-positive Burkitt lymphoma lines. The glycopeptides from these two lines were susceptible to mild acid treatment, in contrast to those from EBNA-positive BLs.

CELLS

47 1

Glycopeptide changes in lymphoblastoid cell lines EBV infection of B lymphocytes results in “ immortalized” cell lines (LBs) and nearly all LBs investigated produced the faster-eluting glycopeptides as compared with normal control lymphocytes. No difference was observed in this respect between “ o l d ” and “ n e w ” and spontaneously in vitro established (U-43909, U-718, U-296, U-61 and U-255) LB lines and LB lines established by means of EBV infection in vitro (Table 11). Besides the occurrence of fast-eluting material on the LB cell surfaces, the size distribution of the glycopeptides on gel filtration was very similar to that of BLs. This similarity between BLs and LBs is further emphasized by the finding of an almost equal partial resistance of their glycopeptides towards neuraminidase digestion and mild acid treatment. Both methods were used earlier to release sialic acid from glycopeptides obtained from rat liver hepatoma cells (Van Beek et al., 1977~).The finding that the size distribution of the glycopeptides of LBs resembles that of BLs rather than of PWM-stimulated B lymphocytes, contrasts to the demonstration of different surface glycoprotein compositions in LB and BL cells by Nilsson et af. (1977~)using surface labelling by the galactose oxidase tritiated sodium borohydride method. The latter method, however, may have the disadvantage of losing some information owing to the necessity of using neuraminidase in the labelling procedure. Moreover, since the faster elution property is determined by the carbohydrate units of the glycoprotein molecule (Buck et al., 1970), examination of whole surface molecules, including the protein part, as performed by Nilsson et al. (19776), may possibly mask the difference under discussion. The elution profile of the glycopeptides of PWM-stimulated B lymphocytes is in agreement with an earlier result on stimulated T lymphocytes (Van Beek et al., 1975). Apparently, growth as such is not responsible for the appearance of the fast-eluting glycopeptides (Van Beek et al., 1975, 19776). It is also unlikely that this group of glycopeptides i s specifically associated with the presence of the EBV genome since EBV-positive as well as EBV-negative BL lines revealed the presence of the fast-eluting glycopeptides on their surface. The occurrence of fast-eluting glycopeptides on the cell surface not only in the neoplastic hematopoietic lines but also in the LB type of cell line is of considerable interest. It is quite possible that the target B lymphocyte, upon EBV infection in vitro, and possibly also in vivo, acquires some phenotypic features commonly expressed by malignant lymphoid cells. It is already established that EBV virus infection leads to the acquisition of the capacity for infinite growth in vitro, a feature commonly encountered by neoplastic human cells. The appearance of fast-eluting glycopeptides may thus be yet another change in these LB cells which they have in common with malignant cells, brought about by the presence of the EBV genome. A third indication that the LB type of cell may represent a partially “ transformed ” B cell is the recent finding by Giovanella et al. (1979) that the LB cells, although not growing subcuta-

472

VAN BEEK ET AL.

neously in adult nude mice, can grow when inoculated intracerebrally. The present findings seem to support the idea that the development of Burkitt’s lymphoma is the result of a step-wise malignant transformation in a n EBV-infected population of B cells where phenotypic changes, i n d u e d by thz presence of EBV g a o n i e in the cells, and subsequent chromosomal alterations, interact and lead to the formation of the eventually autonomously growing BL tumor (Nilsson et al., 19776); Kelin and Klein, 1977).

ACKNOWLEDGEMENTS

The technical assistance of Mrs. A. Westman, Mrs. A. Snellman, Mr. J. Breekveldt and Miss E. de Bakker is gratefully acknowledged. We thank Dr. T. Splinter, Dr. H. Loos and Miss A, van Bzek (Central Laboratory of the Netherlands Red Cross Blood Transfusion Servicz, Amsterdam) for providing us with various cAl lines and leukocyte concentrates. The study was supported by grants from the Swedish Cancer Society.

MODIFICATIONS DES GLYCOPROTEINES DE SURFACE DANS DES LIGNEES DE CELLULES HEMATOPO~ETIQUESH U M A I N E S EBV-POSITIVES ET EBV-NEGATIVES

II a deja Ctt demontre que, par marquage differentiel au fucose de nombreuses cellules normales et cellules tumorales homologues, suivi de largage et de degradation proteolytiques, on obtient des glycopeptides qui, en filtration sur gel, presentent une caracteristique interessante, en ce sens que I’on observe, pour les cellules tumorales, une augmentation des glycopeptides eluant rapidement. Cette technique a ete appliquee aux glycoproteines de la surface de differentes lignees d s cellules hematopoietiques humaines. II s’agissait d e lignees lymphoblasto’ides porteuses de virus d’Epstein-Barr (EBV), d’origine apparemment non neoplasique, de lignees de lymphonie de Burkitt mslin portant le genome EBV, d’autres types de lymphomes EBV-nkgatifs, de Ieucemie et de myelome. En comparaison des lymphocytes peripheriques normaux, les lignees lymphoblastoides et les differents types de lignees d’origine mslignc c-rtaine portaient, a de rares exceptions pres, les glycopeptides t4 elution rapide sur la surface des cellules. I I en est conclu l ) que la canversion mdigne des cellules lympholdes humaines in vivo est g&nh,ralement,mais pas obligatoirement, associke a une modification spkifique de la composition des glycopeptides, et 2) que l’infection par I’EBV des lymphocytes B n’entraine pas seulement une immortalisation in vitro, phenomene deja bien connu, inais aussi, en rligle genkrale, le m6me type d’alteration des glycopeptides que pour les lignkes nkoplasiques. l l a C t e pxsible de distinguer plusieurs categories d e lignex hemitopo’ietiques d’apres l’effet que le pretraitement des glycopeptides A la neuraminidase ou avec un acide leger exerqait sur leur comportement chromatographique subsequent.

REFERENCES

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