Int. J. Cancer: 41,436-441 (1988) 0 1988 Alan R. Liss, Inc.

Publication of the International Union Against Cancer Publication de I’Union Internationale Contre 18 Cancer

QUALITATIVE AND QUANTITATIVE DIFFERENCES BETWEEN BUFXITT LYMPHOMA AND LYMPHOBLASTOID CELL LINES IN THE EXPRESSION OF MEMBRANE ECTO-NUCLEOTIDASES AND IN THE RESPONSE TO AGONISTS OF THE A2-TYPE ADENOSINE RECEPTOR Wolf GUTENSOHN and Heidi JAHN Institut Jiir Anthropologie und Humangenetik der Universitat Miinchen, Arbeitsgruppe Biochemische Humangenetik, Goethestrasse 31, D 8OOO Munich 2, FRG.

The activity of ecto-nucleotidases has been determined on intact cells from Burkitt lymphoma (BL) and lymphoblastoid cell lines established in vitro (LCLs). BL cells never express ecto-5‘-nucleotidase (5‘-N) and exhibit only low levels of ecto- ATPase activity, whereas LCL-cells usually express both en- zymes to varying degrees. There is a certain correlation (r = 0.75) between 5’-N and ecto-ATPase. When cAMP for- mation in response to agonists of the A2-type (stimulating) adenosine receptor is measured in the same cell lines there is no correlation with the expression of ecto-nucleotidases. Within pairs of BL and LCL cell lines derived from the same donor, an inverse relationship between ecto-nucleotidases and the response to the adenosine receptor agonist N-ethyl-car- boxamido-adenosine (NECA) is observed. BL cells show a good response to NECA, whereas this is low or absent in LCL cells. Treatment of cells which exhibit both 5’-N and the adenosine receptor with specific polyclonal or monoclonal antibodies against the enzyme does not impair the function of the recep- tor. Antisera against peptides of the membrane antigen BNLF I-MA, coded by the EBV-genome, do not co-precipitate 5’-N out of detergent extracts of LCL-cells. In both cases 5’-N cannot be closely associated with other membrane compo- nents. The differences between BL and LCL cells in ecto- nucleotidases and the adenosine receptor appear to be fairly stable phenotypical markers, independent of other parame- ters and suitable for use in distinguishing these cells in all populations.

Differences between Burkitt-lymphoma-derived cell lines (BL) and lymphoblastoid lines established by in vitro EBV infection (LCL) have been observed for over a decade (see Nilsson, 1979, for review of early results). Differences en- compass cell morphology, motility, growth characteristics, malignancy, karyotype and a number of surface markers and functional properties. However, a more detailed study of such differences at the molecular level has only recently become possible. This was achieved by using panels of specific mono- clonal antibodies (Rowe et al., 1985), by studying differential sensitivity to MHC-restricted T-cell cytotoxicity (Rooney et al., 1985; Torsteinsdottir et al., 1986) or by performing direct qualitative and quantitative analysis of surface components coded either by the viral (LYDMA or BNLF 1-MA) or the cellular genomes (MHC-products) (Modrow and Wolf, 1986; Modrow et al., 1987).

We now report differences in the expression of 2 membrane ecto-enzymes, ecto-5’-nucleotidase and ecto-ATPase, espe- cially in pairs of BL lines and LCLs established from the same donor. Marked differences in ecto-5 ’ -nucleotidase activity were used to test a hypothesis. Indirect evidence had suggested that ectod ’ -nucleotidase and adenosine receptors, although differ- ent molecules, could act as a functional unit within the cell membrane (Dornand et al., 1980; Bruns, 1980). The question was whether there is any correlation between the ecto-enzyme activity of the cells and their response in terms of cyclic AMP- formation to agonists stimulating Az-type adenosine receptors,

MATERIAL AND METHODS

Cells were grown in RPMI 1640 with 10% fetal calf serum, I u / d penicillin and 100 p g / d strepto- 2 mM glutamhe,

mycin, with passages every 2 to 3 days. Cells were usually harvested in the late logarithmic or stationary growth phase.

Ecto-5’-nucleotidase (5 ‘-N) was tested under isotonic con- ditions on intact cells or using a non-isotonic system for mem- brane extracts and a radiochemical assay (Gutensohn et al . , 1980). 5‘-N activity is defined as that part of total AMPase which may be inhibited by 200 p~ a,P-methylene-adenosine- diphosphate (AOPCP). A radiochemical test for ecto-ATPase on intact cells has also been described (Gutensohn and Rieger, 1986).

Incubation of cells and subsequent determination of cAMP followed the procedure of Hamprecht et al. (1985) with major modifications. After harvesting, the cells were resuspended in incubation medium A (DMEM/HEPES + glucose) at 37°C (Hamprecht et al., 1985). Cells (5 X lo6) in a total volume of 1 ml were incubated with the various effectors for 10 min with shaking. A final concentration of 0.5 mM of compound Ro 20- 1724 (kindly provided by Hoffmann-La Roche, Grenzach, FRG, was used throughout to block phosphodiesterase activ- ity. The reaction was stopped by adding trichloroacetic acid to a final concentration of 5 % , After centrifugation, cAMP from the supernatant was partially purified by chromatography on small cation-exchange columns (Clark and Seney, 1976), ly- ophilized and redissolved in water. The final determination of cAMP was again performed according to Hamprecht et al. (1985); however, cAMP not fixed to the binding protein was removed by adsorption to charcoal.

Polyclonal and monoclonal antisera against human 5 ’ -N had been obtained previously (Gutensohn et al., 1980; Kummer et al., 1984). Rabbit antisera against peptide 1-18 and peptide 252-263, respectively, of BNLF 1-MA were provided by S. Modrow (Modrow and Wolf, 1986). Immunoprecipitation was performed as described by these workers except that 1 % sul- fobetaine 14 was added to the lysis buffer and EDTA was omitted.

RESULTS

Measurements of surface enzyme activities on B-cell lines (Gutensohn et al . , 1980; Gutensohn and Rieger, 1986) have suggested a qualitative difference in the expression of 5’-N between Burkitt-lymphoma-derived (BL) and in vitro estab- lished cell lines (LCLs). Whereas the BL lines never express the enzyme, all other lines show various degrees of 5’-N activity. The search for systematic differences was extended to other cell lines and this finding was used to test the hypoth- esis of a possible connection with the adenosine receptor.

For this purpose we established a system for measuring CAMP-formation in cells. In preliminary experiments we had

Received: July 24, 1987 and in revised form September 19, 1987.

BIOCHEMICAL SURFACE MARKERS IN B-CELL LINES 437

found that maximal cAMP levels were reached after 10 min. All the data are thus based on 10-min incubations of cells in the presence of various effectors. In addition to adenosine (Ado) and its analogues (see below), we also tested the re- sponse of various cell lines to 5’-AMP. We had expected a consistent difference between 5’-N-positive and -negative cells. This was not the case, however. In fact, the 5I-N-negative BL- line Raji produced considerable amounts of cAMP in response to 5‘-AMP in the medium (Fig. 1). On the other hand, when 5 ’-N-positive lines were compared, in some lines CAMP-for- mation showed a better response to 5’-AMP than to adenosine and in others the opposite was true (data not shown). This may be explained by differential expression of non-specific ecto- phosphatases, which we had previously demonstrated in B-cell lines using paranitrophenylphosphate as a substrate (Guten- sohn et al., 1980). These are also capable of breaking down 5’-AMP to adenosine. In our standard assay, with complete repression of 5‘-N by AOPCP, various levels (depending on the particular cell line) of residual AMPase-activity were usu- ally observed. Moreover, in both 5’-N-positive and -negative lines the influence of S‘-AMP on CAMP-formation is clearly mediated by its extracellular breakdown to adenosine (Fig. 1) . Addition of adenosine deaminase (ADA) to the incubation

1 Raji (5-N

3

cAMP t ~MOI/IO~CGIISI

!

. 0 5 10

Effector [pMI

medium abolishes the effects of adenosine as well as of 5’- AMP. This is dependent on the concentration of the enzyme added.

In a first series of experiments a number of cell lines were studied independently. This is summarized in Table I. Con- cerning 5’-N our suggestion (Gutensohn and Rieger, 1986) was fully confirmed. Whereas the BL lines do not express 5’- N, all LCLs exhibit different levels of enzyme activity. Basal production of cAMP in the cell lines listed in Tables I and 11 ranged from 0-150 pMol/107 cells. All cells in Table I show a response to adenosine and usually (with some exceptions) a comparable response to prostaglandin El (PGE1). However, it can already be seen that the BL-line Raji produces the highest amounts of CAMP. On the other hand, isoproterenol had only a weak effect or none at all, indicating that these cells usually lack ,&receptors.

Pairs of BL-lines and LCLs derived from the same donor were also compared with one another (Table 11). Two cell lines, established by infecting cells of donor PR with virus from donors LOU and WEW, respectively, are also included. The non-metabolizable adenosine analogues 5 ’(N-ethyl) carboxamido-adenosine (NECA) and N6-(L-2-phenyliso-

300

cAMP lpMol/107cellsl

200

100

/ +ADA

FIGURE 1 - Influence of adenosine deaminase in the medium on cAMP formation in cells. (a) Cells of the BL line Raji were incubated with the indicated concentrations of adenosine (circles) or 5‘-AMP (triangles) in the absence (closed symbols) or presence (open symbols) of 2 U of adenosine deaminase from calf intestine. (b) ND cells were incubated with 5’-AMP and with increasing concentrations of adenosine deaminase. All other conditions as described in “Material and Methods”.

438 GUTENSOHN AND JAHN

TABLE I - ECTOENZYME ACTIVITIES AND RESPONSE TO VARIOUS EFFECTORS IN INDlVIDUAL CELL LINES

cAMP formation in response to Ado Isoproterenol PGE, Ecto-5’-N

(nMol/hr X lo6 cells) (0.3 W )

Cell line (10 W ) (200 PM)

0’ + + + 2 - Raji TK+ (BL) Jiyoye (BL) 0 ND3

6 .850 .9 + (+) - Staudte Regina 13.4+0.1 833 L 8.3k0.6 + ND 12.7k1.3 ++ (+) - JABA 6.1 +0.6 + (+) -

ND - IDU 78 9 . 4 5 1.3: ++ - + -

+ - VABA 3.5k0.5 + PR + Eli BLX 22.6 f 2.6 ND ND ND

~ _ _ _ _ _ _ _ ~

‘From Gutensohn and Rieger (1986).- ’+ = <200 pMol/107 cells increase over basal level; + + = 200-500 pMol/107 cells increase over basal level; + + + = >500 pMol/107 cells increase over basal level.- 3ND = Not determined.

TABLE I1 - ECTOENZYME ACTIVITIES AND RESPONSE TO A,-RECEPTOR AGONISTS IN PAIRS OF CELL LINES

&to-nucleotidases CAMP-formation in res nse to 5 PM effector Cell line Origin EBV (nMol/hr x lo6 cells) (pMol/lO’cehfZ

Ecto-5‘-N Ecto- ATPase NECA PIA

BL 64 E’ + 0 158558 677.6 547.2 516.0 k29.8 IARC 549 7.650.2 2,627k527 56.4k45.8 0 BL 67 E + 0 302k 121 96.4k4.0 7.4k14.4 IARC 309 10.2k1.6 1,555 +270 0 143.6 k67.6 WEW 1 BL E + 0 6605110 338.8523.0 87.2k55.6 WEW 1 LCL 17.0k2.7 ND 0 0 PR + WEWl BLX 35.5 k 1.2 1,626 5250 0 17.2k8.2 LOU BL NonE‘ + 0 440* 130 322.8k41.2 360.6 k 34 .O LOU LCL 41.3k5.0 3,118 5445 0 0 PR + LOU BLX 9.4+ 1.2 1,943 5498 167.6 k22.4 6.8514.4

38.8k 12.4 BL 2 NonE - 0 494k 159 65 .O 5 3 1.8 IARC 304 0 456k91 538.4k58.4 119.053 1.2 ‘E = endemic; NonE = non-endemic.-’Increase over basal level.

propy1)adenosine (PIA) were used to further characterize adenosine receptors. The “stimulating” A2-type adenosine re- ceptor is expected to be more sensitive to NECA than to PIA. Dose-response curves determined for NECA and PIA for all the cell lines listed in Table I1 establish this point. The dose response for one of the pairs of cell lines and for one line established by the virus of the same donor is illustrated in Figure 2 . The following conclusions can be drawn from the data in Table 11:

(1) Our previous observations on 5’ -N expression are con- firmed. All BL-lines are negative for 5’-N whereas LCLs show enzyme activity (with one exception, IARC 304).

(2) Expression of ecto-ATPase is not “all or none” as with 5’-N but quantitatively follows 5’-N activities. There is a certain correlation between the 2 enzyme activities (r = 0.75), although not quite as marked as in our previous study (Guten- sohn and Rieger, 1986).

(3 ) On comparison of cAMP formation responses to NECA and PIA, respectively, the adenosine receptor for most lines in Table I1 appears to be of the A2-type. There are indeed some exceptions, but this was not studied in detail. However, upon comparing the responses to NECA within pairs of cell lines, an inverse relationship to the expression of the ecto-nucleoti- dases is clearly seen. All BL lines show a positive response, although varying within a wide range. In contrast, in most LCLs the response to NECA is absent or very low. A remark-

able exception is, again, IARC 304 in which we find neither 5 ’ -N activity nor an increase in ecto-ATPase over the corre- sponding line BL 2, but a much higher response to NECA.

Our data lead to one main conclusion: In a number of cell lines both 5’-N and a stimulating (Az) adenosine receptor are expressed (see Table I). In the membrane of these cells, en- zyme and receptor may even be functionally related. But whatever the nature of this relationship may be, there is cer- tainly not a coordinate but rather an inverse regulation of the expression of enzyme and receptor as shown by the examples in Table 11.

Two additional sets of experiments were performed and both speak against a strong association of 5’-N with other mem- brane components on the surface of the cells. The first series showed that polyclonal or monoclonal antibodies against 5’- N, in concentrations sufficient to inhibit enzyme activity al- most completely, are unable to influence the response of intact cells to adenosine or 5’-AMP. For the 5’-N-positive line IDU 78 this is shown in Figure 3 . The fact that even the response to 5‘-AMP remains unaltered after blockade of 5’-N activity seems surprising at first sight. However, here again the effect may be completely blurred by the action of non-specific ecto- phosphatases. A similar experiment using the 5‘-N-negative line Raji had an identical outcome (data not shown). We conclude that the neighborhood of 5 ‘-N and adenosine receptor in the cell membrane cannot be close enough to allow an enzyme-bound antibody to block access to the receptor.

BIOCHEMICAL SURFACE MARKERS IN B-CELL LINES

,200 PRIWEW-I BLX (5’-N 35.5)

0 PIA

~TECA

439

’ 200 WEW-1 LCL (5‘-N 19.3)

f NECA U F l A b * re%

WOO WEW-1 EL (5’-N 0)

cAMP I~MOI/IO~C~IISI

0 5 10 Effector IpMI

FIGURE 2 - CAMP-formation in 3 cell lines (see Table 11) in response to NECA and PIA. Test conditions as described in “ M Methods”.

IDU 78 (5’-N 9.0)

CAMP Ipkkl/lO~CellSl

Pol y clo na I Monoclonal

rial and

No Serum Controlserum Antiserum NoSerum Controlserum Antiserum

FIGURE 3 - Influence of polyclonal or monoclonal anti-S’-N-sera on CAMP-formation in an LCL. Cells of IDU 78 were pre-incubated for 1 hr with the following additions: No serum, polyclonal control or anti-5‘-N serum, titer 1:8; monoclonal mouse control or anti-S’-N ascites, titer I: 10. Basal CAMP-production (Contr.) and response to 10 PM adenosine and 10 C(M 5’-AMP were measured by standard procedure described in “Material and Methods”.

BL lines and LCLs can also be distinguished by the differ- ential expression of membrane proteins (BNLF 1-MA) en- coded by the same reading frame of the EBV genome. As shown by Modrow and Wolf (1986), BL cells only synthesize a smaller polypeptide which lacks the first 138 aminoterminal residues of the full protein, which is produced by LCLs only. To test the hypothesis that expression of 5’-N in LCLs may somehow be linked to BNLF 1-MA, polyclonal antisera against membrane protein peptides 1-18 and 252-263 were used. With none of the 5 different antisera was it possible to co-precipitate 5’-N out of a detergent extract of the 5’-N-positive cells of line LOU-LCL. As a control within the same experiment, a

polyclonal anti-5’-N-serum completely removed the enzyme from the extract. It follows that there is no association of 5’-N with BNLF 1-MA strong enough to resist detergent extraction.

DISCUSSION

Details of our experimental set-up are considered first. We had to select a means of blocking phosphodiesterases in order to observe effects on cAMP formation in intact cells. Com- pound Ro 20-1724 is perhaps not optimal for this purpose. However, some other known phosphodiesterase inhibitors (for example, isobutyl-methyl-xanthine) act also as antagonists of

440 GUTENSOHN AND JAHN

the adenosine receptor and therefore were not suitable for our study. When CAMP-formation is taken as the sole parameter, in case of no response to a particular effector one cannot decide whether this is due to an absence of the receptor or rather to a lack of adenylate cyclase activity. However, our main question was to see whether the chain of ectonucleoti- dases and an adenylate-cyclase-coupled adenosine receptor would generally act together within the membrane of an indi- vidual cell to produce a specific intracellular signal. For that purpose it seemed sufficient to measure the strength of that signal (CAMP) formed in the intact cell under given condi- tions. To avoid side-effects caused by transport and intracel- lular metabolism of the effector, as would be the case for adenosine, the non-metabolizable adenosine analogues NECA and PIA were used for experiments summarized in Table 11.

Any straightforward interpretation of our observations en- counters several obstacles. First, ecto-nucleotidases or the adenosine receptor as molecules have clearly defined func- tions, i. e. , as enzymes or adenylate-cyclase-coupled mem- brane receptors, respectively; the biological significance of these molecules for a lymphoid cell that carries them is, however, unknown. We do not know in which particular en- vironment or at which particular stage in its life cycle the B- cell could profit from being able to break down extracellular purine-nucleotides or to respond to adenosine as a specific signal. Thus, the differences in ectoenzymes or in response to adenosine between BL and LCL cells reported here, can only be considered as another set of phenotypical markers.

Second, the stages of B-cell maturation which could be regarded as the normal phenotypic counterparts of BL and LCL cells have not been defined with certainty. This is best exemplified by the various interpretations given for BL cells. BL lines may be regarded as rather immature B cells, at least less mature than LCL cells. Some BL lines of sporadic origin show progression towards a somewhat more “lymphoblas- toid” phenotype with increasing number of passages in vitro (Rowe et al., 1985). However, one of these lines (LOU-BL) tested in our study at a later passage had not acquired 5‘-N activity. The BL cell might also represent a “suspended mem- ory cell’’, i .e. , a clonally expanded B-cell on the verge of becoming a long-lived B-memory cell, just prevented from returning to the GO stage of the cell cycle by the Iglmyc translocation (Ehlin-Henriksson et al., 1987). Uncertainties about normal cell counterparts may also explain some of the differences between our observations and those of Bonnafous et al. (1982), who found similar responses of adenylate cyclase to NECA in various mouse and human lymphocyte subpopu- lations, greatly differing in 5’-N activities. This stresses once more the independence of enzyme and receptor.

Another possibility for differences between cell lines would be that both 5’-N and the adenosine receptor might exist in inducible and non-inducible forms. This has not been de- scribed, however, in any other system, either for the enzyme or for the receptor. Moreover, we were unable to demonstrate any latent enzyme activity in 5I-N-negative cell lines (Guten- sohn et al., 1980) or any significant shifts in 5’-N activity during stimulation of normal peripheral lymphocytes (AndrCe et al., 1987). Even if 5’-N were inducible by the effectors used, we can assume that the cell phenotype must be stable during the short duration (10 min incubation) of our test. Biosynthesis of 5‘-N and transit to the plasma membrane has been estimated to take at least 20 min in a hepatocyte system

(van den Bosch et al., 1986). However, this would not exclude long-term effects on B-cell precursors in vivo.

In addition to these difficulties in interpretation, a number of experimental observations must be considered. Among blast cells in acute leukemias or in blast crises of CML, CALLA- positive cells are distinguished by high or even extremely high 5’-N activity, whereas other blast cells ( e . g . , in AML or T- ALL) lack the enzyme (Gutensohn and Thiel, 1981; Gutensohn et al., 1983). The reverse is observed with B-cell lines in this study: CALLA-negative LCLs express 5’-N and CALLA- positive BL lines do not.

As for phenotypical markers, independent observations dur- ing normal B-cell maturation have only been published about 5’-N (Bastian et al., 1984; Thompson et al., 1986). 5’-N activity in peripheral blood lymphocytes increases markedly during the first 6 months of life. This is mainly due to a remarkable rise in B-cell enzyme activity (about 5-fold), com- prising an increase in percentage of 5’-N-positive cells (from 32 to 69%) as well as in enzyme activity per positive cell. This increase precedes the ability of the B-cells to synthesize IgG in vitro. If we compare our data on ectoenzymes in B-cell lines with this maturation scheme in normal B-lymphocytes, we rather favor the concept that BL-cells devoid of 5’-N represent less mature forms.

The differences in ecto-nucleotidases and probably also in the adenosine response that we have observed between BL and LCL cells appear to be fairly stable and reliable phenotypical markers, independent of other parameters:

1. There is no dependence on the virus. This is evident from the pairs of lines shown in Table I1 where in some cases the LCL was established by donor EBV and in others by an independent strain of virus. The various transfectants of the EBV-negative lines Ramos and BJAB are indeed also negative for 5’-N (Gutensohn et al., 1980).

2. BL-cells lack ecto-5‘-N and in our hands respond to agonists of the adenosine receptor regardless of their origin. We have examined BL-lines derived from endemic and spo- radic cases and EBV-positive as well as -negative cells.

3. We believe that 5‘-N expressed on LCLs is a product coded by the cell genome. Although this has not been studied systematically, complete cross-reactivity of the lymphoblastoid enzyme with polyclonal or monoclonal antibodies against hu- man placental 5’-N was established, whenever we tested it. In addition, the failure of antisera against BNLF 1-MA to co- precipitate the enzyme is an indication that expression of 5’-N on LCL-cells is independent of virus-coded membrane proteins.

Finally, we stress that there is no coordination between ecto- nucleotidases and the adenosine receptor at the level of expres- sion. This speaks against a close functional relationship of these molecules within the membrane.

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsge- meinschaft. We also acknowledge the generous support of Prof. H. Wolf (Pettenkofer-Institut, Munich) and his collabo- rators Dr. S. Modrow and Dr. W. Jilg in providing cell lines and antisera, as well as the opportunity for valuable dis- cussions.

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