Int. J. Cancer: 51,949-955 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer Publication de I‘Union Internationale Contre le Cancel
EXPRESSION OF THE EPSTEIN-BARR VIRUS (EBV)-ENCODED MEMBRANE PROTEIN LMPl IMPAIRS THE IN VITRO GROWTH, CLONABILITY AND TUMORIGENICITY OF AN EBV-NEGATIVE BURKITT LYMPHOMA LINE Laura cUOM01.3, Torbjorn RAMQUIST’, Pankaj TRIVEDI’,Fred WmG2, George KLEIN’and Maria G. MASUCC11,4 ‘Department of Tumor Biology, Karolinska Institute, Box 60 400, 104 01 Stockholm, Sweden; and 2Departmentsof Medicine, Microbiology and Molecular Genetics, Harvard Medical School, 75 Francis Street, Boston, M A 02115, USA. In a previous study on several independently established Epstein-Barr virus (EBV)-converted sublines of the EBVnegative Burkitt lymphoma (BL) line BL41, we found that expression of the virally encoded membrane protein LMPl was accompanied by reduced agarose clonability and tumorigenicity. In order to investigate whether LMPl can induce these phenotypic changes by itself, we have now studied the growth in suspension culture, the clonability in agarose and the tumorigenicity in immunosuppressed and SClD mice of 4 LMPItransfected sublines of BL41 that carry the gene under the control of the ZnS04-inducible metallothionein promoter. Expression of LMPl at levels comparable to those detected in EBV-transformed lymphoblastoid cell lines (LCL) correlated with impairment of growth in suspension and reduction of clonability and tumorigenicity. Only minor changes were obsewed in transfectants expressing low LMPl levels. Upregulation of LMP I by ZnS04 treatment of the low LMPl clone MTLMS was accompanied by a slowing down of proliferation, increased cell clumping and decreased clonability. The results suggest that expression of LMPl at levels which are compatible with immortalizationof normal B-cells antagonizes the ability of BL cells to grow in vitro and in vivo, and illustrate a possible mechanism by which down-regulation of this viral antigen may favor tumorigenicity in EBV-carrying BLs.
o 1992 Wiley-Liss,Znc.
Epstein-Barr virus (EBV)-carrying human B-cell lines fall into 2 phenotypically distinct categories. Lymphoblastoid cell lines (LCL) derived from EBV-transformed normal B cells have an immunoblastic phenotype. They express B-cell activation markers such as CD23 and CD39 (Rowe et al., 1985) and do not express the CD10 and CD77 markers which characterize a subpopulation of germinal-center B cells (EhlinHenrikson and Klein, 1984). LCLs also express the adhesion molecules LFA-la, LFA-lb, LFA-3 and ICAM-1 and grow in vitro in large clusters (Patarroyo et al., 1986). EBV-carrying as well as EBV-negative Burkitt lymphoma (BL) cells display in vivo the opposite phenotype. They are CD10- and CD77positive and express very few B-cell activation markers and adhesion molecules or none at all (Patarroyo et al., 1988). As long as they remain true to their original phenotype, operationally defined as group-I (grI) BLs, they grow in vitro as free cells. During serial in vitro propagation, most EBV-carrying BL lines drift towards a more “LCL-like” phenotype (grII and grIII BLs) (Rowe et al., 1987). The LCL and grI BL prototypes also differ in the expression of EBV antigens. Eight viral gene products, the nuclear antigens EBNA-1,2,3,4,5 and 6 and the membrane proteins LMPl and terminal protein (TP), are regularly detected in LCLs (Longenecker and Keiff, 1990) while grI BLs express only EBNA-1 (Rowe et al., 1987). The causes of this phenotypic heterogeneity and its influence on the in vivo growth potential of EBV-carrying B-cells are poorly understood. It is likely that the 8 viral antigens expressed in LCLs cooperate in the process of B-cell immortalization but only little information is available on their function. EBNA-1 is required for maintenance of the viral episomes (Yates et al., 1984). EBNA-2 is needed for activation of resting B-cells and (can induce the expression of activation markers in some
EBV-negative B-cell lines (Wang et al., 1987). EBNA-2 exhibits a B-blast-specific pattern of expression since it has never been detected in EBV carrying epithelial tumors, transfected cell lines or somatic cell hybrids that lack B-cell characteristics (Contreras-Salazar et al., 1989). EBNA 3, 4, 5 and 6 are similarly and probably coordinately expressed in B-blasts only, but their function is unknown. LMPl can act as an oncogene in established lines of rodent fibroblasts (Baichwal and Sugden, 1988; Wang et al., 1985) and immortalized human keratinocytes (F5hraeus et al., 1990). In transfected EBV negative BL cells high LMPl expression is associated with extensive phenotypic changes like growth in tight clumps, increased cell size, and plasma membrane ruffling, up-regulation of adhesion molecules and of the B-cell activation markers CD23 and CD39 (Wang et al., 1988). We have previously reported that the EBV-induced shift of originally EBV-negative BL cells towards a more “LCL-like” phenotype is occasionally associated with reduction of agarose clonability and tumorigenicity (Torsteinsdottir et al., 1989). This occurred in 1 out of 7 EBV-converted sublines of the BL41 line, designated BL41/95, which showed an extensive phenotypic shift. The other 6 converted sublines showed limited phenotypic changes and remained clonable and highly tumorigenic. Only BL41/95 expressed detectable LMPl in spite of similarly high levels of the 6 EBNAs in all 7 converted sublines. These findings have promoted the present study, aimed at determining whether reduced clonability and tumorigenicity are true effects of LMPl expression in BL cells. This was examined by comparing the in vitro growth, agarose clonability and tumorigenicity in immunodeficient mice of 4 transfected sublines of BL4l with low and high LMPl expression. MATERIAL AND METHODS
Cell lines The EBV-negative BL line BL41 (Lenoir et al., 1985) was cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 kg/ml streptomycin and 10% heat-inactivated fetal calf serum (complete medium). BL41 cells were transfected by electroporation with pSV2gptbased expression plasmid carrying the LMPl coding sequence linked to the human metallothionein type-2 gene promoter as described (Wang et al., 1988). LMP1-expressing clones and clones transfected with the pSV2gpt vector alone were maintained in complete medium supplemented with 150 pg/ml of xanthine, 10 pg/ml of hypoxanthine and 0.5 pg/ml of mycophe3Present address: Istituto di Patologia Generate, Universita La Sapienza, viale Regina Elena 324,00161 Roma, Italy. 4Towhom correspondence and reprint requests should be sent. Fax: 46 (08) 330498. Received: January 17,1992 and in revised form March 20,1992.
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nolic acid (MPA). All cell lines were found to be Mycoplasmafree by 3 independent detection methods. Immunoblottingand immunodotting Total cell extracts were prepared by resuspending 5 x lo7 cells in 1 ml of 20 mM Tris buffer (pH 7.5) containing 1% Nonidet P-40, 1% Na deoxycholate, 0.1% SDS, 2 mM phenylmethylsulphonyl-fluoride, 4 mM EDTA and 4 mM NaCI. The cell suspensions were sonicated and centrifuged for 20 min at 10,000 g . Supernatants (20 1.1) were resuspended in 80 p1 SDS-PAGE sample buffer and electrophoresed in 7.5% polyacrylamide gels (Laemmli, 1970). Molecular weight determinations were made by running high- and low-molecular-weight standards (BioRad, Richmond, CA) in the same gels. Immunoblotting was performed as described (Towbin et al., 1979). The blots were probed with MAb S12 directed against an antigenic determinant on the carboxyterminal part of the LMPl molecule (Mann et aL, 1985). For quantitative measurement of LMP1, total cell extracts were serially diluted in phosphatebuffered saline (PBS) starting with a concentration of 5 x lo6 cells/ml. One hundred 1. were blotted on nitrocellulose filters pre-wetted in PBS by using a dot-blot apparatus (BioRad) under mild suction. After drying in air, the nitrocellulose sheets were stained with Ponceau-S, to ascertain that equal amounts of proteins had been loaded for each cell line, and subsequently probed with the S12 MAb. Arbitrary LMPl units were defined as the reciprocal of the highest dilution of cell extracts that could still give a positive reaction. Cloning in soft agarose Peripheral blood lymphocytes, obtained by Ficoll/Isopaque gradient centrifugation of heparinized blood from healthy volunteers, were used as feeder layers. PHA-pulsed PBLs (1 p,g/ml for 1 hr at 37°C) were irradiated with 3,000 rads and mixed with 0.45% w/v agarose (Sea-plaque agarose, Marine Colloids, FMC, Rockland, ME) heated and diluted in RPMI 1640 supplemented with 10% FCS. Three ml/dish of the cell suspension (1.5 X lo6 cells/dish) were distributed in 35 x 10-mm Petri dishes (Lux tissue culture dishes with 2-mm grids (Lab Tek, Miles, McLean, VA). After incubation at room temperature for 1 hr, when the underlayer had solidified, the dishes were incubated at 37°C in a 5% C 0 2 atmosphere for 24 hr. Three ml of a 0.35% w/v agarose solution in complete medium were mixed with the cells at 40°C and poured on top of the agarose underlayer. On each dish 1,000 cells were seeded. Cloning efficiency was estimated after 7-8 days of culture at 37°C. A group of more than 20 cells was scored as a clone. Where indicated, the cells were maintained in culture for 10 days in medium containing 75 FM ZnS04 before the cloning assay. Tumorigenicity (a) In immunosuppressed mice. Thymectomized mice, 3-4 weeks old, were injected i.p. with 200 mg/kg cytosine arabinoside and, 48-72 hr later, irradiated with 800 rad. Twenty-four hours after irradiation, the mice were injected S.C. in the lower flank with a single inoculum of 2 x lo7 cells from exponentially growing cultures in a total volume of 0.2 ml. Tumor development was followed by weekly observations. Three different tumor diameters were measured and the geometric mean was calculated. The mean tumor load for each group of mice was calculated by adding the mean tumor diameters and dividing them by the number of mice. (b) In SCZD mice. CB-17 SCID mice were inoculated S.C. with lo7 cells. Tumor sizes were measured as described above. RESULTS
Effect of LMPl expression on growth in suspension cultures Four LMP1-transfected sublines of BL41, designated MTLM-2, MTLM-4, MTLM-5 and MTLM-11, and 2 vectorcarrying control lines gpt-1 and gpt-2 were included in this
series of experiments. A 63-kDa band, corresponding to full-size LMPl transcribed from the EDLl promoter in B95-8 cells, was detected in immunoblots of total cell extracts probed with the S12 MAb in the 4 LMPl transfectants but not in the untransfected and vector-transfected cells (Fig. 1). The intensity of the LMPl band varied among the transfectants. It was weaker in MTLM-4 (indicated in Fig. 1by an arrow), intermediate in MTLM-5 and strongest in MTLM-2 and MTLM-11. A quantitative evaluation of LMPl expression was obtained by probing dot blots of serially diluted samples with S12. The ranges of LMPl units/106 cells, estimated in 6 experiments in which the cell lines were tested in parallel, are shown in Table I. The same ranking order of expression was observed throughout the experiments. MLTM-2 and MTLM-11 expressed LMPl at the same level as the EBV-transformed LCL included in each experiment as positive control. MTLM-5 had a 4-times lower LMPl expression and a further 4-fold reduction was observed in MTLM-4. A n inverse correlation was observed between the level of LMPl expression and the proliferative capacity of the cell lines in suspension cultures. Representative growth curves recorded during 3 consecutive in vitro passages of BL41, the vectortransfected gpt-1 and gpt-2, and 2 LMPl transfectants MTLM-4 and MTLM-11, with low and high LMPl expression respectively are shown in Figure 2. The cell lines were seeded at
FIGURE 1 - LMPl expression of control and transfected cells. Immunoblots of total cell extracts were probed with the S12 MAb. TABLE I - COKRELAI ION BETWEEN LMPl EXPRESSION A N D DOUBLIUC; TIME IN SUSPENSION CULTURES
Cell line
LMPl units
Doublin time
(rangel'
lhrB
BL4l gpt-1 gpt-2 MTLM 4 MTLM 5 MTLM 2 MTLM 11
0 0 0
21.8 20.8 21.6 26.0 25.O 28.0
4-16 1644 64-128 64-128
36.0
'Dot blots of total cell extracts were probed with the S12 MAb. LMP1 units were defined as the reciprocal of the highest dilution that could still give a positive reaction. The range of LMPl units measured in 6 experiments performed with each cell line are shown. The EBV-transformed LCLs included in each experiment as controls expressed > 128 LMPl units.-*The doubling time was calculated as: 200 x 24 DT = mean % recovery The denominator was calculated from the percentage recovery on the second and the third days after seeding on 3 consecutive re-seedings.
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IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
I
0
24
40
72
0
24
40
72
24
0
40
72
HOURS FIGURE 2 - Growth kinetics of untransfected, vector-transfectedand LMP1-transfected clones. Each point in the graph represents cell number/ml counted daily. Three consecutivein vitro passages are represented. The cells were re-seeded at a concentration of 2 x 105/ml every 4th day. BL41,O; gpt-1, 0 ;gpt-2,O; MTLM-4, A; MTLM-11, A.
2 x lo5 cells/ml in 25-cm2flasks and re-seeded every 72 hr at the original concentration. The number of viable cells, assessed by Trypan-blue exclusion, was counted daily under the microscope. The weakly LMP1-expressing MTLM-4 and the untransfected and the vector-transfected controls reached a cell concentration of approximately 1.4 x 106/mlafter 72 hr. In contrast, the strongly LMP1-expressing clone MTLM-11 attained during the same period a cell concentration of only 0.8 x 106/ml. All cultures contained less than 5% dead cells. The average doubling time of untransfected, vector-transfected and LMP1-expressing clones was calculated from the cell recovery recorded during logarithmic phase of the growth curves (Table I). LMPl expression was associated with a dose-dependent increase in the doubling time.
Effect on cloning in semi-solid agarose As shown in Table 11, the LMP1-transfected clones showed a significantly lower agarose clonability than the vectortransfected clones and untransfected parents. The MTLM-4 line that expressed LMPl at the lowest level cloned as efficiently as the controls. MTLM-5, which had an intermediate LMPl expression, showed a less marked reduction of clonability. In the pSV2gpt construct the LMPl gene is linked to the metal-lothionein promoter which is inducible by ZnS04. In order to investigate the effect of increased LMPl levels within the same cell line, the vector and LMP1-transfected clones were cultured for 7 days in the presence of 150, 75 and 35 pM ZnS04. The treatment induced a dose-dependent increase in LMPl expression in the MTLM-5 clone as detected by Western blots (Fig. 3a) and dot blots (Fig. 3b). Cells cultured in the presence of 75 pM ZnS04 had a 4- to 8-times higher LMPl expression compared to untreated cells (Table 11); they grew in large clumps (Fig. 4c-d) and showed a significant slowing-down of cell proliferation which was not accompanied by reduced cell viability. The same treatment did not affect the growth or morphology of the 2 BL4l-vector carrying sublines gpt-1 and gpt-2 (Fig. 4a-6). Higher doses of ZnS04 induced a further increase in LMPl expression but were toxic for both LMPI and vector-carrying cells. The treatment did not up-regulate LMPl in the low-LMP1 clone MTLM-4 and had only minor effects on the MTLM-2 and MTLM-11 clones which constitutively express high levels of the protein (not shown). Pre-treatment with 75 pM ZnS04 did not affect the cloning efficiency of the gpt-1 and gpt-2 lines (Table 11) while the
TABLE I1 - CLONING EFFICIENCY OF LMPI-TRANSFECTED AND CONTROL CELLS
Cell line
BL 41 gut-1 gpt-2 MTLM2 MTLM 11 MTLM4 MTLM5
Cloning efficiency (96)'
LMPl units/106
Untreated
ZnSOd2
Untreated
ZnSOa2
78.6 f 5.1 77.0 & 6.2 75.6 6.7 20.3 2 2.7 21.0 2 3.4 73.0 f 8.0 57.1 2 7.2
ND 73 r 5.0 94 f 4.2 ND ND ND 14 & 2.8
0 0 0 128 128 16 32
ND 0
*
0
ND ND ND 128
*
'Percentage of colonies out of 1,000 cells seeded. Mean SE of 3 experiments. Each experiment was performed in triplicate.2ZnS04treatment was performed by culturing the cells for 7 days in medium containing 75pM ZnS04.The cells were washed twice and resuspended in ZnS04-free medium before ~loning.-~LMPl units/106 cells were calculated as the reciprocal of the highest dilution that gave a detectable dot-blot signal with titration series starting from 5 x lo5 cells per spot. The numbers represent the average of 3 experiments where LMPl units/106 cells were determined just before the cloning assay. clonability of the MTLM-5 line was reduced from 57% to 14%. Cloning assays performed in the continuous presence of ZnS04were not informative because the compound was found to abolish cloning of both vector- and LMP1-carrying cells even at doses which were suboptimal for LMPl induction. Effect on tumorigenicity (a) In immunosuppressed mice. The tumorigenic potential of BL41, the vector-carrying gpt-1 line and 2 representative LMP1-transfected clones, MTLM-4 and MTLM-2, with low and high LMPl expression respectively, was tested in mice immunosuppressed by neonatal thymectomy, cytosine arabinoside treatment and irradiation. (Table 111, Fig. 5). The strongly LMP1-expressing line MTLM-2 grew in a significantly lower proportion of animals (25%) than the untransfected BL41 (56%), the vector-transfected line gpt-1 (66%) and the weakly LMPl expressing line MTLM-4 (65%). The tumors which arose after inoculation of the untransfected, vector-carrying and MTLM-4 lines reached an average diameter of 4-4.5 mm 2 weeks after inoculation (Fig. 5). In contrast, the MTLM-2 line gave rise to significantly smaller tumors, with an average diameter of 1mm. All tumors reached their peak size approximately 2 weeks after inoculation and subsequently regressed
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DISCUSSION
FIGURE3 - Effect of ZnS04 treatment on LMPl expression of the MTLM-5 clone. The cells were cultured for 7 days in medium containing the indicated concentrations of ZnS04. LMPl units were defined as the reciprocal of the highest dilution of cell extracts that could still give a positive reaction.
due to the partial immune recovery known to occur in immunosuppressed recipients of this type. The tumors induced by the MTLM-2 clone regressed faster and more completely than those induced by the BL41, gpt-1 and MTLM-4 lines. (b) In SCID mice. The parental line BL41 and the vectortransfected gpt-l and gpt-2 clones were tumorigenic in SCTD mice and gave rise to progressively growing tumors which reached an average diameter of 20 mm within 3-4 weeks (Fig. 6). In accordance with their longer doubling time in suspension culture (Table I), the weakly LMPI-expressing clone MTLM-4 grew slightly more slowly, and the strongly LMP1-expressing clone MTLM-2 grew much more slowly than the control cells. It took more than 6 weeks for the MTLM-2 line to reach an average diameter of 5 mm. The B95-8-virus-converted BL41/95 line, which expresses high levels of LMPl and has reverted towards a less malignant phenotype (Torsteinsdottir et aL, 1989), behaved like the strongly LMP1-expressing transfectant (Fig. 6).
The present results confirm and extend our earlier observation that expression of the EBV-encoded membrane protein LMPl and the associated shift of BL cells towards a more activated immunoblastic phenotype correlate with reduced agarose clonability and tumorigenicity. As reported earlier for the effect of LMPl on the expression of B-cell activation markers and adhesion molecules (Cuomo et al., 1990; Wang et aZ., 1988) and the induction of stimulatory capacity in allogenic MLCs (Cuomo et al., 1990), the impairment of BL-cell growth appeared to be dose-dependent. Hammerschmidt et al. (1989) have shown that LMPl is toxic to rodent fibroblasts and human B-lymphoblastoid cell lines when expressed at high levels. Analysis of LMPl deletion mutants suggested that the toxic effect is not an experimental artefact and is probably associated with the same biochemical activity as required for transformation of rodent cells (Martin and Sugden, 1991). It is unlikely that the impaired cell growth observed in our transfectants is due to a toxic effect because the percentage of dead cells was similar in control and LMPI-expressing cultures. Moreover, the amount of LMPI constitutively expressed in the MTLM-2 and MTLM-11 lines, and attained in MTLM-5 after optimal ZnS04 induction, did not exceed that regularly detected in EBV-transformed LCLs. The mechanism of action of LMPl is unknown. Its rhodopsin receptor-like structure (Mann et aL, 1985), cytoskeletal association (Wang et aL, 1988), membrane patching and rapid turnover (Martin and Sugden, 1991) are consistent with the possibility that LMPl may act as a ligand-independent receptor. The relationship between these properties and the phenotypic effects associated with LMPl expression are not understood. The reduced agarose clonability of the MTLMJ line under conditions in which, due to the withdrawal of ZnS04 and short half-life of the protein, LMPl expression may rapidly return to relatively low levels, suggests that at least some of the LMPl effects are long-lasting. The reduced tumorigenicity of the strongly LMPI-expressing transfectants seems paradoxical at first sight because this viral membrane protein could transform established rodent fibroblast lines (Baichwal and Sugden, 1988; Wang et al., 1985) and immortalized human keratinocytes (FHhraeus et aZ., 1990). Our findings indicate that LMPl may have an opposite effect on the tumorigenicity of cells, depending on the cell lineage. In epithelial cells, LMPl expression is associated with reduced adhesiveness and impaired cytokeratin expression (Fihraeus et aL, 1990). These changes are indicative of a differentiation block of the type that is frequently associated with invasiveness, metastasis and other parameters of tumor progression (Behrens et al., 1989). The LMPI-induced changes in B cells are more akin to immunoblast activation, which is normally associated with increased adhesion. This was clearly documented in our experiments by the induction of cluster formation in the MTLM-5 transfectant following up-regulation of LMPl by ZnS04 treatment (Fig. 4). Previous results, from our group and others, have led to the assumption that LMPl expression may counteract the tumorigenicity of BL cells by enhancing their susceptibility to immunological control mechanisms. LMPl may provide target epitopes for immune recognition, as suggested by its capacity to sensitize transfected EBV-negative BL lines to EBV-specific CTLs (Murray et al., 1988) and to induce rejection of non-immunogenic mouse mammary carcinoma lines in syngeneic hosts (Trivedi et aL, 1991). In addition, the phenotypic changes associated with LMPl expression may facilitate the interaction with immunocompetent cells. We have shown that LMPI-expressing BL cells conjugate more readily with unprimed T cells and induce more intense proliferative responses in allogeneic MLCs (Cuomo et al., 1990). The present results suggest that LMPl expression may counteract the tumorigenicity of BL cells also by affccting their growth potential. The impaired growth of our
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IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
FIGURE4 - Effect of increased LMPl levels on cell morphology and cluster formation. The vector-carrying line gpt-2 (a, b) and MTLM-5 clone (c, d) were cultured for 7 days in medium with (b, d) or without (a, c) 75 KM ZnS04.
Cell line
BL 41 EDt-1 Lpt-2 MTLM 2 (high LMP1) MTLM 4 (low LMP1) BL41195 (high LMP1)
Take incidence (9%)' Immunosuppressed
44178 56 16124 166r nd' ' 4/16 (25) 17126 65 0125 { O d
SCID
:$:
414 (100' 3110 (B{ 414 (100 2/10 (201
'Number of mice which developed tumors within 2 weeks over the total number of inoculated mice. The inoculum dose was 2 X lo7 cells in immunosuppressed mice and lo7 in SCID mice. Significant reductions are underlined.-*Cumulative results of all experiments performed with these cell lines were taken from Torsteinsdottir et al. (1989). transfectants in suspension cultures and in agarose is particularly noteworthy because it provides a biological explanation for the observation that in vitru EBV conversion of EBVnegative BLs is a cumbersome procedure that, when successful, often results in converted sublines with low or no LMPl expression (Torsteinsdottir et al., 1989). GrI BLs grow more rapidly in vitru than EBV-transformed LCLs and phenotypically similar grIII BLs which express high levels of LMPl (Gregory et al., 1990). The slow growth of LCLs has been attributed to their dependence on autocrine growth factors (Gordon et al., 1984). It was speculated that growth in cell clusters would make it possible to attain a critical local concentration of these factors. This scenario is in line with the assumption that EBV immortalizes B lymphocytes by triggering a cell-division program normally used in mitogen- and
WEEKS
FIGURE5 - Kinetics of tumor growth in immunodefective mice. Mean tumor load after inoculation of LMP1-transfected clones and control lines in immunodefective mice. The mean tumor load at different time joints was calculated by adding the individual tumor diameters and dividing their sum by the total number of animals inoculated according to the formula: d l + d2+ . . . dn mTL = n The inoculum was 2 x lo7 cellslmouse and tumor growth was followed for 5 weeks. BL41,O; gpt-1, A;MTLM 2, A; MTLM 4,O.
antigen-induced blasts. LMPl appears to play a critical role in the activation or maintenance of this program. The autonomous growth of BL cells is stimulated by the constitutive activation of a translocated c-myc gene, juxtaposed to transcrip-
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CUOMO E T A L .
v
0
1
2
3
4
5
6
induced phenotypic changes could override the proliferative signal imposed by t h e activated c-myc without interfering with t h e expression of t h e oncogene. This is consistent with our previous finding that the B95-&induced reversion of tumorigenic phenotype in the BL41/95 cell line was not accompanied by c-myc down-regulation (Torsteinsdottir et al., 1989). O u r results further emphasize the diversity of EBV-targetcell interactions. The virally encoded LMPl is probably involved in t h e immortalization of normal B-cells a nd can transform established fibroblasts an d keratinocytes, yet it suppresses t h e tumorigenicity of BL cells. Perhaps the most intriguing implication of these findings is the demonstration that the same potentially transforming protein can act either as an oncogene, or as a tumor-suppressor gene, depending on t he lineage an d differentiation window of the target cell.
WEEKS AFTER INOCULATION
FIGURE6 - T u m o r growth in CB-17 SCID mice. The mean tumor load was measured at different time points as described in the legend to Figure 5. tionally active immunoglobulin sequences. I t is likely that EBV-transformed a n d activated-myc-driven B cells use at least partially different proliferative programs. This assumption is corroborated by t h e work of Henderson et al. (1991) showing that LMPl up-regulates th e expression of th e bcl-2 oncogene which was shown t o promote cell survival rather t h an cell growth (Hockenbery et al., 1990). Conceivably, t he LMP1-
ACKNOWLEDGEMENTS This investigation was supported by PHS grant 2ROI C A 30264 awarded by t h e National Cancer Institute, D H H A , a nd by grants from t h e Swedish Cancer Society a n d Swedish Medical Association. P.T. is t h e recipient of a fellowship awarded by t h e Cancer Research Institute an d Concern Foundation, Los Angeles, CA. M.G.M. is supported by a fellowship from t h e Concern I1 Foundation, LA. We thank D. Thorley-Lawson for providing t h e S-12 MAb. Ms. K. Kvarnung, Ms. E. Ragnar, Ms. M.L. Solberg, Ms. M. Hagelin a nd Mr. K. A n d e r s o n provided skillful technical assistance.
REFERENCES LENOIR,G.M., VUILLAUME, M. and BONNARDEL, C., The use of lymphomatous and lymphoblastoid cell lines in the study of Burkitt’s lymphoma: a human cancer model. Vol. 60, pp. 309-318, ZARC Publicafions~IARC>Lyon, LONGENECKER, R. and KEIFF,E., A second Epstein-Barr virus membrane protein (LMP2) is expressed in latent infection and colocalizes with LMP1. J. Krol., 64,2319-2326 (1990). MANN,K.P., STAUNTON, D. and THORLEY-LAWSON, D.A., EpsteinBarr virus encoded protein found in plasma membranes of transformed ce1ls.J. viral,, 55,71&720 (1985). MARTIN,J. and SUGDEN, B., Transformation by the oncogenic latent CUOMO,L., TRIVEDI,P., WANG,F., WINBERG,G., KLEIN,G. and membrane protein correlates with its rapid turnover, membrane MASUCCI, M.G., Expression of the Epstein-Barr virus (EBV) encoded localization, and cytoskeletal association. J. Virol., 65, 3246-3258 membrane antigen (LMP1) increases the stimulatory capacity of (1991). EBV-negative B lymphoma lines in allogeneic mixed lymphocytes MURRAY, R.S., WANG,D., YOUNG,L.S., WANG,F., ROWE,M., KEIFF, cultures. Europ. J. Zmmunol., 20,2293-2299 (1990). and RICKINSON, A.B., Epstein-Barr virus specific cytotoxic T cell EHLIN-HENRIKSSON, B. and KLEIN,G., Distinction between Burkitt’s E. recognition of transfectants expressing the virus-coded latent memlymphoma subgroups by monoclonal antibodies: relationship between brane protein LMP1. J. virol., 62,3747-3755 (1988). antigen expression and type of chromosomal translocation. Znt. J. PATARROYO, M., BEAITY, P.G., NILSSON,K. and GAHMBERG, C., Cancer, 33,459-463 (1984). R., RYMO,L., RHIM,J.S. and KLEIN,G., Morphological Identification of a cell surface glycoprotein mediating cell adhesion in FAHRAEUS, transformation of human keratinocytes expressing the LMPl gene of EBV immortalized normal B cells. Znt. J. Cancer, 38,539-547 (1986). PATARROYO, M., PRIETO,J., ERNBERG,I. and GAHMBERG, C.G., Epstein-Barr virus. Nature (Lond.), 345,447449 (1990). GORDON, J., LEY,S.C., MELAMED, M.D., ENGLISH, L.S. and HUGHES- Absence or low expression of leukocyte adhesion molecules CD11 and JONES, N.C., Immortalized B-lymphocytes produce B-cell growth CD18 on Burkitt lymphoma cells. Znt. J. Cancer, 41,901-907 (1988). factor. Nature (Lond.), 310,145-147 (1984). ROWE,M., ROWE,D.T., GREGORY, C.D., YOUNG,L.S., FARRELL, P.J., RUPANI,H. and RICKINSON,A.B., Differences in B cell growth GREGORY, C.D., ROWE,M. and RICKINSON, A.B., Epstein-Barr virusB-cell interactions in phenotypically distinct clones of a Burkitt’s phenotype reflect novel patterns of Epstein-Barr virus latent gene expression in Burkitt’s lymphoma cells. EMBOJ., 6,2743-2751 (1987). lymphoma line. J. gen. Virol., 71,1481-1495 (1990). A.B., LENOIR,G.M., RUPANI, HAMMERSCHMIDT, W., SUGDEN,B. and BAICHWAL, V.R., The trans- ROWE,M., ROONEY,C.M., RICKINSON, lorming domain alone of the latent membrane protein of Epstein-Barr H., Moss, D.J., STAIN,H. and EPSTEIN,M.A., Distinctions between virus is toxic to cells when expressed at high levels. J. Virol., 63, endemic and sporadic forms of Epstein-Barr virus positive Burkitt’s 2469-2475 (1989). Znt. J. Cancer, 35.435441 (1985). lymphoma. -~ HENDERSON, S., ROW M., GREGORY,C.D., CROOM-CARTER, D., TORSTEINSDOTTIR, S., ANDERSON,M.L., AVILA-CARINO, J., EHLINA.B., Induction of bcl-2 expres- HENRIKSSON, WANG,F., KIEFF,E. and RICKINSON, B., MASUCCI, M.G., KLEIN,G. and KLEIN,E., Reversion sion by Epstein-Barr virus latent membrane protein-1 protects in- of tumorigenicity and decreased agarose clonability after EBV converfected B cells from programmed cell death. Cell, 65,1107-1115 (1991). sion of an IgH/myc translocation-carrying BL line. Int. J. Cancer, 43, HOCKENBERY, D., NUNEZ,G., MILLIMAN, C., SCHREIBER, R.D. and 273-278 (1989). KORSMEYER, S.J., Bcl-2 is an inner mitochondria1 protein that blocks TOWBIN, H., STAEHELIN, T. and GORDON, J., Electrophoretic transfer programmed cell death. Nature (Lond.), 348,334336 (1990). of proteins from polyacrylamide gels to nitrocellulose sheets: proceLAEMMLI, U.K., Cleavage of structural proteins during the assembly of dures and some applications. Proc. nut. Acad. Sci. (Wash.), 76, the head of bacteriophage T4. Nature (Lond.), 227,680-685 (1970). 4350-4354 (1979). BAICHWAL, V.R. and SUGDEN, B., Transformation of BALB 3T3 cells by the BNLF-1 gene of Epstein-Barr virus. Oncogene, 2, 461-467 (1988). J., MAREEL,M.M., VANROY,F.M. and BIRCHMEIER, W., BEHRENS, Dissecting tumor cell invasion: epithelial cells acquire invasive properties after the loss of uvomurulin-mediated cell-cell adhesion. J. Cell Biol., 108,2435-2447 (1989). CONTRERAS-Sm4ZAK B., G. and MAsUcCI, M.G., Host cell dependent regulation Of growth-transformation-associated EpsteinBarr virus antigens in somatic cell hybrids. J. Virol., 63, 2768-2772 ,,nnn, (IYOY).
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IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
TRIVEDI,P., MASUCCI,M.G., WINBERG,G. and KLEIN, G., The Epstein-Barr virus encoded membrane protein LMPl but not the nuclear antigen EBNA-1 induces rejection of transfected murine mammary carcinoma cells. In?.J. Cancer, 48,794800 (1991). D, and mIFF, E,, An EBV membrane protein WANG,D,, LIEBOWITZ, expressed in immortalized lymphocytes transforms established rodent cells. Cell, 43,831-840 (1985). WANG,D., LIEBOWITZ, D., WANG,F., GREGORY, C., RICKINSON, A.B., LARSON, R.2 SPRINGER, T. and KIEFF, E.3 EPstein-Barr virus latent infection membrane protein alters the human B-lymphocyte pheno-
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type: deletion of the amino terminus abolishes activity. J. viral., 62, 41734184 (1988). WANG,F., GREGORY, c.D., ROWE,M,, R I C ~ N S OA.B., N , W ~ GD.,, H., KISHIMOTO, T. and KIEFF,E., EpsteinBIRKENBACH, M., KIKUTANI, Barr virus nuclear antigen 2 specifically induces expression of the CD23. proc. Acad. sci. (Wash.), 84, B-cell activation 3452-3456 (1987). YATES,J., WARREN,N., REISMAN,D. and SUGDEN,B., A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. nut. ,&ad. Scj. (Wash.), 81,380&3810 (1984).
Publication of the International Union Against Cancer Publication de I‘Union Internationale Contre le Cancel
EXPRESSION OF THE EPSTEIN-BARR VIRUS (EBV)-ENCODED MEMBRANE PROTEIN LMPl IMPAIRS THE IN VITRO GROWTH, CLONABILITY AND TUMORIGENICITY OF AN EBV-NEGATIVE BURKITT LYMPHOMA LINE Laura cUOM01.3, Torbjorn RAMQUIST’, Pankaj TRIVEDI’,Fred WmG2, George KLEIN’and Maria G. MASUCC11,4 ‘Department of Tumor Biology, Karolinska Institute, Box 60 400, 104 01 Stockholm, Sweden; and 2Departmentsof Medicine, Microbiology and Molecular Genetics, Harvard Medical School, 75 Francis Street, Boston, M A 02115, USA. In a previous study on several independently established Epstein-Barr virus (EBV)-converted sublines of the EBVnegative Burkitt lymphoma (BL) line BL41, we found that expression of the virally encoded membrane protein LMPl was accompanied by reduced agarose clonability and tumorigenicity. In order to investigate whether LMPl can induce these phenotypic changes by itself, we have now studied the growth in suspension culture, the clonability in agarose and the tumorigenicity in immunosuppressed and SClD mice of 4 LMPItransfected sublines of BL41 that carry the gene under the control of the ZnS04-inducible metallothionein promoter. Expression of LMPl at levels comparable to those detected in EBV-transformed lymphoblastoid cell lines (LCL) correlated with impairment of growth in suspension and reduction of clonability and tumorigenicity. Only minor changes were obsewed in transfectants expressing low LMPl levels. Upregulation of LMP I by ZnS04 treatment of the low LMPl clone MTLMS was accompanied by a slowing down of proliferation, increased cell clumping and decreased clonability. The results suggest that expression of LMPl at levels which are compatible with immortalizationof normal B-cells antagonizes the ability of BL cells to grow in vitro and in vivo, and illustrate a possible mechanism by which down-regulation of this viral antigen may favor tumorigenicity in EBV-carrying BLs.
o 1992 Wiley-Liss,Znc.
Epstein-Barr virus (EBV)-carrying human B-cell lines fall into 2 phenotypically distinct categories. Lymphoblastoid cell lines (LCL) derived from EBV-transformed normal B cells have an immunoblastic phenotype. They express B-cell activation markers such as CD23 and CD39 (Rowe et al., 1985) and do not express the CD10 and CD77 markers which characterize a subpopulation of germinal-center B cells (EhlinHenrikson and Klein, 1984). LCLs also express the adhesion molecules LFA-la, LFA-lb, LFA-3 and ICAM-1 and grow in vitro in large clusters (Patarroyo et al., 1986). EBV-carrying as well as EBV-negative Burkitt lymphoma (BL) cells display in vivo the opposite phenotype. They are CD10- and CD77positive and express very few B-cell activation markers and adhesion molecules or none at all (Patarroyo et al., 1988). As long as they remain true to their original phenotype, operationally defined as group-I (grI) BLs, they grow in vitro as free cells. During serial in vitro propagation, most EBV-carrying BL lines drift towards a more “LCL-like” phenotype (grII and grIII BLs) (Rowe et al., 1987). The LCL and grI BL prototypes also differ in the expression of EBV antigens. Eight viral gene products, the nuclear antigens EBNA-1,2,3,4,5 and 6 and the membrane proteins LMPl and terminal protein (TP), are regularly detected in LCLs (Longenecker and Keiff, 1990) while grI BLs express only EBNA-1 (Rowe et al., 1987). The causes of this phenotypic heterogeneity and its influence on the in vivo growth potential of EBV-carrying B-cells are poorly understood. It is likely that the 8 viral antigens expressed in LCLs cooperate in the process of B-cell immortalization but only little information is available on their function. EBNA-1 is required for maintenance of the viral episomes (Yates et al., 1984). EBNA-2 is needed for activation of resting B-cells and (can induce the expression of activation markers in some
EBV-negative B-cell lines (Wang et al., 1987). EBNA-2 exhibits a B-blast-specific pattern of expression since it has never been detected in EBV carrying epithelial tumors, transfected cell lines or somatic cell hybrids that lack B-cell characteristics (Contreras-Salazar et al., 1989). EBNA 3, 4, 5 and 6 are similarly and probably coordinately expressed in B-blasts only, but their function is unknown. LMPl can act as an oncogene in established lines of rodent fibroblasts (Baichwal and Sugden, 1988; Wang et al., 1985) and immortalized human keratinocytes (F5hraeus et al., 1990). In transfected EBV negative BL cells high LMPl expression is associated with extensive phenotypic changes like growth in tight clumps, increased cell size, and plasma membrane ruffling, up-regulation of adhesion molecules and of the B-cell activation markers CD23 and CD39 (Wang et al., 1988). We have previously reported that the EBV-induced shift of originally EBV-negative BL cells towards a more “LCL-like” phenotype is occasionally associated with reduction of agarose clonability and tumorigenicity (Torsteinsdottir et al., 1989). This occurred in 1 out of 7 EBV-converted sublines of the BL41 line, designated BL41/95, which showed an extensive phenotypic shift. The other 6 converted sublines showed limited phenotypic changes and remained clonable and highly tumorigenic. Only BL41/95 expressed detectable LMPl in spite of similarly high levels of the 6 EBNAs in all 7 converted sublines. These findings have promoted the present study, aimed at determining whether reduced clonability and tumorigenicity are true effects of LMPl expression in BL cells. This was examined by comparing the in vitro growth, agarose clonability and tumorigenicity in immunodeficient mice of 4 transfected sublines of BL4l with low and high LMPl expression. MATERIAL AND METHODS
Cell lines The EBV-negative BL line BL41 (Lenoir et al., 1985) was cultured in RPMI 1640 medium supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 kg/ml streptomycin and 10% heat-inactivated fetal calf serum (complete medium). BL41 cells were transfected by electroporation with pSV2gptbased expression plasmid carrying the LMPl coding sequence linked to the human metallothionein type-2 gene promoter as described (Wang et al., 1988). LMP1-expressing clones and clones transfected with the pSV2gpt vector alone were maintained in complete medium supplemented with 150 pg/ml of xanthine, 10 pg/ml of hypoxanthine and 0.5 pg/ml of mycophe3Present address: Istituto di Patologia Generate, Universita La Sapienza, viale Regina Elena 324,00161 Roma, Italy. 4Towhom correspondence and reprint requests should be sent. Fax: 46 (08) 330498. Received: January 17,1992 and in revised form March 20,1992.
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nolic acid (MPA). All cell lines were found to be Mycoplasmafree by 3 independent detection methods. Immunoblottingand immunodotting Total cell extracts were prepared by resuspending 5 x lo7 cells in 1 ml of 20 mM Tris buffer (pH 7.5) containing 1% Nonidet P-40, 1% Na deoxycholate, 0.1% SDS, 2 mM phenylmethylsulphonyl-fluoride, 4 mM EDTA and 4 mM NaCI. The cell suspensions were sonicated and centrifuged for 20 min at 10,000 g . Supernatants (20 1.1) were resuspended in 80 p1 SDS-PAGE sample buffer and electrophoresed in 7.5% polyacrylamide gels (Laemmli, 1970). Molecular weight determinations were made by running high- and low-molecular-weight standards (BioRad, Richmond, CA) in the same gels. Immunoblotting was performed as described (Towbin et al., 1979). The blots were probed with MAb S12 directed against an antigenic determinant on the carboxyterminal part of the LMPl molecule (Mann et aL, 1985). For quantitative measurement of LMP1, total cell extracts were serially diluted in phosphatebuffered saline (PBS) starting with a concentration of 5 x lo6 cells/ml. One hundred 1. were blotted on nitrocellulose filters pre-wetted in PBS by using a dot-blot apparatus (BioRad) under mild suction. After drying in air, the nitrocellulose sheets were stained with Ponceau-S, to ascertain that equal amounts of proteins had been loaded for each cell line, and subsequently probed with the S12 MAb. Arbitrary LMPl units were defined as the reciprocal of the highest dilution of cell extracts that could still give a positive reaction. Cloning in soft agarose Peripheral blood lymphocytes, obtained by Ficoll/Isopaque gradient centrifugation of heparinized blood from healthy volunteers, were used as feeder layers. PHA-pulsed PBLs (1 p,g/ml for 1 hr at 37°C) were irradiated with 3,000 rads and mixed with 0.45% w/v agarose (Sea-plaque agarose, Marine Colloids, FMC, Rockland, ME) heated and diluted in RPMI 1640 supplemented with 10% FCS. Three ml/dish of the cell suspension (1.5 X lo6 cells/dish) were distributed in 35 x 10-mm Petri dishes (Lux tissue culture dishes with 2-mm grids (Lab Tek, Miles, McLean, VA). After incubation at room temperature for 1 hr, when the underlayer had solidified, the dishes were incubated at 37°C in a 5% C 0 2 atmosphere for 24 hr. Three ml of a 0.35% w/v agarose solution in complete medium were mixed with the cells at 40°C and poured on top of the agarose underlayer. On each dish 1,000 cells were seeded. Cloning efficiency was estimated after 7-8 days of culture at 37°C. A group of more than 20 cells was scored as a clone. Where indicated, the cells were maintained in culture for 10 days in medium containing 75 FM ZnS04 before the cloning assay. Tumorigenicity (a) In immunosuppressed mice. Thymectomized mice, 3-4 weeks old, were injected i.p. with 200 mg/kg cytosine arabinoside and, 48-72 hr later, irradiated with 800 rad. Twenty-four hours after irradiation, the mice were injected S.C. in the lower flank with a single inoculum of 2 x lo7 cells from exponentially growing cultures in a total volume of 0.2 ml. Tumor development was followed by weekly observations. Three different tumor diameters were measured and the geometric mean was calculated. The mean tumor load for each group of mice was calculated by adding the mean tumor diameters and dividing them by the number of mice. (b) In SCZD mice. CB-17 SCID mice were inoculated S.C. with lo7 cells. Tumor sizes were measured as described above. RESULTS
Effect of LMPl expression on growth in suspension cultures Four LMP1-transfected sublines of BL41, designated MTLM-2, MTLM-4, MTLM-5 and MTLM-11, and 2 vectorcarrying control lines gpt-1 and gpt-2 were included in this
series of experiments. A 63-kDa band, corresponding to full-size LMPl transcribed from the EDLl promoter in B95-8 cells, was detected in immunoblots of total cell extracts probed with the S12 MAb in the 4 LMPl transfectants but not in the untransfected and vector-transfected cells (Fig. 1). The intensity of the LMPl band varied among the transfectants. It was weaker in MTLM-4 (indicated in Fig. 1by an arrow), intermediate in MTLM-5 and strongest in MTLM-2 and MTLM-11. A quantitative evaluation of LMPl expression was obtained by probing dot blots of serially diluted samples with S12. The ranges of LMPl units/106 cells, estimated in 6 experiments in which the cell lines were tested in parallel, are shown in Table I. The same ranking order of expression was observed throughout the experiments. MLTM-2 and MTLM-11 expressed LMPl at the same level as the EBV-transformed LCL included in each experiment as positive control. MTLM-5 had a 4-times lower LMPl expression and a further 4-fold reduction was observed in MTLM-4. A n inverse correlation was observed between the level of LMPl expression and the proliferative capacity of the cell lines in suspension cultures. Representative growth curves recorded during 3 consecutive in vitro passages of BL41, the vectortransfected gpt-1 and gpt-2, and 2 LMPl transfectants MTLM-4 and MTLM-11, with low and high LMPl expression respectively are shown in Figure 2. The cell lines were seeded at
FIGURE 1 - LMPl expression of control and transfected cells. Immunoblots of total cell extracts were probed with the S12 MAb. TABLE I - COKRELAI ION BETWEEN LMPl EXPRESSION A N D DOUBLIUC; TIME IN SUSPENSION CULTURES
Cell line
LMPl units
Doublin time
(rangel'
lhrB
BL4l gpt-1 gpt-2 MTLM 4 MTLM 5 MTLM 2 MTLM 11
0 0 0
21.8 20.8 21.6 26.0 25.O 28.0
4-16 1644 64-128 64-128
36.0
'Dot blots of total cell extracts were probed with the S12 MAb. LMP1 units were defined as the reciprocal of the highest dilution that could still give a positive reaction. The range of LMPl units measured in 6 experiments performed with each cell line are shown. The EBV-transformed LCLs included in each experiment as controls expressed > 128 LMPl units.-*The doubling time was calculated as: 200 x 24 DT = mean % recovery The denominator was calculated from the percentage recovery on the second and the third days after seeding on 3 consecutive re-seedings.
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IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
I
0
24
40
72
0
24
40
72
24
0
40
72
HOURS FIGURE 2 - Growth kinetics of untransfected, vector-transfectedand LMP1-transfected clones. Each point in the graph represents cell number/ml counted daily. Three consecutivein vitro passages are represented. The cells were re-seeded at a concentration of 2 x 105/ml every 4th day. BL41,O; gpt-1, 0 ;gpt-2,O; MTLM-4, A; MTLM-11, A.
2 x lo5 cells/ml in 25-cm2flasks and re-seeded every 72 hr at the original concentration. The number of viable cells, assessed by Trypan-blue exclusion, was counted daily under the microscope. The weakly LMP1-expressing MTLM-4 and the untransfected and the vector-transfected controls reached a cell concentration of approximately 1.4 x 106/mlafter 72 hr. In contrast, the strongly LMP1-expressing clone MTLM-11 attained during the same period a cell concentration of only 0.8 x 106/ml. All cultures contained less than 5% dead cells. The average doubling time of untransfected, vector-transfected and LMP1-expressing clones was calculated from the cell recovery recorded during logarithmic phase of the growth curves (Table I). LMPl expression was associated with a dose-dependent increase in the doubling time.
Effect on cloning in semi-solid agarose As shown in Table 11, the LMP1-transfected clones showed a significantly lower agarose clonability than the vectortransfected clones and untransfected parents. The MTLM-4 line that expressed LMPl at the lowest level cloned as efficiently as the controls. MTLM-5, which had an intermediate LMPl expression, showed a less marked reduction of clonability. In the pSV2gpt construct the LMPl gene is linked to the metal-lothionein promoter which is inducible by ZnS04. In order to investigate the effect of increased LMPl levels within the same cell line, the vector and LMP1-transfected clones were cultured for 7 days in the presence of 150, 75 and 35 pM ZnS04. The treatment induced a dose-dependent increase in LMPl expression in the MTLM-5 clone as detected by Western blots (Fig. 3a) and dot blots (Fig. 3b). Cells cultured in the presence of 75 pM ZnS04 had a 4- to 8-times higher LMPl expression compared to untreated cells (Table 11); they grew in large clumps (Fig. 4c-d) and showed a significant slowing-down of cell proliferation which was not accompanied by reduced cell viability. The same treatment did not affect the growth or morphology of the 2 BL4l-vector carrying sublines gpt-1 and gpt-2 (Fig. 4a-6). Higher doses of ZnS04 induced a further increase in LMPl expression but were toxic for both LMPI and vector-carrying cells. The treatment did not up-regulate LMPl in the low-LMP1 clone MTLM-4 and had only minor effects on the MTLM-2 and MTLM-11 clones which constitutively express high levels of the protein (not shown). Pre-treatment with 75 pM ZnS04 did not affect the cloning efficiency of the gpt-1 and gpt-2 lines (Table 11) while the
TABLE I1 - CLONING EFFICIENCY OF LMPI-TRANSFECTED AND CONTROL CELLS
Cell line
BL 41 gut-1 gpt-2 MTLM2 MTLM 11 MTLM4 MTLM5
Cloning efficiency (96)'
LMPl units/106
Untreated
ZnSOd2
Untreated
ZnSOa2
78.6 f 5.1 77.0 & 6.2 75.6 6.7 20.3 2 2.7 21.0 2 3.4 73.0 f 8.0 57.1 2 7.2
ND 73 r 5.0 94 f 4.2 ND ND ND 14 & 2.8
0 0 0 128 128 16 32
ND 0
*
0
ND ND ND 128
*
'Percentage of colonies out of 1,000 cells seeded. Mean SE of 3 experiments. Each experiment was performed in triplicate.2ZnS04treatment was performed by culturing the cells for 7 days in medium containing 75pM ZnS04.The cells were washed twice and resuspended in ZnS04-free medium before ~loning.-~LMPl units/106 cells were calculated as the reciprocal of the highest dilution that gave a detectable dot-blot signal with titration series starting from 5 x lo5 cells per spot. The numbers represent the average of 3 experiments where LMPl units/106 cells were determined just before the cloning assay. clonability of the MTLM-5 line was reduced from 57% to 14%. Cloning assays performed in the continuous presence of ZnS04were not informative because the compound was found to abolish cloning of both vector- and LMP1-carrying cells even at doses which were suboptimal for LMPl induction. Effect on tumorigenicity (a) In immunosuppressed mice. The tumorigenic potential of BL41, the vector-carrying gpt-1 line and 2 representative LMP1-transfected clones, MTLM-4 and MTLM-2, with low and high LMPl expression respectively, was tested in mice immunosuppressed by neonatal thymectomy, cytosine arabinoside treatment and irradiation. (Table 111, Fig. 5). The strongly LMP1-expressing line MTLM-2 grew in a significantly lower proportion of animals (25%) than the untransfected BL41 (56%), the vector-transfected line gpt-1 (66%) and the weakly LMPl expressing line MTLM-4 (65%). The tumors which arose after inoculation of the untransfected, vector-carrying and MTLM-4 lines reached an average diameter of 4-4.5 mm 2 weeks after inoculation (Fig. 5). In contrast, the MTLM-2 line gave rise to significantly smaller tumors, with an average diameter of 1mm. All tumors reached their peak size approximately 2 weeks after inoculation and subsequently regressed
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DISCUSSION
FIGURE3 - Effect of ZnS04 treatment on LMPl expression of the MTLM-5 clone. The cells were cultured for 7 days in medium containing the indicated concentrations of ZnS04. LMPl units were defined as the reciprocal of the highest dilution of cell extracts that could still give a positive reaction.
due to the partial immune recovery known to occur in immunosuppressed recipients of this type. The tumors induced by the MTLM-2 clone regressed faster and more completely than those induced by the BL41, gpt-1 and MTLM-4 lines. (b) In SCID mice. The parental line BL41 and the vectortransfected gpt-l and gpt-2 clones were tumorigenic in SCTD mice and gave rise to progressively growing tumors which reached an average diameter of 20 mm within 3-4 weeks (Fig. 6). In accordance with their longer doubling time in suspension culture (Table I), the weakly LMPI-expressing clone MTLM-4 grew slightly more slowly, and the strongly LMP1-expressing clone MTLM-2 grew much more slowly than the control cells. It took more than 6 weeks for the MTLM-2 line to reach an average diameter of 5 mm. The B95-8-virus-converted BL41/95 line, which expresses high levels of LMPl and has reverted towards a less malignant phenotype (Torsteinsdottir et aL, 1989), behaved like the strongly LMP1-expressing transfectant (Fig. 6).
The present results confirm and extend our earlier observation that expression of the EBV-encoded membrane protein LMPl and the associated shift of BL cells towards a more activated immunoblastic phenotype correlate with reduced agarose clonability and tumorigenicity. As reported earlier for the effect of LMPl on the expression of B-cell activation markers and adhesion molecules (Cuomo et al., 1990; Wang et aZ., 1988) and the induction of stimulatory capacity in allogenic MLCs (Cuomo et al., 1990), the impairment of BL-cell growth appeared to be dose-dependent. Hammerschmidt et al. (1989) have shown that LMPl is toxic to rodent fibroblasts and human B-lymphoblastoid cell lines when expressed at high levels. Analysis of LMPl deletion mutants suggested that the toxic effect is not an experimental artefact and is probably associated with the same biochemical activity as required for transformation of rodent cells (Martin and Sugden, 1991). It is unlikely that the impaired cell growth observed in our transfectants is due to a toxic effect because the percentage of dead cells was similar in control and LMPI-expressing cultures. Moreover, the amount of LMPI constitutively expressed in the MTLM-2 and MTLM-11 lines, and attained in MTLM-5 after optimal ZnS04 induction, did not exceed that regularly detected in EBV-transformed LCLs. The mechanism of action of LMPl is unknown. Its rhodopsin receptor-like structure (Mann et aL, 1985), cytoskeletal association (Wang et aL, 1988), membrane patching and rapid turnover (Martin and Sugden, 1991) are consistent with the possibility that LMPl may act as a ligand-independent receptor. The relationship between these properties and the phenotypic effects associated with LMPl expression are not understood. The reduced agarose clonability of the MTLMJ line under conditions in which, due to the withdrawal of ZnS04 and short half-life of the protein, LMPl expression may rapidly return to relatively low levels, suggests that at least some of the LMPl effects are long-lasting. The reduced tumorigenicity of the strongly LMPI-expressing transfectants seems paradoxical at first sight because this viral membrane protein could transform established rodent fibroblast lines (Baichwal and Sugden, 1988; Wang et al., 1985) and immortalized human keratinocytes (FHhraeus et aZ., 1990). Our findings indicate that LMPl may have an opposite effect on the tumorigenicity of cells, depending on the cell lineage. In epithelial cells, LMPl expression is associated with reduced adhesiveness and impaired cytokeratin expression (Fihraeus et aL, 1990). These changes are indicative of a differentiation block of the type that is frequently associated with invasiveness, metastasis and other parameters of tumor progression (Behrens et al., 1989). The LMPI-induced changes in B cells are more akin to immunoblast activation, which is normally associated with increased adhesion. This was clearly documented in our experiments by the induction of cluster formation in the MTLM-5 transfectant following up-regulation of LMPl by ZnS04 treatment (Fig. 4). Previous results, from our group and others, have led to the assumption that LMPl expression may counteract the tumorigenicity of BL cells by enhancing their susceptibility to immunological control mechanisms. LMPl may provide target epitopes for immune recognition, as suggested by its capacity to sensitize transfected EBV-negative BL lines to EBV-specific CTLs (Murray et al., 1988) and to induce rejection of non-immunogenic mouse mammary carcinoma lines in syngeneic hosts (Trivedi et aL, 1991). In addition, the phenotypic changes associated with LMPl expression may facilitate the interaction with immunocompetent cells. We have shown that LMPI-expressing BL cells conjugate more readily with unprimed T cells and induce more intense proliferative responses in allogeneic MLCs (Cuomo et al., 1990). The present results suggest that LMPl expression may counteract the tumorigenicity of BL cells also by affccting their growth potential. The impaired growth of our
953
IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
FIGURE4 - Effect of increased LMPl levels on cell morphology and cluster formation. The vector-carrying line gpt-2 (a, b) and MTLM-5 clone (c, d) were cultured for 7 days in medium with (b, d) or without (a, c) 75 KM ZnS04.
Cell line
BL 41 EDt-1 Lpt-2 MTLM 2 (high LMP1) MTLM 4 (low LMP1) BL41195 (high LMP1)
Take incidence (9%)' Immunosuppressed
44178 56 16124 166r nd' ' 4/16 (25) 17126 65 0125 { O d
SCID
:$:
414 (100' 3110 (B{ 414 (100 2/10 (201
'Number of mice which developed tumors within 2 weeks over the total number of inoculated mice. The inoculum dose was 2 X lo7 cells in immunosuppressed mice and lo7 in SCID mice. Significant reductions are underlined.-*Cumulative results of all experiments performed with these cell lines were taken from Torsteinsdottir et al. (1989). transfectants in suspension cultures and in agarose is particularly noteworthy because it provides a biological explanation for the observation that in vitru EBV conversion of EBVnegative BLs is a cumbersome procedure that, when successful, often results in converted sublines with low or no LMPl expression (Torsteinsdottir et al., 1989). GrI BLs grow more rapidly in vitru than EBV-transformed LCLs and phenotypically similar grIII BLs which express high levels of LMPl (Gregory et al., 1990). The slow growth of LCLs has been attributed to their dependence on autocrine growth factors (Gordon et al., 1984). It was speculated that growth in cell clusters would make it possible to attain a critical local concentration of these factors. This scenario is in line with the assumption that EBV immortalizes B lymphocytes by triggering a cell-division program normally used in mitogen- and
WEEKS
FIGURE5 - Kinetics of tumor growth in immunodefective mice. Mean tumor load after inoculation of LMP1-transfected clones and control lines in immunodefective mice. The mean tumor load at different time joints was calculated by adding the individual tumor diameters and dividing their sum by the total number of animals inoculated according to the formula: d l + d2+ . . . dn mTL = n The inoculum was 2 x lo7 cellslmouse and tumor growth was followed for 5 weeks. BL41,O; gpt-1, A;MTLM 2, A; MTLM 4,O.
antigen-induced blasts. LMPl appears to play a critical role in the activation or maintenance of this program. The autonomous growth of BL cells is stimulated by the constitutive activation of a translocated c-myc gene, juxtaposed to transcrip-
954
CUOMO E T A L .
v
0
1
2
3
4
5
6
induced phenotypic changes could override the proliferative signal imposed by t h e activated c-myc without interfering with t h e expression of t h e oncogene. This is consistent with our previous finding that the B95-&induced reversion of tumorigenic phenotype in the BL41/95 cell line was not accompanied by c-myc down-regulation (Torsteinsdottir et al., 1989). O u r results further emphasize the diversity of EBV-targetcell interactions. The virally encoded LMPl is probably involved in t h e immortalization of normal B-cells a nd can transform established fibroblasts an d keratinocytes, yet it suppresses t h e tumorigenicity of BL cells. Perhaps the most intriguing implication of these findings is the demonstration that the same potentially transforming protein can act either as an oncogene, or as a tumor-suppressor gene, depending on t he lineage an d differentiation window of the target cell.
WEEKS AFTER INOCULATION
FIGURE6 - T u m o r growth in CB-17 SCID mice. The mean tumor load was measured at different time points as described in the legend to Figure 5. tionally active immunoglobulin sequences. I t is likely that EBV-transformed a n d activated-myc-driven B cells use at least partially different proliferative programs. This assumption is corroborated by t h e work of Henderson et al. (1991) showing that LMPl up-regulates th e expression of th e bcl-2 oncogene which was shown t o promote cell survival rather t h an cell growth (Hockenbery et al., 1990). Conceivably, t he LMP1-
ACKNOWLEDGEMENTS This investigation was supported by PHS grant 2ROI C A 30264 awarded by t h e National Cancer Institute, D H H A , a nd by grants from t h e Swedish Cancer Society a n d Swedish Medical Association. P.T. is t h e recipient of a fellowship awarded by t h e Cancer Research Institute an d Concern Foundation, Los Angeles, CA. M.G.M. is supported by a fellowship from t h e Concern I1 Foundation, LA. We thank D. Thorley-Lawson for providing t h e S-12 MAb. Ms. K. Kvarnung, Ms. E. Ragnar, Ms. M.L. Solberg, Ms. M. Hagelin a nd Mr. K. A n d e r s o n provided skillful technical assistance.
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IMPAIRMENT OF BL CELL GROWTH AND TUMORIGENICITY BY LMPl
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