Int. J. Cancer: 35,251-256 (1985) @ 1985 Alan R. Liss, Inc.

CAPACITY OF B-LYMPHOCYTIC LINES OF DIVERSE TUMOR ORIGIN TO PRODUCE AND RESPOND TO B-CELL GROWTH FACTORS: A PROGRESSION MODEL FOR B-CELL LYMPHOMAGENESIS John GORDON’.*, Pierre A MAN, Anders ROSEN. Ingemar ERNBERG, Barbro EHLIN-HENRIKSSON and George KLEIN Depat-tment of Turnor Biology, Kurolinsku Institute, Stockholm, Sweden: und ‘ Mechanisrns in Tuniour Itnrniiriirv, M.R. C. Centre, Cumbridge, UK.

Human cell lines established from cases of acute lymphoblastic leukemia, lymphosarcoma, Burkitt’s lymphoma and multiple myeloma and representing stages of B-lymphocyte development ranging from pre-6 through to plasma cells, were assessed for their ability to produce and respond to 6-cell growth factors (BCGF). All Btell lines studied were found to be con- stitutive producers of a growth activity which assisted the S-phase entry of normal activated 6-cells and pro- vided growth support for lymphoblastoid cells trans- formed by Epstein-Barr virus. Furthermore, all lines responded by enhanced proliferation to supernatants from a BCGF-producing T-cell hybridoma. Not all lines, however, displayed autostimulation to their own supernatants and no tumor 6-cell line appeared totally dependent on soluble factors for its growth. Non-tu- morigenic 6-cell lines, by contrast, revealed a strict dependency on homologous growith factor for their continued proliferation in suspension culture. The findings support a progression model of lymphoma- genesis based upon the utilization, production and, ultimately, emancipation from growth-promoting sol- uble factors.

Normal B-lymphocytes transformed by Epstein-Barr virus (EBV) and malignant cell lines harboring the EBV genome have been found to produce an autostim- ulatory activity resulting in an autocrine loop (Blazar et al., 1983; Gordon et al., 1984~) . Autocrine growth stimulation, whereby a cell both releases and responds to a growth-promoting substance, first described for certain tumors of fibroblastic origin (Delarco and To- daro, 1978), may represent a major route to cell im- mortality and thus, potentially, malignancy. In the case of fibroblasts, two classes of transforming growth fac- tors have been reported with their action centered on the receptor for epidermal growth factor (Assoian et ul., 1984). For other transformed cell types, different molecules have been reported to perform an autostim- ulatory function (Kaighn et al. , 198 I ; Uittenbogaart and Fahey. 1982; Schrader and Crapper, 1983). The recent demonstration of homology between the product of the sis-oncogene and platelet-derived growth factor provides a molecular rationale for the phenotypic change nece.:cary for an autocrine loop (Waterfield et al . , 1983).

Not surprisingly, for transformed B-cells, the auto- stimulatory activity has been found to be a 9-cell growth factor (BCGF) (Gordon et al., 1984b). B-cell growth factors, conventionally obtained from T-cell sources, fail to activate resting (Go) B-cells but fulfill a physiological role by assisting the S-phase entry of B lymphocytes “excited” through signals delivered via their surface membrane immunoglobulin (reviewed by Howard and Paul, 1983; Kehrl et ul., 1984).

The aim of the present study was to survey the production of BCGF among B-cell lines derived from tumors of diverse clinico-pathological origin, includ-

ing those lacking the EBV genome, and from sources other than Burkitt’s lymphoma. In addition, the re- sponse of established B-cell lines to a conventional source of BCGF was studied. What emerged was a series of findings to support a progression model of B- cell lymphomagenesis based upon a diminishing re- quirement of cells for external and endogenous growth factors.

MATERIAL A N D METHODS

Cell lines The origins of the tumor lines used in this study are

referred to in Table I. The establishment, cloning and maintenance of EBV-transformed lymphoblastoid lines from normal 9-cells have been described elsewhere (Gordon et al., 1984~) . All cell lines were grown routinely in RPMI 1640 supplemented with 8-10% fetal calf serum, penicillin/streptomycin (50 ug/mI) and L-glutamine (200 m), and maintained in culture by partial discarding of used medium and feeding twice weekly. All lines were free of mycoplasma at the time of study.

Generution of conditioned media In order to standardize conditions for the generation

of growth factors and responder cells, the following procedure was adopted. Cells were taken from their normal growth regime, washed, resuspended at 2 X 10s/ml in full serum-supplemented RPMI 1640 and grown for 48 hr at 37°C in a humidified 5% COz atmosphere. Following thorough washing in RPMI in 1640, cells were reseeded at 2 X 1OS/ml and grown for a further 24 hr in Iscove’s modified Dulbecco’s medium (IMDM: Gibco, Grand Island. NY) contain- ing delipidated bovine serum albumin (Gibco) at 1 mg/ ml, soybean lipids (Gibco) at 20 ug/ml, 30% iron- saturated transferrin (Gibco) at 30 ug/ml, L-glutamine (200 mM), penicillin/streptomycin (50 ug/ml) and 2- mercaptoethanol (5 x l O P 5 ~ ) . Supernatants at this stage were cleared of cells by centrifugation, passed through a 0 . 4 5 - ~ m filter and used immediately as a source of growth factors. Growth factor assays

Two independent assays were deployed in order to assess BCGF activity in conditioned media (CM). The first of these made use of the differential sensitivity to

2To whom reprint requests should be sent at the Department of Immunology, The Medical School. Vincent Drive, University of Birmingham, B15 2TJ. UK.

Received: October 9 , 1984 and in revised form November 16, 1984

252 GORDON E T A L

TABLE I - B-CELL STIMULATING ACTIVITY FROM B-CELL TUMOR LINES

Stimulating activity for B cells’: Cell line Origin’ Reference EBv’ Igt High Low Lympho-

density density blastoid

Jijoye BL P3HR-I BL

P3HR-1 BL ASNP

Raji BL Namalwa BL Seraphine BL LY67 BL

JI BL

Ramos BL BL2 BL Bjab “BL-like” SPAD-3 “BL-like” SPAD-4 “BL-like” Balm-1 ALL

U-698 LS

266BL Myeloma Molt-4 Hybridoma X PPD K562 CGL

Pulvertaft (1965) Hinuma and Grace

(1967) Klein ef al. (1978)

Epstein et al. (1966) Klein ef al. (1972) Klein et al. (1975) Bemheim er al.

Bornkamm et al.

Klein ef al. (1976) Lenoir er al. (1982) Klein et ali (1974) See below See below’ Minowada et a!.

(1981)

(1980)

(1977) Nilsson and Sund-

strom (1974) Nilsson er al. (1970) Rosen et al. (1984)

Lozzio and Lozzio 11975)

spk 1.2 cpk 0.9

cpk 1.0

cpk 1.0 sph 1.3

sph 0.8

sp6k 0.8

syh 1.2

sph 1.0 sph 1.0 spk 1.3 - 1 .O - 0.7

spk 0.8

spk 0.6

cck 0.9 - 1.2

- 0.9

8.4 2.8

10

7.0 9.5 5.2

11

14

6.4 12 13 8.1 5.3

14

5.7

14 11

1 .o

13 7.8

6.2

13 15 10 18

21

9.3 21 14 16 1 1 15

15

22 24

1.7

‘EL = Burkitt’s lymphoma; ALL = acute lymphoblastic leukemia; LC = lymphosarcoma; CGL = chronic granulocytic leukemia; Bjab derived from African patient with a lymphoma histologically resembling BL but lacking the typical or variant translocation invariably associated with this disease: SPAD lines derived from a “pre-AIDS” patient with an L3 lymphoma and sharing the cytological and cytogenic features of BL.-?EBV status determined by staining for the EBV nuclear antigen (EBNA). EBNA staining performed at frequent intervals confirmed the fixed EBV status of the permanent lines used in this study. t s = surface; c = cytoplasmic; SPAD lines Ig-negative but with rearranged heavy chain locus. -’Normal B-cells plated at 5 x lo4 per micro-well; lymphoblastoid cells from LCLNc plated at 2 x lo3 per micro-well; CM added to 25 % in 200 ul.

BCGF of small‘resting B-cells and of large, low-den- sity B-cells considered to be “pre-activated” in viva. This method has been described in detail elsewhere (Gordon et al . , 1984b). Briefly. it consists of first preparing, by negative selection, B-enriched lynipho- cyte fractions from tonsils obtained at routine tonsil- lectomy. Such preparations were > 97% surface immunoglobulin positive and < 0-5 % E-rosette posi- tive. Fractionation on discontinuous Percoll gradients yielded “high density” cells which were collected be- low 60% and at or above 70% Percoll while “low- density” cells comprised those banding at or above a 50% gradient. The second assay exploited the obser- vation that. in the absence of BCGF and below a critical cell density, EBV-transformed lymphoblasts cease to proliferate (Gordon et al., 1984~). For both assays, thoroughly washed cells were plated in a final volume of 200 pI of IMDM (with serum-free supple- meyts) in flat-bottomed micro-wells (growth area 0.32 cm-) and grown for 72hr in the presence or absence of 25 % CM. Transformed lymphoblastoid cells were plated at 2,000 cells per well while normal cells were seeded at 5 X lo4 /well. DNA synthesis was assessed by pulsing wells for 8hr prior to termination of culture with 0.5 $3 of ’H-Thymidine (Tdr) (76 C i h m o l ) . All determinations were performed in quadruplicate with standard deviations always within 15% of the mean.

Factor-dependency of cell lines Cells which had been grown for 24hr under serum-

free conditions for factor generation were washed ex- tensively and plated at serial dilutions in flat-bottomed

micro-wells and grown for 72hr in 200 pi of protein- supplemented IMDM in the presence or absence of 25% C M from autologous combinations, Raji cells or the Molt-4 x PPD T-cell hybridoma. DNA synthesis was assessed as for growth factor assays.

Stimulation and growth enhancement indices were calculated from the formula:

cpm ‘H-Tdr uptake in presence of CM cpm ‘H-Tdr uptake in absence of CM

RESULTS

Production of B-cell growth factors by turnor B-cell lines

Table I lists the derivation. EBV status and immu- noglobulin profile of the lines used in this study. All conditioned media generated serum-free from tumor B-cell lines contained an activity capable of supporting the S-phase entry of low density, presumably “pre- activated”, tonsillar B-lymphocytes. Resting tonsillar B-cells, recovered from the high-density fractions, failed to respond by significant DNA synthesis to any of the serum-free supernatants. A second assay, utiliz- ing EBV-transformed lymphoblastoid cells plated at low density, confirmed the presence of a stimulating activity for activated B-cells in all B-cell supernatants tested (Table I ) .

In neither assay did supernatants from the non- lymphoid K562 line exhibit B-cell stimulating activity. Other independent studies have confirmed that the pro- duction of growth factors for B-cells is restricted to tumor lines of B-cell origin (Blazar et al., 1983; Uit-

B-LYMPHOCYTE LINES AND BCGF 253

tenbogaart and Fahey. 1982). While some of the B-cell lines matched a BCGF-producing T-cell hybridoma in their ability to provide stimulating activity for B-cells, others yielded partial responses only. It would be mis- leading, however, to reach a quantitative assessment of BCGF activity from these data as many lines addi- tionally released factors which were inhibitory for DNA synthesis. These negative factors were mini- mized by the dual precautions of using relatively low cell numbers and short incubation periods in which to generate CM (see “Material and Methods”). While the actual figures obtained presumably reflect the net effect of stimulatory and inhibitory signals, it is clear that all tumor B-cell lines, irrespective of their origin, released a growth-promoting activity for B-cells into their extracellular environment.

Response of tiinlor lines t o conditioned media All of the tumor B-cell lines responded by enhanced

DNA synthesis when cultured for 3 days with serum- free medium conditioned by the Molt4 X PPD T-cell hybridoma (Table 11). The degree of enhancement de- pended to some extent on the number of responder cells plated but at 1,000 cells per well it ranged from 2-fold to 7-fold for the lines tested. None of the lines responded by enhanced growth to K562 supernatants.

Despite the production of a B-cell stimulatory activ- ity by all tumor B-cell lines, not every line responded by increased proliferation to its autologously condi- tioned medium. The ability to respond to autologous supernatants failed to correlate with the amount of B- cell stimulating activity determined in the independent assays (Table I). Rather, responsiveness appeared to be an inherent feature of individual lines as a standard source of B-cell-derived BCGF, supplied by Raji su- pernatants, revealed a similar pattern to that observed when using conditioned medium in autologous combi- nations with tumor cells (Table 11). K562 cells failed to respond to any of the sources of BCGF, B- or T- cell-derived, or indeed to its autologous supernatant, by enhanced DNA synthesis.

TABLE 11 - RESPONSE OF B-CELL TUMOR LINES TO C M i

Growth enhancement of lines lo CM h i m

Cell line Autologous Moll-4 Rail X K562

ppn ..I

Jijoye P3HR- I P1HR- 1

ASNP Raj i Namalwa Seraphine LY67 J I Rarnos BL2 Bjab

SPAD-4 Balm- 1

266BL K562

SPAD-3

U-698

1.8 1 .0 1 .0

2.0 0.9 I .9 I .8 2.2 2.1 4.4 2.4 1.7 1.8 1.7 1.1 0.9 0.9

~~

2.6 3.3 0.9 2.0 0.9 2.3

2.0 2.1 1.2 3.0 I .6 2.5 2.3 3.7 3.0 2.9 1.8 nd 4.6 6.8 2. I 4.2 1.9 1.8 1.7 2.1 2.4 2.4 0.8 3.3 1.4 2.1 0.7 1 .o

0.6 1 .0 0.7

I .2 0.8 0.7 1 . 1 0.6 0.9 1.3 1 . 1 0.8 0.9 0.9 0.6 0.9 0.8

‘Cell& plated at 10’ per micro-well. CM added to 25% in 200 pi

2 t JI

\

;\i, ,L

01- - L--t--r 4 2 1 0 5 4 2 1 0 5 4 2 1 0 5

Celtvb.esl xlo-3

FIGURE 1 - Factor-dependent growth of B-cell tumor lines. Cells were grown for 3 days in 200 p1 following plating at in- dicated numbers in micro-wells and pulsed with 0.5 pCi of ’H-Tdr for the final 8hr. Counts are means of quadruplicate samples and represent the uptake of ’H-TdrlIOOO cells. 0 , Control; 0, 25% autologous conditioned medium.

Dependency of B-cell lines on B-cell growth factors A previous study revealed that, under serum-free

conditions and in the presence of transferrin, EBV- transformed B-lymphoblasts require a high cell density for sustained growth (Gordon et al., 1984~). This requirement could be replaced by autologous condi- tioned medium, Tumor B-cell lines were compared with typical lymphoblastoid lines for their density- dependent growth in liquid culture.

Some tumor lines, as represented in Figure la, failed to demonstrate a cell-density-dependence for their con- tinued proliferation in serum-free media. Lines exhib- iting this pattern of growth included all those which failed to respond to autologous supernatants by en- hanced proliferation and some which responded only modestly. The remaining tumor B-cell lines revealed a diminished proliferation as cells were diluted in culture

254 GORDON ET AL.

TABLE 111 - GROWTH PATTERNS OF B-CELL LlNES

Degree of density-dependent cell growth’

LOW Intermediate High GE to auto-CM GE to auto-CM GE to auto-CM

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

Namalwa Ramos Seraohine BL2 LCL,,, 266BL Bjab Jijoyk L C L ~ ~ U698 SPAD-3 Raii LCL, P3HR-1 SPAD4 LY67 P3HR- 1 Balm-1 ASNP JI ‘Cell lines were classified as displaying low. intermediate or high

densitydependent cell growth from dilution curves. “Low” includes those lines which, when diluted in culture over the range of 4.000 to 500 per well in 200 pl, lost not more than 50% of the proliferative capacity exhibited at the higher cell number. Intermediate lines lost no more than 80% while highly dependent lines lost >80% of their proliferative poten- tial when diluted over the range. Classification according to growth en- hancement (GE) relates to augmentation in %Tdr uptake occurring in the presence of autologous supernatants at 25% final concentration. -GE <IS; +GE, >1.5 <3; + + + G E , 2 3 (at 1.000 per well). Lympho- blastoid cell lines (LCL) were established as described previously and were carried in culture from 3 months to more than 2 years.

but, over the range studied, none exhibited a critical cell density below which no proliferation occurred. All cell lines in this category displayed growth en- hancement to autologous supernatants such that the decline in proliferative capacity observed on cell dilu- tion was restored (Fig. lb). Table I11 provides a sum- mary of the observed growth patterns for all B-cell lines studied. It should be noted that none of the tumor lines resembled lymphoblastoid cell lines which were 5 x 1 0 ~

1 oL

5x10‘

$ E, I m

102

5x10’

102 L J

So00 4000 2000 1000 500 250

Cells per well

FIGURE 2 - Response of B-lymphoblastoid cells to T-cell supernatants. Cells from an EBV-positive lymphoblastoid line L C L N ~ were plated at indicated cell numbers in 200 p1 in the presence of 25% medium conditioned by autologous cells (m). Molt4 x PPD hybridoma (a), lectin-stimulated ton- sillar T-cells ( A ) or control medium only (0). Following 3 days in culture. cells were pulsed with 0.5 pCi 3H-Tdr for the final 8hr. The absolute incorporation of 3H-Tdr is illustrated.

all included in the category, signifying an absolute requirement for high cell density for their continued growth (Fig. lc). Similarly, while all lymphoblastoid lines responded dramatically to autologous superna- tants, among all the tumor lines only BL2 revealed a particularly high response.

The results depicted in Figure 2 demonstrate that the density-dependent decline in lymphoblastoid cell growth could be compensated for not only by autolo- gous CM but also by the more conventional BCGF sources of the T-cell hybridoma and lectin-stimulated tonsillar T-lymphocytes (Gordon et al., 1984b).

DISCUSSION

The ectopic or constitutive expression of gene prod- ucts guiding cellular proliferation may represent a ma- jor route to cellular immortality and, subsequently, malignancy (reviewed by Heldin and Westermark, 1984). For B-cells infected with EBV, immortalization appears to result, at least in part, from the induction of a B-cell growth factor which is autostimulatory for the transformed cells. In the present study, a number of tumor B-cell lines of diverse origin, including several lacking the EBV genome, were examined for their capacity to produce and respond to BCGF; all were found to exhibit both properties. This raises the possi- bility that, under appropriate conditions of activation and without the intervention of EBV, BCGF produc- tion is a normal phenotypic property of B-cells. The recent claim that Staphylococcus aureus (Cowan Strain I) can activate B-cells directly without the need for exogenous growth factors lends some support to this notion (Falkoff et af., 1983). Such apparently acces- sory cell-independent activators, however, fail to sus- tain their mitogenic influence unless cultures are continually replenished with T-cell-derived products (Muraguchi and Fauci, 1982). Clearly then, even if normal B-cells do produce a BCGF (which has yet to be shown) EBV remains unique in being the only known B-cell activator which, by itself, is capable of achieving the immortalized state through continued growth factor release and auto-utilization.

The capacity of EBV to liberate cells from their normally strict growth factor requirements is readily reflected in the number of in vitro tumor Iines of B- cell origin which are associated with the EBV genome. For those cases in which EBV is not implicated, some other factor, as yet unknown, may have replaced the virus as the causal agent. Alternatively, EBV may serve to increase the probability of the final tumori- genic events occurring which, on stochastic grounds, will arise occasionally in non-infected target cells. Whichever is the case, the possibility is raised of EBV becoming redundant or, at most, surplus to the immor- talized state of the fully malignant cell. While the findings presented in this communication are compati- ble with such a notion, an alternative hypothesis exists.

The most consistent feature of BL, whether associ- ated with EBV or not, is a specific chromosomal trans- location involving the c-myc oncogene and one of the 3 immunoglobulin loci (reviewed by Mein, 1983). This genetic aberration probably results in a loss of normal control over c-myc transcription (Nishikura et al., 1983; Rabbitts et al., 1984). The product of the c- myc gene is a DNA-binding protein (Persson and Leder, 1984) which in normal quiescent cells is acti- vated during competence induction by mitogens (Ar- melin et al., 1984). Competence corresponds to a state

255 B-LYMPHOCYTE LINES AND BCGF

of excitation in which cells are ready to receive and respond to soluble growth-promoting factors. The con- sequence of deregulated c-myc expression in BL cells, rather than superseding the transforming influence of EBV, may in fact be a stabilization of the “immortal- ized” state through heightened and maintained respon- siveness to the virally-induced growth factor. We have noted previously, and confirmed here, that the “im- mortalized” phenotype of EBV-transformed, but non- tumorigenic, LCL is a relative property dependent on a high cell concentration which, in turn, reflects a strict requirement for autostimulatory BCGF. The net result of depriving these cells of sufficient growth factors is cell death. In striking contrast, in none of the tumor lines studied did simple dilution arrest cell growth. Indeed, it is well established that, by compar- ison with LCL which are notoriously difficult to clone, BL cells can be readily cloned in a semi-solid agar support. It is also of interest that, whereas BL cells tend to grow as single cells in suspension culture, LCL display a distinctive clumping pattern (Nilsson and Klein, 1982). These phenotypic differences could be interpreted as reflecting different levels of dependency on locally-produced growth factors.

Progression Model for B-lymphomagenesis

B C G F

4

m 5

0 b -i

I- - * 2. Immortal

I - 0

c 4. Tumc r

FIGURE 3 - Progression model of B-cell lymphomagenesis based upon growth factor production and dependency. (1) Activated normal B-cells, displaying an absolute dependence on exogenous BCGF for their continued growth, are trans- formed by EBV (and possibly other unknown etologic agents) to cells producing their own BCGF. (2) Immortalized B-cells, while producing their own BCGF, continue to display a high requirement for it, exemplified by the need for a high cell density to sustain growth. (3,4) Conversion to the tumori- genic phenotype is accompanied by a progressive decrease in dependence on autogenous growth factors. Such cells either utilize their growth factors more efficiently or have devel- oped alternative, non-soluble factor-dependent mechanisms for autonomous growth.

The model of B-cell lymphomagenesis outlined in Figure 3 is aimed at accommodating the results and speculations presented. The scenario predicts that the evolution of B-cell tumors represents either a more efficient utilization of autostimulatory products or a decreased dependence upon soluble growth factors. Both notions are compatible with the model if it is considered as depicting the apparent requirement for a BCGF at each stage of tumor development. Whereas the first transition to a tumorigenic phenotype repre- sents some emancipation from or a more efficient utilization of autostimulatory BCGF, the final stage of malignancy dictates that a single cell can develop au- tonomously in the absence of cooperation from its clonal companions. Such a state is presumably repre- sented by those tumor lines which showed no diminu- tion of cellular proliferation on dilution in liquid culture and additionally failed to respond by growth enhance- ment to supernatants containing homologous BCGF.

In view of the above considerations, it is interesting that all tumor B-cell lines, including those displaying no enhancement to autologous supernatants, demon- strated an increased proliferative response to T-cell- derived BCGF. Not only does this show that all cells maintain the machinery to respond to external growth signals but it also suggests that the T-cell-derived prod- uct may differ from the B-cell derived BCGF. Alter- natively, the distribution of negative and positive growth factors may differ among the various sources. It is also unclear at present whether the growth-pro- moting activity from Burkitt and lymphoblastoid lines is coded by the EB viral genome. Although the finding of such activity from lines persistently lacking signs of EBV infection would argue against that possibility, studies at the molecular level are clearly warranted.

Finally, we wish to make it clear that, despite the evidence presented, constitutive BCGF production need not be the only, nor indeed the major, route to the immortalization step in the neoplastic transforma- tion of B-lymphocytes. Established cell lines are highly selected examples of B-cell malignancies with the basis of selection quite possibly related directly to the prop- erty examined in this study. Nevertheless, the ability to identify, in even a small subgroup of the malignant B-cell spectrum, a potential mechanism for tumorigen- esis may have far-reaching implications particularly since a similar model has recently and independently been constructed for some T-lymphocyte malignancies (Haas rt al., 1984). Furthermore, our findings on B- cell tumors are highly reminiscent of those of Furth, who showed that hormone-dependent, or “condi- tioned”, neoplasms could progress to “autonomous” or hormone-independent tumors without necessarily losing their responsiveness to the growth stimulus (Furth, 1953). It would now seem appropriate to study freshly-isolated material from B-cell tumors, particu- larly the growth fractions, for the production of, re- quirement for, and response to B-cell growth factors.

ACKNOWLEDGEMENTS

We wish to thank the Swedish Cancer Society, the Medical Research Council (UK) and the NIH (US) (Grant 2 ROI CA 14054-02) for financial support.

256 GORDON ET AL.

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