[CANCER RESEARCH 48. 5411-5416. October 1. 1988]

Stimulation of Tumor Cell Growth in Humans by a MononuclearCell-derived Factor

George Sandru,1 Peter Veraguth, and Beda M. Stadier

Radiobiology Laboratory, Department of Radiotherapy [G. S., P. V.], and Institute of Clinical Immunology fB. M. S.J, University of Berne, Inselspital, Haus 28 d,3010 Berne, Switzerland

ABSTRACT

Serum-free supernatants of peripheral blood mononuclear cell culturessignificantly stimulated |3H|thymidine incorporation of human hemato-

poietic and nonhematopoietic tumor cell lines. For assay we used a lownumber of tumor cells per well and medium enriched with dithiothreitol-treated fetal calf serum. The growth-stimulatory activity was detected inthe supernatant of peripheral blood mononuclear cell culture within thefirst 24 h and decreased thereafter. Treatment of mononuclear cells withOKT3 monoclonal antibodies and rabbit complement decreased onlymoderately the factor production while treatment with anti-Leu-M2 andrabbit complement decreased it significantly. Supernatants of concana-valin A-stimulated peripheral blood mononuclear cell cultures enhancednonsigniflcantly | '11|fliymitliiic incorporation by tumor cell cultures unlessantibodies against tumor necrosis factor a and -y-interferon were addedto the supernatants. Growth-stimulatory activity was heat inactivatedpartially at 60°Cand totally at 80°C.It was abolished at pH 2.5 within

2 h as well as by treatment with dithiothreitol and partially lost bydialysis.

INTRODUCTION

Regulatory cytokines isolated from activated leukocytes suchas IFN-7,2 TNF-a, or -ß,and TGF-/3 are known to inhibit

growth of cancer cells. These cytokines are well characterizedand their complementary DNAs are cloned. However, therehave been only few reports concerning stimulation of tumorcell growth by leukocyte products. Such stimulation by lymph-oid cells has been suggested previously by results obtained byboth the in vitro microcytotoxicity assay (1-3) and the in vivoWinn test (4,5). This led to the concept of "immunostimulationof tumor cell growth" which claims that immune lymphocytes

may not kill but rather stimulate proliferation of tumor cells(6). On the other hand, lymphoid cells stimulate growth ofmany cell types. Thus, T-lymphocytes secrete the autocrinegrowth factor IL-2 (7), B-cell growth factors (8), and colony-stimulating factors for hematopoietic stem cells (9). Granulo-cyte-macrophage colony-stimulating factors also stimulate pla-cental cell growth (10). Monocyte/macrophages secrete IL-1which is comitogenic for T-lymphocytes (11) and also potentiates PDGF-induced fibroblast proliferation (12). Moreover,monocytes or macrophages secrete colony-stimulating factors(9, 13), as well as factors different from IL-1 (12) that enhancegrowth of fibroblast and mesenchymally derived cells such aschondrocytes, smooth muscles, osteoblasts, and endothelialcells (14-16). Recent work also indicated that activated monocytes express c-sis protooncogene and release a mediator show-

Received3/1/88;revised6/10/88;accepted7/1/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.2The abbreviations used are: IFN-f, -y-interferon; OKT3, monoclonal antibod

ies reacting with human T-lymphocytes; anti-Leu-M2, monoclonal antibodiesreacting with monocytes; Con A, concanavalin A; MNC, peripheral blood mono-nuclear cells; TNF-tt and -ß,tumor necrosis factors a and 0; TGF-a and -/3,transforming growth factors a and 0; IL-1, interleukin 1; IL-2, interleukin 2;PDGF, platelet-derived growth factor; DTT. dithiothreitol; PCS, fetal calf serum;SH, sulfhydryl; PBS, phosphate-buflered saline (137 IHMNaCl-6.6 mM Na2HPO4-1.5mMKH2PO4.

ing PDGF-like activity which competes for receptor bindingwith PDGF (17). Also, a PDGF homologue which interactswith anti-PDGF antibodies was reported to be produced byactivated rat alveolar macrophages (18). In addition, tumor-associated macrophages have been reported to stimulate cloningof tumor cells in soft agar (19, 20) and a soluble factor has beenfound to be responsible for this effect (20). Thus, it would beof interest to clarify under what conditions lymphoid cells maysecrete inhibitory cytokines for tumor cell growth or producemerely growth factors.

In this paper, we studied the influence of conditioned mediafrom MNC and Con A-stimulated MNC cultures on in vitrogrowth of human hematopoietic and nonhematopoietic tumorcell lines, as assessed by a [3H]thymidine incorporation assay.

Using only a small number of cells per well and a low PCSconcentration or dithiothreitol-treated PCS in the assay, wefound that supernatants from nonstimulated MNC culturessignificantly enhanced tumor cell growth. On the contrary,supernatants from Con A-stimulated MNC cultures did notstimulate or stimulated only nonsignificantly tumor cell growthunless monoclonal antibodies against IFN-7 or TNF-a wereadded to the assay.

MATERIALS AND METHODS

The following tumor cells were used in this paper: U937, a humanhistiocytic lymphoma cell line; HL-60, a human promyelocytic leukemia cell line; K562, a human chronic myelogenous leukemia cell line;Jurkat. a human T-cell leukemia cell line; IM1', an immunoglobulin-secreting human B-cell line; WIL-2 NS, a non-immunoglobulin-secret-ing human B-cell line; SW-620, a cell line derived from a lymph nodemetastasis of colon adenocarcinoma; HeLa, an epithelioid cervix carcinoma cell line; and HT-1080 human fibrosarcoma cell line (ATCC,CCL 121). Also, two fibroblast cell lines have been used: Hs-27,newborn human foreskin (ATCC CRL 1634); and NRK-49F, normalrat kidney fibroblast (ATCC CRL 1570).

The tumor cells were grown in 10% FCS-enriched RPMI 1640 at37°Cin 5% CO2-containing water-saturated atmosphere. The cell lines

were maintained in culture and split twice a week. The tumor cellsgrowing in monolayer culture (SW 620, HeLa, Hs-27, NRK-49F) weretrypsinized with 0.025% trypsin (Gibco) in Puck's solution.

|3H|Thymidine Incorporation Assay. Tumor cells were suspended in5% DTT-treated FCS-enriched RPMI 1640 and adjusted at 105/ml.

Cell suspension (25 «1)and 50 »/Iof either medium or supernatants ofMNC or Con A-pulsed MNC cultures per well were incubated for 3days. [3HJThymidine (0.375 uCi; NEN, Boston, MA) per well added

24 h before cell harvesting. Cells growing in monolayer cultures weretrypsinized before being harvested. The dried filters containing cellswere counted in a beta counter.

Preparation of Supernatants of MNC or Con A-pulsed MNC Cultures.Peripheral blood mononuclear cells obtained from healthy volunteers(Swiss Red Cross) were isolated by Ficoll-Hypaque centrifugation of1:1 diluted blood and then suspended in serum-free RPMI 1640 at 3 x106/ml and cultured for 24 h. Supernatants obtained by centrifugation(250 x g) were concentrated 20-fold on an Amicon membrane (M,5000 cutoff) and kept at -80°Cuntil use. The 24-h preincubated MNC

were pulsed for 3 h with 0.5 mg/ml concanavalin A in 5% FCS-enrichedmedium, then washed with a-D-methyl mannoside (0.1 M), and further

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MNC-DERIVED TUMOR CELL GROWTH ACTIVITY

cultured for 24 h in FCS-free medium. Supernatants obtained bycentrifugation were concentrated and frozen as described above.

In some experiments, MNC were preincubated with either OKT3monoclonal antibodies (Ortho) and rabbit complement or anti-Leu-M2monoclonal antibodies (Becton-Dickinson) and rabbit complement(fresh rabbit serum, 1:5 final dilution). Both antibodies were added ata concentration of 0.5 >ig/ml.

Heat Inactivation of Supernatants. Samples of MNC culture supernatants were heated in a water bath at 56°C,60°C,or 80°Cduring 30

min before assay.Sensitivity to Low pH and DTT Treatment. pH was lowered to 2.5

with l M HC1 and Supernatants were left at room temperature for 2 h;then pH was adjusted to 7.2 with l M NaOH before assay. Othersamples were incubated for l h at room temperature with 0.065 Mdithiothreitol and then dialyzed overnight against RPMI 1640 at 4°C

while being stirred. Other supernatant samples were only dialyzed.Dialyzation was done using Spectrapor membrane tubing (A/, 6000cutoff).

Treatment of Supernatants with Anti-TNF-a or Anti-IFN-7 Antibodies. Monoclonal anti-TNF-a antibodies were used in the form of ahybridoma supernatant diluted 1:100 for final use (a gift from Dr. W.A. Buurman, Limberg University, Maastricht, The Netherlands).

Rabbit anti-TNF-a antibodies (Genzyme) exhibited 5x10' neutralizing units/ml. A 1:1000 final dilution was used. Monoclonal anti-IFN-7 antibodies (Bio-Science) were obtained from the supernatant of ahybridoma cell line at 4.4 x IO4neutralizing units/ml. It was used in a

1:100 final dilution. The antibodies were mixed with Supernatants ofMNC or Con A-pulsed MNC cultures and incubated for 2 h at 4"C

before assay.Treatment with Dithiothreitol of FCS. For inactivating growth factors

containing SH-bridges, FCS was treated with DTT as described previously (21). In brief FCS was incubated with 100 mM DTT (Sigma) for2 h at room temperature while being stirred and then dialyzed for 24 hat 4°Cagainst PBS (pH 7.4) by using Spectrapor membrane tubing (M,

6000 cutoff). Then, 0.5% iodoacetamide was added for another 2 h atroom temperature under constant stirring, followed by dialysis for 2days against PBS and another day against PBS containing 0.9 mMCaCl2 and 0.5 mM MgCl2.

RESULTS

Serum-free Supernatants from 24-h MNC cultures increased[3H]thymidine incorporation of tumor cells from human tumorcell lines. The assay consisted of a 3-day culture of an initial2500 tumor cells per well in medium supplemented with MNCculture Supernatants and DTT-treated FCS.

When native FCS was used in the assay, consistent stimulation of tumor cell growth by MNC culture Supernatants wasobtained only at serum concentrations below 2% as shown inFig. 1 (top). Progressive doubling of FCS concentration inmedium resulted in a steady enhancement of tumor cell proliferation, but in the presence of MNC culture Supernatants in adose-related reduction of proliferation at serum concentrationsgreater than 2%. Thus, FCS and the supernatant activity actedin synergy only at low serum concentration. This, however, wasnot observed with dithiothreitol-treated FCS, suggesting that aSH-containing serum component was responsible for the inhibitory effect. As shown in Fig. 1 (bottom), tumor cells proliferatedpoorly in medium enriched with dithiothreitol-treated FCS.However, if such cultures were supplemented with Supernatantsfrom MNC cultures, [3H]thymidine incorporation increasedsignificantly. Thus, DTT-treated FCS was used subsequently inall experiments for detecting the growth-stimulatory activity oftumor cells in this system.

On the other hand, the growth-enhancing activity of MNCculture Supernatants was more significant if low numbers oftumor cells per well at the onset of culture were used. At higher

1»

14

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fi

o) o» na u

•-2•„uo

„-41-0

-T-•-«toir*-±<r*-

='--r-ir4rtr-tr-4-%

DTT FCS 0 Öl O* US U S 10 IO

Fig. 1. Effect of Supernatants from nonstimulated MNC cultures on |'II|

thymidine incorporation of tumor cells cultured in the presence of increasingconcentrations of FCS (top) or of dithiothreitol-treated FCS (DTT-FCS) (bottom)U937 tumor cells (2.5 x lO'/well) were cultured in either medium (D) or MNCculture Supernatants (fR). [3H]Thymidine, 0.375 /iCi, was added 24 h before cellharvesting. Treatment of FCS with dithiothreitol was described in "Materials andMethods." Bars, SD.

Table 1 Influence of tumor cell density on expression of MNC-derivedgrowth-stimulatory activity

(3H]Thymidine incorporation (cpm)

Experiment0 Cells/well* Medium MNC-Sup' S.I.''

1.SW6202.

U9372.5

xIO36.25xIO31.25xIO42.5xIO43.75

xIO37.5xIO31.5x IO4315.3

±436,282.0±41125,480.0

±64458,713.5±1,015878.0+

1196,950.0±1,0781

8,51 8.0 ±1,3745,671.3

±28318,985.0±48855,859.6±72595,511.2+95715,

164.0±84933,676.0±1,85455,044.0±2,63818321.61753

" The tumor cells were cultured for 3 days in 1.66% dithiothreitol-treated FCS-

enriched RPMI 1640 in Microtest II plates.* [3H]Thymidine (0.375 ».Ci/well;NEN was added 24 h before cell harvesting.

For harvesting SW 620 cells were trypsinized.' Serum-free supernatant of MNC cultures was prepared as described in

"Materials and Methods" and used as supplement for tumor cell cultures (50%).'' S.I., stimulation index: the ratio of the mean cpm of MNC-Sup-supplemented

cultures to the mean cpm of control cultures.

numbers of cells per well, the difference in [3H]thymidine incorporation between control cultures and conditioned medium-enriched cultures became smaller in terms of stimulatory index(Table 1).

As for most lymphokine production schemes we arbitrarilyharvested culture Supernatants after 24 h of incubation. Wetherefore next studied the kinetics of factor production. Fig. 2shows that the highest level of activity was found after 24 h ofculture and that this decreased significantly after 48 h (Fig. 2).

The experiments using mononuclear cells pretreated witheither OKT3 or anti-Leu-M2 monoclonal antibodies and complement suggested that the growth-stimulatory activity is produced mainly by Leu-M2+ cells. This is .in agreement with

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fj

UM7 HLM SW410

Fig. 2. Kinetics of the growth-stimulatory activity production. Stimulation oftumor cell growth by MNC culture supernatants of 24 h (g), 48 h (ÜB),72 h (•),96 h ( |8'|), and 120 h (•)as compared to medium (D). Tumor cells were suspendedin 5% dithiothreitol-treated FCS-enriched RPMI 1640 at Id" ml. Cell suspension

(25 fil/well + either medium or conditioned media (SO *il) were incubated for 3days at 37°C.[3H]Thymidine (0.375 (iCi/well) was added 24 h before the end of

the cultures. For harvesting, SW 620 tumor cells were previously trypsinized.cpm represent mean ±SD (bars) from 6 individual cultures.

Fig. 3. Influence of treatment with OKT3 or anti-Leu-M2 monoclonal antibodies and rabbit complement upon growth-stimulatory activity production byMNC cultures. Tumor cells were cultured in medium (D) or supematants fromnative MNC cultures (ft)) or OKT3 + complement-pretreated MNC cultures (O),or anti-Leu-M2 -I-complement-pretreated MNC cultures (•).Treatment of MNCwith monoclonal antibodies and rabbit complement as well as tumor cell cultureswere done as described in "Materials and Methods." Bars, SD.

recent observations in a soft agar assay for tumor cell cloning(22). However, as can be seen in Fig. 3, unfractionated MNCcultures usually produced significantly higher growth-stimulatory activities than the corresponding MNC cultures lacking in

OKT3+ cells. This suggested that T-cells can also produce orregulate production of this growth stimulatory factor. This ideawas strengthened also by our observations that X-irradiation ofmononuclear cells before performing the cultures significantlyincreased the yield of activity in the supernatant (data notshown).

Interestingly, supernatants from Con A- or LPS-stimulatedMNC cultures were less effective in inducing proliferation inthis system. These data suggested that unstimulated mononuclear cells may release a factor(s) capable of enhancing thegrowth of various tumor cell lines of hematopoietic origin suchas U937, HL-60, Jurkat, IM-9, Wil-2 NS, K562, as well as ofnonhematopoietic origin such as SW 620 and HeLa. Some celllines (K562, HeLa) were only nonsignificantly enhanced intheir proliferation as can be seen in Fig. 4. MNC culturesupernatants also moderately stimulated [3H]thymidine incorporation of TH 1080 fibrosarcoma, Hs-27 foreskin fibroblast,and NRK-49 F cell lines (Table 2).

That supernatants of Con A-pulsed a-D-methyl mannoside-washed MNC cultures only poorly enhanced tumor cell proliferation, in contrast to nonstimulated MNC cultures, might bedue to the presence of growth-inhibitory cytokines (23). Indeed,when monoclonal antibodies directed to IFN-7 or to TNF-awere added to supernatants from Con A-stimuIated MNC cultures before assay, [3H]thymidine incorporation increased sig

nificantly. As can be seen in Fig. 5, a synergistic stimulatoryeffect was observed when both antibodies were supplied tocultures, confirming previous observations that IFN-7 andTNF-a have synergistic antiproliferative effects on certain tu

mor cell lines (24). This suggested that the supernatant activitywhich enhances growth may be counteracted by both IFN-7and TNF-a. Thus lectin-stimulated MNC cultures actuallyproduced higher levels of growth-inducing activity, which wasnot apparent when TNF-a and IFN-7 were not inhibited byspecific antibodies. Moreover, monoclonal antibodies to IFN-7 and TNF-a increased moderately but significantly the growth-stimulatory effect from supernatants of nonstimulated MNCcultures, suggesting that small amounts of these cytokinesmight also be produced by nonstimulated MNC cultures buttheir effect is actually hidden by the growth-stimulatory factor^) (Fig. 6).

We next attempted to characterize this growth-inducing activity. As can be seen in Fig. 1A, the enhancing activity fortumor cell growth was totally inactivated by incubation at pH2.5 for 2 h. The biological activity seems to be heat sensitivesince it was destroyed partially at 56°Cand completely at 80°C.

Treatment with dithiothreitol of conditioned medium totallyabolished growth-stimulatory activity for tumor cells, suggesting that disulfide bridges are required for biological activity.Dialyzation against medium of MNC culture supernatants decreased growth-stimulatory activity (Fig. IB).

Further, we investigated the role in this effect of some wellcharacterized cytokines such as IL-1, IL-2, IL-6, and colony-stimulating factors. As can be seen in Table 3, IL-6 moderatelystimulates [3H]thymidine incorporation of the SW 620 tumor

cell line but is inactivated by neither heating nor incubation atlow pH. Also, supernatants of MNC cultures did not increase[3H]thymidine incorporation of mouse thymocytes or Con A-

pulsed peripheral blood mononuclear cells, suggesting that II,1 and IL-2 activities were lacking. Moreover, monoclonal antibodies directed to IL-1, when mixed with MNC culture supernatants, did not impair but moderately increased [3H]thy-

midine incorporation of SW 620 tumor cells. This suggestedthat IL-1 might not play a significant role as a growth-stimu-

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Fig. 4. Influence on tumor cell growth ofsupernatants from nonstimulated MNC cultures (B) or from MNC cultures stimulatedby Con A (M) or by Escherichìacoli lipopoly-saccharide (•)as compared to medium (O).[3H]Thymidine incorporation assay was doneas described in "Materials and Methods."

Bars, SD.

11

10

n-

le-

I«uo- n

w

( l

mIM-« J->" «m-l «541

Table 2 Stimulation offìbroblastcell ¡inegrowth by MNC culture supernatants

Experiment123Cells"HT-

1080Hs-27NRK-49FCultureMediumSup.MNCMedium

Sup.MNCMedium

Sup. MNCcpm*1137

±802639 ±1871744

±203445 ±348380

±781388 ±71S.I.C2.32.03.6P"<0.001<0.001<0.001

"HT-1080, human Fibrosarcoma cell line; Hs-27, newborn human foreskinfibroblast cell line; NRK-49F, normal rat kidney fibroblast cell line. The cellswere suspended in 5% dithiothreitol-treated FCS-enriched RPMI 1640. Cellsuspension (25 ¿il)and of medium or MNC culture supernatant (Sup. MNC) (50,1!)were incubated for 3 days at 37'C. |3H]Thymidine (0.375 nCi/well was added

after 48 h of culture. The cells were trypsinized before harvesting.* Mean ±SD of the cpm from 6 identical cultures.1S.I., stimulatory index.'' Student's r test between mean values from stimulated and control cultures.

latory activity in this system. Also, supernatants of nonstimulated MNC cultures did not induce colony formation on humanbone marrow cells cultured in soft agar, suggesting that colony-stimulating factors might not be responsible for a growth-stimulatory effect on tumor cells (Table 3).

DISCUSSION

This study shows that in vitro growth of some human hema-topoietic and nonhematopoietic tumor cell lines was enhancedby supernatants from MNC cultures but not significantly influenced by supernatants from Con A-stimulated MNC cultures.

However, supernatants of Con A-stimulated MNC culturesexhibited even greater growth-stimulatory activity than thatfrom MNC culture supernatants when mixed with monoclonalantibodies directed to IFN-7 or TNF-a.

Two conditions were required to demonstrate growth-stimulatory activity for tumor cells, namely, a small number of cellsper well and low serum concentration in the assay. By increasingthe number of tumor cells per well, [3H]thymidine incorporation

increased both in cultures containing only medium and in thoseenriched with conditioned medium decreasing thus the rate ofproliferation. The increased proliferation in medium alone mayreflect an autocrine stimulatory mechanism but this should befurther verified. By increasing the concentration of PCS in themedium, tumor cell proliferation was proportionally enhanced,

HL60

Fig. 5. Blocking biological activity of IFN--y or TNF-a by monoclonal antibodies increases growth-enhancing activity of Con A-stimulated MNC culturesupernatants. The tumor cell cultures were done in medium (D), Sup. MNC (M ),Sup. MNC. Con A (HI),Sup. MNC. Con A -I-anti-IFN--x monoclonal antibodies(•),Sup. MNC. Con A + anti-TNF-a (151),Sup. MNC Con A + anti-IFN-f +anti-TNF-a monoclonal antibodies (II). 25 j/l tumor cell suspension UOVml in5% DTT-FCS-enriched RPMI 1640) + 50 nl medium or supernatant per wellwere incubated for 3 days at 37°C.[3H)Thymidine (0.375 (iCi/well) was added 24

h before cell harvesting. Bars, SD.

but in the presence of conditioned medium, increasing PCSconcentrations decreased [3H]thymidine incorporation. How

ever, this was not observed if PCS was previously treated withdithiothreitol, suggesting that a SH-containing factor was re-

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30-,

937

Fig. 6. Increasing of the stimulatory activity for tumor cell growth in supernatants of nonstimulated MNC cultures by monoclonal antibodies, ant ill N -or anti-TNF-a. Tumor cell cultures were done as described in Fig. 5. Medium(D), Sup. MNC (I I), Sup. MNC + anti-IFN-7 (•),Sup. MNC -I-anti-TNF-a(o), Sup. MNC + anti-IFN-7 + anti-TNF-a (•),Sup. MNC Con A, ( 4 Sup.MNC Con A + anti-IFN-7 (El), Sup. MNC Con A + anti-TNF-a (|||f). Sup.MNC Con A -I-anti-IFN-7 + anti-TNF-a monoclonal antibodies ( H ). Bars, SD.

sponsible for the growth-inhibitory effect. One candidate for aserum factor which inhibits tumor cell proliferation in thepresence of conditioned medium, is TGF-0 which has beenreported to either inhibit or stimulate proliferation in the presence of other growth factors (25). TGF-/3 is found in importantamounts in serum and is released from «-granulesof platelets

during coagulation (26).The growth-stimulatory activity for tumor cells presented in

this paper may be biologically distinct from a number of wellcharacterized factors. Thus, IL-6, which stimulates proliferation of various cell lines of B-cell, T-cell, or non-B-non-T-cellorigin (27) as well as colony formation of some hematopoieticstem cells (28), induces only poorly [3H]thymidine incorpora

tion of SW 620 tumor cells (Table 3). The different sensitivityto heating and low pH suggested that IL-6 is not involved ingrowth-stimulatory activity from this system. On the otherhand, the supernatants of nonstimulated MNC cultures whichenhance proliferation of tumor cells are ineffective in the thy-mocyte assay. Monoclonal antibodies directed to IL-1, whenadded to supernatants of MNC cultures, increased moderately

!«

It

II

16

¿I

jFig. 7. Influence of low pH, heating, dithiothreilol treatment (A), or dialyza-

tion (B) upon the stimulatory activity for tumor cell growth from supernatants ofMNC cultures Tumor cell cultures were performed in medium (D), Sup. MNC(i ]), Sup. MNC-preincubated at pH 2.5 (I ), Sup. MNC heated at either 56'C(m) or 60'C (I I), or 80"C (El), Sup. MNC pretreated with dithiothreitol TO,Sup. MNC dialyzed against medium (•).Tumor cell cultures for [3H]thymidineincorporation were done as described in "Materials and Methods." Bars, SD.

Table 3 Effect of other cytokines on induction of tumor cell growth

Experiment1Assay"[3H]dThdCellsSW620Culture*Medium

IL-6IL-6, 80°CIL-6, pH 2.5cpmc142.7

±18434.8 ±75834.4 ±52

1,001.8 ±162

2345[3H]dThd[3H]dThd[3H]dThdColony

formationSW620Mouse

thymocytesCon

A-pulsedBone

marrowMedium

Sup. MNCSup. MNC +a-IL-1Medium

Sup. MNCSup. MNC. ConAMedium

Sup. MNCSup. MNC. ConAMedium

Sup. MNCSup. MNC. Con A75.5

±241,683.0 ±3102,1 72.3±56651.3

±1731.0 ±9

2,802.0 ±4633,970.7

±1063,920.9 ±143

17,047.6±3460

2.2 ±146.4 ±9

* [3H]dThd, 3 days [3H)thymidine incorporation assay using either 2500 SW620 tumor cells per well in 1.66% dithiothreitol-treated FCS-enriched RPMI1640 or lO'/well mouse thymocytes in 10% FCS-enriched medium, or 10*/wellCon A-pulsed a-D-methyl mannoside-washed mononuclear cells in 10% FCS-enriched medium. Colony formation, granulocyte-macrophage colony formationassay (performed in the Hematological Central Laboratory, Inselspital) was doneusing mononuclear bone marrow cells from the iliac crest of healthy volunteersin 3% agar and 20% FCS-enriched McCoy's 5A modified medium. Values

represent numbers of colonies (>50 cells) after 2 weeks of culture.b Recombinant IL-6, 1 *ig/ml. In some cultures IL-6 was previously heated for

30 min at 80°Cor incubated for 2 h at pH 2.5 at room temperature. Sup. MNC,serum-free MNC culture supernatant. In some cultures Sup. MNC was mixed100:1 with ami 11 I monoclonal antibodies (a-IL-1). Sup. MNC. Con A, serum-free supernatant of Con A-pulsed a-D-methyl mannoside-washed MNC cultures.

' Mean ±SD from 4-6 individual cultures.

[3H]thymidine incorporation of SW 620 tumor cells (Table 3).

This is in agreement with some recent observations showingthat IL-1 both inhibits tumor cell growth (29, 30) and exhibitssynergistic antiproliferative effects with other cytokines (31,32). Our results suggest also that MNC-derived growth-stimulatory activity for tumor cells is distinct from CSFs since no

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significant colony formation was found for human bone marrowcells when supernatants of nonstimulated MNC cultures wereused in this assay. TGF-/3, which is produced by a variety ofcells including lectin-activated MNC but only in minuteamounts by nonstimulated MNC (33), inhibits cell proliferation(25, 34). However, in the presence of TGF-a or of EGF, TGF-ßinduces anchorage-independent growth of nonneoplastic in

dicator cells in soft agar (25). Our preliminary observationssuggest that conditioned media from MNC cultures in thepresence of FCS can also stimulate anchorage-independentgrowth of NRK-49F cells in soft agar (not shown) being similarto monocyte-derived growth factor which has been recentlyreported to exhibit this function (20, 22). However, monocyte-derived growth factor was resistant to dithiothreitol treatmentand to dialyzation (20). Furthermore, the kinetics of the factorproduction is different. Under serum-free conditions, monocyte-derived growth factor was detected only after 48 h and wasmaximal after 5 days (20). Therefore, we will further investigatethe nature of this growth-stimulatory activity for tumor cells.Despite all the dissimilarities described here, it might be analready known factor, but our data suggest it to be a new entity.The fact that this factor is produced without intentional stimulation and is counteracted by IFN--y and TNF-a acting on the

same tumor cell makes it a potential regulator of in vivo growthof tumor cells.

ACKNOWLEDGMENTS

We thank Christen Franziska and Mathys Daniel for excellent technical assistance. We are grateful to Dr. W. A. Buurman, from LimbergUniversity, Maastricht, The Netherlands, for providing the monoclonalanti-TNF-a. We thank also the following persons and institutions fromBerne for kindly offering us the tumor cell lines: Drs. Jungi and Morel(Tiefenauspital); F. Bettens (Institute of Immunology); A. Ziemiecki(Ludwig Institute): K. Blaser (Institute of Chemistry); and R. Schindler(Institute of Pathology).

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1988;48:5411-5416. Cancer Res   George Sandru, Peter Veraguth and Beda M. Stadler  Cell-derived FactorStimulation of Tumor Cell Growth in Humans by a Mononuclear

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