[CANCER RESEARCH 47, 5382-5385, October 15, 1987)
Modulation of the Growth of Transformed Cells by Human Tumor
Necrosis Factor-«and Interferon-7
Gail D. Lewis,1 Bharat B. Aggarwal, Thomas E. Eessalu, Barry J. Sugarman,2 and H. Michael Shepard
Departments of Pharmacological Sciences ¡G.D. L., B. J. S., H. M. S.J and Molecular Immunology [B. B. A., T. E. E.J, Genentech, Inc.,
South San Francisco, California 94080
ABSTRACT
Recombinant human tumor necrosis factor-a (ri lui NT-«)inhibited
growth of the cervical carcinoma cell line, ME-180*â„¢,at doses greater
than 50 units/ml, but stimulated the growth of these cells at low doses
(0.1-10 units/ml). ME-180"*0 variants selected for resistance to the
cytotoxic effects of rHuTNF-o retained the ability to be growth stimu
lated at all concentrations tested. ME-ISO**"cells and the rHuTNF-oresistant Ml -ISO""' variants possessed equivalent steady state numbers
of TNF-a receptors with similar Afdvalues. Recombinant human interferon-T (rHu!FN--y) augmented the rHuTNF-a-induced cytotoxic re
sponse of ME-ISO"â„¢cells and overcame the resistance of the MK-180 '
variants to rHuTNF-a cytotoxicity. In separate experiments we were
able to show that the number of TNF-a binding sites on both rHuTNFa-sensitive and -resistant M K-180"â„¢
cells was similar and was increased
by treatment with rHuIFN-7. These results suggest that the growth
stimulation of tumor cells mediated by rHuTNF-a can be dissociated
from the cytotoxic response and that these responses are not related to
the number or affinity of TNF-a binding sites.
INTRODUCTION
The cytotoxic effects of activated macrophages are mediated
through a number of factors including interleukin 1(1), arginase
(2, 3) and reactive oxygen intermediates, such as Superoxide
anión and hydrogen peroxide (3, 4). TNF-a3 has also been
shown to be a major effector of macrophage-induced tumor cell
cytotoxicity (3, 5-7). This monokine is known to induce hemorrhagic necrosis of Meth A sarcomas in vivo (8-10), to inhibit
the growth of various transformed cells in vitro (11-13), and to
enhance the antiproliferative activity of IFN-7 in vitro (13-16)
and in vivo (17). In addition to these anticellular and antitumor
effects, TNF-a can inhibit lipid biosynthesis (18, 19), activate
polymorphonuclear
neutrophils (20-22), and augment the
growth of normal diploid fibroblasts (13, 23).
In the present work, we investigated the responses to
rHuTNF-a of ME-180neo cervical carcinoma cells and of MEjgQneo variants selected for resistance to rHuTNF-a-induced
cytotoxicity. The results of these studies show that (a) depend
ing on its concentration, rHuTNF-a can either stimulate or
inhibit tumor cell growth, (b) sensitivity to rHuTNF-a is not
proportional to steady state receptor number, and (c) induction
of TNF-a binding sites by rHuIFN--y may correlate with the
synergistic reduction in cell growth observed in the presence of
both cytokines on either rHuTNF-a-sensitive
or -resistant
clones of ME-180nco tumor cells.
MATERIALS
AND METHODS
Cytokines. Highly purified recombinant DNA-derived HuTNF-a (9)
and HuIFN-T (24) were prepared at Genentech, Inc. The specific
Received 8/8/86; revised 4/15/87; accepted 7/21/87.
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 in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1To whom requests for reprints should be addressed, at Department of
Pharmacological Sciences, Genentech, Inc., 460 Point San Bruno Boulevard,
South San Francisco, CA 94080.
2 Present address: AMGEN, Thousand Oaks, CA 91320.
'The abbreviations used are: TNF, tumor necrosis factor, IFN, interferon;
r 11».recombinant human; HTR, human tumor necrosis factor resistant.
activities of these cytokines were 5 x IO7 units/mg for rHuTNF-a and
4 x IO7 units/mg for rHuIFN-f. The specific activity of rHuTNF-a
was determined in an L-M-cell cytotoxicity assay in the presence of
actinomycin D (25). The specific activity of rHuIFN-> was determined
by a cytopathic effect inhibition assay using encephalomyocarditis virus
infection of A549 cells. Titers of rHuIFN-y were standardized against
the NIH reference standard No. Gg 23-901-530.
Cell Lines. ME-180 cervical carcinoma cells were obtained from the
American Type Culture Collection (Rockville, MD). Clones of ME180 cells selected for resistance to neomycin (ME-ISO1*0) after trans
fection with pSVEne°Bal6(26) were grown in McCoy's 5A media
(GIBCO, Grand Island, NY) supplemented with 10% (v/v) heat-inac
tivated fetal bovine serum (Armour, Kankakee, IL), 80 units/ml peni
cillin G, 80 Mg/ml streptomycin sulfate, and 400 pg/ml geneticin (G418 sulfate; GIBCO). rHuTNF-a-resistant
variants were cloned by
subculturing ME-180"" cells in media containing 5000 units/ml
rHuTNF-a. Individual colonies were isolated using cloning cylinders
(Bélico
Glass, Inc., Vineland, NJ) and expanded for further character
ization. Cell lines were routinely tested for and found free of Mycoplasma infection.
Incubations with Cytokines. Ml-:-Iso1"'" cells were treated with
rHuTNF-a alone or in combination with rHuIFN-T and stained with
crystal violet for determination of cell viability as previously described
(13, 27, 28). To determine changes in cell number after incubation with
rHuTNF-a, the monolayers were washed with phosphate-buffered sa
line, detached with trypsin, and viable cells were counted by trypan blue
exclusion using a hemocytometer. In both types of assays, each group
consisted of 4-8 replicates and the coefficient of variance was less than
10%.
In some experiments, [3H]thymidine incorporation into DNA was
measured. For this assay, cells were seeded into 96-well plates (IO4
cells/well) and incubated for 72 h with different concentrations of
rHuTNF-a. [3H]Thymidine (80 Ci/mmol; New England Nuclear, Bos
ton, MA) was added (1 ¿/Ci/well)for the last 4 h of the incubation
period and the cells were harvested as previously described ( 13). Eight
replicates were used for each treatment group and the coefficient of
variance was less than 10%. Experiments measuring changes in relative
percentage of viability, cell number, or [3H]thymidine incorporation
were performed at least 4 times. The data shown are those of repre
sentative experiments.
Receptor Binding Experiments. lodinated rHuTNF-a was prepared
as previously described (29). For '"I-labeled rHuTNF-a binding stud
ies, ME-180"0 or ME-180~°-HTR (2 x 10s cells/well) were grown
overnight in 12-well plates (Costar, Cambridge, MA). After washing
the monolayers once with phosphate-buffered saline, rllull-'N--, (5000
units/ml) or media alone were added to the cultures. After 18 h, the
cells were washed once with media and incubated for 2 h at 37*C with
2 x IO5cpm '"I-labeled rHuTNF-a (-800 mCi/^moI) in the presence
or absence of a 100-fold molar excess (Fig. 3), or with a range of
concentrations (Fig. 4), of unlabeled rHuTNF-a. The monolayers were
then washed 3 times with media and solubilized with 2% sodium
dodecyl sulfate for determination of cell-bound radioactivity. Specific
binding of I25l-labeled rHuTNF-a represents the difference between
total and nonspecific binding. Each experiment was performed 3 times
and all measures were made in triplicate.
RESULTS
Response of ME-18011*0Cells to rHuTNF-a. Changes in the
growth of rHuTNF-a-sensitive
and -resistant ME-ISO"10 cells
after a 72-h incubation with various concentrations of rHuTNF-
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MODULATION
OF TUMOR CELL GROWTH BY rHuTNF-a AND rHuIFN--y
a are shown in Fig. 1. The ME-180nco parental cells exhibited
a biphasic growth response to rHuTNF-a. Concentrations
greater than 50 units/ml had an antiproliferative effect, whereas
low doses (0.09-21 units/ml) enhanced the growth of these
cells. The inset to Fig. 1 shows that the same growth response
pattern occurred when cell number was determined by counting
viable cells in a hemocytometer. rHuTNF-a-resistant
MEISO1*0variants (ME-180ne°-HTR10, -25, -30, -31) treated with
the same concentrations of rHuTNF-a demonstrated a broad
optimum response (30-60% above controls) which decreased
to slightly more than control levels by 5000 units/ml.
In separate experiments, changes in [3H]thymidine incorpo
Table 1 Effect ofrHuTNF-a
on the growth ofME-l8O~° and ME-l8(T°-HTRil
tumor cells
ME-180~°cells were incubated for 72 h with rHuTNF-a. [3H]Thymidine (1
pCi/well) was added for the last 4 h of the incubation period and the cells were
harvested for determination of [3H]thymidine incorporation. The data are ex
pressed as a percentage of [3H]thymidine incorporated into DNA compared to
control cells (n = 8 for each group). Mean cpm ±SE for [3H]thymidine incorpo
ration into control cells was 58,813 ±7,539 for ME-180~°and 56,769 ±3,420
forME-180~°-HTR31.
| '111l'h> inuline incorporation
rHuTNF-a(units/ml)00.090.250.762.296.8620.5861.73185.19555.561,666.675,000.00ME-180~°100141
ration were measured to corroborate the different growth re
sponses of ME-180neo cells to rHuTNF-a (Table 1). Increased
[3H]thymidine uptake correlated with the enhanced growth
responses of ME-180neo and ME-180neo-HTR31 as measured
by staining (Table 1 and Fig. 1). Similar results were obtained
with the other ME-ISO1"0 variants (data not shown). Likewise,
the antiproliferative effect of higher doses of rHuTNF-a on
ME-ISO1*0 cells was reflected by a decrease in DNA synthesis.
The data in Table 1 and Fig. 1 indicate that the maximum
proliferative responses to rHuTNF-a of both ME-180neo and
ME-180neo-HTR31 cell lines occurred at less than 10 units/ml.
Although the magnitude of increase or decrease in cell growth
varied somewhat between experiments (compare Fig. 1 and
Table 1), these results show that by 2 independent methods the
changes in proliferation of ME-180neo cells in response to
rHuTNF-a are also reflected by changes in DNA synthesis.
Characterization of rHuTNF-a-Resistant
Ml -ISO" ' Tumor
Cells. As shown in Fig. 2, rHuIFN-7 inhibited the proliferation
of rHuTNF-a-sensitive (Fig. 2A) and -resistant (Fig. 2B) tumor
cells. However, only the ME-ISO1"0 parental line was sensitive
to the cytotoxic effects of rHuTNF-a. Treatment of ME-180nco
cells with combinations of rHuTNF-a and rHuIFN-7 had an
enhanced antiproliferative effect similar to that reported pre
viously for the ME-180 parent line (Fig. 2A; Ref. 13). rHuTNF«-inducedproliferation of ME-180"™-HTR31 was abrogated by
rHuIFN-7 (Fig. 2B). In addition, some combinations of
rHuTNF-a
and rHuIFN-7 (e.g., 5, 50, or 500 units/ml
rHuTNF-a plus 0.1, 0.5, or 5 units/ml rHuIFN--y) resulted in
cytotoxicity greater than that observed with rHuIFN--y alone.
These results suggest that under some conditions exposure of
ME-lSO^-HTRSl
to rHuIFN-^ overcomes the rHuTNF-a-
160
«
140
0.1
120
100
ID
JS
(D
cc
5
50
500
Fig. 2. Enhancement of the antiproliferative effect of rHuTNF-a by rHuIFNy on tumor cells. ME-180~° cells (A), and the rHuTNF-cr-resistant ME-180~°
variant, HTR31 (B), were incubated for 72 h with rHuTNF-a alone (•),rHuIFNy alone (•),or various concentrations of rHu!FN--x plus 0.1 (O), 0.5 (D), 5 (A),
50 (x), or 500 (0) units (f/)/ml rHuTNF-a. The monolayers were then washed
with phosphate-buffered saline and stained with crystal violet for determination
of cell viability.
I
s.
0.5
rHuTNF-a or rHulFN-y (U/ml)
80
60
40
20
0.1
10
100
1000
10000
Concentration rHuTNF-a (U/ml)
Fig. 1. Effect of rHuTNF-a on the growth of rHuTNF-a-sensitive
and
-resistant tumor cells. ME-180~°or ME-180~°-HTR tumor cells were incubated
with various concentrations of rHuTNF-a for 72 h and stained with crystal violet
for determination of viable cell number. Points, mean of 8 replicates; the coeffi
cient of variance was less than 10%. Inset, changes in ME-180"â„¢cell number in
response to rHuTNF-a as measured by counting viable cells using trypan blue
dye exclusion. U, units.
resistant phenotype. Other rHuTNF-a-resistant ME-ISO1*0var
iants responded to combinations of rHuTNF-a and rHuIFN-f
in a similar fashion (data not shown).
Modulation of TNF-a Receptors by rHuIFN-y. Further ex
periments were undertaken to determine whether there was a
correlation between changes in the number of TNF-a binding
sites and the various responses of ME-180neo tumor cells to
combinations of rHuTNF-a, either in the presence or absence
5383
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MODULATION
OF TUMOR CELL GROWTH BY rHuTNF-a AND rHuIFN-x
of TNF-a receptors (1300 and 1000, respectively) and that the
receptor number in both cell types was increased 1.5-fold by
preincubation with 5000 units/ml rHuIFN--y. The results of
these TNF-a binding experiments (Figs. 3 and 4) demonstrate
that the resistance of the ME-ISO^-HTR variants to rHuTNFa is not due to a lack of TNF-a receptors. In addition, the
increase in rHuTNF-a binding sites by pretreatment with
rHuIFN-7 in ME-1801"0 and the ME-180neo-HTR cell lines
suggests one mechanism which may explain the enhanced cytotoxicity observed in the presence of both cytokines.
2000
\
1000
I
1
m
J
DISCUSSION
ME-180
HTR25
HTR30
HTR31
Fig. 3. Effect of rHuIFN-y on binding of '"I-labeled rHuTNF-a to ME-180"0
and ME-180"~-HTR tumor cells. After incubation for 18 h in the absence (O) or
presence (O) of 5000 units/ml rHuIFN-% the monolayers were washed and
incubated for 2 h with '"I-labeled rHuTNF-a plus media alone or a 100-fold
molar excess of unlabeled rHuTNF-a. The data are specific binding of "M-labeled
rHuTNF-a, mean cpm ±SE (bars).
„ 3000
0001
0-01
0.1
10
[rHuTNF-a],
10
nM
0001 001
0.1 1.0 10
[rHuTNF-a], nM
100
Fig. 4. Competition of '"I-labeled rHuTNF-a binding by various concentra
tions of unlabeled rHuTNF-a in ME-180~°cells (A) or in the rHuTNF-a-resistant
ME-1801*0 variant, HTR31 (B). Cells received no pretreatment (•,O) or were
pretreated for 18 h with 5000 units/ml rllull N -. {•.
O). Insets, Scatchard plot
analyses of 12!I-labeled rHuTNF-a binding to ME-180~> cells (A) and ME-180~>HTR31 (/>')under the same conditions.
of rHuIFN-7. Binding of I25l-labeledrHuTNF-a to ME-1801"0
cells and the rHuTNF-a-resistant ME-1801"0variants is shown
in Fig. 3. The number of rHuTNF-a binding sites on the
variants was similar to the parental cell line, with the greatest
difference seen on ME-180neo-HTR30, which has approxi
mately one-half the number of rHuTNF-o binding sites as
compared to the ME-1801"0parent. Treatment of the 4 MEISO^-HTR variants and the parental cell line with 5000 units/
ml rHuIFN-7 for 18 h enhanced the binding of labeled
rHuTNF-a 1.5- to 2-fold.
Since the rHuTNF-a-resistant variants did not appear to
differ substantially from the parental cell line with regard to
IM L binding sites, further experiments were undertaken to
determine if the affinity of the TNF-a receptor for its ligand
was altered in the rHuTNF-a-resistant cells. 125I-labeled
rHuTNF-a competition binding assays were performed on MEISO"â„¢
cells and the rHuTNF-a-resistant variant, HTR31. The
data in Fig. 4 show that the I25I-labeledrHuTNF-a binding
isotherms for ME-1801"0 and HTR31 are similar, and that
pretreatment of both cell types with rHuIFN-7 increased the
binding of labeled rHuTNF-a. Scatchard plot analyses of these
curves (Fig. 4, insets) indicate that the affinity constants for
both cell types are similar (Kt = 0.1-0.2 nM). These data also
show that ME-1801"0and HTR31 possess equivalent numbers
The results presented here demonstrate that the growth en
hancing activity of rHuTNF-a occurs not only on normal cells
but also on tumor cells which are sensitive or resistant to the
cytotoxic effects of this monokine. ME-1801"0tumor cells sen
sitive to the cytotoxic effects of rHuTNF-a exhibited a biphasic
response. Concentrations of rHuTNF-a greater than 50 units/
ml inhibited the growth of these cells, whereas lower doses
actually enhanced cell growth. The biphasic effect of rHuTNFa on cell growth was also observed in murine L-929 cells, NIH
3T3 fibroblasts, and B16-F10 melanoma cells (data not shown).
The maximum growth stimulation of ME-1801"0cells selected
for resistance to rHuTNF-a-induced cytotoxicity was similar
to that reported for normal fibroblasts (13, 23). The results of
binding studies using I25l-labeled rHuTNF-a indicated that
there were no substantial differences in the steady state levels
of TNF-a receptors between the ME-1801"0parental cells and
the rHuTNF-a-resistant ME-1801"0 variants. These findings
are similar to previous reports showing that the different growth
responses of various cell types appear to be unrelated to TNFa receptor number or affinity (13, 30, 31).
The mechanisms involved in the enhancement of rHuTNFa-induced cytotoxicity by rHuIFN-7 are unclear. It is possible
that this augmented cytotoxic response is related to induction
by rHuIFN-7 of a different type of TNF-a receptor. This
hypothesis is supported by our finding that ME-1801"0
rHuTNF-a-resistant variants have levelsof TNF-a binding sites
similar to the parental cell line and that rHuTNF-a binding is
increased by treatment with rHuIFN-7. Only in the presence
of rHuIFN-7 do the rHuTNF-a-resistant variants become sen
sitive to the cytotoxic effects of rHuTNF-a. This result has
important clinical significance since it provides a rationale for
combination therapy with rHuTNF-a and rHuIFN-7 for pro
gressively growing tumors which have developed resistance to
macrophage (TNF-a)-mediated tumor cell cytotoxicity (5, 32).
In summary, rHuTNF-a is distinguishable from most other
well-characterized growth factors by its ability to both enhance
and inhibit the growth of the same tumor cell type in a dosedependent manner. Although transforming growth factor-/?also
stimulates or inhibits cell proliferation, these activities have
been shown only on different cell types. For example, growth
of mesenchymal cells is stimulated by transforming growth
factor-/•>,
whereas cells of epithelial origin are growth inhibited
(33-35). An unusual example of a growth factor either stimu
lating or inhibiting cell growth as a function of concentration
is the action of epidermal growth factor on A431 human
epidermoid carcinoma cells (36). The response of ME-1801"0
carcinoma cells to rHuTNF-a differs from that of normal
fibroblasts, which respond only by enhanced proliferation, and
suggests that tumor cells have acquired multiple response path
ways to this monokine. The mechanism(s) underlying this bi
phasic response, either in the case of rHuTNF-a or epidermal
growth factor, is unknown.
5384
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MODULATION
Our ability to isolate rHuTNF-a-resistant
OF TUMOR
CELL GROWTH
ME-180neo tumor
cell clones, which have lost their capacity to respond to the
cytotoxic effects of rHuTNF-a while retaining their response
to the growth-promoting activity of this cytokine, further indi
cates that there must be 2 pathways of response to rHuTNF-a
in these tumor cells. Because TNF-a is a major component of
macrophage-mediated tumor cell cytotoxicity (5-7), our results
suggest that a possible mechanism of tumorigenesis in vivo
could be the selection in situ within developing tumors of
subpopulations of tumor cells resistant to TNF-a. Enhanced
proliferation induced by TNF-a of tumor cells resistant to its
cytotoxic activity would confer a selective growth advantage to
these cells. The macrophage, which may be an important early
negative regulator of tumor cell proliferation (32, 37), would
then actually potentiate tumor growth. In fact, previous work
suggests that tumor growth in vivo can be enhanced by intratumoral macrophages (38, 39). Moreover, in in vitro studies,
adherent cells were shown to increase tumor cell growth as
measured by colony formation (40). It will be of considerable
interest to use genetic variants selected to be defective in the
cellular response pathways to TNF-a as a means of further
dissecting the mechanisms controlling tumor cell proliferation.
15.
16.
17.
18.
19.
20.
21.
22.
23.
ACKNOWLEDGMENTS
The authors wish to thank Dr. David W. Martin, Jr. and Barbara
Levinson of Genentech, Inc. for assistance with the initial stages of this
project, and Dr. Peter Lengyel of Yale University for helpful discus
sions.
24.
25.
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Modulation of the Growth of Transformed Cells by Human
Tumor Necrosis Factor- α and Interferon-γ
Gail D. Lewis, Bharat B. Aggarwal, Thomas E. Eessalu, et al.
Cancer Res 1987;47:5382-5385.
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